Liposomes equipped with poly(N-isopropyl acryl amide)-containing coatings as potential drug carriers

Abstract

Polymer coated liposomes are promising drug delivery candidates. Herein, we report on the coating of liposomes with mixed films containing poly(dopamine) (PDA) and different types of poly(N-isopropyl acryl amide) (pNiPAAm). Their potential as drug carriers from solution in the presence of shear stress or in a substrate-mediated manner using macrophages is assessed. Although we found no difference in the cell mean fluorescence (CMF) when applying shear stress when employing fluorescently labeled liposomes, macrophages exposed to liposomes coated with a mixture of PDA and highly-branched pNiPAAm (L^D/HB) exhibited significantly higher CMF after 2.5 h compared to liposomes coated with only PDA (L^D) or with a mixture of PDA and aminated pNiPAAm (L^D/pNH₂). The coated liposomes did not affect the cell viability in the time frame tested, but the application of shear stress reduced the number of surface-adherent macrophages. L^D, L^D/pNH₂ or L^D/pHB could be immobilized to poly(L lysine) pre-coated silica substrates. All substrates were equally suited for macrophages to adhere. The CMF of the adhering macrophages was found to be independent of the coating of the surface-immobilized liposomes when using fluorescently labeled liposomes in the coatings. Taken together, we demonstrate that mixed PDA-based coatings can be used to affect the interaction of liposomes with macrophages with potential in drug delivery.

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Introduction

Liposomes are considered in diverse areas of biomedicine, from biosensing¹ to drug delivery.² They can be administered from solution or in a substrate-mediated manner, and are promising candidates in emerging fields like therapeutic cell mimicry.³ Approaches involving liposomes are so manifold and diverse due to the advantages of these lipid based carriers including their ease of assembly and biocompatibility, their ability to encapsulate hydrophilic and hydrophobic cargo and the multiple ways of lipid modification for targeting or PEGylation. Polymer coating of liposomes is considered as a powerful approach to improve the stability of the liposomes, to tune the control over the cargo retention and release, or to allow for subsequent lipid-independent surface modification. However, depositing polymer layers onto liposomes without lipid-modification remains challenging with only a few successful approaches reported in the literature.⁴ The sequential deposition of interacting polymers/biomolecules has been considered.^5,6 However, this approach suffers from the practical drawback that the liposomes need to be washed/purified after each adsorption step. Alternatively, liposomes have been successfully coated with thick layers by polymerization of the liposome surface including demonstrated pH or temperature sensitivity of the polymer/lipid assemblies.^7–9 We recently demonstrated that poly(dopamine) (PDA)^10,11 can self-polymerize onto liposomes,¹² equipping them with a layer that could be easily further modified via amines or thiols.¹³ PDA as such is not responsive to external stimuli, but we and others have recently shown that functional building blocks could be copolymerized with dopamine (DA).^14–24 Specifically important in the context of current report are our prior findings which confirmed that mixed films of PDA/aminated poly(N-isopropylacrylamide) (pNiPAAm-NH₂)¹⁴ and PDA/highly-branched pNiPAAm (pNiPAAm-HB)¹⁶ could be assembled. pNiPAAm is an interesting building block to consider since the addition of this polymer equips the PDA-based films with temperature responsiveness. However, mixed PDA-based coatings have not yet been considered as coatings for liposomes, but it would be an interesting approach to equip these popular nanocarriers with functional coatings.

An important aspect to consider when characterizing carriers for drug delivery applications is the platform employed to screen them ex vivo. This is necessary due to the fact that every intravenously administered drug delivery vehicle which travels in the blood circulation not only is in contact with the blood components by also with the constituent flow-dynamic environment. Microfluidic chips have recently started to emerge as a platform with superior ability to mimic the in vivo situation over the often used well-plate approach. Assessing the interaction of drug carriers with cells while considering shear stress has led to interesting findings which are expected to help narrow down the potential lead delivery candidates prior to animal experiments which are expensive and ethically controversial. Only a limited number of studies have so far assessed the potential of drug carriers in the presence of shear stress.²⁵ Results from these efforts include the uptake of drug carriers by endothelial cells in the context of transfection^26,27 and targeting.^28–30 The former aspect was found to increase in the presence of shear stress, while the latter decreased. Studies with other cell lines are even more scarce. In one study, macrophages were found to exhibit enhanced phagocytosis of serum opsonized latex beads.³¹ We have recently demonstrated that low shear stress affected the uptake of charged liposomes by myoblasts and the subsequent therapeutic response.³² Further, we showed that the PEGylation efficiency of liposomes was dependent on the cell line (hepatocytes vs. myoblasts) used, the shear stress applied and the exposure time.¹³ Shear stress in the context of substrate-mediated drug delivery (SMDD) is often neglected despite its importance, since implanted devices are likely to contact body fluidics. In this context, only bio-catalytic coatings for the local conversion of prodrugs and the subsequent delivery to adhering hepatocytes have been characterized in the presence of shear stress.³³

Herein, we report on the coating of liposomes with PDA-based films and their potential as drug carriers in solution exposed to shear stress or in a substrate-mediated manner (Scheme 1). Specifically, we (i) confirmed the assembly of PDA, PDA/pNiPAAm-NH₂ or PDA/pNiPAAm-HB coated liposomes (L^D, L^D/pNH₂ or L^D/pHB), (ii) assessed the interaction of L^D, L^D/pNH₂ and L^D/pHB with macrophages in the presence of shear stress in terms of uptake/association and cytotoxicity, (iii) compared the adsorption behavior of L^D, L^D/pNH₂ or L^D/pHB onto PLL coated surfaces at 22 °C and 37 °C, and (iv) visualized macrophage adhesion to PLL/L^D(L^D/pNH₂ or L^D/pHB) coated substrates and evaluated the ability of the adhering cells to internalize fluorescent lipids in a substrate-mediated manner.

Scheme 1 Schematic illustration of liposomes coated with PDA, PDA/pNiPAAm-NH₂ or PDA/pNiPAAm-HB (L^D, L^D/pNH₂ or L^D/pHB) and their administration to macrophages either from solution under the influence of shear stress (i) or in a substrate-mediated manner (ii).

Experimental

Materials

Aminated poly(N-isopropylacrylamide) (pNiPAAm-NH₂, MW = 2500 Da), dopamine hydrochloride (DA), tris(hydroxymethyl)aminomethane (TRIS), sodium chloride (NaCl), ethanol, chloroform, and poly(L lysine) (PLL, 40 000–60 000 Da) were purchased from Sigma-Aldrich. Highly-branched pNiPAAm (pNiPAAm-HB) was synthesized using a prior reported protocol.¹⁶ Zwitterionic lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and fluorescent lipids 1-oleoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-glycero-3-phosphocholine (NBD-PC) were purchased from Avanti Polar Lipids, USA.

Two types of TRIS buffer were use throughout all the experiments: TRIS¹ consisted of 10 mM TRIS (pH 8.5) and TRIS² consisted of 10 mM TRIS and 150 mM NaCl (pH 7.4). The buffer solutions were made with ultrapure water (Milli-Q gradient A 10 system, resistance 18 MΩ cm, TOC < 4 ppb, Millipore Corporation, USA).

Unilamellar liposome stock solutions were prepared by evaporation of chloroform mixed with 2.5 mg lipids under vacuum for at least 1 h. This was followed by hydration into 1 mL TRIS¹ buffer and extrusion through 100 nm filters (11 times). For fluorescently labeled liposomes, 4 wt% of NBD-PC was added to the lipid mixture.

Liposome coating

Zwitterionic liposomes were coated with DA/pNiPAAm-NH₂ in 10/1 molar ratios. Specifically, 4 mg mL⁻¹ DA stock solution in TRIS buffer was prepared and diluted to 2 mg mL⁻¹ using TRIS¹ buffer or pNiPAAm-NH₂ solution (5.3 mg mL⁻¹). Typically, 200 μL of liposome stock solution was mixed with 200 μL DA/pNiPAAm-NH₂ solution leading to a final DA concentration of 1 mg mL⁻¹. After 70 min coating time, the samples were dialyzed (14 kDa MWCO) against TRIS² for 3 h yielding L^D/pNH₂. For coating of the liposomes with pNiPAAm-HB, 200 μL liposome stock solution was mixed with 200 μL DA/pNiPAAm-HB solution (2/1 wt%) leading to a final DA concentration of 1 mg mL⁻¹. After 70 min coating time, the samples were dialyzed (100 kDa MWCO) against TRIS² for 1 h yielding L^D/pHB. Liposomes were coated with PDA (1 mg mL⁻¹) for 30 min and dialyzed (14 kDa MWCO) against TRIS² for 3 h yielding L^D. As controls, the liposomes were mixed with either pNiPAAm-NH₂ solution (1.3 mg mL⁻¹) or pNiPAAm-HB (1 mg mL⁻¹) and let to incubate. All samples were continuously shaken during the coating process.

The diameter, polydispersity index (PDI) and ζ-potential of the samples was determined at different time points by measuring in a dynamic light scattering (DLS) instrument (Zetasizer nano, Malvern Instruments) using a material refractive index of 1.590 and a dispersant (water at 25 °C) refractive index of 1.330. Within this paper, samples with a PDI > 0.4 were considered aggregated and were discarded.

For the cell experiments, fluorescently labeled liposomes were coated according to the above described protocol. Further, to ensure that similar amounts of carriers were delivered to the cells in each independent repeat, the fluorescent intensity was assessed using a multiplate reader. When mixing the coated liposomes with cell media, always the same amount of ‘fluorescence’ was added.

Cryo-electron microscopy (cryo-TEM)

Cryo-TEM samples were prepared by adsorbing 4 μL of the different liposome suspensions onto Quantifoil R3.5/1 holey carbon film mounted on 300 mesh copper grids (Quantifoil Micro Tools GmbH, Jena, Germany). Prior to adsorption, the grid was rendered hydrophilic by glow discharge in a reduced atmosphere of air for 10 s. The specimen was applied and after 1 min incubation on the surface, the grid was blotted and quick-frozen in liquid ethane using a Vitrobot automated plunging device (FEI Company, Eindhoven, The Netherlands).

Frozen grids were transferred to liquid nitrogen before loading into a Gatan 626 cryo-holder (Gatan, Pleasanton, CA, USA). The cryo-holder was then inserted into the stage of a Philips CM200 FEG TEM (FEI company) operated at 200 kV. Imaging was performed at cryogenic temperatures (approx. −170 °C) in low-dose, bright-field mode. Electron micrographs were recorded digitally on a TVIPS 4k × 4k CMOS Camera (TVIPS GmbH, Gauting Germany) at given defocus values of −2.5 μm.

Quartz crystal microbalance with dissipation monitoring (QCM-D)

QCM-D measurements (Q-Sense E4, Sweden) were used to analyze the interaction of the differently coated liposomes with PLL coated surfaces. Silica-coated crystals (QSX300, Q-Sense) were cleaned by immersion in a 2 wt% sodium dodecyl sulfate solution overnight and rinsing with Milli-Q water. Afterwards, the crystals were blow-dried with N₂, treated with UV/ozone for 20 min and mounted into the liquid exchange chambers of the instrument. When a stable baseline in TRIS² buffer was obtained, PLL solution (1 mg mL⁻¹) was introduced into the measurement chamber and let to adsorb until the surface was saturated. Then, the chamber was rinsed with TRIS² buffer and the pre-coated crystals were exposed to either L^D, L^D/pNH₂ or L^D/pHB solutions. When the surface was saturated, the measurement chamber was rinsed with TRIS² buffer. The frequency and dissipation measurements were monitored at 22 ± 0.02 °C or 37 ± 0.02 °C. Normalized frequencies using the third harmonic are presented.

Cell work

RAW264.7 mouse macrophage where used for all the experiments and were cultured in 75 cm² culture flasks in medium (DMEM with 4500 mg L⁻¹ glucose, sodium pyruvate and sodium bicarbonate supplemented with 10% fetal bovine serum, 50 μg mL⁻¹ penicillin and 50 μg mL⁻¹ streptomycin) at 37 °C and 5% CO₂.

The statistical significance used to compare the distribution was determined using a two-way ANOVA with a confidence level of 95% (α = 0.05), followed by a Tukey's multiple comparison posthoc test (* = p < 0.05) for all cell experiments.

Solution-based experiments

RAW267.4 cells were seeded at a density of 82 000 cells per channel in 120 μL medium into closed perfusion chambers (μ-slide VI^0.4 ibitreat, Ibidi GmbH, Munich, Germany) and allowed to attach in a humidified incubator for 24 h at 37 °C and 5% CO₂. Thereafter, 7.5 mL of cell media containing the L^D, L^D/pNH₂ or L^D/pHB in the desired concentration was added to the reservoir (syringe) of the ibidi pumping system. As static controls, cells were exposed to the same liposome containing media in the perfusion channels (τ₀ = 0 dyn cm⁻²). Subsequently, the perfusion chamber was connected to a syringe pump and placed in the humidified incubator (37 °C and 5% CO₂). The flow experiments were performed at a shear stress of τ₄ = 4 dyn cm⁻² for 2.5 h at 37 °C and 5% CO₂. After this time, the channels were flush-washed twice with PBS (120 μL), followed by the addition of 60 μL of trypsin for 5 min to detach the cells from the surface. The cells were harvested by washing the channels with PBS (100 μL) 2× and the cell mean fluorescence (CMF) was analyzed with a BD Accuri® C6 flow cytometer using an excitation wavelength of 488 nm. At least 2000 cells were analyzed. The autofluorescence of cells has been subtracted and the control cells have been gated out in all the presented results. All cell experiments were performed in at least three independent repeats.

The cell viability of the cells was assessed after their exposure to shear stress by replacing the liposome cell medium with 120 μL of a mixture containing 100 μL cell medium and 10 μL Cell Counting Kit-8 assay solution. After 2 h of incubation at 37 °C and 5% CO₂, the absorbance was measured using the multi-mode plate reader. The experiments were performed in three independent experiments and the results were normalized to the cells incubated in cell medium only under static conditions.

Substrate-mediated administration

The substrates were prepared by sonication of 9 mm glass slides for 10 min in ethanol, rinsed with ultrapure water and dried with N₂ followed by 30 min UV/ozone treatment. Then, the samples were coated with PLL (1 mg mL⁻¹ in TRIS², 10 min), rinsed with TRIS², and finally the coated liposomes were let to adsorb for 40 min and rinsed again with TRIS². The samples were transferred into a new 48 well plate with sterile PBS and UV-sterilized for 30 min. 50 000 cell per well were seeded and let to adhere for 3 h or 24 h at 37 °C and 5% CO₂ before washing with PBS and the addition of 50 μL trypsin to detach the cells. The cells were then collected by washing with PBS 3×. The CMF was analyzed using the flow cytometer. The autofluorescence of cell cultured on PDA coated glass slides was subtracted and the control cells have been gated out in all the presented results. All cell experiments were performed independently, repeated at least three times.

For light microscopy imaging of the macrophages, cells were fixed using a solution of 4% paraformaldehyde in water (5–7 min) followed by three washing steps using PBS. Two different imaging techniques were employed: differential interference contrast (DIC) images were taken with an Olympus CKX41 microscope and fluorescence images of the cells were taken with an Axiovert microscope coupled to an LSM 700 confocal laser scanning module (Zeiss, Germany).

Results and discussion

Liposomes coating with pNiPAAm-containing films

With the aim to equip zwitterionic liposomes with PDA-based mixed films, the liposomes were mixed with a DA, a pNiPAAm-NH₂ and a DA/pNiPAAm-NH₂ (Fig. 1a) or a DA, a pNiPAAm-HB and a DA/pNiPAAm-HB (Fig. 1b) solution and the change in diameter over time was monitored by DLS. In the absence of DA, no change in liposome diameter was observed, indicating that no polymer was deposited. On the other hand, when DA was present, the diameter increased with time, suggesting the deposition of a polymer coating. We would like to note that the quantitative increase in diameter might be misleading due to the deposited black PDA-based coating which potentially could adsorb the laser light and by doing so, affected the outcome of the DLS measurements. This aspect has been observed previously.^12,13 Importantly, we also showed that PDA coated liposomes could be further modified with PEG using the interaction of amines with PDA.¹³ What is more, we have previously confirmed the coating of surface immobilized liposomes with PDA and PDA/pNiPAAm-HB by QCM-D.¹⁶ Further, the adsorption of liposomes onto silica particles followed by the deposition of PDA yielded structurally intact capsosomes upon silica core removal, demonstrating the assembly of a stable PDA shell onto of liposomes.³⁴ All these aspects taken together strongly point towards the coating of the liposomes.

Fig. 1 Liposome coating using DA/pNiPAAm-NH₂ (a) or DA/pNiPAAm-HB (b) including the control with DA and pNiPAAm-NH₂(HB) only.

In order to stop the reaction, TRIS² buffer was added and the unbound/unreacted DA and pNiPAAm was removed using dialysis, yielding similarly sized assemblies (ESI Table S1†). From now on, the coated liposomes will be referred to as: L^D for liposomes coated with PDA, L^D/pNH₂ for liposomes coated with DA/pNiPAAm-NH₂ and L^D/pHB for liposomes coated with DA/pNiPAAm-HB. The three types of coated liposomes were visualized using cryo-TEM (Fig. 2). The three types of coated liposomes looked similar, exhibiting a predominantly spherical shape and structural integrity. For further characterization, the ζ-potentials of L^D, L^D/pNH₂ and L^D/pHB were measured. As expected, L^D had the lowest ζ-potential. Adding pNiPAAm-NH₂ or pNiPAAm-HB to the coating increased the ζ-potential of the liposomes, a trend that has previously been observed for capsules consisting of PDA, PDA/pNiPAAm-NH₂ or PDA/pNiPAAm-HB.¹⁵ The change in ζ-potential together with our prior findings strongly supported the deposition of a mixed polymer layer on the liposomes.

Fig. 2 Cryo-TEM images of L^D (left), L^D/pNH₂ (middle) or L^D/pHB (right). The scale bars are 100 nm. The ζ-potential values (average/standard deviation) of the samples measured in 10 mM Tris, pH 7.4 are noted on top of the images.

Solution-based administration considering shear stress

With the aim to understand the potential of these polymer coated liposomes for drug delivery applications, we compared the interaction of L^D, L^D/pNH₂ and L^D/pHB with macrophages under static conditions (τ₀ = 0 dyn cm⁻²) and under the influence of shear stress (τ₄ = 4 dyn cm⁻²) mimicking the dynamic in vivo conditions in the capillaries. Macrophages were chosen since these are important cells in the context of drug delivery. Macrophages will experience different shear stresses and respond differently to this mechanical stimuli depending whether they are circulating or tissue resident. Since the fundamental knowledge of macrophages interacting with potential drug carriers in the presence of shear stress is by far missing, we chose to start with the above mentioned shear stress and a rather short incubation time of 2.5 h. The employed the RAW264.7 mouse macrophages were a good choice for this purpose since they are a widely accepted immortalised model cell line for immune cells. We are interested in understanding the cell–drug carrier interaction from the fundamental point of view. Therefore, we are not specifically aiming at either targeting or uptake prevention depending on the coating at this point in time. We would like to note that while the coating of the liposomes was performed at room temperature, the subsequent cell work was done at 37 °C and 5% CO₂.

The cell mean fluorescence (CMF) of macrophages (Fig. 3a) and the uptake efficiency (ESI Fig. S1†) upon exposure to L^D, L^D/pNH₂ and L^D/pHB with incorporated fluorescent lipids for 2.5 h were monitored by flow cytometry. First, when comparing the CMF and uptake efficiency for a specific type of coated liposomes with or without applied shear stress, no significant difference was found at τ₀ or τ₄. This is in agreement with our previous findings when comparing the CMF of myoblasts exposed to zwitterionic or negatively charged liposomes in the presence or absence of shear stress.³² While applying shear stress did not affect the CMF or uptake efficiency, the type coating of the liposomes did. The CMF of macrophages exposed to L^D/pHB was significantly higher than for cells incubated with L^D or L^D/pNH₂ at τ₀ and τ₄. This finding likely reflected the different ζ-potential of the coated liposomes, where the more negatively charged carriers L^D and L^D/pNH₂ were less associated with the cells. CLSM images of macrophages incubated with L^D, L^D/pNH₂ and L^D/pHB at τ₀ and τ₄ confirmed the internalization of the fluorescent cargo (Fig. 3b). In all cases, the fluorescence was homogenously distributed in the cytosol of the cells, and it was excluded from the nuclei. Control images of cells which were not exposed to any fluorescent liposomes had no fluorescent signal associated with the cells (results not shown), demonstrating that the observed fluorescence in Fig. 3 originated from internalized fluorescent lipids.

Fig. 3 Interaction with macrophages considering shear stress: (a) CMF of macrophages upon exposure to L^D, L^D/pNH₂ or L^D/pHB for 2.5 h at τ₀ or τ₄. (n = 3, *p < 0.05) (b) CLSM images of macrophages after 2.5 h incubation with L^D, L^D/pNH₂ or L^D/pHB at τ₀ or τ₄. The scale bars are 10 μm.

Understanding the effect on the cell viability due to their exposure to the coated liposomes and/or the shear stress in a quantitative manner is an important aspect when biomedical applications are considered.

Overview bright field images of the macrophages taken after the exposure to the coated liposomes at τ₀ and τ₄ and macrophages in the absence of the coated liposomes showed similar cell densities and cell morphologies for all the tested conditions (ESI Fig. S2†). Further, the cell viability of macrophages incubated for 2.5 h with or without L^D, L^D/pNH₂ and L^D/pHB at τ₀ or τ₄ was quantitatively compared (Fig. 4). There was no significant difference between pristine cells and cells exposed to L^D, L^D/pNH₂ or L^D/pHB at τ₀ or τ₄, indicating that none of the coated liposomes was inherently affecting the viability of the macrophages. However, it should be noted that the number of pristine cells at τ₄ was lower compared to τ₀. This reduction by ∼50% could not be observed in the microscopy images. Our chosen cell viability kit was measuring the dehydrogenase activities in cells. Therefore, the results point towards an interesting effect in a metabolic pathway involving this enzyme induced by the applied shear stress that was not affecting the observed cell number.

Fig. 4 The cell viability of macrophages after incubation with or without L^D, L^D/pNH₂ or L^D/pHB for 2.5 h at τ₀ or τ₄. The results are normalized to cells in the absence of any coated liposomes at τ₀.

Substrate-meditated administration

Substrate-mediated drug delivery (SMDD) is an approach to deliver active compounds to adhering cells or to cells in the close proximity.^35,36 Trapping therapeutic compounds in thin polymer film can be challenging especially for small hydrophobic cargo or fragile proteins due to uncontrolled leakage and loss in functionality, respectively. Using drug deposits in the form of cyclodextrins, micelles, polymersomes or liposomes has turned out to be a promising strategy to overcome these issues.³ We and others^37,38 have trapped liposomes in polymer films assembled either via the layer-by-layer technique or using PDA-based films and demonstrated interaction with adhering cells. So far, we have either sequentially deposited the liposomes and the PDA^16,39 or mixed the liposomes with the DA solution to adsorb the mixed coatings in one step.⁴⁰

We aimed to understand if L^D, L^D/pNH₂ and L^D/pHB could potentially be used to deliver therapeutic compounds from the surface to adhering cells. In a first step, we compared the ability of the three types of coated liposomes to adsorb onto PLL pre-coated crystals at 22 °C and 37 °C using QCM-D. The frequency changes Δf (Fig. 5) and dissipation changes ΔD (ESI Fig. S3a†) showed that a similar amount of intact L^D and L^D/pNH₂ were deposited at both tested temperatures. The observed changes in Δf and ΔD were similar to values we have previously observed when depositing liposomes mixed with DA, pointing not only towards the assembly of intact liposomes, but also towards the formation of a dense layer.⁴⁰ On the other hand, L^D/pHB exhibited a temperature dependent adsorption behavior. At 22 °C, a similar Δf was observed as for L^D and L^D/pNH₂. However, at 37 °C, Δf was ∼66% lower. We have previously shown that the phase transition for this pNiPAAm-HB was between 30 and 40 °C.¹⁶ The increasing hydrophobic nature of pNiPAAm-HB at 37 °C seemed to affect the mixed coating and hindered the interaction of L^D/pHB with the PLL pre-coated crystals. With the aim to having similar amounts of surface immobilized L^D, L^D/pNH₂ and L^D/pHB, 22 °C was chosen as the adsorption temperature for the following cell experiments. We would like to note that films with immobilized L^D/pHB at 22 °C were also found to be stable upon increasing the temperature to 37 °C (ESI Fig. S3b†).

Fig. 5 Frequency change Δf of PLL pre-coated crystals upon exposure to L^D, L^D/pNH₂ and L^D/pHB at 22 °C or 37 °C.

The next goal was to assess the potential of these liposome-coated surfaces in SMDD. First, we compared the ability of macrophages to adhere to PLL pre-coated substrates coated with PDA, L^D, L^D/pNH₂ or L^D/pHB after 3 h and 24 h adhesion time (Fig. 6a). The macrophages were found to adhere equally well to all the tested surfaces at both time points. Further, the cell adhesion to the liposome-coated surfaces was comparable to PDA coatings (ESI Fig. S4†). This observation demonstrated that the presence of the liposomes was not negatively affecting the macrophage adhesion. Also, there was no difference in cell adhesion observed depending on the type of polymer used to coat the liposomes. This is in agreement with our previous findings using liposome-containing films assembled in a sequential manner and varying the type of capping layer (PDA, PDA/pNiPAAm-HB or pNiPAAm-HB) for the liposomes.¹⁶ The successful macrophage adhesion makes these coatings potentially useful in SMDD.

Fig. 6 (a) Representative bright field images and (b) CMF and uptake efficiency of macrophages adhering to PLL pre-coated substrates modified with L^D, L^D/pNH₂ and L^D/pHB after 3 h and 24 h adhesion time. The scale bars are 50 μm. (n = 3, *p < 0.05).

Following on, we aimed to compare the different coatings in their ability to delivery cargo embedded into the coated liposomes to adhering macrophages. We would like to note that while the assembly of the liposomes coated substrates was performed at 22 °C, the subsequent cell work was done at 37 °C and 5% CO₂. As a proof-of-concept, we chose a fluorescent lipid as a model cargo embedded in the surface-immobilized coated liposomes. The CMF and the uptake efficiency of the fluorescent lipids by adhering macrophages were measured after 3 h and 24 h by flow cytometry (Fig. 6b). There were no significant differences for the CMF or uptake efficiency depending on the coating of the liposomes. However, both aspects were significantly lower after 24 h compared to 3 h cell adhesion time, probably due to depletion of the surface from the lipids and/or cellular processing of the fluorescent lipids. We have prior found a similar effect in decreasing CMF and uptake efficiency when considering sequentially deposited coatings of PLL/liposomes/PDA.³⁹ The addition of a polymer capping layer would likely allow for prolonged cargo delivery to the adhering macrophages.⁴¹ Although no significant difference depending on the coatings was found, these results demonstrate that coated surface immobilized liposomes could be used in SMDD.

Conclusions

We reported on the use of L^D, L^D/pNH₂ or L^D/pHB as drug carriers from solution or in SMDD for macrophages. The coating of the liposomes with different films has been demonstrated. The CMF of macrophages exposed to L^D, L^D/pNH₂ or L^D/pHB was independent of the applied shear stress, but L^D/pHB led to significantly higher CMF values compared to the other samples. The cell viability remained unaffected by the coated liposomes, but τ₄ reduced the number of adhering macrophages. Similar Δf of PLL pre-coated crystals upon exposure to L^D and L^D/pNH₂ were monitored by QCM-D at 22 °C and 37 °C. On the other hand, Δf measured for crystals exposed to L^D/pHB at 22 °C was ∼66% higher than for adsorption at 37 °C. Macrophages adhered equally to L^D, L^D/pNH₂ and L^D/pHB coated substrates. There was no significant difference found depending on the coating of the liposomes when considering the CMF and the uptake efficiency, but both parameters were lower after 24 h compared to 3 h cell adhesion time.

In summary, the three types of coated liposomes have the potential to act as drug carriers for different types of administration pathways, and the composition of the coating could be employed to affect the cell response.

Acknowledgements

This work was supported by a Sapere Aude Starting Grant from the Danish Council for Independent Research, Technology and Production Sciences, Denmark. We acknowledge Associate Prof. R. Meyer (iNANO, Aarhus University, Denmark) for access to CLSM, and Associate Prof. A. Zelikin (Department of Chemistry, Aarhus University, Denmark) for access to the DLS, plate reader and flow cytometer.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Table of diameter and PDI of L^D, L^D/pNH2 and L^D/pHB, uptake efficiency of L^D, L^D/pNH₂ and L^D/pHB by macrophages, overview bright field images of macrophages exposed to L^D, L^D/pNH₂ and L^D/pHB, and dissipation changes ΔD for QCM-D measurements. See DOI: 10.1039/c4ra07720b

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