PhospholipidâBlock Copolymer Hybrid Vesicles with Lysosomal

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Phospholipid – Block Copolymer Hybrid Vesicles with Lysosomal Escape Ability Wei Zong, Bo Thingholm, Fabian Itel, Philipp Sebastian Schattling, Edit Brodszkij, Daniel Mayer, Steffen Stenger, Kenneth N Goldie, Xiaojun Han, and Brigitte Städler Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01073 • Publication Date (Web): 18 May 2018 Downloaded from http://pubs.acs.org on May 19, 2018

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Phospholipid – Block Copolymer Hybrid Vesicles with Lysosomal Escape Ability Wei Zong,a,b,‡ Bo Thingholm,b,‡ Fabian Itel,b Philipp S. Schattling,b Edit Brodszkij,b Daniel Mayer,c Steffen Stenger,c Kenneth N. Goldie,d Xiaojun Han,a* Brigitte Städlerb* a

State Key Laboratory of Urban Water Resource and Environment, School of Chemistry and

Chemical Engineering. Harbin Institute of Technology, 92 West Da-Zhi Street, Harbin, 150001, China b

Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wieds Vej 14, 8000

Aarhus, Denmark c

Institute for Medical Microbiology and Infection Control, University Hospital Ulm, Ulm,

Germany d

Center for Cellular Imaging & Nano Analytics, Biozentrum, University of Basel, Basel,

Switzerland.

KEYWORDS: vesicles, lipids, block copolymer, lysosome, macrophages

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ABSTRACT. The success of nanoparticulate formulations in drug delivery depends on various aspects including their toxicity, internalization and intracellular location. Vesicular assemblies consisting of phospholipid and amphiphilic block copolymers are an emerging platform, which combines the benefits from liposomes and polymersomes while overcoming their challenges. We report the synthesis of poly(cholesteryl methacrylate)-block-poly(2-(dimethylamino) ethyl methacrylate) (pCMA-b-pDMAEMA) block copolymers and their assembly with phospholipids into hybrid vesicles. Their geometry, their ζ-potential, and their ability to adsorb onto polymer coated surfaces were assessed. Giant unilamellar vesicles were employed to confirm the presence of both, the phospholipids and the block copolymer in the same membrane. Further, cytotoxicity of selected hybrid vesicles was determined in RAW 264.7 mouse macrophages, primary rat Kupffer cells and human macrophages. The internalization and lysosomal escape ability of the hybrid vesicles was confirmed using RAW 264.7 mouse macrophages. Taken together, our findings illustrate that the reported hybrid vesicles are a promising complementary drug delivery platform to existing liposomes and polymersomes.


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INTRODUCTION Nanoformulations have and will improve how therapeutic molecules are administered to patients affected by diverse medical conditions as recently reviewed by Torchilin.1 Polymer micelles,2 liposomes,3 hydrogel nanoparticles,4 or mesoporous silica5 are just the main stream assemblies considered as drug carriers. Although these different drug delivery platforms are envisioned to overcome challenges involved with conventional drug formulations, clinical success stories are rare. Their suboptimal performance can be attributed to the diverse biological barriers that the drug carriers have to overcome to facilitate beneficial therapeutic impact as recently discussed in detail by Ferrari and coworkers.6 Such barriers include opsonization and subsequent sequestration by the mononuclear phagocyte system (MPS), limited and/or non-specific cellular internalization or inability to escape from endosomal and lysosomal compartments among others. Generally, different strategies were derived for intracellular delivery of therapeutic cargo including the trafficking out of the lysosomes/endosomes into the cytosol as recently reviewed by Torchilin and co-workers.7 Endosomal/lysosomal escape approaches8-9 include the use of fusogenic lipids or polymers, cell penetrating peptides, or cationic nanocarriers employing the proton-sponge effect (also known as pH-buffering effect). The latter example, originating from poly(ethylenimines) (PEI),10 is an interesting approach which is based on polymeric acidresponsive systems activated by the acidifying endosomes.11 These polymers contain tertiary amines, which become protonated inside the acidified environment of endosomes. In this context, the polymers buffer the targeted drop in pH, leading to continuous proton translocation, passive chloride ions entry and water influx. This induces a high osmotic pressure, which causes swelling and eventually rupturing of the endosomes, thus releasing the endosomal content into the cytosol. Apart from PEI, poly(amidoamine),12-13 histidines (i.e., the imidazole ring),14-15 and 3 ACS Paragon Plus Environment

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poly(N,N-dimethylaminoethyl methacrylate) (pDMAEMA),16 among others were used as building block in polymeric drug carriers as recently reviewed by Ge et al.17 Tertiary amine methacrylate-based block copolymers represent a category of polymers with unique properties and advantages for diverse applications in nanomedicine as extensively discussed in the review by the Liu group.18 Homo- and block copolymers of pDMAEMA are promising examples in this context due to their simple synthesis in various, well-defined compositions and architectures allowing to control their properties yielding polymers with e.g., (potentially) lower cytotoxicity or controlled biodegradation as illustrated in the comprehensive review by Agarwal et al.19 Recent examples for the assembly of pDMAEMA containing copolymers include temperatureinduced polymeric assemblies20-22 and the formation of nanometer-sized polyplexes.23-24 All these reports rely on polymers only which often put rather stringent design criteria on the copolymer to allow for the assembly of vesicles and the (hydrophilic) cargo loading procedures can be elaborate. Therefore, hybrid vesicles, composed of a mixture of lipids and polymers, are emerging to combine the advantages of liposomes and polymersomes while overcoming their shortcomings as discussed in two comprehensive reviews.25-26 These new types of membranes have considerably diversified the possibilities to assemble vesicular carriers for diverse applications in bionanotechnology and nanomedicine. For instance, the lipid to polymer ratios used to assemble the hybrid vesicles was found to modulate the drug release profiles27-28 or the in vitro and in vivo toxicity.29 Recent efforts also illustrated that magnetic actuation30 or phospholipase A231 could be employed to control the calcein release from hybrid vesicles. Hybrid vesicles were also used to demonstrate the relevance of shear stress when testing the uptake efficiency by cells, i.e., shorter hydrophobic extensions led to their higher internalization by RAW264.7 mouse macrophage with applied shear stress.32 In another effort, 4 ACS Paragon Plus Environment

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asymmetric giant hybrid vesicles were assembled exhibiting a lipid and polymer inner/outer leaflet.33 From a different perspective, recognition events between ligands (biomolecules, drug carriers etc.) and (functionalized) membranes are important occurrences for the fundamental understanding of biological process or targeted drug delivery. Lipid – polymer membranes provide an interesting model system in this context. As an example, the Binder group has illustrated that the binding of cholera toxin B to ganglioside GM1 led to changes in the membrane morphology of hybrid giant unilamellar vesicles assembled from 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine and poly(isobutylene)-block-poly(ethyleneoxide).34 In another report, they showed that the supramolecular recognition between triazine-functionalized polymers in hybrid vesicles and thymine- modified nanoparticles caused the polymer to be selectively removed from the assembly causing vesicle destruction via membrane rupture.35 Hybrid vesicles were also shown to significantly improve the efficiency of cell surface receptor targeting in cell culture and in an animal model compared to polymersomes.36 In a different context, the reconstitution of membrane proteins, especially transmembrane proteins, into model membranes has diverse applications in nanobiotechnology.37 Therefore, the addition of membrane proteins to lipidpolymer membranes has been explored on planar films38-39 and vesicles.40 In the latter case, the reconstitution of the membrane protein Cyt bo 3 into hybrid vesicles composed of 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine (POPC) and poly(butadiene-b-ethylene oxide) showed lower initial enzyme activity but improved long-term stability compared to reconstitution into POPC liposomes.41 In another effort, the permeability of polymersomes, liposomes and hybrid vesicles was compared.42 It was found that the hybrid vesicles exhibited the highest permeability, which could be further increased by incorporating ionophores and the ion channel gramicidin A.


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Hybrid vesicles have been successfully employed to generate ATP by integrating two enzymes (ATP synthase and bo3 oxidase) into their membrane.43 Herein, we report the assembly of phospholipid – polymer amphiphile hybrid vesicles with ability to escape lysosomes (Scheme 1). Specifically, we i) synthesized two pDMAEMA-blockpCMA diblock copolymers with different number of DMAEMA units, ii) characterized the assembly of these polymers with phospholipids into hybrid vesicles, iii) assessed the cytotoxicity of the hybrid vesicles in comparison to liposomes in immortalized and primary macrophages, and iv) compared hybrid vesicles and liposomes in terms of cellular uptake efficiency and lysosomal escape ability using immortalized macrophages.

Scheme 1. Cartoon illustrating the vesicular assembly consisting of lipids and an amphiphilic block copolymer with a poly(cholesteryl methacrylate) (pCMA) and a pDMAEMA block, interacting with macrophages.


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EXPERIMENTAL SECTION Materials. Sodium chloride (NaCl), poly(L-lysine) hydrobromide (PLL, MW 40-60 kDa), poly(methacrylic acid) sodium salt (PMA, MW 18.6 kDa), 4-(2-hydroxyethyl)piperazine-1ethane-sulfonic acid (HEPES), phosphate buffered saline (PBS), chloroform anhydrous (≥ 99%), ethanol, 2-(dimethylamino)ethyl methacrylate (DMAEMA), 2-(dodecylthiocarbonothioylthio)-2methylpropionic acid N-hydroxysuccinimide ester (NHS-CTA), 4′,6-diamidino-2-phenylindole (DAPI), paraformaldehyde (PFA), cell counting kit-8 (CCK-8), polyethylene glycol-tertoctylphenyl ether (Triton X-100), sodium dodecyl sulfate (SDS), trypsin-EDTA 0.25%, and tetrahydrofuran (anhydrous) (THF) were purchased from Sigma-Aldrich. Triethylamine and 5[(2- aminoethyl)amino] naphthalene-1-sulfonic acid sodium salt were acquired from Alfa Aesar. Azobisisobutyronitrile (AIBN) was commercially available from Merck KGaA. Methanol and concentrated hydrochloric acid was received from VWR. 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC), 1palmitoyl-2-oleoylphosphatidylserine (POPS), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-myristoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3phosphocholine (NBD-PC) and Texas Red™ 1,2-dihexadecanoyl-sn-glycero-3phosphoethanolamine (Texas Red-DHPE) were purchased from Avanti Polar Lipids. LysoTracker® Red DND-99, Amplex® Red reagent, dimethylsulfoxide (DMSO), horseradish peroxidase (HRP), stabilized hydrogen peroxide, reaction buffer (0.5 M potassium buffer containing 0.25 M NaCl, 25 mM chloric acid and 0.5% Triton® X-100 at pH 7.4), cholesterol oxidase from Streptomyces, cholesterol esterase from Pseudomonas and a cholesterol reference standard were purchased from Thermo Fisher Scientific. Six channel ibi-treated µ-slides VI0.4 were acquired from iBidi®. Vanillin reagent was purchased from Cell Biolabs, Inc.


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Two types of buffers were used unless noted otherwise: HEPES1 consisting of 10 mM HEPES at pH 7.4 and HEPES2 consisting of 10 mM HEPES and 150 mM NaCl at pH 7.4. The buffer solutions were made using ultrapure water (18.2 MΩ cm−1 resistance) provided by an ELGA Purelab Ultra system (ELGA LabWater, Lane End, UK). Block Copolymer Synthesis and Characterization Synthesis of cholesteryl methacrylate (CMA). Cholesterol (8.0 g, 0.020 mol) was dissolved in 100 mL THF and 30 mL trimethylamine was slowly added. Methacryloyl chloride (3.3 mL, 0.034 mol) in 20 mL THF was added dropwise to the stirred solution. The solution was heated until reflux and was stirred overnight. The product was purified by precipitation in 1.5 N hydrochloric acid in methanol. 7.8 g (0.017 mol, 85%) CMA was obtained as colorless powder. RAFT polymerization of poly(cholesteryl methacrylate) (pCMA). CMA (300 mg, 0.660 mM), AIBN (1 mg, 0.006 mM) and NHS-CTA (30 mg, 0.065 mM) were dissolved in 1.5 mL toluene. After purging the solution for 2 min with argon, the reaction vial was subjected to three evacuation cycles and subsequently refilled with argon after each cycle. The polymerization tube was placed in a preheated oil bath at 80°C and stirred for 8 h. The polymerization was quenched by exposure to oxygen. After the solution was cooled down, the polymer was purified by three consecutive precipitation steps in acetone and re-dissolving in chloroform. NMR (400 MHz, CDCl3) δ (ppm): 5.27 (-(CH2)2-C=CH-), 4.43 (-O-CH-(CH)2-), 2.89 and 2.82 (NHS), 2.23-0.61 (Chol skeleton).


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RAFT polymerization of pCMA-block-poly(2-(dimethylamino) ethyl methacrylate) (P1 and P2). For the block extension, pCMA was used as a macro-chain transfer agent. pCMA (50 mg, 0.010 mM), AIBN (0.2 mg, 0.001 mM) and DMAEMA (373 mg, 2.372 mM) were dissolved in 1 mL toluene. After purging the solution for 2 min with argon, the reaction vial was subjected to three evacuation cycles and subsequently refilled with argon after each cycle. The polymerization tube was placed in a preheated oil bath at 80°C and stirred for 8 h. The polymerization was quenched by exposure to oxygen. After the solution was cooled down, the polymer was purified by three consecutive precipitation steps in hexane and re-dissolving in chloroform. NMR (400 MHz, CDCl3) δ (ppm): 5.27 (-(CH2)2-C=CH-), 4.19 (-O-CH2-(CH2N(CH3)2), 2.92 (O-CH2-CH2-N(CH3)2), 2.35 (-O-CH2-CH2-N(CH3)2), 1.84 (DMAEMA backbone -CH2-), 1.50 – 0.68. (Supporting Information Figure S1) RAFT polymerization of labeled pCMA-block-poly(2-(dimethylamino) ethyl methacrylate) (P3). For the block extension, pCMA was used as a macro-chain transfer agent. pCMA (58 mg, 0.009 mM), AIBN (0.14 mg, 0.0009 mM), DMAEMA (212 mg, 1.35 mM), and fluorescein omethacrylate (FlMA, 10 mg, 0.0249 mmol) were dissolved in 0.5 mL toluene and 0.1 ml DMF. The solution was purged with Ar for 1 hr. The polymerization flask was placed in a preheated oil bath at 80°C and stirred overnight. The polymerization was quenched by exposure to oxygen. After the solution was cooled down, the polymer was purified by two consecutive precipitation steps in hexane and re-dissolving in chloroform. NMR (400 MHz, CDCl3) δ (ppm): 5.27 (-(CH2)2-C=CH-), 4.19 (-O-CH2-(CH2N(CH3)2), 2.92 (O-CH2-CH2-N(CH3)2), 2.35 (-O-CH2-CH2-N(CH3)2), 1.84 (DMAEMA backbone -CH2-), 1.50 – 9 ACS Paragon Plus Environment

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0.68. 6.46 (1H, Ar), 6.61 (2H, Ar), 6.77 (2H, Ar), 7.18 (2H, Ar), 7.62 and 7.66 (2 H, Ar), 8.00 (1 H, Ar) Vesicle Assembly. Polymer-lipid hybrid vesicles and liposomes were assembled using the film rehydration method. Varying amounts of the lipid stock solution (POPC, 25 mg mL-1 in CHCl3) and polymer stock solution (10 mg mL-1 in CHCl3), keeping a total mass of 1 mg constant, were added into a round-bottom glass flask. The solvent was evaporated under vacuum for at least 1 h, followed by hydration at 37 ℃ with 1 mL HEPES1 or HEPES2 buffer solution and extrusion through 200 nm polycarbonate filters at room temperature. For fluorescent samples, 100 µL NBD-PC (1 mg mL-1 in CHCl3) was added to the round bottom flask prior to solvent evaporation. If needed, 0.2 mg of the POPC lipids were replaced with positively charged POEPC lipids or negatively charged POPS lipids. From now on, the hybrid assemblies will be denoted as PxLy, where Px represents either polymer P1 or P2 and y indicates if high (H) or low (L) amounts of lipids were added. Further, liposomes using 0.3 mg and 0.7 mg lipids were assembled as control samples. These control liposomes are referred to as Lcz with cz indicating the concentration of lipids used e.g., Lc1 and Lc0.3 refers to liposomes assembled from 1 mg mL-1 lipids and 0.3 mg mL-1 lipids, respectively. Further, L refers to POPC lipids only while L+ and Lindicates assemblies with 20 wt% POEPC and 20 wt% POPS, respectively, in the total amount of added lipids. The detailed composition of the different assembled hybrid vesicles and liposomes can be found in Supporting Information Table S1. The diameter, polydispersity index (PDI) and ζ-potential of the assemblies were analyzed by dynamic light scattering (DLS, Zetasizer Nano ZS Malvern Instruments) using a material refractive index of 1.590 and a dispersant (water at 25 °C) refractive index of 1.330. For ζpotential measurements, the assemblies were prepared with HEPES1 buffer. Further, the stability 10 ACS Paragon Plus Environment

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of selected assemblies was obtained by DLS and ζ-potential measurements. In this case, the samples were measured at different time points and stored at 37 °C. Within this paper, samples with a PDI>0.4 were considered aggregated and were discarded. The lipid and cholesterol content of the assembled hybrid vesicles was quantified by the Lipid Quantification Kit (Cell Biolabs, Inc.) and Amplex® Red Cholesterol Assay Kit (Thermo Fisher Scientific), respectively. Non-extruded POPC liposomes (LML, 0.0 - 12.5 mg mL-1 lipids) were used as a reference curve (fitted using a linear regression) for the lipid quantification. The lipids were quantified following the protocol of the supplier. Briefly, 15 µL (1.0 mg mL-1) of hybrid vesicle stock solution or LML standards were transferred into an Eppendorf tube. 150 µL 18 M sulfuric acid was added to each tube and the samples were incubated at 90°C for 10 min. Subsequently, the samples were transferred to 4°C for 5 min. 100 µL of each sample were transferred into a standard 96-well plate. 100 µL of Vanillin Reagent, solubilized at 37°C for 30 min, was added to each well and mixed carefully. Finally, the samples were incubated at 37°C for 30 min and the absorbance was measured at λ = 540 nm using a multimode plate reader (PerkinElmer EnSight). Absorbance values from a HEPES2 buffer control were subtracted for background correction. The LML standard curve was used to translate the measured absorbance values into a lipid concentration. Three independent repeats were performed. To quantify the cholesterol content in the hybrid vesicles, 100 µL hybrid stock solution (1.0 mg mL-1 in HEPES2 buffer) was mixed with 600 µL HEPES2 buffer. 300 µL DMSO was added to this solution to obtain a hybrid concentration of 0.1 mg mL-1 with 30% (v/v) DMSO. Subsequently, 50 µL of sample was added to each well in a black 96-well OptiPlate. 50 µL of a working solution consisting of 300 µM Amplex® Red reagent, 2 U mL-1 horseradish peroxidase, 2 U mL-1 cholesterol oxidase and 0.8 U mL-1 cholesterol esterase diluted in the supplied reaction 11 ACS Paragon Plus Environment

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buffer (0.1 M potassium phosphate, 0.05 M NaCl, 5 mM cholic acid, 0.1% Triton® X-100 at pH 7.4) was added to each well. The samples were incubated at 37°C for 30 min, protected from light. Finally, the fluorescence was measured using a multimode plate reader (excitation/emission λ = 545/590 nm). The values from a HEPES2 buffer control was subtracted to correct for background fluorescence. The cholesterol standard (0.0 - 1.0 µg mL-1) supplied by the kit was used to obtain a reference curve (fitted with a linear regression) to convert the measured fluorescence intensity values into a cholesterol concentration. Three independent repeats were performed.

Cryo-Transmission Electron Microscopy (cryo-TEM). Samples for cryo-TEM were prepared in HEPES2 buffer. 4 µL of the sample suspension was adsorbed onto lacey carbon film mounted on 300 mesh copper grids (Ted Pella, Inc, Redding, Ca, USA). Prior to adsorption, the grid was rendered hydrophilic by glow discharge. The specimen was applied and after 1 min incubation on the surface, the grid was blotted and quick-frozen in liquid ethane using a Leica EM GP automated plunging device (Leica Microsystems, Vienna, Austria). The frozen grids were transferred under liquid nitrogen and loaded into a Gatan 626 cryo-holder (Gatan, Pleasanton, CA, USA). The cryo-holder was then inserted into the stage of a FEI Talos transmission electron microscope (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 CETA 16M 4k x 4k CMOS Camera (FEI Company) at given defocus values of approximately –2.5 µm. Image magnification was at nominal 57 000× with a pixel size at the camera level of 2.59 Å. Electron dose for imaging was maintained at 20 e.Å-2.


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At least 80 assemblies were measured when evaluating the membrane size from the images.

Giant Unilamellar Vesicles (GUVs). GUVs were fabricated using the electro-formation method. Indium tin oxide (ITO)-coated glass coverslips (25 × 45 mm2) were cleaned in ethanol and water each for 15 min by sonication and then dried under a stream of nitrogen. The corresponding lipid-P3 mixtures were dissolved in chloroform at a total concentration of 5 mg mL-1. 30% or 70% (w/w) P3 and 0.5% (w/w) TR-DHPE was added to the mixture to fluorescently label the GUVs. 7.5 µL of the lipid-polymer mixture was deposited on the ITO electrode surface using a needle by spreading the solution carefully back and forth (6 ×), followed by drying under vacuum for 2 h. The lipid-polymer-coated ITO electrodes were separated by a rectangular polytetrafluoroethylene (PTFE) spacer with a length, width and height of 35 mm, 25 mm and 2 mm, respectively, and the chamber was filled with pure water. An ACelectric field (5 V, 10 Hz) was applied for 4 h at 60 °C to generate the GUVs. The GUVs were transferred to a vial and stored at 4 °C before imaging. The morphology of the GUVs was verified using a fluorescence microscope (Nikon 80i, Japan).

Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D). The interaction of the hybrid vesicles and liposomes with PLL or PLL/PMA pre-coated surfaces was measured using QCM-D (Q-Sense E4, Sweden). Silica-coated crystals (QSX300, Q-Sense) were cleaned in a 2 wt% SDS solution overnight and rinsed with ultrapure water. Then, the crystals were dried in a stream of nitrogen, exposed to UV/ozone for 30 min and mounted into the chambers of the QCM-D instrument. The frequency changes (∆f) and dissipation changes (∆D) were monitored 13 ACS Paragon Plus Environment

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at 20 ± 0.02 ℃. After a stable baseline was obtained in HEPES1 buffer, a PLL solution (1 mg mL-1 in HEPES1 buffer) was introduced into the chambers and let to adsorb. When the surface was saturated, the chambers were rinsed with HEPES1 buffer. The pre-coated PLL crystals were exposed to a hybrid vesicle or liposome stock solution until the surface was saturated. A layer of PMA (1 mg mL-1 in HEPES1 buffer) was adsorbed onto the PLL pre-coated crystals prior to the exposure to assemblies containing positive lipids. Normalized ∆f and ∆D using the third harmonic are presented.

Cell Culture Experiments. The immortalized RAW 264.7 mouse macrophage cell line and primary rat Kupffer cells were purchased from European Collection of Cell Cultures. RAW 264.7 cells were cultured in 75 cm2 culture flasks in cell medium (Dulbecco’s Modified Eagle’s Medium with 4500 mg L-1 glucose, sodium pyruvate and sodium bicarbonate supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 µg mL-1 streptomycin and 50 µg mL-1 penicillin (all from Sigma-Aldrich)) at 37 ℃ and 5% CO2. Human monocytes were isolated from buffy coat preparations from healthy, anonymous blood donors from the blood bank of the Red Cross Baden Wuerttemberg by adherence to plastic. The human monocytes were differentiated into human macrophages using granulocyte macrophage colony stimulating factor (100 ng ml-1) for 4-5 days. For all the cell work, the (theoretical) total amount of DMAEMA units was kept constant. The number of DMAEMA units was estimated by estimating the polymer content in the hybrid vesicles using their different molecular weights (Table 1). The number of DMAEMA units was obtained by multiplying the number of polymer molecules by the degree of polymerization. 14 ACS Paragon Plus Environment

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Further, the estimation of the lipid incorporation efficiency was used to correct the number of DMAEMA units present in the individual samples, assuming that the polymer to lipid ratio remained constant. Cell Viability. RAW 264.7 cells were seeded in a 96-well plate (50 000 cells per well) and allowed to adhere overnight at 37 ℃ in 5% CO2. The cells were washed twice with 100 µL PBS and exposed to cell medium containing hybrid vesicles with a (theoretical) total amount of DMAEMA units between 0 and ~1350×1013 per well for 5 h. A hybrid vesicle concentration corresponding to ~450 ×1013 DMAEMA units was used to assess the cell viability after 24 h. A maximum of 10 µL vesicle sample was added per well. After the incubation time, the cells were washed twice with fresh cell medium and 110 µL cell medium containing 10 µL Cell Counting Kit-8 solution (CCK-8, Dojindo) was added to each well. The cells were incubated for 2 h at 37 °C in 5% CO2. Then, 100 µL of the solution from each well was transferred to a new 96-well plate and analyzed using a multimode plate reader by measuring the absorbance at 450 nm. Three independent repeats were performed for all samples. Primary rat Kupffer cells were transferred to a 15 mL falcon tube containing 9 mL (4 ℃) Kupffer Monoculture Medium (KMM) immediately after being thawed and placed on ice. The cells were centrifuged at 500 g for 5 min and suspended in 1-2 mL KMM. 50 000 cells were seeded per well in a 96 well plate and the KMM was exchanged after 4-6 h and 24 h prior to the experiments. The viability experiments were performed using the protocol outlined for the RAW 264.7 cells with an incubation time of 5 h using P2LL and P2LL+ hybrid vesicles (DMAEMA units ~450×1013 and ~1350×1013). The Kupffer cell viability experiments were performed twice using the same batch of cells with duplicates of each sample, i.e., no independent repeats were conducted. The human macrophages (2×105 per well) were cultured with P2LL for 5 h and then 15 ACS Paragon Plus Environment

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stained with Annexin V-FITC/propidium iodide (Becton Dickinson) to detect apoptotic cells. At least 100 000 cells per sample were acquired by flow cytometry and analyzed using FlowJo® Software (Becton Dickinson). All samples were set up in duplicates. The experiments were repeated 3 times using cells from different donors. Uptake Experiments. RAW 264.7 cells were seeded in a 96-well plate (50 000 cells per well) and allowed to adhere overnight at 37 ℃ in 5% CO2. The cells were then washed twice with 100 µL PBS and incubated in cell medium containing hybrid vesicles or liposomes for 5 h and 24 h. The (theoretical) added liposome concentration was 0.1 mg mL-1 lipids and the (theoretical) added hybrid vesicle concentration corresponded to ~450×1013 DMAEMA units per well. The cells were washed twice in PBS buffer, and 100 µL trypsin-EDTA (5 min at 37 ℃) was used to detach them. Trypsin was neutralized with 200 µL cell medium before analysis by flow cytometry (Accuri C6 Flow Cytometer, BD Biosciences) using an excitation wavelength of 488 nm. At least 2000 cells were analyzed and at least three independent repeats were performed for all the reported flow cytometry results. The auto-fluorescence of the cells was subtracted by analyzing control cells where no sample was added. In addition, the cell mean fluorescence was normalized to the fluorescence of the individual hybrid vesicle or liposome stock solution. The fluorescence of a given volume of the stock solution was measured using a multiplate reader and the obtained flow cytometry values were normalized to correct for the assumed concentration variations. This normalization approach assumes that NBD-PC lipids were evenly distributed in the lipid bilayer of the vesicles independent on their composition. Lysosomal Escape. 100000 RAW 264.7 cells were seeded per channel in a 6 channel ibi-treated iBidi® µ-slide VI0.4. The cells were allowed to adhere overnight at 37 ℃ in 5% CO2. The cells were washed twice with PBS and incubated for 1.5 h and 5 h with cell medium containing 75 nM 16 ACS Paragon Plus Environment

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LysoTracker® Red DND-99 and P2LL, or P2LL+ and 50 nM LysoTracker® Red DND-99 and Lc1 or Lc1+ (all samples containing 4 wt% NBD-PC), respectively, at 37 ℃ in 5% CO2. The (theoretical) added liposome concentration was 0.1 mg mL-1 lipids and the (theoretical) added hybrid vesicle concentration corresponded to ~450×1013 DMAEMA units per well. Before adding to the cell culture, the samples were diluted to approximately the same fluorescence intensity measured at 460 nm / 534 nm (ex. / em.) using the sample with the lowest fluorescence as a reference in an attempt to expose the cells to comparable amounts of fluorescent vesicles. 95 µL HEPES2 buffer and 5 µL vesicle stock solution was added to wells (black OptiPlate-96) to measure the different fluorescence intensities. After incubation, the cells were washed twice with PBS. 120 µL PFA (4 %) was added to each channel and incubated for 10 min at room temperature. The slide was then washed 3× with PBS and incubated with DAPI (1 mg mL-1 diluted 750 times in ultra pure water) for 1.5 h. The use of Triton-X was avoided as this interfered with the LysoTracker®. Finally, the cells were washing 3× in PBS and 120 µL PBS was added to each channel for storage. The images were taken using a Zeiss LSM700 confocal laser scanning microscope (CLSM). The settings for the red, green and blue channel were kept constant. In order to quantify the co-localization between the hybrid vesicles or liposomes with the lysosomes, the total Pearson correlation coefficient (PCC) was determined using the Coloc 2 plug-in in ImageJ. Background subtraction (10 pixel ball pen size) was performed before analysis for all the images. Two images from two independent repeats were used.

RESULTS AND DISCUSSION Polymer Synthesis. Radical addition fragmentation chain transfer (RAFT) polymerization belongs to the controlled radical polymerization techniques, allowing the synthesis of well17 ACS Paragon Plus Environment

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defined polymers with narrow molecular weight distribution. Further, polymers polymerized by RAFT feature the intrinsic ability to be extended by another block in a classical chain extension polymerization, as a consequence of the high end group fidelity of the so-called chain transfer agent (CTA). In this context, RAFT polymerization was the method of choice to synthesize the amphiphilic block copolymers presented herein (Scheme 2). Poly(cholesteryl methacrylate) (pCMA) was chosen as the hydrophobic block since cholesterol is a biomolecule that exhibits self-assembly properties and diverse strategies are available for its conjugation to polymers and the subsequent assembly into supramolecular structures.44 In turn, pDMAEMA was chosen as the hydrophilic block due to its lysosomal escape capabilities.

Scheme 2. Chemical structure of the amphiphilic block copolymer pCMA-block-poly(2(dimethylamino) ethyl methacrylate) (pDMAEMA) synthesized by RAFT polymerization. The utilized chain transfer agent was 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid Nhydroxysuccinimide ester.

First, poly(cholesteryl methacrylate) (pCMA) was synthesized utilizing an Nhydroxysuccinimide (NHS) ester modified CTA. The purified polymer was then employed as 18 ACS Paragon Plus Environment

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macro-CTA in order to add a second block based on DMAEMA. This synthetic approach was utilized to synthesize two amphiphilic polymers with two different ratios of the hydrophilic and hydrophobic extension (Table 1). P1 features a 1:1 weight ratio between the pCMA and pDMAEMA. In contrast, the hydrophilic block in P2 was extended to 23 000 g mol-1, which corresponds to a 1:5 weight ratio, in order to facilitate an enhanced solubility of the polymer. P3 is a fluorescently labeled block copolymer where fluorescein o-methacrylate (FlMA) was copolymerized with DMAEMA. Its architecture was aimed to be similar to P2, but both the hydrophobic and hydrophilic blocks were slightly higher in molecular weight. Table 1. Overview of the properties of the synthesized pCMA macro RAFT agents and the AB block copolymers P1 and P2 obtained after the extension with pDMAEMA. Abb. Hydrophobic

Mn

Hydrophilic

Mn

extension

(kDa)

DPNMR

DPNMR

Extension

(kDa)

P1

pCMA

5

11

pDMAEMA

4

25

P2

pCMA

5

11

pDMAEMA

23

150

P3

pCMA

6.7

15

p(DMAEMA-coFlMA)

38/2.8

233/7

Hybrid Vesicle Assembly and Characterization. Phospholipids are needed in addition to P1 or P2 for the assembly of hybrid vesicles. Mono-unsaturated zwitterionic 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC) lipids were chosen since cholesterols prefers to associate with saturated acyl chains.45-46 Further, in contrast to saturated lipids like 1,2-dipalmitoyl-sn-glycero3-phosphocholine (DPPC), POPC has a transition temperature (Tm) of ~ -2 oC, which makes it more convenient to assemble into liposomes since no heating is required. 1-palmitoyl-2-oleoylsn-glycero-3-ethylphosphocholine (POEPC) and 1-palmitoyl-2-oleoylphosphatidylserine (POPS) 19 ACS Paragon Plus Environment

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were selected as the matching lipids to POPC with positively and negatively charged head groups, respectively. In order to assemble hybrid vesicles containing P1 or P2 and phospholipids, different amounts of lipids and block copolymers were mixed in chloroform. These solutions were used for hybrid vesicle assembly via the rehydration method where in the first step, the chloroform is evaporated leaving behind a dried film consisting of the lipids and block copolymers attached to a round bottom flask. In the second step, it is important that the majority of the dried film was released from the flask during the rehydration step to be able to vary the composition of the hybrid vesicles. Films comprising of P1 or P2 with at least 30 wt% lipids could be detached without visible material left on the flask in HEPES1 or HEPES2 buffer, yielding a milky solution which was subsequently extruded through 200 nm membranes. Specifically, if only polymer was present in the dried film, no detachment during the rehydration step was observed i.e., no pure polymer assemblies could be obtained. From now on, the hybrid assemblies will be denoted PxLy, where Px represents the type of polymer and y indicates if high (H) or low (L) amounts of lipids were added. Further, control liposomes using different concentrations of lipids were made and referred to as Lcz with cz indicating the concentration of lipids used e.g., Lc1 and Lc0.3 refers to liposomes assembled from 1 mg mL-1 lipids and 0.3 mg mL-1 lipids, respectively. The superscript “+” and “–” indicates the addition of positive- and negative-charged lipids, respectively. This simplified labeling of the different assemblies was chosen for clarity. Table S1 (Supporting Information) provides a detailed overview over the used amounts of block copolymer and lipids for the different assemblies. First, the size and polydispersity (PDI) of the different assemblies were analyzed by DLS and their ζ-potential was determined (Table 2). As expected, all the assemblies consisting of lipids 20 ACS Paragon Plus Environment

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only had similar diameters with equally low PDI with the expected ζ-potential values. We would like to note that the latter parameter was only a measure for the highest lipid concentration, since no differences were expected when lower overall lipid concentrations were used for liposome assembly. On the other hand, the success of the hybrid vesicle assembly depended on the type of lipids used and the ratio of lipids to block copolymer. When using the zwitterionic lipids (POPC), P1LH and P1LL yielded in non-aggregated assemblies with narrow PDI. As expected, decreasing the amount of lipids led to higher ζ-potentials due to the presence of larger amounts of positively charged block copolymer. P2LL could be successfully assembled, while P2LH led to large aggregates with high polydispersity within the first 2 h after extrusion. We speculate that this might be due to membrane instabilities due to phase separation leading to uncontrolled rearrangements into lipid-polymer aggregates. Membrane instabilities i.e., phase separation and budding, in hybrid giant unilamellar vesicles were previously observed when low amounts of polymer were using during assembly.47-49 Although we did not observe phase separation and budding in hybrid giant unilamellar vesicles assembled from POPC lipids and P2 within 2 h (Supporting Information Figure S2), nano-sized vesicles might exhibit different membrane stabilities, leading to the aggregates we observed. Further, P2LL exhibited a higher ζ-potential compared to P1LL, indicating the presence of higher amounts of DMAEMA units due to the high molecular weight hydrophilic chains in the former case. Further, similar results were obtained when positive lipids were added to the assemblies. P1LH+, P1LL+ and P2LL+ were successfully assembled, while P2LH+ aggregated. Further, the ζ -potential of Lc1+ was higher compared to the ζ-potential of P1LH+ indicating the addition of the polymer to the assembly. The decreasing ζpotential reflected the higher amounts of block copolymer present in P1LL+ and P2LL+ compared to P1LH+. Finally, when negative lipids were used, none of the tested combinations with P1 led to 21 ACS Paragon Plus Environment

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good quality assemblies. P2LH- was impossible to detach from the glass surface during the rehydration process, and only P2LL- led to assemblies with the expected diameter, PDI and positive ζ-potential. These results were not surprising considering the opposing charges of the block copolymers and lipids. P2LL- likely contained a sufficiently high amount of block copolymer to make the positive charge the dominating entity, also confirmed by the ζ-potential measurements. However, since the stability of P2LL- was low, i.e., sample aggregation was observed within 3 days, this assembly was not further considered. Table 2. Overview over the diameter, PDI and ζ-potential of different liposomes and hybrid vesicles. Diameter/STD Assembly

ζ-potential/STD PDI/STD

[nm]

[mV]

Lc1

229.1/10.1

0.14/0.04

-4.4/1.6

Lc0.7

229.4/10.2

0.12/0.05

NA

Lc0.5

224.4/9.0

0.12/0.02

NA

Lc0.3

201.8/21.3

0.15/0.04

NA

P1LH

214.0/15.2

0.09/0.05

0.5/1.9

P1LL

172.8/25.5

0.12/0.04

12.9/4.3

P2LH

1473.3/605.5

0.83*/0.27

NA

P2LL

230.0/33.7

0.18/0.06

24.2/7.7

Lc1+

179.4/13.5

0.12/0.02

59.9/1.8

Lc0.7+

180.6/8.9

0.12/0.03

NA

Lc0.5+

192.6/31.1

0.13/0.03

NA

Lc0.3+

190.5/13.3

0.12/0.02

NA

P1LH+

210.2/17.9

0.10/0.02

49.5/2.7 22

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P1LL+

198.4/27.9

0.11/0.02

20.5/2.6

P2LH+

442.2/178.9

0.74*/0.36

NA

P2LL+

180.8/15.0

0.14/0.03

29.8/1.3

Lc1-

212.4/45.6

0.16/0.07

-45.7/8.9

Lc0.7-

204.5/17.5

0.17/0.03

NA

Lc0.5-

210.1/34.9

0.14/0.03

NA

Lc0.3-

206.2/40.6

0.19/0.03

NA

P1LH-

1072.2/283.5

0.68*/0.28

NA

P1LL-

1119.9/384.2

0.76*/0.27

NA

P2LH-

NA

NA

NA

P2LL-

178.2/13.7

0.23/0.04

21.6/1.1

*Sample aggregation (i.e., diameter > 300 nm and/or PDI > 0.4)

Following on, the lipid content in the assembled hybrid vesicles P1LL, P1LH and P2LL was estimated and compared to liposomes (Table 3). Specifically, multilamellar liposomes (LML) assembled via the dehydration/rehydration method using 2.5 mg POPC lipids and 1 mL HEPES2 buffer solution without extrusion were used to obtain a standard curve (Supporting Information Figure S3a) assuming that in this case 100% of the initially added lipids were assembled into liposomes. The lipid incorporation efficiency of Lc1 was close to 100%, whereas efficiencies of P1LL, P1LH and P2LL were 38%, 36% and 67%, respectively, suggesting that the hybrid vesicle assembly was more efficient when the used block copolymer was less hydrophobic due to the longer pDMAEMA extension of P2. Additionally, the cholesterol content was estimated, using a cholesterol standard sample provided by the Amplex® Red Cholesterol Assay Kit to obtain a standard curve (Supporting Information Figure S3b). The cholesterol incorporation was


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detectable, but very low for all hybrid vesicles. The reason for the low amount of detected cholesterol was likely due to the fact that pCMA was in the membrane of the hybrid vesicles and therefore difficult to access by the cholesterol oxidase from the detection kit. Attempts to disintegrate the assemblies in aqueous environment using DMSO and sonication prior to cholesterol detection had limited success. However, when comparing the three tested hybrid vesicles to each other, P1LH contained ~50% less cholesterol units than P1LL, and P2LL was estimated to contain the lowest number of cholesterol units. Although the concentration were very low, the qualitative ratios between the samples was as expected, indicating that varied amounts of polymers were incorporated into the different hybrid vesicles. Table 3. Overview of the theoretical and experimental lipid and cholesterol amounts in Lc1, P1LL, P1LH and P2LL. Lipids Assembly

Theo.

1 -1

[mg mL ]

Exp.2

Cholesterol Efficiency

Exp.

Efficiency

Ave./STD

Ave./STD

[µg mL-1]

[%]

Theo.

Ave./STD3

Ave./STD

[mg mL-1]

[%]

[µg mL-1]

Lc1

1.00

0.97/0.08

97/8

NA

NA

NA

P1LL

0.30

0.11/0.02

38/7

1.654

0.04/0.00

2.4/0.1

P1LH

0.70

0.25/0.07

36/10

0.709

0.02/0.00

2.9/0.2

P2LL

0.30

0.20/0.02

67/7

0.532

0.02/0.00

3.5/0.1

1

Theoretical amount (Theo.), 2Experimental amount (Exp.), 3Average/standard deviation (Ave./STD)

Following on, the goal was to identify the nature of the assemblies i.e., if micelles or vesicles were present, using cryo-TEM (Figure 1). The analysis of the morphologies of the hybrid 24 ACS Paragon Plus Environment

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assemblies (P1LL, P1LL+, P2LL, and P2LL+) revealed that only vesicles were present with sizes ranging from 20 - ~400 nm. However, the larger sized assemblies (> 100 nm) showed many multilamellar or vesicle-inside-vesicle structures, especially for P1LL and to lesser extend for P2LL (Figure 1a and 1c). The additional charge on the membrane of samples P1LL+ and P2LL+ likely caused the bilayer to separate from each other (Figure 1b and d), yielding in more unilamellar assemblies. Membrane thicknesses were found to be between 5.8 and 6.7 nm, thus, thicker than the membrane of a pure POPC lipid bilayer (5.4 ± 0.5 nm). In addition, the membrane thicknesses of the samples containing P1 (P1LL = 6.0 ± 0.7, P1LL+ = 5.8 ± 0.5) were thinner than the ones containing P2 (P2LL = 6.7 ± 0.5, P2LL+ = 6.4 ± 0.7). The difference in membrane thickness likely reflects the different lengths of the pDMAEMA block i.e., the higher molecular weight of P2 over P1, since the pCMA block is the same for both polymers. In addition, the bilayer structure was visible in all four samples, which showed that the lipids still form the main membrane and the block copolymers are most probably simply attached via the cholesteryl moieties.


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Figure 1. Representative cryo-TEM images of different polymer-lipid hybrid vesicles: a) P1LL, b) P1LL+, c) P2LL, and d) P2LL+. The insets show a close-up of the bilayer structure of hybrid vesicle membranes. (Scale bar: 10 nm) The stability of the liposomes Lc1 and hybrid vesicles P2LL at 37 °C was assessed using ζpotential measurements and DLS over 15 days (Figure 2). The ζ-potential of P2LL increased by 10 mV within the first 48 h from 15 mV to 25 mV, where it remained constant, while the value for Lc1 remained constant from the beginning (~-5 mV). In contrast to other reports,50-51 no change in ζ-potential due to hydrolysis of the pDMAEMA block was observed. However, in these reports, only pDMAEMA homopolymers free in solution were tested. Overall, together with the diameter and PDI of the vesicles, which also remained constant for both samples, the hybrid vesicles were found to be stable for at least 2 weeks.


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Figure 2. ζ-potential (a) and DLS measurements (b) of Lc1 and P2LL over time incubated at 37 °C. With the aim to visualize that, the membrane of the hybrid vesicles was a mixture of amphiphilic block copolymers and lipids, giant unilamellar vesicles (GUVs) from P3 and POPC lipids at two different ratios were generated and visualized by fluorescence microscopy. Although GUVs are much larger than the ~200 nm hybrid vesicles, and the difference in membrane curvature might affect the distribution of the lipids and block copolymers,52 GUVs still provide relevant information on the lipid – block copolymer arrangement in the bilayer.49 GUVs assembled from P3 and POPC exhibited a homogenously distributed fluorescence originating from Texas RedDHPE lipids and P3 in the membrane, indicating mixing of the lipids and polymer chains at the given resolution for both tested P3 to POPC ratios (Figure 3a). In order to induce demixing in 27 ACS Paragon Plus Environment

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this system, the membrane composition was changed i.e., 17 wt% cholesterol was added. The addition of cholesterol to lipid-polymer hybrid vesicles was previously shown to alter the domain-size of the constituents.53 The addition of this small amount of cholesterol led to a rearrangement of the membrane into lipid- and polymer-rich domains for both of the tested P3 to POPC ratios (Figure 3b). It was estimated from fluorescence microscope images that ~95% of hybrid vesicles underwent phase separation for this triple component system (Supporting Information Figure S4). Control GUVs were assembled using only POPC lipids and cholesterol yielding a homogenous distribution of the red fluorescence (Figure 3c). This control experiment confirmed that the phase separation in the membrane was due to the polymer and not due to the cholesterol. We would like to note that it was not possible to distinguish the domain sizes between the two different P3 to POPC ratios. These experiments confirmed the presence of both the block copolymers and the lipids in the membrane of the same GUV. Since our current efforts aimed at the assembly of vesicles with homogenous membranes, P2 and POPC mixtures were used for the subsequent experiments.


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Figure 3. Fluorescent microscopy images of GUVs assembled from P3, lipids and cholesterol (Chol) at different weight ratios: a) P3:POPC = 3:7 (i) and 7:3 (ii), b) P3:POPC:Chol = 3:7:2 (i) and 7:3:2 (ii) and c) POPC:Chol = 7:2 (i) and 3:2 (ii); (a) green: FlMA in P3; red: Texas redDHPE, I: green channel, II: red channel, III: overlay). Scale bars are 20 µm. In a next step, the ability of the nano-sized hybrid vesicles and liposomes to adsorb onto a precoated surface was assessed by QCM-D as an indirect confirmation of their different surface properties. The changes in frequency (∆f) and dissipation (∆D) of the polymer pre-coated crystals upon exposure to different hybrid vesicles was monitored. PLL and PLL/PMA were used as precursor layers to ensure that the hybrid vesicles based on zwitterionic lipids (Figure 4a) and hybrid vesicles containing positively charged lipids (Figure 4b) were electrostatically interacting with the crystal surface without rupturing. First, the PLL pre-coated crystals were exposed to liposomes assembled using decreasing amounts of zwitterionic lipids (Lc1 to Lc0.5 to Lc0.3) to understand the change in ∆f and ∆D due to the lower amount of liposomes present. It should be noted that Lc0.3 corresponded to liposomes made from the lowest lipid concentration used in the hybrid vesicle assembly. As expected, ∆f for crystals exposed to Lc1 was ~3× higher than ∆f for crystals incubated with Lc0.3. ∆D was less than ~3× lower in the latter case probably due to the non-linear relationship between energy dissipation and the liposome density on the surface i.e. rather low density liposome layers dissipate large amounts of energy. On the other hand, exposure of PLL pre-coated crystals to P1LH led to ∆f and ∆D similar to Lc1 suggesting that the presence of the block copolymer did not hinder the hybrid vesicle deposition. Also, incubation with P1LL resulted in comparable ∆f and ∆D to Lc0.5. However, P2LL led to 29 ACS Paragon Plus Environment

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lower ∆f and ∆D even when compared to Lc0.3. This finding illustrated that P2 in the assembly affected the surface properties of the assemblies i.e., the positive charges of the hybrid vesicles and the PLL pre-coated crystal likely repelled each other. This effect was more pronounced when P2 was used, due to the higher molecular weight of the positively charged hydrophilic extension which was more efficient in rendering the hybrid vesicle’s surface positively charged than P1. This explanation was supported by the ζ-potential results where P2LL was found to have the most positive surface charge. In a next step, 20 wt% of zwitterionic lipids in the assemblies were replaced with positively charged lipids yielding P1LH+, P1LL+ and P2LL+. ∆f and ∆D of PLL/PMA pre-coated crystals due the electrostatically driven deposition of these hybrid vesicles in comparison to Lc1+, Lc0.5+ and Lc0.3+ were analyzed (Figure 4b). Exposing the polymer pre-coated crystals to Lc1+, Lc0.5+ and Lc0.3+ led to the expected liposome concentration dependent decrease in ∆f and ∆D. When the crystals were exposed to the positively charged hybrid vesicles, the ∆f and ∆D values were similar to Lc1+ and Lc0.5+ despite the lower amount of lipids in the hybrids samples. The ∆f and ∆D of P1LH+ (0.7 mg/mL lipids) was comparable to that of Lc1+ (1.0 mg/mL lipids), whereas the ∆f and ∆D of P1LL+ and P2LL+ (0.3 mg/mL lipids) lay between Lc1+ (1.0 mg/mLlipids) and Lc0.5+ (0.5 mg/mL lipids). These observations strongly suggest the presence of block copolymers in the hybrid vesicles in all three cases. Taken together, the QCM-D results confirmed the assembly of hybrid vesicles.


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Figure 4. Adsorption of hybrid vesicles: Frequency changes ∆f (i) and dissipation change ∆D (ii) of PLL (a) and PLL/PMA (b) pre-coated crystals upon exposure to P1LH, P1LL or P2LL and P1LH+, P1LL+ or P2LL+ hybrid vesicles, respectively. For comparison, ∆f and ∆D of the crystals due to exposure to liposomes assembled from different lipid concentrations (Lc1(+), Lc0.5(+) and Lc0.3(+)) are shown.

Interaction of Hybrid Vesicles with Macrophages Cell Viability. The first aspect considered in the biological evaluation was the cell viability of RAW 264.7 mouse macrophages upon exposure to hybrid vesicles for 5 h. In order to compare the cell viability results to the hybrid vesicle composition, the number of DMAEMA units the cells were exposed to in the different samples was estimated. This estimation was based on the results from the lipid quantification, assuming that the polymer-lipid ratio remained constant. Employing the estimated molecular weight and the dilution factor, the total number of DMAEMA units the cells were exposed to was calculated. First, when incubating the RAW 264.7 cells with hybrid vesicles P1LH, P1LL and P2LL, no significant difference in cell viability 31 ACS Paragon Plus Environment

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was observed for the tested conditions, i.e., the different amounts of DMAEMA units and the 5 h incubation time (Figure 5ai). On the other hand, significantly lower RAW 264.7 cell viability was measured upon exposure to P2LL+ compared to the other hybrid vesicles (Figure 5aii). We would like to note that no significant decrease in viability was observed when the cells were exposed to liposomes (Supporting Information Figure S5), indicating that the observed differences were due the presence of P1 and P2. Further, RAW 264.7 cells were incubated with P2LL and P2LL+ exhibiting ~450×1013 DMAEMA units for 24 h and 48 h without observing a significantly negative effect on the cell viability (Figure 5b). This concentration of P2LL and P2LL+ was chosen because there was no significant reduction in RAW 264.7 cell viability observed after 5 h.


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Figure 5. a) Short-term cell viability: The effect on the RAW 264.7 cell viability upon exposure to hybrid vesicles (i) P1LH, P1LL and P2LL, as well as (ii) P1LH+, P1LL+ and P2LL+ for 5 h is shown. The concentration is expressed in (theoretical) amounts of DMAEMA units incorporated into hybrid vesicles. b) Long-term cell viability: The effect on the RAW 264.7 cell viability


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upon exposure to P2LL and P2LL+ (both ~450×1013 DMAEMA units) after 5 h, 24 h and 48 h is shown. (n = 3, *p < 0.05).

Following on, primary rat Kupffer cells and freshly isolated human monocytes differentiated into macrophages were considered to assess the effect of the hybrid vesicles on their viability (Figure 6). Kupffer cells are specialized macrophages located in the liver and play a key role in the removal of substances including nanomaterials delivered to the liver via the blood.54 Kupffer cells incubated with P2LL did not show significant lower viability depending on the amount of DMAEMA units in the tested time frame. Human monocytes circulate in the peripheral blood and differentiate into macrophages when trafficking into tissue and organs. Hybrid vesicles had a striking effect on their cell viability i.e., human macrophages exposed to samples with DMAEMA units above 500 exhibited a more than 50% reduced viability. This possibly reflects the enhanced vulnerability of human macrophages cultured under in vitro conditions.


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Figure 6. The effect on the cell viability of and primary rat Kupffer cells and human macrophages upon exposure to P2LL exhibiting different amounts of DMAEMA units for 5 h. The data points are normalized to untreated cells.

Uptake Efficiency. The amount of internalized assemblies is an important consideration when applications in drug delivery are envisioned. Therefore, the normalized cell mean fluorescence (nCMF) of RAW 264.7 mouse macrophages was analyzed by flow cytometry after the cells were exposed to fluorescently labeled liposomes and hybrid vesicles for 5 h (Figure 7a and Supporting Information Figure S6a). The nCMF was obtained by normalizing the flow cytometry data to the fluorescence of the individual samples as a measure to correct for the potentially different amounts of vesicles formed during the assembly when using the different compositions. This normalization step also assumed that the fluorescent lipids were homogenously distributed among all assemblies in a sample. First, all tested hybrid vesicles containing zwitterionic lipids exhibited a significantly higher nCMF compared to Lc1. On the other hand, when considering positive lipids containing assemblies, only P1LL+ possessed a significantly higher uptake compared to Lc1+. However, it should be noted that expectedly, Lc1+ led to higher nCMF than Lc1, as positive charged vesicles exhibit stronger affinity for the negatively charged cell membrane. The lowest uptake efficiency was found for Lc1 (~60%) while all the other samples led to ~100 % uptake efficiency, illustrating an initial homogenous distribution of the vesicles in the cell culture. Taken together, these results have shown that the different material properties of the hybrid vesicles compared to the liposomes led to different uptake behavior by RAW 264.7 mouse 35 ACS Paragon Plus Environment

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macrophages. Further, the previous observed reduction in cell viability could not solely be attributed to the higher amounts of internalized hybrids. While P2LL+ was found to have the most significant negative effect on the cell viability, cells exposed to P1LL+ had the highest nCMF, showing that the design of the building blocks could influence the biological response. Following on, the nCMF and uptake efficiency of the RAW 264.7 macrophages due to incubation with two selected hybrid vesicles (P2LL and P2LL+) was assessed over time (Figure 7b and Supporting Information Figure S6b). These samples were chosen since they exhibited similar nCMF after 5 h. No significant differences in nCMF were observed for either of the hybrid vesicles after the measured time points. Further, the uptake efficiency remained constant at 100% over the assessed time period, preserving the homogenous distribution of the hybrid vesicles over 24 h.


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Figure 7. Uptake: a) nCMF of RAW 264.7 cells upon incubation with fluorescently labeled hybrid vesicles (left: P1LH, P1LL and P2LL; right: P1LH+, P1LL+ and P2LL+) and liposomes (Lc1 and Lc1+) for 5 h. b) nCMF of RAW 264.7 cells upon incubation with fluorescently labeled P2LL and P2LL+ for 5, 16 and 24 h. (n = 3, *** p ≤ 0.001) Cytosolic Placement Since pDMAEMA was previously used to facilitate lysosomal escape, the hybrid vesicles were tested for their ability to enter the cytosol in comparison to liposomes. To this end, CLSM was employed to visualize the fluorescent signal originating from internalized hybrid vesicles and liposomes and the LysoTracker Red DND-99-labeled lysosomes (Figure 8). Specifically, Lc1 and Lc1+ were compared to P2LL and P2LL+, respectively. A 5 h incubation time for Lc1 and Lc1+ was 37 ACS Paragon Plus Environment

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required compared to 1.5 h for P2LL and P2LL+ to make the fluorescence detectable in CLSM. Despite the different incubation time and in agreement with the flow cytometry data, the fluorescence signal of the cells due to internalized hybrid vesicles (labeled in green in Figure 8) was higher compared to cells exposed to liposomes. Cells incubated with P2LL+ had the highest fluorescence signal confirming the flow cytometry results. Additionally, in an attempt to quantitatively compare the colocalization of the fluorescence signals, the total Pearson correlation coefficient PCC was determined. The fluorescence signal in cells exposed to Lc1 and Lc1+ exhibited a high degree of colocalization with the red-labeled lysosomes, illustrated by PCC close to 1. This indicated retention of Lc1 and Lc1+ in the lysosomes. In contrast, the PCC was lower for images of cells exposed to the hybrid vesicles, suggesting lower colocalization between the fluorescence from the lysosomes and the hybrid vesicles i.e., cytosolic origin of the hybrid vesicle fluorescence was likely. However, it was impossible to conclude whether the hybrid vesicles remained intact upon internalization and lysosomal escape or if the assemblies disintegrated into their lipid and block copolymer building blocks.


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Figure 8. Lysosomal escape: Representative color split (left and middle) and merged (right) CLSM images of RAW 264.7 mouse macrophages incubated with Lc1 (i) and Lc1+ (iii) for 5 h as well as P2LL (ii) and P2LL+ (iv) for 1.5 h (green: NBD lipids in the assemblies, red: LysoTracker® Red DND-99 stained lysosomes, PCC: Pearson correlation coefficient). The scale bars are 20 µm. (n = 2)

CONCLUSIONS The synthesis of two different pDMAEMA-pCMA diblock copolymers and their successful assembly into hybrid vesicles with phospholipids was reported. The concentration- and timedependent cytotoxicity of these hybrids in comparison to liposomes was assessed using RAW 264.7 mouse macrophages, primary Kupffer cells and human macrophages. The negative effect 39 ACS Paragon Plus Environment

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on the cell viability was most severe in the latter case. Further, the internalization of selected hybrid vesicles by RAW 264.7 mouse macrophages was found to be highest for P1LL+ hybrid vesicles. Finally, the hybrid vesicles were more successful in escaping the lysosomes than liposomes. Taken together, the cell culture results of the discussed pDMAEMA-pCMA containing hybrid vesicles illustrated that these types of assemblies have the potential to broaden the portfolio of vesicular nanocarriers for cytosolic drug delivery.

ASSOCIATED CONTENT Supporting Information. Non-essential figures and descriptions that complement the findings and discussion of the manuscript: NMR spectra, table with detailed composition of the used hybrid vesicles and liposomes, calibration curves for lipid and cholesterol quantification, GUV overview images, and liposomes cell viability experiments. AUTHOR INFORMATION Corresponding Author *[email protected], *[email protected] Author Contributions ‡These authors contributed equally. ACKNOWLEDGMENT


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This work was supported by a visiting PhD student grant from the Graduate School of Science and Technology, Aarhus University, Denmark (W. Z.) and a Lektor Starting Grant from the Aarhus University Research Foundation, Denmark. REFERENCES 1.

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PhospholipidâBlock Copolymer Hybrid Vesicles with Lysosomal

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