Enhanced drug delivery to melanoma cells using PMPC-PDPA polymersomes

Enhanced drug delivery to melanoma cells using PMPC-PDPA polymersomes

Cancer Letters 334 (2013) 328–337 Contents lists available at SciVerse ScienceDirect Cancer Letters journal homepage: www.elsevier.com/locate/canlet...

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Cancer Letters 334 (2013) 328–337

Contents lists available at SciVerse ScienceDirect

Cancer Letters journal homepage: www.elsevier.com/locate/canlet

Original Research Article

Enhanced drug delivery to melanoma cells using PMPC-PDPA polymersomes Carla Pegoraro a,b,c,d,e,f,1, Denis Cecchin a,b,c,d,1, Lorena Simon Gracia d,h, Nicholas Warren a,b,c,d,g, Jeppe Madsen a,b,c,d,g, Steven P. Armes g, Andrew Lewis i, Sheila MacNeil c,e,f, Giuseppe Battaglia a,b,c,d,⇑ a

The Krebs Institute, University of Sheffield, Sheffield, UK The Centre of Membrane Interaction and Dynamics, University of Sheffield, Sheffield, UK The CRUK/YCR Sheffield Cancer Research Centre, University of Sheffield, Sheffield, UK d Department of Biomedical Sciences, University of Sheffield, Sheffield, UK e Department of Materials Science and Engineering, University of Sheffield, Sheffield, UK f Kroto Research Institute, University of Sheffield, Sheffield, UK g Department of Chemistry, University of Sheffield, Sheffield, UK h Institute for Research in Biomedicine, University of Barcelona, Barcelona, Spain i Biocompatibles UK Ltd., Farnham, UK b c

a r t i c l e Keywords: Polymersomes Melanoma Targeted delivery Doxorubicin

i n f o

a b s t r a c t We present the efficient and stable encapsulation of doxorubicin within pH sensitive polymeric vesicles (polymersomes) for intracellular and nuclear delivery to melanoma cells. We demonstrate that PMPC25PDPA70 polymersomes can encapsulate doxorubicin for long periods of time without significant drug release. We demonstrate that empty polymersomes are non-toxic and that they are quickly and more efficiently internalised by melanoma cells compared to healthy cells. Encapsulated doxorubicin has a strong cytotoxic effect on both healthy and cancerous cells, but when encapsulated it had a preferential effect on melanoma cells indicating that this formulation can be used to achieve an enhanced drug delivery to cancerous cells rather than to the healthy surrounding cells. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction High molecular weight block copolymers that self-assemble into highly organised nanoscale structures have an increasing potential for biomedical applications such as non-invasive diagnostics and therapeutic tools [1–3]. In the field of cancer therapy, block copolymers have been used to develop drug carriers capable of enhancing the therapeutic effects of various compounds, of allowing for dosage control and at the same time of diminishing the possible adverse side effects, such as toxicity to the healthy surrounding tissues [4–6]. To do this it is necessary for the nanoscale structures to efficiently encapsulate and retain the therapeutic molecule, to release it at the correct time and place and to exhibit mechanical and chemical stability. Due to the high level of synthetic control over the physical chemistry, amphiphilic block copolymers have been easily tailor-made for these specific applications [1,7]. These structures, when exposed to an aqueous environment, self assemble in such a way as to ⇑ Corresponding author at: Department of Chemistry, University College London, Sheffield, UK. Tel.: +44 1142222305. E-mail addresses: g.battaglia@sheffield.ac.uk, [email protected] (G. Battaglia). 1 These authors have equally contributed to this paper and are jointly first authors. 0304-3835/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2013.02.007

inhibit the exposure to water of the apolar blocks and increase that of the polar blocks according to the hydrophobic effect [8–10]. Polymeric vesicles, also known as polymersomes, that form as a result of such a self-assembly process, can encapsulate both hydrophilic and hydrophobic molecules, they can be chemically functionalised for an active targeting mechanism, they can increase the pharmaco-dynamic and -kinetic profiles of drugs, cellular uptake and in vivo stability [6,11–15]. Compared to liposomes, they are characterised by high membrane stability, tuneable permeability and higher circulation times. This is due the higher molecular weight of polymer chains compared to lipids. Once assembled into a bilayer-like membrane structure these chains are characterised by a high level of entanglement, which overall enhances their toughness and mechanical stability. Drug encapsulation within polymersomes can be effectively controlled by the hydrophobic chemistry of the polymeric membrane and the consequent drug/ polymer affinity. For water soluble molecules permeability across polymersome membranes and consequently the ability of polymersomes to retain a drug is directly proportional to the solubility of the drug within the hydrophobic membrane [16]. This means that retention and a controlled release profile can be chemically engineered [17].

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Anti-cancer drug applications are often severely limited due to their non-selectivity and therefore toxicity to surrounding healthy tissues. The development of a delivery system capable of enhancing anti-cancer drug encapsulation and reducing leakage as well as increasing intracellular uptake and achieving a controlled and targeted delivery to the tumour site would greatly increase their efficacy [18–20]. Doxorubicin (DOXO), which intercalates into the DNA helix in cell nuclei, has for the last 50 years been one of the most commonly used anti-cancer drugs. Among some of the side effects associated with it are drug resistance, dose dependent cardiotoxicity and myelosuppression [3,21–23]. Significant progress has been made towards the investigation of alternative forms of DOXO administration that reduce these undesired side effects and increase tumour targeting. Various groups have developed synthetic or natural polymer-DOXO conjugates [24,25], dendrimers [26], physically encapsulated or covalently bound DOXO polymeric micelles [18,27], DOXO loaded liposomes [20,28,45] and magnetic microspheres [29]. For the most part these systems are characterised by good storage stability, low systemic toxicity, biodegradability and localised drug release. The advantage in using specific polymersome formulations lies in the high drug encapsulation, stability and intracellular delivery via the endocytotic pathway that could potentially reduce drug resistance. Her e we present the encapsulation and delivery to melanoma cells of DOXO within poly(2-(methacryloyloxy)ethylphosphorylcholine)-co-poly(2(diisopropylamino)ethylmethacrylate) (PMPC25-PDPA70) pH sensitive polymersomes. Polymersomes have demonstrated slow chain exchange dynamics with low rates of dissociation, which gives greater stability and retention of the encapsulated drug [15,17]. This polymersome formulation forms stable vesicles at physiological conditions that can dissociate within milliseconds around pH 6.4 [30]. This property is fundamental for cytosolic delivery to cells via endocytosis. The endosome lumen is acidic and this causes the dissociation of the vesicles with a sudden increase in osmotic pressure due to the increase in number of species present (each polymersome is composed of many polymer chains plus the encapsulated aqueous cargo). This increase in pressure lyses temporally the endosome membrane and releases into the cytosol the encapsulated species. Previous work has demonstrated that this formulation is not up taken by cells that are not characterised by the endocytotic pathway (e.g. red blood cells) and that it is successful at delivering plasmid DNA, antibodies, antigens and various therapeutic molecules without affecting cell viability or inducing inflammation [31–35]. Importantly, PMPC25-PDPA70 polymersomes can easily and efficiently encapsulate hydrophilic molecules like DOXO. In this study we show that by using doxorubicin hydrochloride, which is a weak amphipathic base [36,37], the drug does not leak out of polymersomes in any significant amount over time and that when encapsulated its cytotoxic effect is grater in melanoma cells compared to healthy cells.

2. Materials and methods 2.1. Polymersome preparation Atom transfer radical polymerisation (ATRP) was used to synthesise both free and Rhodamine labelled (excitation = 540 nm, emission = 600 nm) PMPC25-PDPA70 copolymers as reported by Du et al. [38]. The copolymer was dissolved in a 2:1 chloroform: methanol solution within an autoclaved glass vial with 10% w/w% of Rhodamine labelled PMPC25-PDPA70 (chloroform and methanol purchased from Fisher Scientific and Sigma–Aldrich UK respectively). The polymer solution was left to evaporate overnight in a dessicator to obtain a uniform film on the inside wall of the glass vial (the vial was sealed with a sterile solvent resistant nylon membrane purchased from Millipore UK Limited). Once all the solvent had evaporated the film was rehydrated in PBS (prepared from PBS tablets purchased from Oxoid Ltd.) at pH 7.4, so as to obtain a 10 mg/ml solution, and left to stir for a minimum of 2 weeks. DOXO encapsulated polymersomes were prepared by stirring the polymer film in a

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drug loaded PBS solution (doxorubicin hydrochloride, MW 579.98, excitation = 470 nm and emission = 590 nm, was purchased from Sigma–Aldrich UK). All the polymersome solutions were purified by preparative gel permeation chromatography using a Sepharose 4B size exclusion column (Sepharose 4B was purchased from Sigma–Aldrich UK). A far red labelled lipid molecular probe (1,10 dioctadecyl-3,3,30 ,30 -tetramethyl indodicarbocyanine, 4-chloro benzene sulphonate salt, MW 1052.8, purchased from Invitrogen) was also encapsulated for imaging purposes by dissolving it in chloroform and adding it to the 2:1 chloroform:methanol solution of PMPC25-PDPA70. Polymersomes with this encapsulated dye were prepared as above.

2.2. Permeability study Drug permeability across polymersome membranes was determined using a Spectra/PorÒ Macrodializer and dialysis membranes characterised by a 3.5 kDa molecular pore (purchased from Spectrumlabs). The dialysis system consists of two chambers with a dialysis membrane disc clamped between them to separate the sample solution from the dialysate. Each chamber can be connected to a peristaltic pump for dynamic dialysis flow via luer fitting ports. Free and polymersome encapsulated DOXO were loaded into the upper chamber (1.7 ml) and the concentration of the permeated drug in the lower chamber was sampled throughout a 24 h period and measured via its UV absorbance at 267 nm using a UV spectrophotometer.

2.3. High-performance liquid chromatography analysis (HPLC) The final concentrations of polymer and drug after purification were determined by HPLC. The system used was an UltiMateÒ 3000 HPLC series provided with a UV–VIS and fluorescence detector and a Chromeleon Data system software purchased from Dionex. The column used was a Jupiter C18 300 A, size 150  4.60 mm, purchased from Phenomenex Inc.

2.4. Dynamic Light Scattering Analysis (DLS) and Transmission Electron Microscopy (TEM) The average size of polymersomes was determined by Dynamic Light Scattering analysis (DLS). A Zetaseizer Nano ZS (Malvern Instruments) with a 633 nm HeNe laser was used. Samples with a minimum volume of 800 ll were placed in polystyrene cuvettes and scanned for 10 s at 173° and averaged over 13 runs. This was repeated three times and then averaged. Particle size and polydispersity were estimated using the CONTIN multiple-pass method. TEM imaging was performed using a FEI TECNAI G2 Spirit transmission electron microscope. Polymersomes samples were placed on carbon-coated copper grids previously glow discharged. Sample excess was removed from the grids by blotting with absorbent paper and stained with a phosphotungesteic acid water solution (0.75% and pH 7) for 5 s.

2.5. Cell culture Human fibroblast isolation was performed from skin biopsies obtained from abdominoplasties or breast reduction operations from patients who gave informed consent. The tissue was used under an HTA Research Tissue Bank licence number 12179. The tissue was cut into thin pieces approximately 0.5 cm2 and incubated overnight at 4 °C in Difco-Trypsin (0.5 g of Difco-Trypsin powder, 0.5 g of D-glucose, and 0.5 m of phenol red in 500 ml of PBS). Following dermis-epidermis separation, the dermis was minced into 1 mm2 pieces and incubated at 37 °C overnight in a collagenase A solution (0.05% w/v% of collagenase A powder in serum-free fibroblast culture medium). The cell suspension was centrifuged for 5 min at 1000 rpm and the pellet was resuspended in medium. Cells were seeded into a T25 flask and incubated at 37 °C with 5% CO2 in a humidified atmosphere. At 80% confluence they were seeded at a density of 105 cells into a T75 flask. Primary dermal fibroblasts were used between passages 4 and 9. Fibroblast culture medium was made up of DMEM high glucose (4500 mg/l glucose), 10% v/v% FCS, 2 mM L-glutamine, 0.625 lg/ml amphotericin B, 100 IU/ml penicillin, and 100 lg/ml streptomycin. HBL cells were derived from a lymph node metastasis of a nodular melanoma and maintained in Ham’s F10 medium supplemented with 5% v/v% FCS, 5% v/v% new born calf serum (NBCS), and 2 mM L-glutamine, 100 IU/ml penicillin plus 100 lg/ml streptomycin. A375-SM cells were a gift from Professor Fidler (USA) via Professor M.J. Humphries (University of Manchester, UK). They were cultured in Eagle’s modified essential media (EMEM) supplemented with 10% v/v% FCS, 2 mM L-glutamine, 100 IU/ml penicillin and 100 lg/ml streptomycin, 1.2 lg/ml amphotericin B, 1.5% v/v vitamin concentrate, 1 mM sodium pyruvate, and 10% v/ v non-essential amino acids. Both cell types were cultured in a humidified atmosphere, 95% air, 5% CO2 at 37 °C. HBL cells were used between passages 15 and 25 and A375-SM cells between 20 and 35. All cell culture medium reagents were purchased from Sigma–Aldrich UK except for the sera, which were purchased from Biowest Biosera UK.

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2.6. Viability assay Cells were plated in 24 well plates and exposed to varying concentrations of empty and loaded polymersome formulations for 24 and 48 h. A 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (MTT) was added to the wells after washing with PBS and left to incubate in a 5% CO2 atmosphere at 37 °C for 40 min (MTT purchased from Sigma–Aldrich UK). The solution was removed and the remaining insoluble formazan was dissolved by adding 100– 200 ll of acidified isopropanol (125 ll of 10 M HCl were added to 100 ml of isopropanol). The eluted dye was transferred to a 96 well plate and the optical density was analysed using a Biotek plate reader set to 540 nm with a reference at 630 nm. All optical densities were normalised to a control sample of cells treated with PBS. The statistical analysis performed on the data was a paired Student’s t-test. 2.7. In vitro uptake Intracellular polymersome and drug uptakes were determined using HPLC and fluorescence activated cell sorting (FACS). For HPLC analysis cells were plated in 96 well plates and treated with known concentrations of empty and loaded polymersome formulations for 5, 10, 20, 30 min and 1, 2, 6 and 24 h. Cells were washed with PBS and incubated overnight at 20 °C. Each well was carefully scraped after the addition of acified water (MilliQ water with 10% Sodium dodecyl sulphate, SDS). The cell suspension was spun in an Eppendorf Centrifuge 5424 and the supernatant was then carefully removed and analysed via HPLC. For the FACS analysis cells were seeded in 24 well plates and exposed to the same polymersome and drug conditions as above. After washing the cells were detached and the cell suspensions were collected and spun down in an Eppendorf Centrifuge 5424 for 5 min. The pellet was resuspended in PBS and analysed using a BD FACS Array (purchased from BD Biosciences). 2.8. Imaging uptake Cells were seeded in 96 well plates and exposed to empty and loaded polymersome formulations for 5, 10, 30 min and 1, 2, 6 and 24 h. At the end of each time point the cells were washed with PBS, fixed with 3.7% formaldehyde and stained with Hoechst (purchased from Invitrogen UK). Imaging was performed using a Carl Zeiss LSM 510 Microscope.

3. Results 3.1. DOXO encapsulation and permeability across polymersome membranes Self-assembly produced polymersomes with average diameters of 100 nm as measured via DLS and TEM analysis (Fig. 1). The presence of DOXO in the solution did not affect polymersome assembly and the average size was not significantly different from empty polymersomes. TEM analysis clearly showed that morphologically both formulations were very similar with average membrane thicknesses of 6 nm (Fig. 1A and B). The micrographs clearly show a monodisperse nanoparticle population. HPLC analysis confirmed that the encapsulation efficiency of DOXO was approximately 39 times higher than the statistical theoretical encapsulation (i.e. the amount that could be encapsulated if the drug concentration within the polymersome were the same as the bulk drug concentration [39]). This indicates a positive interaction between the drug and the polymer, which lead to an average number of drug molecules per Polymersome of 10,500. As shown in Fig. 1C, this is an average value of the number of drug molecules that 100 nm sized polymersomes can theoretically contain within their core, as calculated using the method developed by Wang et al. [39]. A positive interaction between DOXO and polymer has already been reported by Lecommandoux and coworkers, who showed that doxorubicin hydrochloride was found both in the hydrophilic core and the hydrophobic corona of biodegradable polymersomes at neutral pH [36]. They found that, depending on the pH, the drug could be partially adsorbed into the polymeric membrane through electrostatic interactions. With increasing hydrophobicity the amount of aggregation increases and consequently the amount of encapsulated drug. Release kinetics are also strongly affected by this process since the drug must first be solubilised and then diffuse across the poly-

meric membrane into the outer medium. The permeability of DOXO across polymersome membranes was determined using a dialysis system composed of two chambers separated by a membrane. Diffusion across it was measured via UV at different time points for free DOXO and for PMPC25-PDPA70 encapsulated DOXO. The free drug can freely cross the dialysis membrane whilst the encapsulated drug must first cross the polymeric membrane before diffusing through the dialysis membrane, which creates a delay effect. Fig. 1D shows the release of both formulations. The total flux of free DOXO shows a fast linear diffusion across the dialysis membrane reaching a plateau after 4 h, while the encapsulated DOXO was not detected in the lower chamber of the dialysis system.

3.2. Cellular viability In all tumours, healthy cells surround cancerous ones and an anti-cancer drug delivery vehicle must ideally increase the effect on the first and minimise it on the latter, either via a preferential uptake or a targeted form of delivery. In vitro cytotoxic effects of polymersomes, DOXO and the combined formulation were analysed via MTT to determine the loss of cell viability in terms of decreased metabolic activity. Fig. 2 shows the percentage of cell viability compared to a control of all three cell types. Empty PMPC25-PDPA70 polymersomes gave little or no reduction in cell viability at the lower concentrations after a 24 h exposure and even at the higher concentration the loss in viability was never more than 20% (the cells most affected were HBLs). Even after 48 h of exposure there was a similar trend. HDF and A375-SM cells showed no significant reduction in viability when exposed to the lowest concentration and HBLs showed a drop to just above 80% of control viability. A higher concentration of polymersomes in all the cell types showed a reduced metabolic activity compared to the control population, but viability did not go any lower than 76%. Cytotoxicity due to free and encapsulated DOXO was both concentration and exposure time dependant (Fig. 2C–F). The IC50 values calculated from these dose response curves are summarised in Fig. 3. After 24 h of exposure the A375-SM cells did not show any substantial loss in metabolic activity for concentrations of the free drug up to 0.17 lM (above that the IC50 value was 5.8 lM). Free DOXO after 24 h had a stronger effect on HBL cells, which even at the lowest concentration of drug exhibited a loss in viability under 80% (IC50 was 0.61 lM). After 48 h of exposure it was clear that the drug effects had increased. A375-SM cells showed relevant levels of toxicity at lower concentrations with an IC50 of 0.5 lM and HBL cells showed a similar trend with a lower IC50 of 0.2 lM. A375-SMs were less sensitive to DOXO but in both cases the melanoma cells exhibited an increased level of toxicity at higher concentrations and exposure times. Normal fibroblast cells demonstrated an intermediate behaviour when exposed to free DOXO: IC50 values of 35.9 lM after 24 h and of 0.48 lM after 48 h. DOXO was subsequently encapsulated within PMPC25-PDPA70 polymersomes and added to the cell culture medium at varying concentrations (control cells were treated with empty polymersomes). The melanoma reduction in cell viability increased compared to the free DOXO exposure and consequently a lower amount of drug was required to obtain a similar level of toxicity (Fig. 2E and F). Even by delivering only 0.29 lM of encapsulated doxorubicin, after 24 h the loss in viability was below 80% for A375-SMs and HBLs and for higher concentrations viability was below 40%. After 48 h all melanoma cells showed a viability of less than 20%. HDFs on the other hand, clearly showed that the free drug had more effect than the encapsulated one, demonstrating a potentially preferred targeting of these delivery vehicles.

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Time (sec) Fig. 1. Transmission electron microscopy of nanoparticles and loading efficiency. (A) Empty PMPC25-PDPA70 polymersomes and (B) DOXO loaded PMPC25-PDPA70 polymersomes formed via film rehydration and stirring are characterised by an on overage monodisperse nanoparticle population with average diameters of 101 nm. The presence of the anti-cancer drug does not impact self-assembly (scale bar 200 nm). (C) Comparison between the average size of PMPC25-PDPA70 polymersomes as measured via DLS and the loading efficiency of DOXO expressed as the number of drug molecules per individual Polymersome as a function of the size. (D) Cumulative release profiles of the free (-s-) and encapsulated (-j-) drug across the dialysis membrane over time.

Fig. 2. Effect on cell viability of polymersome encapsulated drugs. MTT after 24 (A) and 48 h (B) exposure to PMPC25-PDPA70 polymersomes. MTT after 24 (C) and 48 h (D) exposure to free DOXO. MTT after 24 (E) and 48 h (F) exposure to DOXO encapsulated PMPC25-PDPA70 polymersomes: (-j-) A375-SM cells, (-N-) HBL cells, (-s-) HDF cells. Data are the mean ± SD of three separate experiments (⁄p < 0.01, ⁄⁄p < 0.05).

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Fig. 3. IC50 values of A375-SM, HBL and HDF cells for free and encapsulated doxorubicin (lM): free doxorubicin exposure after 24 h (black), encapsulated doxorubicin exposure after 24 h (black striped pattern), free doxorubicin exposure after 48 h (white), encapsulated doxorubicin exposure after 48 h (white striped pattern). Data are the mean ± SD of three separate experiments (⁄p < 0.01).

3.3. Cellular uptake of PMPC25-PDPA70 polymersomes Cellular uptake of fluorescently labelled PMPC25-PDPA70 polymersomes was studied via FACS, HPLC and confocal microscopy (CLSM). PMPC25-PDPA70 polymersomes were added to fresh cell culture medium at a concentration of 1 mg/ml and incubated for specific amounts of time. FACS shows that HBL melanoma cells be-

gan to show significant uptake after 2 h of incubation and after 6 h more than 90% of all the cells had internalised PMPC25-PDPA70 polymersomes (Fig. 4A). A375-SMs demonstrated an even faster uptake, which is clearly visible after 30 min (86% of cells have internalised polymersomes after only 2 h of incubation). HDF uptake of PMPC25-PDPA70 polymersomes has been already thoroughly studied by our group and for an 80% uptake 6 h of incubation are required [4,31,40]. Fig. 4B shows the number of Rhodamine labelled PMPC25-PDPA70 polymersomes internalised by an individual cell as a function of the incubation time as measured via HPLC. A375-SMs had a fast and highly efficient uptake whilst the HBL melanoma had a much slower uptake. For confocal microscopy, cells were cultured both individually and in co-culture with HDFs to compare uptake kinetics, incubated with Rhodamine labelled PMPC25-PDPA70 polymersomes and imaged after fixation. Fig. 4C shows the CLSM images of polymersome uptake for the individual melanoma cells. They show the nuclei staining obtained with Hoechst (blue), and the fluorescently labelled polymersomes (red). HBLs exhibited very little uptake after 1 h compared to A375-SMs and only some of the cells exhibited any fluorescence in the red channel. After 2 h, fluorescence was more apparent and most of the uptake could be clearly seen between 6 and 24 h of incubation. A375-SMs began to exhibit some fluorescence in the red channel after only 30 min of incubation. After 2 h the cell outline could be visualised via the red fluorescence and between 6 and 24 h most of the uptake had occurred. To visualise cellular uptake in the co-cultures, A375-SMs and HBLs were initially incubated with non-toxic far-red dye encapsulated polymersomes to clearly distinguish them in CLSM (see Sup-

Fig. 4. Polymersome uptake kinetics of melanoma cells. Rhodamine labelled PMPC25-PDPA70 polymersomes were incubated over time with the two melanoma cell types and analysed via FACS to show the percentage of cells containing polymersomes (A), via HPLC to show the number of polymersomes per individual cell (B) and via CLSM to visualise uptake as a function of exposure time (C), (nuclei are stained with Hoechst and shown in blue while polymersomes are shown in red, scale bar 20 lm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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porting information), washed and then co-cultured with HDFs. These co-cultures were then incubated as before with Rhodamine labelled PMPC25-PDPA70 polymersomes. Fig. 5 shows the uptake of polymersome by the melanoma cells together with fibroblasts. The melanoma cells stained with the far-red dye are shown in white, the cell nuclei were stained with Hoechst and shown in blue whilst PMPC25-PDPA70 polymersomes are shown in red. A275-SMs, due to their higher affinity with polymersomes (Fig. 4), exhibited uptake after 1 h whilst HDFs only began to show internalisation after 6 h of incubation (as expected by our previous studies). Similarly, HBLs showed a faster uptake that commences between 1 and 2 h. Potentially this demonstrates that PMPC25-PDPA70 polymersomes are preferentially internalised by melanoma cells compared

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to healthy HDFs and that the side effects associated with free DOXO would be reduced if in the encapsulated formulation. In the next section we will discuss free and encapsulated DOXO delivery. 3.4. Cellular uptake of DOXO Cellular uptake was analysed using two methods, FACS and HPLC. For FACS analysis all the cells were cultured at high cell densities due to the loss that can naturally occur during sample preparation and exposure to drugs such as DOXO. Free and encapsulated doxorubicin were added and incubated in the cell culture medium at equivalent concentrations. FACS analysis was

Fig. 5. Polymersome uptake into melanoma fibroblast co-cultures. A375-SM and HBL cells were treated with a far-red dye (shown in white) to distinguish them from healthy HDFs and after co-culturing they were incubated with PMPC25-PDPA70 polymersomes (shown in red) and imaged at specific time points (nuclei are stained with Hoechst and shown in blue, scale bar 20 lm). For the controls see supplementary information. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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4. Discussion The stability of a drug and of the delivery vehicle is one of the paramount requirements for potential pharmaceutical products with regards to their long-term storage and scalable processing.

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highly affected by the cytotoxic effect of the drug. In both cases, exposure to DOXO caused cell death and therefore detachment from the tissue culture plastic. This means that the cells more likely to be considered as a positive event for the FACS analysis (because they have internalised the largest amount of drug) are lost during the various washing steps. FACS under these circumstances was not able to count all of the relevant events and could only give an approximate idea of the uptake kinetics. A375-SM and HDF cells showed no significant free DOXO uptake and HBLs only after 24 h of incubation (Fig. 6A). Encapsulated DOXO was taken up more efficiently by the melanoma cells (HBL after 2 h and A375-SM after 1 h of incubation) compared to the free drug but for HDFs it was unaffected (Fig. 6B). This kind of analysis can be considered quantitatively reliable only when a sufficient number of positive events are counted (how many cells have internalised the drug). However in our case this was never successfully achieved due to cell loss. All that could be observed is that, as with PMPC25-PDPA70 polymersomes, the A375-SM cell line had the fastest uptake kinetics when exposed to a polymersome formulation and that for all cell types exposure to DOXO had a cytotoxic effect. A more quantitative analysis can be obtained via HPLC since the loss of cells can be corrected for. As with empty polymersome uptake, HPLC showed that A375-SMs had a faster uptake compared to HBLs and HDFs for both free and encapsulated DOXO (Fig. 6C). Free DOXO had a fast internalisation reaching a plateau after 6 h. HBLs reached a plateau at a similar time point but uptake was not as effective as with A375-SMs. HDFs exhibited a reduced internalisation of the free drug compared to the melanoma cells (up to 8 orders of magnitude). The encapsulated formulation showed a drastic increase in uptake (two orders of magnitude) for A375SM and HDF cells whilst for HDF there was no relevant difference between the two formulations. CLSM confirmed the HPLC uptake results (Fig. 7). When incubated with encapsulated DOXO, HBLs exhibited very little uptake after 2 h, after 6 h doxorubicin was surrounding the cell nuclei and was then within the cell cytosol. After 24 h of incubation most of the cells had died and detached. There was clearly some doxorubicin in the cell cytosol of A375SM cells after 30 min and co-localisation between the nuclei and DOXO began to be observed after 1 h of incubation (indicating that polymersomes have released the drug within the cytosol and that DOXO has intercalated with DNA inside the nucleus of the cell). Between 2 and 6 h of incubation most of the cells had taken up the drug and the level of co-localisation between the drug and the nuclei had increased (although after 24 h most of the cells died and detached). HDFs demonstrated cytosolic delivery, if not nucleic, of encapsulated DOXO after 6 h of exposure, which then mostly disappeared after 24 h. There is a small amount of co-localisation between DOXO and the edge of the cell nuclei after 6 h but it was not as evident as with A375-SM cells. Free DOXO delivery did not show as substantial a cytosolic delivery for the melanoma cells as with the encapsulated formulation (only A375-SM cells have a clear fluorescence signal after 6 h of incubation which then increased after 24 h) and nearly all of the cells were still attached to the cell culture plastic. HDFs on the contrary exhibited a very marked nucleic delivery after 2 h of exposure demonstrating the stronger effect that the free drug has compared to the encapsulated drug on healthy cells and vice versa on the melanoma cells.

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Time (min) Fig. 6. Uptake of DOXO by FACS and HPLC. Free and PMPC25-PDPA70 polymersomes encapsulated DOXO where incubated with the three cell types at equivalent concentrations and analysed via FACS as the number of cells containing the drug (A and B) and HPLC, expressed as the mass of drug (ng) per individual cell (C): (–j–) A375-SM free DOXO, (–N–) HBL free DOXO, (–s–) HDF free DOXO, (-j-) A375-SM encapsulated DOXO, (-N-) HBL encapsulated DOXO and (-s-) HDF encapsulated DOXO.

Importantly if the polymersome membrane were permeable and the encapsulated drug were able to diffuse across it over time then it could potentially affect all cells indiscriminately. If however the drug remains within the delivery vehicle and can only be released by lowering the pH during endocytosis, then it will be delivered only within the cell cytosol and will not affect other cells. In this study all cell types are capable of endocytosis but the cancerous cells were able to take up quantitatively more polymersomes and they did this faster than the healthy cells. In most cancer therapies, collateral damage to the healthy surrounding tissues is considered a necessary part of the process. If there is some degree of selectivity by melanoma cells taking up proportionately more polymersomes and hence drug compared to normal cells, then these side effects could be reduced. The dialysis study therefore

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Fig. 7. Imaging of doxorubicin cellular delivery. Free and PMPC25-PDPA70 polymersomes encapsulated DOXO were incubated with the three cell types at equivalent concentrations for increasing amount of time. Cell nuclei were stained with Hoechst and are shown in blue while DOXO is shown in red. Co-localisation shows in white where the fluorescence signals associated with the cell nuclei and with DOXO are found together (scale bar 20 lm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

was specifically designed to determine the permeability of PMPC25-PDPA70 polymersome membranes to DOXO. The total flux of the drug is the result of the flux across the dialysis membrane and the drug released by each polymersome. If there were a difference in the diffusion profiles between the free and the encapsulated drug then the release from the polymersome could be expressed as the difference between the total flux and the flux across the dialysis membrane. Assuming that diffusion follows Fick’s first law then the permeability coefficient could be calculated as a function of the ratio between the amount of drug that has diffused after reaching equilibrium and the concentration gradient. In our case doxorubicin was not detected in the lower chamber of the dialysis system when encapsulated (Fig. 1C), it was only able to permeate across the dialysis membrane when free in solution. This clearly demonstrates that when trapped within polymersomes the

drug is not able to leak out of the delivery vehicle and that encapsulation is a stable process for as long as 8 h. Doxorubicin hydrochloride is a weak amphipathic base, which means that during the self assembly process, the molecule will be encapsulated within the hydrophilic core of polymersomes, but also partially trapped within the hydrophobic membrane [46]. Consequently, the permeation across the membrane will be drastically reduced and the encapsulation enhanced compared to doxorubicin formulations that are entrapped only within the aqueous lumen. PMPC25-PDPA70 polymersomes were taken up very quickly by the melanoma cells with very little toxicity even at high concentrations. When DOXO was encapsulated within them, intracellular and nuclear delivery to HBLs and A375-SMs was increased by two orders of magnitude compared to the equivalent concentration of free drug. An ideal anticancer treatment consists of a

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targeted delivery with minimal collateral damage to the healthy cells localised around the cancer. Both FACS and HPLC analysis confirmed that the uptake kinetics of the cancer cells were generally much faster than those of HDFs. This raised the possibility that when placed in a co-culture, the cancerous cells would internalise proportionally more of the polymersomes and therefore more of the encapsulated therapeutic molecule. Preferential uptake of PMPC-PDPA polymersomes was clearly visualised in the CLSM analysis of the co-culture system. For both melanoma types polymersomes were visible in the cytoplasm after 1 h compared to HDFs. Recent work in our group has demonstrated that PMPCPDPA uptake is regulated by the interaction with specific receptors that are overexpressed in cancerous cells, and consequently explain why they have a preferential uptake compared to HDFs (I. Canton, M. Avila et al. in preparation). When incubated with cells, free DOXO can penetrate the plasma membrane in a non-specific way via both electrostatic and hydrophobic interactions [41]. Once inside the cytoplasm it is then translocated to the nucleus by the formation of a specific proteasome complex that actively targets the nucleus [42]. Binding to proteasomes inhibits regulatory proteins that control cell growth and can therefore cause apoptosis. Once inside the nucleus DOXO dissociates from the proteasome and intercalates with DNA, for which it has a higher binding affinity, thereby inhibiting nucleotide synthesis [43]. For a DOXO delivery system to be considered as a practical alternative it should enhance intracellular and nuclear delivery while at the same time counterbalancing the effect of drug resistance due to transporters such as P-glycoprotein that are actively involved in eliminating external substances from the internal cellular environment [44]. Shoichet and co-workers have reported the intracellular accumulation of a DOXO surface conjugated nanoparticle (DOXO-NP) based on a furan functionalised amphiphilic copolymer (poly(TMCC-co-LA)-g-PEG-furan) coupled with antibody targeting ligands [27]. They observed that the antibody formulation had a significantly higher intracellular accumulation compared to the DOXO conjugated nanoparticle. As with free DOXO, the nanoparticle based system can form a complex with proteasomes and can be selectively transported to the cell nucleus. DOXO-NP did accumulate in the cell cytoplasm but compared to the ligand formulation, which enhances the internalisation mechanism by the addition of a receptor mediated endocytosis process, the amount of delivered drug within the cells was not as high. Compared to free DOXO however the cytotoxic effect of these ligand-NPs was less. With PMPC-PDPA polymersomes we also have an active receptor mediated endocytosis that enhances cellular uptake together with a very fast release rate [4]. Many polymer-based drug complexes suffer from their inability to quickly degrade and release the drug once they have been internalised. Without this process DOXO cannot form the proteasome complexes and quickly be transported to the nuclei. Once PMPCPDPA polymersomes are inside the endosome it is only a matter of a few milliseconds for the drug to be released in its free form within the cytoplasm. Considering the amount that is taken up and the time required for the intracellular release to occur, it is easy to understand how there is both a highly efficient intracellular delivery and a marked accumulation of the drug to the nuclear region. Compared to free DOXO, PMPC-PDPA polymersomes are actively and quickly taken up by cells (in particular melanoma cells). Consequently the amount of drug delivered within the cells is higher than that which can passively permeate across the plasma membrane and it can therefore overcome the effects of drug resistance since there is enough that does not get expelled and is still able to target the nucleus (the free drug is more easily removed by the melanoma cells since there is less of it and it is internalised over longer periods of time). This is confirmed by the IC50 values of the encapsulated drug in melanoma cells that are considerably

lower compared to the free formulation. To have the same cytotoxic effect less drug is required in the encapsulated formulation. HDFs have slower uptake kinetics and to achieve the same percentage of cell death as with the melanoma cells larger amounts of drug are required and importantly, when encapsulated, exposure time no longer appears to have a strong impact (Fig. 3). HPLC also confirmed the increased potential of encapsulating DOXO inside PMPC-PDPA polymersomes compared to free drug. A375-SMs and HBLS were characterised by fast DOXO uptake during the first 6 h of incubation, which was up to two orders of magnitude more efficient than the free drug formulation (Fig. 6C). Fibroblasts however did not demonstrate an increased uptake and with both formulations the amount actually delivered was relatively the same (although with the encapsulated drug the cytotoxic effects were diminished compared to the free drug). CLSM showed that when the drug is encapsulated, there is high cellular internalisation (especially in the melanoma cells) and that A375-SMs have a high level of co-localisation between the nuclei and the drug. A375-SMs were less affected by the drug (as can be seen from the cell viability experiments) but for both melanoma cell types the encapsulated DOXO achieved cell death and after 24 h most of the cells had detached. HDFs demonstrated a certain amount of internalisation but most of the cells even after 24 h were still proliferating. This is due to the fact that uptake is slower compared to melanoma cells and consequently HDFs uptake less drug in the same period of time (in terms of ng of internalised drug per cell there is a difference of up to 8 orders of magnitude between HDFs and the melanoma cells) and can more effectively expel it via the membrane protein pumps. CLSM images of free drug uptake showed that with the same DOXO concentration as the encapsulated formulation there is not such a clear uptake for the melanoma cells compared to HDFs. When encapsulated, DOXO is more cytotoxic to the melanoma cells compared to healthy cells and it is more selectively delivered to the former. In conclusion we have developed a pH sensitive polymersome system capable of efficiently encapsulating doxorubicin. We have demonstrated that the encapsulated formulation has a stronger toxic effect on melanoma cell lines compared to the free drug due to the active cellular internalisation. Importantly, this formulation is preferentially taken up by cancerous cells compared to normal fibroblasts, thereby promoting a potentially selective and enhanced therapeutic strategy for melanoma treatment. Acknowledgments We would like to thank Biocompatibles UK for support of the project and the EPSRC for funding the White Rose Doctoral Training Programme of Leeds and Sheffield Universities, which supported Carla Pegoraro.

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