Detection of Phase Transition in Photosensitive Liposomes by

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Detection of Phase Transition in Photosensitive Liposomes by Advanced QCM Lauri Viitala, Tatu Lajunen, Arto O. Urtti, Tapani Viitala, and Lasse Murtomäki J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.5b04042 • Publication Date (Web): 04 Jun 2015 Downloaded from http://pubs.acs.org on June 9, 2015

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The Journal of Physical Chemistry

Detection of Phase Transition in Photosensitive Liposomes by Advanced QCM

Lauri Viitala1, Tatu Lajunen2, Arto Urtti2, Tapani Viitala2, Lasse Murtomäki1*

1

Aalto University, Department of Chemistry, PO Box 16100, FI-00076 Aalto, Finland

2

University of Helsinki, Faculty of Pharmacy, PO Box 56, FI-00014 University of Helsinki, Finland

*Corresponding author: Email: [email protected] tel. +358 50 5706352

ACS Paragon Plus Environment


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Abstract

In this work an impedance-based quartz crystal microbalance (QCM) is used to detect heat induced changes in the viscoelastic properties in the films of adsorbed liposomes. Liposomes are bound to a polymer-modified QCM surface, and heat is induced in the bilayer via light absorption into gold nanoparticles (GNPs) embedded in the liposomes. Due to very rapid heat transfer at the nanoscale, nanoparticles can reside either in the liposome cavity or within the bilayer to cause changes in the lipid viscoelasticity. The changes are observed as changes in the film relaxation time as well as by mapping the measured resonance frequency vs. resistance. The QCM results indicate that viscoelastic changes occur throughout the vesicle layer, possibly causing fusion between the liposomes. The ultimate goal of the work is to develop a smart drug delivery system for the eye, whereby a drug loaded in the liposome can be released in a controlled manner by light triggering.

Keywords: liposomes, quartz crystal microbalance, gold nanoparticles, light triggering


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1. Introduction Various approaches to develop safe and efficient nanoparticle based drug delivery systems (DDS) with external control of localization and drug release have been studied in the past few decades1, 2. Magnetic and electric fields are often used for the transport (e.g. electrophoretic transdermal drug delivery). However, in order to achieve best therapeutic effect with minimum side effects, sitespecific DDSs and DDSs for intracellular delivery are needed.3 The most accessible route through the cell membrane for DDSs, such as liposomes, is via the endosomal pathway. For intracellular activation, smart DDS could be engineered to react to the pH drop during the endocytosis4. Many drugs are sensible to pH changes and other conditions in their surroundings (i.e. redox conditions). Drug carriers may protect the drug during transport process. A DDS must also be biocompatible, biodegradable, and elongate the clearance time but, at the same time, it should be effective in drug release at the target site. Thus, a DDS that is mute until activation by external trigger may improve drug efficacy, and reduce the off-target effects4. Liposomal drug carriers can be used for drug encapsulation and they can be functionalized with targeting ligands (e.g. peptides, aptamers, antibodies) to enable site-specific drug delivery.3 Liposomes are often coated with polyethylene glycol (PEG) to increase the biocompatibility and prolong circulation time of the liposomes in blood3, 5. Furthermore, the liposomes can be functionalized, also with materials that render them sensitive to the external stimuli. These materials are, for instance, gold nanoparticles (GNPs) that can be triggered by illumination. During the illumination, most of the absorbed energy is converted to heat by the GNPs6-8 and part of the energy is emitted as photoluminescence 9. From a practical point of view, photoluminescence effect occurs only in the case of gold clusters with sizes d < 5 nm9. Hence, in the case of liposomes with encapsulated GNPs the excess energy is dissipated as heat into the surrounding lipid bilayer. As the thermal motion is increased in the system, permeability of the lipid bilayer is increased and drug may be released8, 10, 11. Further, collisions between the liposomes may cause liposome fusion during the process12. Diseases in the posterior part of the human eye are difficult to treat, because the eye drops do not deliver drug adequately to the retina and choroid in the back of the eye13. After systemic administration blood-retina barrier prevents drug delivery to the retina. Therefore, drugs are often administered as intravitreal injections13. Liposomal drug delivery to the retina and choroid is a promising approach in several ways. Firstly, it was shown previously that small liposomes can target somehow to the retinal pigment epithelium after topical administration14.



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Secondly, liposomes can prolong the action of intravitreal drugs15. Thirdly, liposomes may be useful in ocular drug targeting after intravenous administration. In all these cases, it is an attractive option to trigger drug release in the back of the eye at defined time and site using external light signal. Previous drug release experiments with light triggering8,

10

and work

running concurrently at the University of Helsinki16 have confirmed that drugs can, indeed, be released from liposomes and delivered into human retina pigment epithelium cell line (ARPE19). Advanced QCM techniques, such as impedance-based QCM or QCM with dissipation monitoring, have become standard methods in the research of lipid films and other thin layers. One interest lies in the lipid vesicle fusion process that may take place during the intracellular delivery17, 18. A lipid bilayer can under specific conditions be readily formed on top of the quartz sensor by vesicle spreading and rupturing. Thus, it is possible to study molecular interactions between, for example, small molecular drugs or nanoparticle based drug delivery systems and the lipid bilayer19-21. However, liposomes can also be bound to the quartz sensor surface without rupturing into a bilayer. For example, liposomes composed of zwitter-ionic lipids typically form a monolayer of nonruptured liposomes on gold and titanium oxide coated quartz sensor surfaces19, 22, 23. Furthermore, a recent study has reported on the issue of surface modification methods to control the lipid surface morphology24, 25, but only a few papers utilize the films of adsorbed liposomes in any applications other than the formation of lipid bilayers by liposome rupturing. A few examples are given by Morita et al.26 who studied the protein-liposome interactions on a carboxythiol modified quartz sensor, and Branden et al. who studied sucrose uptake through transmembrane melittin pores into liposomes attached to a surface plasmon resonance sensor surface through DNA hybridization.27 In this paper, light-induced changes in the physical properties of liposomes encapsulated with gold nanoparticles are studied. To our knowledge, we are the first to report that such a phenomenon can be detected with advanced QCM. The work is based on three key ideas. First, the quartz crystal is coated with a hydrophilic polymer to enable controlled liposome binding. Second, the adhesion of liposomes can be studied by mapping the ∆f and ∆R data together. Third, upon illumination, the GNP encapsulated in the liposomes induce a phase change in the adsorbed liposomes that can be detected from the ∆f and ∆R map or from normal Kelvin-Voigt-based QCM data modelling.


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2. Experimental 2.1 Preparation and Characterization of Liposomes Lipid solutions (25 mg/ml of lipid diluted in chloroform) were purchased from Avanti Polar Lipids. First, 1,2-distearoyl-sn-glycero-3-phosphocholine

(DSPC) and 1,2-dipalmitoyl-sn-glycero-3-

phosphocholine (DPPC) were added to a test tube in different ratios. In a typical experiment, 5 mg of lipids with mass ratio of 1:9 DSPC:DPPC was used. Chloroform was evaporated under a stream of nitrogen at room temperature. Next, 0.25 mg (5 mg/ml in deionized water) of Nanopartz™ Accurate™ Spherical Gold Nanoparticles (Nanopartz, USA, diameter 10 nm) were added on top of the lipid film right before addition of a buffer solution containing 20 mM Hepes and 140 mM NaCl pH 7.4. The total volume of the solution was 0.5 ml. The lipid solution was then sonicated briefly at 60 °C so that the lipid film was totally hydrated. Liposomes were then extruded eleven times (T ≈ 60 °C) through two-stacked polycarbonate membranes with a pore size of 200 nm. After the last extrusion, the syringe was rapidly cooled down under running water and the content was diluted with buffer solution. Finally, the solution contained ca. 0.4 mg/ml of lipids.

Cryogenic transmission electron microscope (Cryo-TEM) samples were prepared according to Kuntsche et al.28 and Iancu et al.29 with small modifications. The vitrification step was done by FEI Vitrobot (FEI, USA). Briefly, an ethane-propane 1:1 mixture were condensed to a liquid-nitrogencooled Vitrobot cup after the temperature on the cup surface was dropped down to around −175 °C. Plasma treated (30 s H2-O2, Gatan Solarus 950, Gatan, USA) TEM grid CF-2/1-2C (Electron Microscopy Sciences, USA) was placed inside the Vitrobot chamber and the humidity was increased to 100 %. 5 µl of liposome suspension, containing 3 mg/ml of DPPC and 0.03 mg/ml of GNPs in the total volume, were pipetted on top of the TEM grid. The sample was then blotted to a filter paper for 1 s and dropped down into the ethane-propane mixture. The grid was moved to the Cryo-TEM sample holder under liquid nitrogen and the imaging was done with FEI Tecnai T12 (FEI, USA) with accelerating voltage of 120 kV. The size distribution analysis was made by a selfmade Matlab script that can be found in the supplementary material.

2.2 Substrate Preparation Gold-coated QCM sensor crystals (5 MHz) were purchased from Q-Sense, Sweden. Sensors were first washed in a boiling solution of 1:1:5 H2O2:NH4OH:H2O for 10-15 minutes. Crystals were then immersed in MilliQ water, rinsed, and dried in a stream of nitrogen. Next, the sensors were



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immersed into a solution of 2 mg of a hydrocarbon chain-polyethylene oxide thiol derivative (C=C10PEOdC10SH) in 1 ml of ethanol. The synthesis of C=C10PEOdC10SH is described in Granqvist et. al.25. After 24 h, the crystals were washed with ethanol and water and dried with a stream of nitrogen. Prior to the QCM experiment, the crystals were immersed to a solution of 20 mM CHAPS hydrate (Sigma Aldrich) for 5 minutes, washed thoroughly with MilliQ water, and dried with nitrogen gas. After the measurement, sensors were immersed in CHAPS hydrate, water, 10 % Hellmanex III (Hellma analytics, Germany), 10 % ethanol and water. Freshly prepared and cleaned re-used quartz sensors were stored in a fridge in MilliQ water.

2.3 QCM Measurement QCM measurements were performed with an impedance-based QCM-Z500 instrument (KSV Instruments, Finland). The measurement system contained a flow channel with a quartz glass lid, thermoelectric heater, a light source (OmniCure S1000, Lumen dynamics) with an incident light wavelength of 365 nm, and a two channel peristaltic pump (Ismatec Reglo, Germany). First, a buffer solution was pumped into the system and heated to 37 °C. The sensor was left to stabilize in a static environment before starting the measurement. Overtones 1st, 3rd, 5th, 7th and 9th were collected during the QCM measurements. The sensor was left to stabilize further if drifting was observed. A baseline for the C=C10PEOdC10SH coated sensor in the buffer solution was measured for 200-500 s. The liposome solution was pumped through the system for 10 minutes. Unbound liposomes and GNPs were washed away with the buffer solution flow for 10 minutes. Thereafter, the pump was switched off and the system was left to stabilize for at least 1000 s. After the stabilization, the sensor surface with the liposomal layer was illuminated for 5 minutes (light output power ca. 0.7 W/cm2) and stabilized for ca. 1500 s before further actions, i.e. next illumination or a washing step. A graphical description of the QCM measurement and a typical response in the QCM frequency are illustrated in Figure 1. The response in QCM frequency vs. time during a typical measurement with liposome adhesion and illumination is shown in the upper left figure. Insets (A)(D) represent different steps: (A) liposomes are introduced to the QCM sensor coated with polymer (C=C10PEOdC10SH) in a stream of buffer solution; (B) excess liposomes and GNPs are washed away; (C) bound supported vesicle layer (SVL) is left to stabilize for a while and (D), the SVL is illuminated with UV light that causes the temperature rise in the GNPs encapsulated in the liposomes. After the re-stabilization, the change in the fluidity of the SVL is detected. For clarity,


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Inset (E) shows the response in QCM frequency when a supported lipid bilayer (SLB) is formed on the QCM sensor. This frequency change is always around −28 Hz. The peak between stages (A) and (B) and the peak in the middle of time scale in Inset (E) are due to switching the pump off and on when the solution is changed from liposome suspension to buffer solution.

Figure 1. Upper images: left, frequency response during liposome adhesion and illumination; right, schematic image of a supported lipid bilayer on QCM sensor and a frequency response during lipid bilayer formation (Inset (E)). Insets (A), (B), (C) and (D) represent different work stages during the liposome film experiment at the respective time windows in the upper left image. Look text for details.

2.4 Data Analysis Advanced QCM results were analysed using the KSV QCM-Z500 software (version 3.4). The routine analyses were performed in the viscoelastic regime by utilizing a Kelvin-Voigt based model, briefly described in the next section. Additionally, self-made Matlab (MathWorks, Inc.) scripts were used to visualize and further analyse the results as described in the section 2.4.2.



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2.4.1 Viscoelastic Modelling in QCM Since the QCM theory and viscoelastic modelling has been described in detail by many authors, only a brief description is given here.30-34 In most cases, the behaviour of the quartz crystal is described with lumped elements the values of which are obtained by transforming the transmission line theory into the Butterworth-van Dyke (BvD) equivalent circuit model. In practice, a QCM piezoelectric element consists of a loaded and an unloaded ( = 0) faces, and the total motional impedance Zm is a sum of these two: = + = + + j +

with

= −

,

(1) (2)

where is the motional capacitance and is the motional inductance. There is also a direct relation between the motional impedance of the load and the mechanical impedance:

≈ = ,

(3)

where is the overtone (1st, 3rd , 5th , 7th, 9th etc.) of the fundamental resonance frequency, , and

=

! " (! and " are the density and shear stiffness of quartz). As the unperturbed part of the

motional impedance remains practically constant, changes in the BvD-fitted parameters are sufficient to describe the entire system, i.e.:

and

= Δ = Re

(4)

&

(5)

= −

'

= Im

From the earlier theoretical point of view, one can regard a viscoelastic film as a propagating medium in terms of the input and the output (i.e. the liquid load) characteristic impedances: , ., 0123 4 5

= * +,- .,/ 0123 4/ 5/ 6, /

-

where * = !* 7, 9* = j:

/ /

;/

Detection of Phase Transition in Photosensitive Liposomes by

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