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Phospholipase A2-Induced Degradation and Release from Lipid-Containing Polymersomes Mudassar Mumtaz Virk, and Erik Reimhult Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03893 • Publication Date (Web): 12 Dec 2017 Downloaded from http://pubs.acs.org on December 13, 2017

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Phospholipase A2-Induced Degradation and Release from Lipid-Containing Polymersomes Mudassar Mumtaz Virk, Erik Reimhult* Institute for Biologically Inspired Materials, Department of Nanobiotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 11, 1190 Vienna, Austria KEYWORDS. Hybrid vesicles, lipopolymersomes, solvent inversion, phopspholipase A2 PLA2, vesicle drug delivery system, lipid domains, amphiphile phase separation

ABSTRACT. Hybrid vesicles, comprising blends of amphiphilic block copolymers and phospholipids, have attracted significant attention recently due to their unique combination of chemical and physical properties. We report a method to make unilanellar hybrid vesicles with diameters of 100 nm by mixing polybutadiene-block-poly(ethylene oxide) and phosphocholine lipids using a combination of solvent inversion and sonication. We show that homogeneous hybrid vesicles are formed when one component is a minor fraction. At compositions with balanced mass fractions, separate populations of similarly sized pure liposomes and hybrid vesicles were observed. We investigate the release kinetics of calcein encapsulated in the lumen as hybrid large and giant unilamellar vesicles (LUV and GUV) of different compositions are exposed to phospholipase A2 (PLA2). PLA2 hydrolyzes lipids, leading to dissolution of lipid domains and provide a trigger for release of calcein as pores are formed. We demonstrate that depending on the polymer mole fraction, block copolymers can either protect or boost the rate of

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lipid degradation and thereby the release rate from nanoscale hybrid vesicles. Strong indications of lipid phase separation into nanoscale domains in LUV are observed. Most importantly, hybrid GUV with lipids in the fluid phase release calcein slowly as lipids in the liquid disordered phase do not phase separate, but they show the fastest release of all blends as LUV. This indicates phase separation on the nanoscale in contrast to on the microscale, but it also indicates retained high mobility of lipids between the nanoscale domains, which is absent for lipids in the gel phase. Our results demonstrate several ways in which nanoscale hybrid vesicles can and should be optimized for PLA2-triggered release of water soluble compounds. INTRODUCTION Liposomes are biodegradable, non-toxic and biocompatible. They are easily internalized by living cells and capable of encapsulating and transporting both hydrophobic and hydrophilic compounds. Therefore, ever since they were developed in the mid-sixties, liposomes are considered to be one of the best drug delivery vehicle systems.1 They have also been studied as theranostic tools for cancer diagnosis and treatments.2-6 However, one of the major drawbacks of liposomes is their poor mechanical and potentially colloidal and biochemical stability in complex fluids, in particular in blood. For example, degradation of liposomes during circulation or in tissue lead to uncontrolled and premature release of encapsulated drugs. Polymersomes, similar in structure to liposomes but assembled from amphiphilic diblock copolymers, display enhanced mechanical stability and concurrent lower permeability compared to liposomes.7 Other membrane properties of polymersomes, such as shear viscosity and lateral diffusion coefficient, also differ from those of liposomes; as a result, the drug release kinetics are significantly different for polymersomes than for liposomes.8-9 The chemical versatility of block


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copolymers makes them easier to adapt to specific applications or to equip them with additional functions than it is to similarly adapt liposomes. For example, properties such as permeability and mechanical stiffness of polymersome membranes can be tuned by simply varying the molecular weight over a large range.7 Just like liposomes, due to their size (~100 nm) and deformability, polymersomes are suited to reach targeted tissue in, e.g., cancer therapy through the enhanced permeability and retention effect.10 However, the drug also has to be released at the destination. The robustness of polymersomes can then be a disadvantage, but their chemical versatility make them suitable for programming of physically or chemically triggered release as reviewed by Palivan et al.11 Only certain triggers have led to the design of polymersomes that can be applied in the human body;11 this limits the selection of polymersome systems that are possible to translate into actual in vivo drug delivery systems and it partially explains the to-date limited number of polymersome systems that have actually gone on to clinical testing. Depending on the type of drug and the route of application, polymersomes have to fulfil the same set of complex requirements required for all drug delivery systems: no/limited toxicity, biodegradability, sufficient blood circulation time (for systemic applications), ability to reach and/or be taken up by target cells, and release of their therapeutic payload specifically at the target destination.12-13 With respect to these criteria, polymersomes are considered to be less biocompatible compared to liposomes, because polymersomes are formed from synthetic building blocks. This leads to slower degradation and also to higher toxicity. Liposomes and polymersomes thus have their respective advantages and limitations. An interesting approach is to combine the mechanical stability and tunability of polymersomes with the biocompatibility and biofunctionality of liposomes by engineering hybrid vesicles in which the membrane comprises both lipids and block copolymers.14-17 In addition to the


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combination of membrane robustness and biocompatibility, it has been shown that such hybrid vesicles are also capable of enhancing the targeting efficiency in drug delivery applications.18 It is also easier to incorporate and accommodate other nanomaterials, such as superparamagnetic iron oxide nanoparticles, in hybrid membranes; this has been exploited to achieve efficient externally triggered release from hybrid lipid/block copolymer vesicles by applying an alternating magnetic field.19 Even though significant progress has been made in producing new responsive hybrid materials, much more research is needed to understand interactions between biological molecules and self-assembled polymers to increase the scope of their application in e.g. drug delivery.11 Degradation of liposomes in vivo can be caused by the direct action of lipases that catalytically degrade lipids, e.g. by cleavage of the fatty acid tail from the lipid headgroup, and thereby lead to permeabilization of the membrane and dissolution of the vesicle. However, lipase degradation can also be used to trigger release of drugs encapsulated in liposomes. For example, phospholipase A2 (PLA2) is an enzyme that hydrolyzes the lipids at the sn-2 position, which leads to the formation of fatty acids and lysophospholipid products, as reviewed by Berg et al.20 PLA2 exists at elevated levels at cancer sites21-26 and has therefore been used to achieve triggered release from lipid-based cancer drug carriers.27-28 Group II PLA2 in healthy serum is 10ng/ml, while the concentration of PLA2 in effusions from cancer patients can rise up to 200ng/ml.21 A liposome drug delivery vehicle would however be susceptible to degradation also at lower enzyme concentration outside tissues with elevated levels of lipase. Hybrid vesicles are vesicles with a membrane comprising of a mixture of block copolymers and lipids. Such vesicles could increase stability to undesired enzymatic and other degradation processes during circulation, while still allowing for enzyme-triggered release through a minor membrane fraction of lipids.


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Blends of lipids and block copolymers are known to mix or phase separate into domains depending on physical properties such as size and stiffness of the polymer chains and melting temperature of the lipids, as well as the relative volume fractions, as reviewed by Le Meins et al.29 Measurements of these phase diagrams have been performed on giant unilamellar vesicles (GUVs), for which phase separation can be observed on the micron scale.14 In most cases lipids are homogeneously distributed throughout the membrane of a hybrid vesicle if the temperature is above the corresponding lipid membrane melting temperature Tm and the lipid and block copolymer de-mix upon cooling below Tm.30 However, it is also possible to create lipid domains above Tm of the lipids by introducing an external driving force to bring all the lipids together within the membrane. For example, Nam et al. achieved this for hybrid vesicles composed of 1palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and copolymer polybutadiene-bpoly(ethylene oxide) (PBD-b-PEO) by biotinylation of one of the constituents and crosslinking with the protein NeutrAvidin.15 In the studies mentioned above, confocal microscopy was used to investigate mixing and demixing in GUVs (10-50 μm) produced via electroformation. However, nanoscale vesicles required for encapsulation and release applications such as drug delivery are much smaller than the micron-sized domains observed in GUVs and it has been debated whether the same phase separation is present within individual nano-sized hybrid vesicles. There are to date very few studies in which the phase behavior of nanoscale hybrid vesicles was studied.17,

31-33

Strong

support for nanoscale phase separation within small and large hybrid vesicles was found, but alternatively two different populations of pure liposomes and pure polymersomes could be observed if phase separation occurs and lead to similar results. Recently, Tuyen et. al. studied the mixing of a flexible poly(dimethyl siloxane) backbone and two poly(ethylene oxide) pendant


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moieties (PEO-PDMS-PEO) of Mw = 3000 g· mol−1 and lipids in nanoscale hybrid vesicles using small angle neutron scattering and time-resolved Förster resonance energy transfer.34 They observed the formation of nanodomains of lipids even when the lipids were in fluid phase. The same membrane composition was previously observed to mix homogeneously in GUVs.14 The same group also studied the mixing behavior of poly(dimethyl siloxane) (PDMS) and poly(ethylene oxide) (PEO) block copolymers with different molar masses combined with POPC in the membranes of giant hybrid vesicles using FRET and fluorescence lifetime imaging microscopy (FLIM). They confirmed the presence of nanodomains in the membrane, which were not visible in confocal images for the same compositions of polymer and POPC.35 These comparison raises severe doubts on the general equivalence of macroscopic and nanoscale phase separation, which is of utmost importance for the design of nanoscale hybrid vesicles for applications such as drug delivery. Release through enzymatic degradation could be expected to be inefficient if lipids are homogeneously distributed and shielded in a majority block copolymer membrane and work best if phase separation occurs in the nanoscale hybrid vesicle membrane. Understanding the link between membrane composition, physical state of the hybrid membrane and its interaction with lipases is therefore a requirement for accurate design of such drug delivery systems. Herein, we report a method of making nanoscale hybrid vesicles by mixing polybutadiene(1200 Da)-block-poly(ethylene oxide)(1000 Da) (PBD-b-PEO) with different weight fractions of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). These hybrid vesicles loaded with the fluorescent dye calcein are used to study the release kinetics via degradation by PLA2, We investigate the effect of lipid phase (below or above

) on release kinetics by using

hybrid vesicles composed of PBD-b-PEO and 30 % w/w of phospholipid with different

that


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are either in the liquid or gel phase at body temperature. The results on nanoscale hybrid vesicles are complemented with confocal microscopy on lipid distribution in giant hybrid vesicles composed of PBD-b-PEO and 30 % w/w of lipids (POPC and DPPC) and lipase triggered release. Our report on the physicochemical properties and degradation of PLA2-responsive hybrid vesicles opens new perspectives on design of more robust phospholipase-triggered release vehicles; it sheds light on the distribution of lipid and block copolymer during formation and application of nanoscale hybrid vesicles and the role of lipid phase separation and mobility in enzymatically triggered release.

EXPERIMENTAL SECTION Materials: Phospholipase A2 (PLA2) from porcine pancreas; Calcein; Superdex 75; Triton X100, Calcium Chloride (CaCl2) and all solvents were purchased from Sigma Aldrich and used as received without further purification. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-Dioleoyl-sn-Glycero-3Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)2000 Da] (Ammonium Salt) (PEG(2)PE) were purchased from Avanti Polar Lipids and used as received without further purification. Polybutadiene(1200 Da)-block-poly(ethylene oxide)(1000 Da) (Mw = 2200g/mole, Ð = 1.09) was obtained from Polymer Source Inc. and used as received. Sample preparation by solvent inversion and sonication: PBD-b-PEO was dissolved in chloroform at a concentration of 25 mg/ml. The block copolymer was mixed with the respective weight percentage of lipid (5 mg total mass of lipid and block copolymer). The chloroform was then removed using rotavapor for 30 min at 60 °C and dried under high vacuum (0.05 mbar)


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overnight. 200 µL of THF were added to the lipid and block copolymers cake. The flask containing lipid and block copolymers in THF was connected to a rotary evaporator and , where

submerged in a water bath (

is the transition temperature between the gel

and liquid crystal phases of the lipid membrane) at 1 atm and stirred for 30 seconds to mix the lipid and block copolymers thoroughly. The suspension of lipid and block copolymer in THF added drop-wise to 1ml of (preheated 60 °C for DPPC to be above

,

) filtered 10 mM

TRIS buffer with 150 mM NaCl at pH 7.4 (TBS) containing 10 mM calcein under magnetic stirring. This produces a turbid solution, which was kept at 60 °C under magnetic stirring for 1520 min. The turbid solution was then immersed in an ultrasonic bath (Elmasonic P30H) and sonicated for 30 minutes (

,

) at frequency of 37 Hz and power of 320 W to form a

translucent solution. After sonication, the sample was further diluted to the final concentration of 2 mg/ml. Gel filtration was used to separate the sample from non-encapsulated calcein and residual THF. A Bio Logic Duo Flow chromatography system, equipped with a UV-detector, a Knauer Smartline RI 2300 detector and a Bio Logic BioFrac collector was used. Samples (2 mL, 2 mg/ml, homogenized) were purified by passing over a FPLC-column (length x diameter: 60 cm x 3 cm, stationary phase: Superdex 75) in TBS with a flow rate of 0.75 mL/min. Fractions of 2 mL containing the desired sample (usually 4 fractions) were identified by UV and RI detection. The concentration of the sample decreased to 0.5 mg/mL by purification. Release - fluorescence measurements: Stock solution of 0.9 mg/ml of PLA2 in TBS was made and then 50 µl of this stock solution was added to the sample (5 min after the measurement was started) of 1 ml to achieve a final concentration of 0.045 mg/ml. Before adding PLA2, 37 µl of CaCl2 from stock solution of 80 mM was also added to the 1ml sample so that the final concentration of CaCl2 was 3 mM in the whole sample. Release of the encapsulated calcein to


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the bulk phase was quantified by recording the increase in sample fluorescence intensity as function of time. Fluorescence intensity was continuously measured with a PerkinElmer LS55 luminescence spectrometer at an excitation wavelength of 495 nm and an emission wavelength of 515 nm and a slit width of 2.5 nm. Release of calcein was calculated as

%

Equation 1

where Ii is the initial fluorescence intensity measured before adding PLA2, IPLA is the fluorescence intensity measured after adding PLA2 to the sample. Itot is the total fluorescence intensity measured after complete lysis of the vesicles by addition of Triton X100. Giant vesicle preparation by electroformation: Giant unilamellar vesicles (GUVs) were formed by electroswelling of the dried mixture of lipids and polymers in 250 mM sucrose and 0.066mg/ml calcein (for calcein loaded giant vesicles) solution at room temperature for POPC and PBD-b-PEO mixture or at 45 °C for DPPC and PBD-b-PEO mixture using a Nanion Vesicle Prep Pro stage (Nanion Technologies GmbH) at 10 Hz and 3 V AC. The samples (containing 0.5 mol-% fluorescently labelled Rhd-PE lipid) were mixed at the desired weight fractions in chloroform at 0.5 mg/ml. 50 µl of the suspension was spread on conducting indium tin oxide (ITO) coated glass slides. The solvent was slowly evaporated in a gentle stream of nitrogen before the slides were dried in high-vacuum for several hours prior to overnight electroformation. Confocal Microscopy: For confocal imaging, the vesicle samples were diluted with the same molar concentration of glucose as the concentration of sucrose during electroformation. PLA2


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and CaCl2 from stock solutions were added to the sample at room temperature, such that the total concentration of PLA2 and CaCl2 was 0.09 mg/ml and 3 mM, respectively, in the whole sample. Confocal imaging was conducted immediately after adding PLA2 using a Leica SP8 confocal laser scanning microscope with a HC PL APO 40x/1.3 oil immersion objective. The z-stacks were processed using the ImageJ software to create 3D projections that visualize the presence of domains.

RESULTS First, confocal microscopy was used to demonstrate the mixing behavior of PBD(1200)-bPEO(1000) and lipids (DPPC and POPC) in GUVs formed by electroformation.30, 36 The images were acquired at room temperature, at which a POPC membrane is in the liquid phase but a DPPC membrane is in the gel phase. Figure 1a shows representative confocal images of calceinloaded (0.066mg/ml) hybrid GUVs composed of 30 % w/w POPC and 0.5 mol-% fluorescently labeled Rhd-PE lipid. The POPC lipids are uniformly distributed throughout the membranes of the vesicles and no domain formation is observed at the micron scale. This is in good agreement with literature.15 However, phase separation is observed in the GUVs composed of 30 % w/w DPPC shown in Figure 1b. 2D and corresponding 3D projections of the GUV (without calcein) are shown in Figure SI 1 and SI 2


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(a)

(b)

Figure 1. (a) 2-D slice through hybrid vesicles composed of PBD(1200 Da)-b-PEO(1000 Da) blended with (a) 30 % w/w POPC and 0.5 % mol fluorescently labeled Rhd-PE lipid (red channel) and (b) 30 % w/w DPPC and 0.5 mol-% fluorescently labeled Rhd-PE lipid. The bright arcs observed in (b) demonstrate the presence of lipid domains, while homogeneous membrane fluorescence is observed in (a). The vesicles incorporate 0.066 mg/ml calcein in the lumen (green channel).

PLA2 degradation of and calcein release from giant hybrid vesicles composed of PBD(1200 Da)-b-PEO(1000 Da) blended with 0.5 mol-% fluorescently labeled Rhd-PE lipid and 30 % w/w POPC or 30% w/w DPPC, and incorporating calcein below the self-quenching concentration, was also investigated using confocal microscopy. PLA2 was added to the sample at a concentration of 0.09 mg/ml at room temperature. When PLA2 is added to the hybrid vesicle, it hydrolyses and cleaves the lipid. The resulting lysolipids have a higher critical aggregation concentration and do not form vesicular membranes. The digestion of the lipid and release of lysolipid is thus expected to create a (transient) pore in the lipid parts of the membrane, through which we expect release of encapsulated calcein. As the lipid domains are digested, the


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remaining membrane minimizes its energy by resealing the vesicle as shown in Scheme 1. A hybrid vesicle is thereby expected to eventually be reduced to a pure polymer vesicle if there is sufficient block copolymer remaining to form a stable polymersome. In a pure lipid part of a membrane, the pore formation and release is expected to be more efficient than in a homogeneous blended lipid and block copolymer membrane, in which only individual lipids can be digested.

Scheme 1. PLA2 hydrolysis of the lipid domains in hybrid vesicles creates small pores into the membrane through which small hydrophilic molecules from the lumen can be released. The nondegradable block copolymer membrane remains and reseals the vesicle membrane.


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The time series of microscopy images in Figure 2A shows that the homogeneous distribution of lipids within the membrane observed on the micron scale for lipids above the membrane remained during exposure to PLA2. However, a shrinking of the vesicle over time is observed. The reduction in size correlates with continuous release of calcein (decrease in calcein fluorescence from the lumen). The final image in the time series shows buckling of the membrane and complete loss of calcein from the hybrid vesicle as the membrane has shrunk below a critical fraction of its original size. Figure 2B shows the time evolution of the average calcein fluorescence intensity in the hybrid vesicle lumen. It should be noted that t = 0 on the xaxis corresponds to the time at which a stable recording of the GUV fluorescence could be started after injection of the PLA2, and not to the time at which the PLA2 was added. The calcein concentration decreases almost linearly, but slowly, as function of time during the PLA2digestion of the vesicle membrane. After 30 min of monitoring, the vesicle membrane apparently ruptures and releases the remaining content, which coincides with the visible buckling seen in the last panel of Fig. 2A. This release kinetics was reproducible and observed in full for many vesicles, two of which were recorded and shown in Figure SI 3. One vesicle showed burst release after only a short observation, which was recorded and shown in Figure SI 3c. For the latter vesicle, it is likely that the vesicle had already reached the stage where we have rapid release, since there was a time-lag between PLA2 injection and the start of the imaging.


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(A) 0 min

7.6 min

15.3min

23 min

33.3 min

35 min

(B)

Figure 2. (A) 2-D slice through a hybrid vesicle taken at subsequent times showing the reduction in size and calcein content as function of time, as well as the final buckling of the membrane, when the vesicle is exposed to PLA2. The vesicle is composed of PBD(1200 Da)-bPEO(1000 Da) blended with 30 % w/w POPC and 0.5 mol-% fluorescently labeled Rhd-PE lipid


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and encapsulating 0.066 mg/ml of calcein. It is exposed to PLA2 at a concentration of 0.09 mg/ml. The white square denotes the area from which the average calcein fluorescence intensity within the vesicle shown in (B) was calculated. (B) Average fluorescence intensity of calcein inside the hybrid vesicle in (A) as function of time in the presence of PLA2 at a concentration of 0.09 mg/ml.

Figure 3 shows the radically different membrane degradation of and calcein release from hybrid vesicles composed of PBD(1200 Da)-b-PEO(1000 Da) blended with 30 % w/w DPPC and 0.5 mol-% fluorescently labeled Rhd-PE lipid. Figure 3A shows the lipid phase separation within the membrane visualized also in Figure 1b that occurs when the lipid fraction in the hybrid vesicle membrane is at a temperature below its

. The lipid domain clearly loses size

and fluorescence intensity over time; the lipid part of the membrane is almost completely destroyed within 10 min of observation. However, all of the calcein is released before a significant shrinking of the lipid domains is registered. In contrast to the slow and continuous release from the POPC-containing vesicle shown in Figure 2B, Figure 3B evidences burst release within seconds for the DPPC-containing hybrid giant vesicle. This observation was also highly reproducible and observed for more than five DPPC hybrid GUVs. It is noteworthy that the release kinetics are completely different in Figures 2 and 3; the time-scale of burst release from phase-separated hybrid GUVs is about three orders of magnitude faster than the slow release observed for homogeneously mixed GUVs.


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(A) 0 sec

58 sec

97 sec

323 sec

413 sec

529 sec

(B)

Figure 3. (A) 2-D slice through a hybrid vesicle taken at subsequent times showing the rapid release of calcein content as function of time and the slower loss of lipid due to enzymatic degradation by PLA2. The vesicle is composed of PBD(1200 Da)-b-PEO(1000 Da) blended with 30 % w/w DPPC and 0.5 mol-% fluorescently labeled Rhd-PE lipid and encapsulating 0.066


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mg/ml of calcein. It is exposed to PLA2 at a concentration of 0.09 mg/ml. The white square denotes the area from which the average calcein fluorescence intensity within the vesicle shown in (B) was calculated. (B) Average fluorescence intensity of calcein inside the hybrid vesicle in (A) as function of time in the presence of PLA2 at a concentration of 0.09 mg/ml.

The results from the GUVs raises the question whether also nanoscale hybrid vesicles can be used for efficient enzymatically triggered release. Enzymatic release would rely on that nanoscale vesicles, a fraction of the size of the observed domains, also form with blended compositions. Furthermore, only phase separated membranes would be expected to show rapid, burst-type release. To investigate this, nanoscale hybrid vesicles comprising PBD(1200 Da)-bPEO(1000 Da) blended with 30 % w/w (56.3 mol%), 50 % w/w (75 mol%) and 70 % w/w (87.5 mol%) DPPC were formed by injection of the amphiphile mixture with THF in buffer followed by sonication. Vesicles filled with dye in the lumen for release experiments were formed in buffer containing self-quenching concentrations of the water soluble fluorescent dye calcein. Pure PBD-b-PEO polymersomes and liposomes as well as hybrid vesicles containing POPC instead of DPPC were also prepared for comparison. The volatile THF is completely removed during the incubation and sonication process as previously demonstrated.37 Dynamic light scattering was performed to measure the hydrodynamic size distribution of vesicles prepared by the solvent inversion and sonication method. The hybrid vesicles, pure liposomes and pure polymersomes exhibit predominantly monomodal volume-weighted size distributions as shown in Figure 4. Pure polymersomes have bigger size (≈50.75 nm, corresponding to peak maximum) compared to pure liposomes (≈28.2 nm) with the same THF-injection preparation method and parameters (sonication power, sonication frequency and time of sonication). The average size


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continuously decreases as the lipid weight fraction (lipid area fraction,

) in the membrane is

increased. Lipid weight fractions are translated into lipid area fractions by using the formula, ⁄ polymers respectively and

where and

headgroup respectively. Assuming

and

represent the mole fractions of lipids and

represent the area per lipid and area per PBD-b-PEO 0.48 nm2

38

for DPPC and

0.76 nm2

39

for

PBD(1200)-b-PEO(1000) we can plot the obtained average size of the hybrid vesicles as function of DPPC lipid area fraction

(calculated for 30%, 50% 70% and 100% w/w DPPC),

as shown in Figure 4b. This can be explained by the higher membrane bending modulus and bulkier hydrophilic part of PBD-b-PEO compared to the phospholipids. A higher membrane bending modulus is expected to lead to a larger thermodynamically favored size of vesicles.40 In practice, vesicle size is determined by the kinetic pathway followed to form them. However, formation of vesicles by sonication follows a similar argument, by which the higher input of energy from sonication is balanced by the amphiphile ability to form vesicle membranes deformed to smaller diameter. The observed slight trend to smaller size with higher lipid fraction can therefore be expected as the formation of smaller vesicles for the same energy input is facilitated due to the lower bending modulus of lipid-rich membranes.


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Figure 4. (a) Examples of volume-weighted size distributions of hybrid vesicles for different weight fractions of DPPC measured by DLS. (b) Diameter of hybrid vesicles as function of lipid (DPPC) area fraction. The error bars show the sample size-corrected standard error of the mean41 of two independent samples.

To study the kinetics of PLA2-triggered release from nanoscale hybrid vesicle dispersions, we incubated hybrid vesicles encapsulating self-quenching concentrations of calcein with PLA2 at a concentration of 0.045 mg/ml at 37 °C and with 3 mM CaCl2 added to the TBS. At room temperature, the activity of PLA2 is much lower than at body temperature (see Figure SI 5). The release can be recorded as an increase in sample fluorescence due to dequenching of the calcein fluorescence as the released calcein is at a concentration below the self-quenching concentration present in the vesicle lumen. Figure 5a shows the release of calcein from hybrid vesicles blended with 30 % w/w of DPPC (blue) or POPC (red). The PLA2 was added 5 min after the measurements was started. No significant release was observed within the first 5 min (Figure 5b) after adding PLA2 in the case


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of DPPC-containing vesicles. This lag phase is longer than what has been reported for PEGylated and pure liposomes in some previous studies.42-43 In these previous studies a very low concentration of PLA2 (150 nM) was used to observe the lag phase. No lag phase was observed for PEGylated (Figure SI 5 blue) and pure liposomes (Figure 5c green) for the concentrations used in our study, which are ~20 times higher than in the reference study above. Thus, we infer that the lower accessibility of the lipid fraction of the membrane in hybrid vesicles compared to for pure liposomes creates an important lag phase for degradation also at high enzyme concentration. The resistance to unwanted degradation is therefore increased under relevant conditions. A low release rate with a half-time rate constant (approximated as the inverse time of achieving 50 % of complete release) of

⁄

1⁄

⁄

0.0110

0.0005 min-1 was observed

after the initial lag phase when vesicles were blended with 30 % w/w DPPC, as shown by the blue solid curve in Figure 5a. 75 % of the total encapsulated calcein could be released; this amount had been released 160 min after the PLA2 was added. The control measurement without addition of PLA2 shows negligible release over the same time period (Figure 5a, blue diamond). The calcein release observed in the presence of PLA2 can therefore fully be attributed to enzymatic degradation of the lipid parts of the vesicle membranes. The effect of lipid phase on release kinetics was investigated by additionally testing hybrid vesicles with 30 % w/w POPC. While a DPPC bilayer is in the gel phase at the experimental temperature of 37 °C, POPC is in the liquid phase. A comparatively high release rate with halftime rate constant of

⁄

1⁄

⁄

0.0297

0.0033 min-1 was observed when the lipids are

in liquid phase. A negligible lag phase (2min) is observed when the lipids are in liquid phase. This data is also shown in Figure 5b.


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Figure 5. (a) Calcein release kinetics for hybrid vesicles blended with 30 % w/w DPPC (blue) and 30 % w/w POPC (red) during exposure to 0.045 mg/ml PLA2 and 3 mM CaCl2 at 37 °C. The corresponding diamond marked lines show the passive release (PR) without addition of PLA2 at the same temperature. The error bars show the sample size-corrected standard error of the mean 41

of 2 (blue) and 5 (red) independent preparations and measurements. (b) A zoom of the first 20

min of panel a. (c) Calcein release kinetics for hybrid vesicles during exposure to 0.045 mg/ml PLA2 and 3 mM CaCl2 at 37 °C for different weight fractions of DPPC. The complete curve for the release kinetics of 30 % w/w DPPC is for clarity only shown in panel (a). The error bars


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show the sample size-corrected standard error of the mean

41

of 2 (black, blue and green), 4

(brown) and 3 (red) independent measurements on different samples. (d) Half-time rate constant ⁄

as function of DPPC weight fractions.

⁄

is shown for the first phase (fast release

kinetics) as red and second phase (slow release kinetics) as black for 50 % w/w of DPPC, which shows biphasic kinetics.

We also performed a series of experiments in which the lipid fraction was varied to further verify that release is due to digestion of the lipid fraction in nanoscale hybrid lipopolymersomes. Figure 5c shows release kinetics for hybrid vesicles exposed to PLA2; the DPPC lipid fraction was varied from 0 to 100 %. First, as expected, no release was observed from pure polymersomes upon adding PLA2 to the sample (Figure 5c, black), and the size distribution of the polymersomes exposed to enzyme remained unchanged (Figure SI 6). A fast rate of calcein release with half-time rate constant

⁄

0.5

0.1 min-1 (Figure 5d) is observed from the

pure DPPC liposomes and complete release of encapsulated calcein was achieved in only 6 min (Figure 5c, green curve). Unexpectedly, for pure POPC liposomes a similar very fast release rate was measured, but only a fraction of the total amount of encapsulated calcein could be released (see Figure SI 7). In between these extremes, we observe a continuous increase in release rate with increasing lipid fraction. At lower lipid fraction in hybrid DPPC/PBD-b-PEO vesicles, there is a trend toward a larger fraction of calcein remaining trapped in vesicles after completed enzymatic degradation, but all lipid-containing preparations show a very high fraction of total release (75-100 %). The release kinetics for hybrid vesicles blended with 50 % w/w DPPC show a clear biphasic release profile (Figure 5c, brown); almost 50 % of the calcein is released 7.5 min after the addition of PLA2 with

⁄

0.7

0.3 min-1, which is followed by a comparatively


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slow release (

⁄

0.044

0.007 min-1) of the remaining encapsulated calcein. The

unexpectedly high release rate for lipid-rich vesicles in the mixed sample is observed also for the 70 % w/w lipid sample, for which complete release was achieved within only 9 min with 0.8

⁄

0.2 min-1.

The hydrodynamic size distribution of vesicles after exposure to PLA2 was compared to their size before exposure; this is shown in Figure 6 for hybrid vesicles composed of 30 % w/w POPC or DPPC, respectively. For both types of lipids the average size of the hybrid vesicles decreases, but larger aggregates also appear in the sample. Size distributions before and after PLA2 exposure for others lipid fractions are shown in SI 8-10.

Figure 6. Volume-weighted size distributions of hybrid vesicles composed of PBD-b-PEO and 30 % w/w POPC (red) or 30 % w/w DPPC (blue) measured before (solid square) and after (dashed diamond) PLA2 addition.


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DISCUSSION The measurements on the GUVs show that on the micron scale lipid phase separation leads to fast and complete lipid degradation as well as burst release of encapsulated compounds. In contrast, homogeneous mixing of lipids and block copolymers – as the microscopy shows for POPC – leads to inefficient and slow release of encapsulated compounds as well as much slower enzymatic degradation of the lipid membrane component. As expected, pure polymersomes do not show any release when exposed to PLA2. However, we observe the opposite relationship between membrane phase behavior and release for nanoscale hybrid vesicles; the release rates are faster when the lipids are in the liquid phase (POPC) than when they are phase separated (DPPC). This raises the question whether the phase behavior is the same for the nano-sized membranes of SUVs and LUVs as for GUVs or if there are other factors at play to explain the radical difference. In either case, the results make clear that release and degradation kinetics observed on micron-sized GUVs cannot automatically be extrapolated to nanoscale vesicles used for most applications. To begin with, the release kinetics of nanoscale hybrid vesicles comprising different lipid weight fractions, can provide us with information about the mixing behavior of lipids and block copolymers in small to large unilamellar vesicles. Monomodal and seemingly homogeneous distributions of hybrid vesicles were formed for 30 % and 70 % w/w lipids. The release kinetics for hybrid vesicles blended with 50 % w/w DPPC show a biphasic release profile (Figure 5c, brown); almost 50 % of the calcein is released within 7.5 min after the addition of PLA2 with ⁄

0.7

0.3 min-1, which is followed by a comparatively slow release (

⁄

0.044

0.007 min-1). The latter is reminiscent of the rate of release for hybrid vesicles blended with a


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lower lipid fraction. There are two plausible interpretations of the observation of two different release rate constants for the 50 : 50 (lipid : polymer) blend. The first is that there are two distinct populations of vesicles. Although a quite monomodal size distribution is observed, there can be two co-existing vesicle populations of similar size but with different compositions: one with high lipid fraction and one with low lipid fraction. The initial release rate constant of the 50 : 50 blend is very high. If this release rate is attributed to half of the vesicle sample as indicated by the total amount released at the higher rate, then the rate constant for release is significantly higher than for the sample of pure lipid composition, lending high credence to the interpretation that lipids and polymers have enriched unevenly into two semi-distinct populations of different compositions. The remaining half of the sample is then by default polymer-rich. Thus, it would seem that a combination of lipid-rich and lipid-poor vesicles are preferred for intermediate lipidto-polymer weight-ratios, while homogeneous mixing within nanoscale vesicles is suppressed. This behavior cannot be observed for GUVs formed by electroformation, for which the large dimensions of the membrane allow the phases to co-exist within a single vesicle, but it is interesting to note that Nam et al. in their work15 observed that GUVs did not form by electroformation in this compositional range. Furthermore, it is interesting to note that the tentative phase separation leading to two populations also significantly affected the encapsulation efficiency compared to the other blends. The average encapsulation efficiency was lower for nanoscale vesicles with lipid : polymer ratios around 50 : 50 and most significantly it showed a large batch-to-batch variation (Figure SI 11). The second tentative interpretation could be that nanoscale vesicles go through a phase transition at low lipid concentration that due to the structural change leads to significantly lower release rate as the concentration threshold for the phase transition is reached. For example, at low lipid fraction such as 30 % DPPC the lipid phase


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separation into large lipid domains might no longer be favored and lipid degradation decreased accordingly. Thus, an initial high release rate for the entire vesicle population could then be decreased to a much lower release rate reminiscent of that for 30 % DPPC hybrid vesicles after action of the lipase. A similar transition in the release rate was not observed for the very fast release kinetics of the 70 % DPPC vesicles, nor in the distribution of lipids in GUVs, which makes this explanation less likely than inhomogeneous distribution of lipids within the vesicle population formed by sonication. Although burst release is observed for the DPPC GUVs, the corresponding SUVs and LUVs show continuous, but fast release. These findings are compatible when one considers that the release experiments on nanoscale vesicles is performed on an ensemble, for which the integration of a statistical distribution of many individual small vesicle burst release events will produce a continuous increase in fluorescence increase. Interestingly, the release kinetics of 30 % w/w POPC small and large unilamellar hybrid vesicles seemed to indicate the formation of nanodomains despite displaying homogeneous distribution of lipids throughout the PBD-b-PEO-rich membrane in GUVs (Figure 1), at least as imaged on the micron scale. The micron-scale homogeneous mixing for this blend has also been reported earlier.34 The release rate was faster for POPC in the liquid phase than for DPPC in the gel phase, as the comparison for the nanoscale vesicles in Figure 5a shows. Complete release was achieved after 80 min, which is almost two times faster than when the same lipid fraction is in the gel phase. This contrasts sharply with the results on GUVs, for which only a slow continuous release was observed for POPC blends and rapid burst release was observed for DPPC blends. A homogeneous distribution as inferred from the microscopy on GUVs should intuitively lead to restricted access of the PLA2 to the sn-2 position on the lipids, due to


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surrounding and sterically blocking brush-like PEO-chains and reduced probability to transfer the enzyme to a neighboring lipid. Additionally, the degradation of single lipids would not be expected to lead to formation of a pore sufficiently large to release calcein. This is compatible with the results observed for GUVs. However, enzymatic membrane degradation and pore formation seems to be promoted rather than inhibited on the nanoscale for the same membrane composition. A possible explanation for the apparent discrepancy is that lipid domains exist on the nanoscale in the PBD-b-PEO / lipid hybrid vesicles even for lipids in the fluid phase, since nanoscale domains would be sufficient for release although they cannot be observed by traditional investigations such as by confocal fluorescence microscopy. Existence of such domains but retaining the higher activity of enzyme to lipids in the fluid phase with easier access to the enzymatic cleavage site, could explain the faster release observed from blended POPC than from blended DPPC hybrid vesicles. Alternatively, one can speculate that the difference could be due to that an enzyme once docked to a vesicle can access and degrade all liquid phase and more mobile lipids in small domains as they can move throughout the entire vesicle membrane. In contrast, for non-mobile (gel-phase) but large domains, an enzyme can only degrade one domain and must then release and dock to another domain to continue to degrade all lipids in a single vesicle. The longer diffusion time-scale applying to enzymes required to dock and undock to lipid domains by 3D bulk diffusion compared to enzymes using 2D diffusion of lipids within the membrane could lower the apparent enzyme activity in hybrid vesicles with gel phase lipids and also contribute to faster calcein release from POPC than from DPPC hybrid vesicles. Regardless of the detailed mechanistic explanation, our results contradict the assumption of equivalence of phase behavior of lipid/block copolymer blends observed in giant unilamellar vesicles and in nanoscale vesicles; only the latter is of importance for applications


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but not directly observable. If the explanation indeed is the formation of fluid phase lipid domains in nanoscale vesicles, then that is different to what is observed for GUVs, which neither showed phase separation in confocal microscopy nor fast calcein release. The main physical parameter that could explain such a difference would be the higher membrane curvature of especially SUVs compared to GUVs. The results presented in Figure 5c also indicate that release rates can be enhanced when there is a polymer component in the membrane, as long as it is a minor fraction of the membrane. Alternatively, there could be a difference in average liposome size and thereby volume-tomembrane area ratio that influences the results. The difference in average vesicle size is small enough to be within the margin of error for 70 % w/w and 100 % lipid fraction vesicles (Figure 4); it is therefore unlikely to explain the results. The 70 % w/w lipid vesicles release at a rate that is higher than the pure liposomes, and a similar release rate at least as high as that of pure liposomes is indicated for a fraction of the 50 % w/w lipid samples. Faster degradation of liposomes containing PEGylated lipids than of pure liposomes has been reported.42-43 It is therefore possible that a fraction of block copolymer in the liposomes could similarly increase the degradation rate of the membrane. Possibly, the PEG aids rather than impairs the ability of the enzyme to associate with the vesicle surface or it increases the enzyme turnover rate once the enzyme has adhered to the membrane. Regardless of the mechanism, it seems a design criterion for hybrid vesicles to improve on stability to lipase degradation is that a majority fraction of polymer is present in the membrane. The data presented above clearly demonstrates that release takes place when the lipid part of a hybrid vesicle membrane is digested by PLA2. This is supported not the least by the absence of release for pure polymersomes and the average release rate as a continuous function of the lipid


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fraction. The enzymatic degradation leads to the formation of lysolipids. Degradation resulting in lysolipids should lead to a loss of the lipid fraction of the polymersomes and leave behind pure polymersomes since lysolipids have a lower critical aggregation constant and do not form vesicles. A decrease in vesicle area and size corresponding roughly to the loss of the lipid fraction should therefore occur. Such a decrease in the average size of the main vesicle population correlating with the lipid fraction was observed (see Figure 6). However, objects larger than the original vesicles also appeared in the samples after exposure to PLA2. These larger objects were not observed for pure polymersomes exposed to PLA2 (Figure SI 6) and they seemed mostly to be absent for pure liposomes. Thus, it is likely that these are aggregates of polymersomes and enzymes induced by the presence of lysolipids. The formation of large aggregates seemed particularly pronounced for hybrid vesicles containing DPPC. Figure 6 shows the results of size reduction for 30 % w/w lipid fraction. We estimated the reduction in vesicle membrane area of the main size peak in this data, by calculating the average liposome area from the diameter corresponding to the main volume-weighted size distribution peak maximum. The area is reduced by 44.4 % and 62.2 ± 5.8 % for hybrid vesicles with 30 % w/w DPPC and POPC respectively. Assuming and POPC38, 44 as well as

0.48 nm2 and

0.63 nm2 for DPPC38

0.76 nm2 for PBD(1200 Da)-b-PEO(1000 Da)39 we expect an

area decrease of 44 % and 52 % for 30 % w/w DPPC and 30 % w/w POPC, respectively. Thus, the decrease in area for 30 % w/w DPPC and 30 % w/w POPC is very close to the expected values and any discrepancy due to that also aggregates are present. The area reduction of 50 %, 70 % and 100 % w/w DPPC after adding PLA2 is shown in Figure SI 8-10. For the 50 % w/w and 70 % w/w lipid fraction samples, we expect a larger reduction in area after enzymatic lipid


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digestion than for the 30 % w/w vesicles. However, an even higher than expected average decrease in membrane area is observed. It has been believed for some time that grafting drug delivery liposomes with PEG inhibits the access of PLA2 to the surface of liposomes and minimizes their degradation. However, it was observed that presence of PEG-grafted lipids in the liposome membrane can decreases the lag time of enzymatic degradation of liposomes by PLA2 and effectively enhance its activity.43 Similar increases of the release rate at low fractions of PBD-b-PEO was observed also for hybrid vesicles, which suggests that the PEG stabilizes and enhances enzyme activity once at the membrane. However, our data on hybrid vesicles with low lipid mole fractions indicate that PEG at high densities in the membrane and attached to a non-degradable scaffold can serve to protect the lipid component from degradation. This should significantly enhance the blood circulation time of hybrid vesicles, while a high release efficiency and fast release rates still are achieved at high PLA2 concentrations expected in target tissue. This seems especially true for hybrid vesicles with lipids in the fluid phase, for which unexpectedly high release rates were observed for nanoscale vesicles, indicative of fluid domain formation in nanoscale hybrid vesicles.

CONCLUSION We have demonstrated a method based on solvent inversion and sonication to create small to large (~50-100 nm in diameter) unilamellar nanoscale hybrid vesicles composed of PBD(1200 Da)-b-PEO(1000 Da) and phospholipids (DPPC or POPC) at varied lipid-to-copolymer ratio. The size of the hybrid vesicles decreased moderately with increasing lipid content in the membrane. Through analysis of DLS data for the size distributions and the release kinetics for


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lipase degradation by PLA2 we can conclude that amphiphile blends with 30 % or 70 % w/w of lipid form quite monodisperse hybrid vesicles. 50 % w/w of lipid tentatively leads to the formation of two separate populations of hybrid vesicles; one of the populations has a lower lipid content than 50 % w/w while the other population possibly comprises almost pure liposomes; however, alternatively the data could indicate a phase transition in the hybrid membranes triggered as lipase degradation pushes the lipid fraction below ~30%, which was only observable for 50 % w/w DPPC-containing vesicles. At 70 % w/w of lipids the release kinetics was faster than for pure liposomes, which together with other observations make us conclude that the presence of a small amount of PEGylated block copolymer enhances enzyme activity, similar to as previously observed for PEGylated liposomes. Additionally, despite a macroscopically homogeneous distribution of lipid in the fluid phase, a faster enzymatically triggered release was observed for POPC than for gel-phase DPPC for nanoscale vesicles, implying phase separation of fluid phase lipids in small and large unilamellar hybrid vesicles, despite that POPC is generally regarded as soluble in PBD(1200 Da)-b-PEO(1000 Da) membranes. This contrasts greatly with the result on GUVs, for which a homogeneous distribution of POPC in the membrane and slow, in contrast to burst release for non-mixing DPPC, was observed. Our results suggest several ways by which hybrid vesicles can be optimized to use PLA2 for triggered drug release, both to improve release rates and to avoid early degradation and release during circulation in the blood stream. A translation to actual drug delivery systems, however, require optimization and demonstration of the same effects also at the much lower lipase concentrations encountered in cancer tissues than used in this investigation.

ASSOCIATED CONTENT


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Supporting Information This material is available at AUTHOR INFORMATION Corresponding Author Erik Reimhult* ([email protected]) Author Contributions MMV and ER planned the work. MMV prepared all the samples and performed all the experiments. The manuscript was written by ER and MMV. All the authors have given the approval to the final version of the manuscript. Funding Sources The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Program (FP/2007-2013)/ERC Grant Agreement no. 310034. ACKNOWLEDGMENT We thank Gianluca Bello for support with the electroformation of GUVs. We also thank the VIBT Imaging Center at the University of Natural Resources and Life Sciences Vienna for providing access to confocal microscopy and Noga Gal for support with the confocal microscopy. We also thank the Reviewers for constructive and insightful comments that improved our original submission. REFERENCES


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