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. Author manuscript; available in PMC: 2017 Apr 17.
Published in final edited form as: J Immunol. 2017 Mar 13;198(9):3596–3604. doi: 10.4049/jimmunol.1601901

Targeting Neutrophilic Inflammation using Polymersome-Mediated Cellular Delivery

James D Robertson *,†,‡, Jon R Ward †,‡, Milagros Avila-Olias *, Giuseppe Battaglia §,¶,2,#, Stephen A Renshaw †,‡,2,#
PMCID: PMC5392731  EMSID: EMS71605  PMID: 28289157

Abstract

Neutrophils are key effector cells in inflammation and play an important role in neutralising invading pathogens. During inflammation resolution, neutrophils undergo apoptosis before they are removed by macrophages, but if apoptosis is delayed, neutrophils can cause extensive tissue damage and chronic disease. Promotion of neutrophil apoptosis is a potential therapeutic approach for treating persistent inflammation, yet neutrophils have proven difficult cells to manipulate experimentally. Here we deliver therapeutic compounds to neutrophils using biocompatible, nanometre-sized synthetic vesicles “polymersomes” that are internalised by binding to scavenger receptors and subsequently escape the early endosome through a pH-triggered disassembly mechanism. This allows polymersomes to deliver molecules into the cell cytosol of neutrophils without causing cellular activation. After optimising polymersome size, we show that polymersomes can deliver the cyclin-dependent kinase inhibitor (R)-roscovitine into human neutrophils to promote apoptosis in vitro. Finally, using a transgenic zebrafish model, we show that encapsulated (R)-roscovitine can speed up inflammation resolution in vivo more efficiently than the free drug. These results show that polymersomes are effective intracellular carriers for drug delivery into neutrophils. This has important consequences for the study of neutrophil biology and the development of neutrophil-targeted therapeutics.

Introduction

Neutrophils are the most abundant leukocyte in mammals and are the first cells recruited to sites of infection. The main role of neutrophils is to remove microorganisms through phagocytosis (1). Internalised microorganisms are rapidly degraded in the phagosome using a destructive cocktail of proteases, antimicrobial peptides and reactive oxygen species (ROS) that are delivered by cytoplasmic granules (2). Killing pathogens intracellularly allows neutrophils to remove microorganisms without causing off-target damage to the host; however, not all microorganisms can be removed by this method, particularly if contact is obstructed by the extracellular matrix (3). Following activation, neutrophils degranulate, expelling their destructive contents into the extracellular environment (4). This degranulation helps ensure that the surrounding area is sterilised, and collapses nearby capillaries and lymphatic vessels to prevent microorganisms from escaping (3). Yet, unsurprisingly, degranulation can cause extensive tissue damage, and so this process must be tightly controlled. Therefore, once an infection has been cleared, neutrophil apoptosis is an important step in preventing further damage to the host (5).

Neutrophil apoptosis can be initiated following phagocytosis of target pathogens, or, in the absence of survival signals, it can occur spontaneously (6). This ensures that neutrophils cease pro-inflammatory functions and allows their toxic contents to be packaged within apoptotic bodies for safe clearance by macrophages. Upregulation of phosphatidylserine and other ‘eat me’ signals during apoptosis helps ensure that neutrophil clearance occurs before the contents are lost through secondary necrosis (7).

Not only does apoptosis prevent neutrophils from responding to pro-inflammatory stimuli, but apoptotic neutrophils can also actively promote inflammation resolution by sequestering cytokines (8) and by converting macrophages to a pro-resolution phenotype following efferocytosis (9). However, apoptosis can be delayed by stimulation with survival factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF), with prolonged neutrophil survival leading to excessive tissue damage, as observed in multiple inflammatory diseases (10–12).

Driving neutrophil apoptosis is a potential therapeutic strategy for the treatment of inflammatory diseases. This can be achieved by drug therapy with Tanshinone IIA or Cyclin-dependent kinase inhibitors (CDKi) (13). The broad spectrum CDKi (R)-roscovitine, for instance, has emerged as a potent inducer of neutrophil apoptosis (14–16) even in the presence of survival factors such as GM-CSF (17). Drug delivery carriers may enable more efficient access to the neutrophil intracellular milieu to help encourage drug signalling processes and limit off target side effects. However, cellular delivery and genetic manipulation of neutrophils has proven difficult. This has not only prevented us from manipulating neutrophils for therapeutic intervention, but has also limited our understanding of the molecular pathways that control neutrophil function. A number of neutrophil transfection techniques have been attempted, but none have become well established (18–21).

In this study poly(2-(methacryloyloxy)ethyl-phosphorylcholine)-co-poly(2-(diisopropylamino) ethyl methacrylate (PMPC-PDPA) polymersomes are explored as drug delivery vectors for neutrophils. Polymersomes were tested for adverse effects on neutrophil viability and IL-8 release, and their size was optimised for efficient cargo delivery. Finally, the ability of polymersomes to encapsulate and deliver (R)-roscovitine into neutrophils was tested in vitro and in a zebrafish model of neutrophilic inflammation.

Materials and methods

Reagents

Unless otherwise stated reagents were purchased from Sigma-Aldrich. Chloroform and methanol were purchased from Fisher Scientific. (R)-roscovitine was purchased from Cambridge Bioscience, GM-CSF from PeproTech and cascade blue from Life Technologies. The IL-8 ELISA kit was purchased from R and D systems.

Polymersome formation

Polymersomes were assembled using the pH-switch or film-rehydration method as described previously (22). The synthesis of PMPC25-PDPA65 and rhodamine 6G-labelled PMPC25-PDPA70 was performed by reversible addition fragmentation chain-transfer polymerisation (RAFT), and atom-transfer radical polymerisation (ATRP) respectively (23, 24). Polymersomes were purified using cross-flow filtration and differential centrifugation as described previously (25). Briefly, polymersomes were purified into different sizes by initially extracting the smallest nanoparticles using spectrum's KrosFlo Research IIi System with a 50nm Hollow fibre filter module, all purchased from Spectrum labs. Followed by differential centrifugation at increasingly higher centrifuge speeds (25).

Polymersome uptake by human neutrophils

Peripheral blood was extracted from healthy donors as approved by the South Sheffield Research Ethics Committee (STH13927). Neutrophils were purified from the peripheral blood of healthy donors using a Percoll density gradient as described previously, typically yielding neutrophil purities of >95% (26, 27). For cytospin apoptosis counts and cytokine ELISAs, neutrophils were further purified using negative magnetic selection, to ensure that contaminating PMBCs did not influence neutrophil responses (28, 29). All other experiments on human neutrophils were using neutrophils purified by using the percoll gradient only. In the initial neutrophil viability and IL-8 ELISAs, polymersomes were not purified to a specific size fraction. Viability was assessed either by observation of nuclear morphology on histochemically stained cytospin preparations according to well-accepted protocols (30), or by flow cytometry of neutrophils stained with Alexa Fluor 647 annexin V and propidium iodide (Biolegend). Flow cytometry was performed with a BD FACSArray Bioanalyzer with 10,000 events captured within the neutrophil gate. Data was analysed using FlowJo software (Tree Star Inc., Oregon, USA). This experiment was repeated with a similar protocol on FaDus purchased from the American Type Culture Collection (Manassas, VA, USA) seeded in 24 well plates and grown for one day in supplemented Dulbecco's modified Eagle's medium (DMEM).

Cytokine release was measured using an IL-8 enzyme-linked immunosorbent assay (ELISA) using matched antibody pairs at optimised concentrations as described previously (31). To assess the delivery of polymersomes encapsulating rhodamine B octadecyl ester perchlorate , neutrophils were incubated with 1mg/ml of CelLuminate for 6 hours or 24 hours and then washed, fixed with 1x Cellfix (Becton Dickinson) and visualised on a Perkin-Elmer UltraVIEW VoX spinning disk confocal microscope with a 514nm laser and a 40x oil immersion lens.

Optimisation of polymersome size

Purified polymersome size fractions were incubated with neutrophils at 37°C for the desired time period. Neutrophils were centrifuged at 300RCF for 3 minutes with a table-top centrifuge (Eppendorf Minispin) and the pellet was then washed with ice cold PBS and this washing step was repeated. Neutrophils were kept on ice before the fluorescence intensity was measured using an LSR II Flow Cytometer (BD Biosciences) with 10,000 events captured within the neutrophil gate and a 450nm violet laser (cascade blue) or a 575nm blue laser (Rho-PMPC-PDPA). The relative median fluorescence intensity (rMFI) was obtained by dividing the MFI of the treated neutrophils by the MFI of the untreated neutrophils and subtracting 1, so that a MFI value equal to the control had an rMFI of 0. Microscopy was undertaken on cells fixed with 1x Cellfix (Becton Dickinson) and visualised with a Perkin-Elmer UltraVIEW VoX spinning disk confocal or a Olympus FV1000 confocal microscope.

Calculation of encapsulated (R)-roscovitine concentration

Following (R)-roscovitine encapsulation, free drug was removed using gel permeation chromatography (32) and the polymersomes were purified to the desired size as described above. To measure the concentration of polymer and encapsulated (R)-roscovitine, Reverse-Phase High-performance Liquid Chromatography RP-HPLC was performed on a Dionex Ultimate 3000 system with an associated UV-Vis spectrophotometer. The sample pH was lowered to 6 using 1M HCL to disassemble the polymersomes. The samples were then passed through a C18 analytical column (Phenomenex Jupiter; 300 Å, 150x4.6mm, 5 μm) and run in a multistep gradient of eluent a: methanol + 0.1% TFA, and eluent b: H2O + 0.1% TFA. The gradient was as follows: 0 min 5%, 6.5 min 5%, 12 min 100%, 14 min 100%, 15 min 5%, 20 min 5%. RP-HPLC UV-Vis calibration curves were used to calculate the concentration of polymer and encapsulated (R)-roscovitine.

Zebrafish husbandry and assays

Zebrafish were raised and maintained according to standard protocols (33) in UK Home Office-approved aquaria at the University of Sheffield Zebrafish Facility in the Bateson Centre. The transgenic Tg(mpx:GFP)i114 line, referred to as mpxGFP, was used in both zebrafish experiments. Injury by tail fin transection was performed as previously described (34). Briefly, 3-day post fertilisation (dpf) mpxGFP zebrafish embryos were immersed in 0.02% 3- amino benzoic acid ethyl ester (tricaine) and then placed onto masking tape on a Petri dish lid. The embryos were injured by complete tail fin transection at the caudal fin with a micro-scalpel (World Precision Instruments). Four hours following injury, zebrafish were visualised under a fluorescent dissecting microscope (Leica MZ10F) and embryos with 20-25 neutrophils at the injury site (posterior to the circulatory loop) were selected, transferred to a 96-well plate and polymersomes or their controls were added to the zebrafish media. Zebrafish were then washed and visualised using a Perkin-Elmer UltraVIEW VoX spinning disk confocal microscope or the number of neutrophils at the injury site was counted under a fluorescent dissecting microscope.

Statistical analysis

Data were analysed using a two-way ANOVA or a one-way ANOVA with Bonferroni post-test (Prism 5.0; GraphPad Software).

Results

Polymersomes are biocompatible intracellular vectors for human neutrophils

In a similar manner to membrane phospholipids, the amphiphilic PMPC-PDPA copolymer contains a hydrophilic group (PMPC) and a hydrophobic group (PDPA) that enables the copolymer to self-assemble into a membrane in aqueous solutions (35). We have shown that these membranes wrap into vesicles, otherwise known as polymersomes, that are capable of encapsulating a variety of molecules, proteins and nucleic acids (36–38). The DPA component of this co-polymer is a weak base with a pKa of 6.4 under physiological conditions. Once internalised, polymersomes are transported into early endosomes where the drop in pH triggers polymersome disassembly, resulting in a sudden increase in the number of charged molecules (39). This leads to an osmotic shock triggering temporary destabilisation of the endosome and release of some of the polymersome cargo into the cell cytosol (38). Importantly, the pKa of the DPA group ensures that this process occurs very early on in the endolysosomal pathway, enabling release of the polymersome cargo without provoking cellular toxicity (39).

The PMPC block is a biocompatible polymer that is highly resistant to protein adhesion, and widely used as a coating for contact lenses, coronary stents and artificial joints (40–42). While PMPC-PDPA are resistant to unspecific protein adhesion, we have shown previously that the phophorylcholine groups have high affinity for scavenger receptor B1 enabling them to bind and become internalised by a variety of cell types, particularly immune cells and cancer cells, which are rich in scavenger receptors (39, 43).

To explore the potential of PMPC-PDPA polymersomes as intracellular delivery vectors for neutrophils, human peripheral blood neutrophils were purified from the blood of healthy donors using a Percoll density gradient and the cells were incubated with polymersomes encapsulating the fluorescent dye rhodamine b octadecyl ester perchlorate (rhodamine b). After 6 hours or 24 hours incubation, the cells were washed and then examined by confocal microscopy revealing delivered rhodamine was dispersed throughout the interior of the cell (Figure 1a, Supplementary Video 1).

Figure 1. Uptake of polymersomes by human neutrophils.

Figure 1

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(a) Confocal photomicrograph of neutrophils incubated with polymersomes encapsulating rhodamine b octadecyl ester perchlorate for 6 hours or 24 hours at a final concentration of 1mg/ml, scale bar = 8μm. (b) Apoptosis assays on primary human neutrophils after incubation with empty polymersomes or GM-CSF (0.01μg/ml) for 4 hours or (c) 20 hours. (d) Interleukin 8 (IL-8) levels assayed by ELISA on human neutrophils after incubation with empty polymersomes or controls for 4 hours or (e) 6 hours. Neutrophils treated with polymersomes were not significantly different from the negative control group in all experiments (one-way ANOVA with post Bonferroni’s multiple comparison test, error bars = SEM from 3 independent experiments, *P<0.05 ***P<0.001).

Extensive toxicity analysis on over 20 different cell types has demonstrated that PMPC-PDPA polymersomes are well tolerated, even at relatively high concentrations (39). However, neutrophils are very sensitive cells and many manipulations cause neutrophil activation or cell death (26).

To ensure that polymersomes alone do not alter neutrophil life span, the rate of neutrophil apoptosis after incubation with 1mg/ml of polymersomes for 4 or 20 hours was measured by observation of nuclear morphology on histochemically stained cytospin preparations according to well accepted protocols (30). Treatment with GM-CSF (0.01μg/ml) resulted in a reduction in neutrophil apoptosis at both time points, but empty polymersomes had no effect on neutrophil apoptosis counts compared with the PBS control (Figure 1b, 1c).

Activation of neutrophils and initiation of their pro-inflammatory response results in the production and release of inflammatory cytokines such as interleukin-8 (IL-8) (44). IL-8 is a potent chemokine that is involved in recruiting neutrophils and co-ordinating the immune response. In order to determine the effect of polymersomes on neutrophil activation, neutrophils were incubated with polymersomes for 4 or 6 hours. Supernatants were extracted and IL-8 concentration was measured by ELISA. Incubation with polymersomes at a concentration of 1mg/ml had no effect on neutrophil IL-8 release compared with the PBS control (Figure 1d, 1e). In contrast, the positive control (lipopolysaccharide, LPS) activated neutrophils and increased the amount of IL-8 release at both time points.

Optimising polymersome size for efficient cargo delivery

Nanoparticle size is known to have a critical effect on numerous aspects of nanoparticle in vivo function including: circulation time, cell binding, internalisation, extravasation and biodistribution (45–47). Controlling the rate of internalisation will influence both the efficiency and specificity of a drug vector for a specific cell type. However, the rate of internalisation can be difficult to predict theoretically because it is also dependent on nanoparticle surface chemistry (39), ligand surface density (48), receptor expression and binding affinity (49), mechanism(s) of receptor-mediated endocytosis (50) and even the degree of nanoparticle interactions on the plasma membrane (51). Thus, the optimal nanoparticle size depends on the specific surface properties of the nanoparticle and the target cell (45) and must be determined experimentally.

Following endosomal release, polymersome-forming copolymer chains integrate with cell membranes (39). As shown in Supplementary Figure 1a, neutrophils incubated with rhodamine-labelled polymersomes (where rhodamine is covently bound to individual copolymer chains) show a strong signal from membranous compartments (Supplementary Figure 1a). The aminocoumarin fluorophore cascade blue is a water-soluble, cell impermeable fluorescent dye often used to measure membrane permeability (52). When polymersomes loaded with cascade blue were incubated with neutrophils the signal is distributed throughout the cell cytosol (Supplementary Figure 1a). To investigate the consequence of polymersome size on internalisation by human neutrophils, rhodamine-labelled polymersomes encapsulating cascade blue were formed by the pH-switch method. By encapsulating cascade blue in rhodamine-labelled polymersomes it was possible to track the amount of polymer internalised by the cell, as well as the amount of delivered cargo.

Rhodamine-labelled polymersomes encapsulating cascade blue were separated into six size fractions using cross-flow filtration followed by differential centrifugation as described previously (25). The concentration of cascade blue and rhodamine-labelled polymer was measured using fluorescence spectroscopy. Human neutrophils were incubated with one of the six purified polymersome size fractions at a final polymer concentration of 0.1mg/ml. At multiple time points throughout the experiment neutrophils were analysed by flow cytometry. Example dot plots are shown in Supplementary Figure 1b.

Incubation with polymersomes resulted in a rapid increase in the normalised neutrophil fluorescence intensity, referred to as the relative median fluorescence intensity (rMFI). The degree of neutrophil fluorescence over time was dependent on the polymersome size (Figure 2a and 2b). Neutrophils treated with the 190nm polymersome fraction appeared to have the highest internalisation of rhodamine labelled polymer and cascade blue. By plotting the data from the final time point only, it can be seen that the amount of polymer internalised by neutrophils escalates with increasing diameter up to 190nm (Figure 2c). This is consistent with previous reports showing that spherical nanoparticle uptake is optimal at a given critical radius that depends on the receptor/ligand affinity and receptor density (53).

Figure 2. Quantified neutrophil rMFI over time after incubation with rhodamine labelled polymersomes encapsulating cascade blue.

Figure 2

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(a) Neutrophil rMFI in the rhodamine channel showing the amount of rhodamine-labelled polymer internalised over time for each of the size fractions. (b) Neutrophil rMFI in the cascade blue channel showing the amount of cascade blue delivered into neutrophils over time for each size fraction. The graphs show that neutrophil fluorescence from the polymer and cascade blue increases over time with a plateau at later time points. The extent of neutrophil fluorescence was dependent on the polymersome size. (c) Neutrophil rMFI after incubation with polymersomes at each size fraction for 600 minutes. (d) rMFI of neutrophils treated with rhodamine-labelled polymersomes and washed after 240 minutes to determine the rate of polymer release (on all graphs error bars = SEM, from 3 independent experiments).

To investigate the rate at which the polymersome signal drops from neutrophils following internalisation, neutrophils were incubated with 190nm polymersomes using the same protocol. After 240 minutes the neutrophils were pelleted by centrifugation, washed and returned to normal media. The neutrophil rMFI was measured at three further time points to determine the rate of polymer release from the cells. Interestingly, once the extracellular polymersomes were removed, neutrophil rMFI rapidly decreased (Figure 2d). This may be due to polymer metabolism or secretion.

The uptake of polymersomes in vivo will depend not only on the rate of internalisation by neutrophils at a given nanoparticle size, but also the relative rate of internalisation by other cell types. Therefore, the size dependent internalisation experiment was repeated using the cell line FaDu for comparison. Once again FaDu cells were incubated with one of six purified polymersome size fractions and were analysed by flow cytometry at multiple time points up to 25 hours. In agreement with the complementary neutrophil experiment, the fluorescence from both the cascade blue channel and the rhodamine channel increased over time and then plateaued (Supplementary Figure 2a, b). However, the effect of size on internalisation was far less marked (Supplementary Figure 2c).

Encapsulated (R)-roscovitine promotes neutrophil apoptosis

The broad spectrum CDKi (R)-roscovitine drives neutrophil apoptosis by down-regulating the survival protein Mcl-1; (14) however, clinical use may be restricted due to off-target effects on non-immune cell populations. To overcome this potential limitation, (R)-roscovitine was encapsulated within polymersomes using the film rehydration method (54). Following (R)-roscovitine encapsulation, polymersomes were purified to a mean diameter of approximately 200nm (Supplementary Figure 3a). Encapsulation of (R)-roscovitine was calculated by reversed-phase high-performance liquid chromatography (RP-HPLC) associated with UV detection (Supplementary Figure 3b, c).

Human neutrophils were incubated for 8 hours with GM-CSF (0.01μg/ml) and 1–30μM of encapsulated (R)-roscovitine or free (R)-roscovitine. Cell viability was measured by flow cytometry with annexin V and propidium iodide staining. Incubation of polymersomes with encapsulated or free (R)-roscovitine resulted in a dose-dependent increase in neutrophil apoptosis, but the rate of apoptosis was not significantly different between these groups (Figure 3a). Example dot plots in Figure 3b demonstrate similar levels of annexin V positive and propidium iodide positive cells were observed in neutrophils treated with polymersomes compared with their controls. Empty polymersomes had no effect on neutrophil viability compared with cells treated with PBS alone (Figure 3c).

Figure 3. Encapsulated and free (R)-roscovitine promote neutrophil apoptosis.

Figure 3

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(a) Dose response line graph showing the percentage of apoptotic neutrophils (annexin V positive) when incubated with increasing concentrations of encapsulated or free (R)-roscovitine for 8 hours. Treatment with roscovitine significantly increased neutrophil apoptosis in both groups (P<0.0001), but encapsulation within polymersomes did not significantly affect apoptosis rates compared with free roscovitine (two way ANOVA, error bars show SEM from 4 independent experiments). (b) Dot plots from flow cytometry of human neutrophils showing annexin V and propidium iodide staining after 8 hours incubation with treatment or controls (GM-CSF at 0.01μg/ml, (R)-roscovitine at 30μM and PMPC-PDPA polymer at 1.15mg/ml). (c) Bar graph showing the percentage of apoptotic neutrophils (annexin V positive) after 8 hours incubation with PBS, DMSO or empty polymersomes. Treatment with empty polymersomes did not significantly alter the rate of apoptosis (GM-CSF at 0.01μg/ml, PMPC-PDPA polymer at 1.15mg/ml) (one-way ANOVA ,error bars = SEM from 4 independent experiments).

Encapsulated (R)-Roscovitine Promotes Inflammation Resolution in an in vivo Zebrafish Model

Previous studies in our lab have shown that polymersomes can improve the delivery of molecules into tissues due to their intrinsic flexibility (55, 56). For instance, PMPC-PDPA polymersomes have enabled the delivery of fluorescent dextran and large immunoglobulins into ex vivo human skin, the delivery of antibiotics into human mucosa and the delivery of small molecules into the central core of a multicellular tumour spheroid (57, 58). Enhanced delivery of (R)-roscovitine through the skin may help in the treatment of inflammatory skin diseases such as psoriasis or severe eczema.

To study the potential benefits of polymersomes in the treatment of neutrophil dominated inflammatory diseases, Zebrafish were employed as an animal model. Zebrafish provide an excellent model for studying the innate immune system because the embryos are transparent and contain neutrophils that can be fluorescently labelled by transgenesis. Zebrafish neutrophils share morphological, biochemical and functional similarity to human neutrophils and we have shown they respond in a similar manner to chemicals known to influence neutrophil function (13, 59). Zebrafish tail fin transection results in sterile inflammation characterised by neutrophil recruitment peaking at 6 hours post injury (hpi) and resolution of neutrophilic inflammation by 24hpi (Renshaw et al., 2006). This provides a relatively cheap, rapid and effective method for testing the potency of anti-inflammatory compounds in promoting inflammation resolution (Wang et al., 2014, Robertson et al., 2014a). The transgenic Tg(mpx:GFP)i114 line specifically labels neutrophils with green-fluorescent protein (GFP), which allows neutrophils to be tracked in vivo using a fluorescence microscope (33). To study polymersome delivery in vivo, 3dpf mpx:GFP zebrafish embryos were subject to tail injury by transection of the tail end and incubated with polymersomes encapsulating rhodamine b for 8 hours. Visualisation of the tail region by confocal microscopy revealed a diffuse rhodamine signal throughout the tail (Figure 4a).

Figure 4. Polymersome mediated delivery of (R)-roscovitine into the zebrafish tail speeds up inflammation resolution.

Figure 4

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3-day post fertilisation zebrafish larvae (mpx:GFP) were subject to tail injury by transection of the tail end. (a) At 4hpi zebrafish larvae were incubated with polymersomes encapsulating rhodamine b octadecyl ester perchlorate (CelLuminate) for 8 hours before the fish were washed and imaged by confocal microscopy. (b) Zebrafish larvae were treated with 30μM free or encapsulated (R)-roscovitine at 4hpi. At 12hpi neutrophils numbers at the injury site were counted using a dissecting microscope. Encapsulated (R)-roscovitine significantly improved inflammation resolution compared with free roscovitine or controls (one-way ANOVA with Bonferroni’s post hoc comparison, ***P<0.001, ****P<0.0001, lines represent the mean +/- SEM from 3 independent experiments).

To compare the ability of encapsulated and free (R)-roscovitine to promote inflammation resolution in vivo, mpx:GFP zebrafish were injured by tailfin transection and treated at 4 hours post injury (hpi) with 30μM free (R)-roscovitine, 30μM of polymersome-encapsulated (R)-roscovitine, or their respective controls. At 12hpi the neutrophils at the site of injury were counted under a fluorescence microscope. As shown in Figure 4b, polymersome-encapsulated (R)-roscovitine significantly reduced the number of neutrophils at the site of injury at 12hpi compared with free (R)-roscovitine or control. These results demonstrate that (R)-roscovitine encapsulated in polymersomes can speed up inflammation resolution in vivo significantly better than free drug.

Discussion

Neutrophils have proven very difficult cells to manipulate and to our knowledge no commercially available vector exists that enables the efficient intracellular delivery of cargo within their short life span and without compromising their viability and activation state. The ability of PMPC-PDPA polymersomes to deliver cargo into neutrophils without detrimental effects may be attributed to a number of features. It is important to emphasize that most transfection agents are based on cationic lipids or polymers forming positively charged nanoparticles that interact strongly with the negatively charged cell membrane favouring binding and delivery (60). This strong interaction, however, leads to unspecific binding to Toll-like receptors, such as TLR4, with consequent activation of leukocytes (61). Therefore, it is not surprising that lipoplex, the most studied non-viral nucleic acid vector, causes systemic inflammation and toxicity when injected into mice (62, 63). Conversely, the neutral charge of the polymersome membrane prevents non-specific interactions between polymersomes and proteins. This property, known as anti-fouling, is crucial for translation of synthetic polymers into the clinic and is responsible for the proven non-immunogenicity of PMPC (41).

It is likely that the rapid internalisation of polymersomes is mediated through a high binding affinity for their target receptors. It has previously been shown that the class B scavenger receptors, scavenger receptors B1 and B2 (SR-BI/SRBII) and CD36 are essential for the internalisation of PMPC-PDPA polymersomes into a number of cell types (43). Indeed, CD36 and SR-BI/SRBII are both expressed by neutrophils, which may explain the fast rate of polymersome internalisation (64, 65).

The accumulation of polymersomes within neutrophils will depend on both the rate of internalisation and the rate of removal. Incubation of human neutrophils with polymersomes resulted in an increase in fluorescence followed by a gradual plateau. Interestingly, when the polymersomes were removed from solution, the fluorescence intensity rapidly declined. This indicates that the polymer is either degraded or released from the cells over time. This may also explain the gradual plateau in fluorescence due to a balance in polymersome internalisation and release. Rapid release of polymersomes following cargo delivery may be beneficial, preventing the unnecessary intracellular accumulation of polymer within the cells, but further work will be necessary to confirm this.

The rate of nanoparticle internalisation is dependent on a number of factors. Internalisation can be driven in part simply from the physical interaction of a nanoparticle with the cell membrane. By modelling membrane wrapping, the Freund group calculated that there is an optimal nanoparticle diameter of approximately 50-60nm that facilitates membrane wrapping in the shortest time period (49). However, the rate of internalisation experimentally varies depending on the binding affinity and the properties of the nanoparticle and its target cell (66–70).

In this study, polymersomes with a diameter of approximately 190nm resulted in the greatest increase in neutrophil fluorescence intensity both when considering the fluorescence from the rhodamine-labelled polymer and encapsulated cascade blue. It should be emphasised that the increase in neutrophil fluorescence intensity from the rhodamine demonstrates that 190nm polymersomes are most efficient in terms of the amount of polymer delivered into the cells, but not the number of polymersomes internalised. This is because the larger polymersomes are formed from a greater amount of polymer and hence more rhodamine is delivered into the neutrophil for every polymersome that is internalised. However, the data highlights that this size of polymersome may be optimal as a drug delivery vehicle for neutrophils.

The rate of polymersome internalisation into FaDu cells compared with neutrophils cannot be compared by fluorescence intensity alone. However, these data show that FaDu cells, unlike neutrophils, appeared to internalise polymersomes at a similar rate at all sizes tested. The reason for this is unclear, but may be a result of the different density of receptors on their cell membrane. Alternatively, the specialised abilities of neutrophils to internalise larger foreign particles may improve their uptake of polymersomes with larger diameters. These data suggest that size will influence the amount of polymersomes that are internalised into neutrophils compared with other cells. When targeting neutrophils, polymersomes with a diameter of about 200nm may be effective while a smaller diameter of approximately 50nm may allow a greater degree of internalisation by cancer cells. (R)-roscovitine encapsulated in polymersomes promoted neutrophil apoptosis in vitro in a dose dependent manner. The ability of encapsulated (R)-roscovitine to promote neutrophil apoptosis was not significantly different to that of free (R)-roscovitine. This suggests that the mass of (R)-roscovitine delivered into neutrophils using polymersomes is similar to the mass of free (R)-roscovitine that enters cells by other means.

Zebrafish provide an excellent model for studying the innate immune system. The transparent embryos contain neutrophils and macrophages, which can be fluorescently labelled by transgenesis and by 3dpf the innate immune system is fully functional allowing its role in infection and inflammation to be understood in the absence of the adaptive immune system that does not fully mature until 1 month post fertilisation (71). The zebrafish has been helpful in the study of neutrophil biology, leading to a number of discoveries in the field, including the role of neutrophil reverse migration in inflammation resolution (72, 73), (34). Using the zebrafish tail transection model, it was shown that (R)-roscovitine encapsulated within polymersomes promoted inflammation resolution significantly quicker than the free drug. Free (R)-roscovitine alone did not significantly reduce neutrophil numbers at the injury site compared with the untreated control, although the mean number of neutrophils was slightly lower (not significant). The improvement of encapsulated (R)-roscovitine compared with the free drug suggests that polymersomes were more efficient at delivering the drug into zebrafish neutrophils than could be achieved by other means.

Figure 4 demonstrates that polymersomes are capable of penetrating deep into the zebrafish tail. Free (R)-roscovitine will likely offer only modest tissue penetration since it has a partition coefficient (logp) of 3.2 and generally only highly hydrophobic molecules are capable of deep tissue penetration, indeed compounds with a logP below 3.8 have relatively low penetration in zebrafish (74). Delivery of (R)-roscovitine using polymersomes helps improve drug tissue penetration and may offer a more selective uptake into immune cells, compared with other cell types(43, 57, 58). Consequently we believe the superior ability of polymersomes to deliver (R)-roscovitine to neutrophils in the zebrafish tail improved the outcome of this model. Polymersomes may prove an effective vector for the local delivery of drugs to neutrophils.

Neutrophil dominated inflammatory diseases are common and there are currently no neutrophil-targeted treatments available. In this study we have shown that PMPC-PDPA polymersomes can effectively deliver molecules into human neutrophils without evoking obvious detrimental effects on neutrophil viability or IL-8 release. These polymersomes successfully encapsulated and delivered (R)-roscovitine to promote neutrophil apoptosis in vitro and help resolve a model of neutrophilic inflammation in vivo. These results have important consequences for the study of neutrophil biology, and the development of neutrophil-targeted therapeutics.

Supplementary Material

Supplementary Figures
Supplementary Video Legend
Video 1
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Acknowledgements

The authors thank Biocompatibles UK Ltd for supplying the PDPA monomer and Prof S. Armes, and Drs J. Madsen and N. Warren for the synthesis of the PMPC-PDPA copolymers. The authors would like to thank the staff of the Bateson centre fish facility for their excellent fish care and assistance with zebrafish husbandry, and the blood donors who have contributed to this work.

The work was supported by an MRC Senior Clinical Fellowship (SAR; reference number G0701932), MRC Centre grant (G0700091) and a MRC Programme Grant to S.A.R. (MR/M004864/1). Microscopy studies were supported by an MRC grant (G0700091) and a Wellcome Trust grant (GR077544AIA). GB acknowledges the funding of the ERC STG grant (MEViC). GB is also recipient of an EPSRC established career fellowship. JDR thanks the MRC and MAO thanks the BBSRC for supporting their PhD scholarships.

Abbreviations used in this article

ANOVA

Analysis of variance

hpi

hours post injury

mpx:GFP

Tg(mpx:GFP)i114

PBS

phosphate buffered saline

TFA

trifluoroacetic acid

Footnotes

Conflict-of-interest disclosure: The authors declare no competing financial interests.

References

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