Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

Summary

This work outlines a protocol to achieve dynamic, non-invasive monitoring of heat transfer from laser-irradiated gold nanoparticles to tBLMs. The system combines impedance spectroscopy for the real-time measurement of conductance changes across the tBLMs, with a horizontally focused laser beam that drives gold nanoparticle illumination, for heat production.

Abstract

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry using tethered bilayer lipid membranes (tBLMs) assembled on gold electrodes. Irradiated modified GNPs, such as streptavidin-conjugated GNPs, are embedded in tBLMs containing target molecules, such as biotin. By using this approach, the heat transfer processes between irradiated GNPs and model bilayer lipid membrane with entities of interest are mediated by a horizontally focused laser beam. The thermal predictive computational model is used to confirm the electrochemically induced conductance changes in the tBLMs. Under the specific conditions used, detecting heat pulses required specific attachment of the gold nanoparticles to the membrane surface, while unbound gold nanoparticles failed to elicit a measurable response. This technique serves as a powerful detection biosensor which can be directly utilized for the design and development of strategies for thermal therapies that permits optimization of the laser parameters, particle size, particle coatings and composition.

Introduction

The hyperthermic performance of irradiated gold nanomaterials offers a new class of minimally invasive, selective, targeted treatment for infections and tumors¹. The employment of nanoparticles that can be heated by a laser has been used to selectively destroy diseased cells as well as providing a means for selective drug delivery^2,3. A consequence of the photothermolysis phenomena of heated plasmonic nanoparticles is damage to the cell membranes. The fluid lipid bilayer membrane is considered a particularly vulnerable site for cells undergoing such treatments because denaturation of intrinsic membrane proteins as well as membrane damage can also lead to cell death⁴, as many proteins are there to maintain the ionic potential gradient across cell membranes. While the ability to determine and monitor heat transfer at the nanoscale is of key interest to the study and application of irradiated GNPs^1,5,6,7, assessment and understanding of the molecular interactions between GNPs and bio-membranes, as well as the direct consequences of the laser-induced heating phenomena of embedded GNPs in biological tissues, are yet to be fully elucidated⁸. Therefore, a thorough understanding of the hyperthermia process of irradiated GNPs remains a challenge. As such, the development of a nanomaterial-electrode interface that mimics the natural surroundings of cells could provide a means by which to undertake an in-depth investigation of the heat transfer characteristics of irradiated gold nanoparticles within biological systems.

The complexity of native cell membranes is one of the significant challenges in understanding the irradiated GNPs interactions in cells. There have been various artificial membrane platforms developed to provide close simple bio-mimetic versions of natural lipid membrane architecture and functionality, including, but not limited to, black lipid membranes⁹, supported planar bilayer membranes¹⁰, hybrid bilayer membranes¹¹, polymer-cushioned lipid bilayer membranes¹² and tethered bilayer lipid membranes¹³. Each artificial lipid membrane model has distinct advantages and limitations with respect to mimicking the natural lipid membranes¹⁴.

This study describes the employment of lipid membrane-coated electrodes as a sensor for assessing gold nanoparticle and lipid membrane interactions, using the tBLM model. The tBLM based biosensor detection scheme provides inherent stability and sensitivity¹³ as tethered membranes can self-repair, unlike other systems (such as membranes formed by patch-clamp or liposomes) in which only a small amount of membrane damage results in their collapse^15,16,17,18. Further, because tBLMs are of mm² dimensions, the background impedance is orders of magnitude lower than patch-clamp recording techniques, which enables a recording of changes in basal membrane ionic flux due to nanoparticle interactions. As a result of this, the present protocol can contrast changes in membrane conductance by bound GNPs that are excited by lasers whose powers are as low as 135 nW/µm².

The system presented here provides a sensitive and reproducible method for determining precise laser parameters, particle size, particle coatings and composition needed to design and develop thermal therapies. This is critical for the refinement of emerging photothermal therapies, as well as offering valuable information for detailed mechanisms of heat transfer within biological systems. The presented protocol is based on previously published work¹⁹. An outline of the protocol is as follows: the first section defines the tBLM formation; the second section outlines how to construct the setup and align the excitation laser source; the final section illustrates how to extract information from the electrical impedance spectroscopy data.

Protocol

1. tBLMs electrodes preparation

1. Preparation of first monolayer coating
   1. Immerse a freshly sputtered gold patterned electrode microscope slide in an ethanolic solution comprised of a 3 mM 1:9 ratio of benzyl-disulfide-tetra-ethyleneglycol-OH "spacer" molecules (benzyl disulfide comprised a four oxygen-ethylene glycol spacer, terminated with an OH group) and benzyl-disulfide (tetra-ethyleneglycol) n=2 C20-phytanyl "tethered" molecules. This creates the first layer coating to which a bilayer can be anchored.
      ​NOTE: The gold electrode is made by evaporating 100 nm, 99.9995% gold (5n5 gold) film onto custom 25 mm x 75 mm polycarbonate slides²⁰.
   2. Incubate electrodes with the first layer at room temperature for at least 1 h.
   3. Rinse the gold electrodes by immersing in copious amounts of pure ethanol over 30 s.
   4. Use the gold electrode slide with the first monolayer directly for the next step or store in a jar full of pure ethanol.
   5. NOTE: To ensure the integrity of the first layer, minimize any direct contact to the gold portions of the slide
2. Assembling the first monolayer coated slide
   1. Carefully take off one coplanar gold electrode slide from its container using tweezers, being sure not to make contact with the patterned areas where the tBLMs will form.
      NOTE: Be mindful to identify the side of the slide onto which the gold is deposited.
   2. Air dry slide for 1 - 2 min in to remove any residual ethanol.
   3. Place gold electrode over a dry surface, ensure the gold electrode is correctly oriented with patterned gold surface facing up.
   4. Peel the transparent adhesive layer cover from a thin laminate and place over the 6 channels to define each well.
   5. Use a pressure roller to release any air between the slide and transparent adhesive layer, as shown in Figure 1A.
      NOTE: The time required for this step will need to be optimized by the researcher. In this protocol, times are ranging from 2-3 min.
   6. Introduce as soon as practicable (within 1-2 minutes) the second lipid bilayer to the assembled first monolayer coated electrode for self-assembly to avoid damaging the first layer.
3. Preparation of second lipid bilayer
   1. Add 6 µL of 3 mM lipids of interest to the first well of the six wells slide. Do not let the edge of the micropipette tip touch the gold surface, which can damage the tethered chemistries on the electrode.
      NOTE: The lipid mixture used in this work consisted of 3 mM 70% zwitterionic C20 diphytanyl-ether-glycero-phosphatidylcholine (DPEPC) and 30% C20 diphytanyldiglyceride ether lipids (GDPE) mixed with 3 mM cholesterol-PEG-Biotin in 50:1 molar ratio.
   2. Introduce 6 µL of the lipid mixture to the other wells with a 10 s gap between each addition.
   3. Incubate each well for exactly 2 min at room temperature before exchanging the lipid mixture over the electrodes with a buffer such as PBS. Space the times for the addition and buffer exchange 10 s apart so each well is incubated with the lipid for exactly 2 min each.
   4. Wash 3 more times with 50 µL of PBS buffer (pH 7.0). Be sure to leave 50 µL of buffer over the electrodes at all times. Do not allow the electrodes to dry.
      NOTE: Displacing the ethanol solvent with the aqueous solution in this way (the solvent exchange method) enables the rapid formation of a single lipid bilayer anchored to the gold electrode via the tethered chemistries.
4. Testing tBLM formation using electrical impedance spectroscopy (EIS) measurements
   1. Insert prepared electrode slide into an AC impedance spectrometer (e.g., Tethapod). Ensure that the spectrometer is connected via a USB port to a computer running the software.
   2. Open the software, click Setup and open Hardware.
   3. Set the hardware settings to use 25 mV peak-to-peak AC excitation.
   4. Set frequencies between 0.1 and 10,000 Hz with two steps per decade for rapid impedance measures press ok.
   5. Click the Setup menu and open Model.
   6. Use an equivalent circuit model that describes the tethering gold electrode as a constant phase element in series with a resistor describing the electrolyte buffer and a parallel resistor-capacitor network to describe the lipid bilayer, and press OK.
   7. Press the Start button in order to start a real-time measurement of membrane capacitance (C_m) and membrane conduction (G_m). C_m values of typical tBLMs should be in the range of 12.5 nF to 15.5 nF for 10% tethered chemistries^21,22.
   8. After running the protocol and finishing the experiment, save the data.
   9. Repeat the measurement with the next well.

2. Laser irradiation

1. Experimental setup
   NOTE: The custom-made system is set up for each tBLM well individually.
   1. Perform experiments in a light-proof box to minimize laser hazardously.
   2. Use an optics table to set up the experiment to reduce unwanted vibrations.
   3. Place the impedance reader, where is the gold slide is connected, on an XYZ stage and elevate such that it sits in the path of the laser source.
   4. Use coarse-fine focusing microscopic gearing to control the height of the laser source to achieve the appropriate precision.
   5. Target the laser path along the longitudinal axis of the electrode slide.
      CAUTION: Always wear suitable laser safety glasses and maintain good laser safety protocols.
   6. Allow the selected tuned laser to stabilize before starting the experiment.
      NOTE: A schematic of the experimental setup is illustrated in Figure 2A.
2. Alignment of laser and gold electrodes
   NOTE: Before beginning, always assess the laser power output using a power-meter to ensure only very low wattages are delivered to the tBLMs.
   1. Adjust either the laser path or the angle of the electrode such that the laser passes through the liquid covering the electrode and is just visible, evenly, at the gold surface.
   2. Adjust the laser beam light position for each experiment by raising or lowering the laser beam source using the fine adjustment while observing changes in membrane conductance.
   3. Lock the knob to secure the position of the laser path when there are no conductance changes are observed.
      NOTE: Increased membrane conductance values will be generated when the laser interacts with the underlying gold electrode. It is, therefore, important to adjust the laser path such that no such interactions are possible.
3. Sample preparation
   1. Prepare the laser beam light alignment (where there is no change in membrane conductance), as shown in Figure 2, position 3.
   2. Add GNPs of interest (functionalized or bare) to the PBS buffer in which the tBLMs are immersed while the laser is switched OFF.
   3. Mix the PBS buffer surrounding the tBLMs gently three times, being careful not to touch the electrode.
   4. Incubate for 5-10 min at room temperature.
   5. Turn the laser ON to irradiate sample, using the correct aligned laser beam light position as seen in Figure 2, position 3.
   6. Use the appropriate combination of GNPs size, shape and concentration with laser light wavelength.
      NOTE: The laser beam of set wavelength should couple to the corresponding GNP plasmon resonance frequency.
   7. Record measured current continuously (real-time measurements).
   8. Perform steps 2.2.1 - 2.3.7, omitting GNP addition for the control experiments.

3. Statistical data analysis and presentation

1. Export the data into a spreadsheet.
2. Extract the membrane conductance parameter versus time.
3. Use the recorded data after setting a laser beam light with the right position and prior to GNPs introduction.
4. Normalize data by dividing the measured membrane conductance over the baseline membrane conductance.
   NOTE: This confirms that relative changes in membrane conduction values elicited by introduced irradiated GNPs.
5. Present data as plots of time (x-axis) versus normalized membrane conduction (y-axis).

4. Predict the amount of localized heat generated in the tBLMs from irradiated nanoparticles (thermal predictive model)

1. Solve the radiation transfer problem according to Dombrovsky²³, in order to calculate absorbed radiation power in irradiated nanoparticle solutions.
2. Calculate the heat generation by incorporating the heat source due to absorbed radiation into the energy equation.
   NOTE: For a detailed explanation of the numerical analysis of heat generation in the tBLMs from irradiated nanoparticles and the nanomaterial-electrode interface, refer to ¹⁹.

Results

The gold substrate upon which tBLMs can be created is shown in Figure 1. A schematic of the experimental setup is presented in Figure 2.

Coplanar gold electrodes, as shown in Figure 1A, are made from 25 mm x 75 mm x 1 mm polycarbonate base substrate with patterned gold arrays. A transparent adhesive layer defines the six individual measuring chambers. The coplanar gold electrode allows the ...

Discussion

This protocol describes the use of tBLM model with a coplanar electrode substrate in conjunction with a horizontal laser alignment set up that enables the real-time electrical impedance recording in response to laser irradiation of gold nanoparticles. The method of EIS recording presented here constructs a minimal list of experiments necessary to provide recording of ion current changes across the membrane, which corresponds to the heat generated by the coupled laser and gold nanoparticle...

Materials

Name	Company	Catalog Number	Comments
30 nm diameter streptavidin-conjugated gold nanoparticles	Cytodiagnostics	AC-30-04-05	This is a streptavidin-conjugated GNPs product ready for use
30 nm diameter bare gold nanoparticles	Sigma-Aldrich	753629	This is a bare GNPs product ready for use
Cholesterol-PEG-Biotin (MW1000)	NANOCS	PG2-BNCS-10k	Dissolved in highly pure ethanol
C20 Diphytanyl-Glycero-Phosphatidylcholine lipids	SDx Tethered Membranes Pty. Ltd.	SDx-S1	1 ml glass vial containing 70% C16 diphytanyl phosphatidylcholine (DPEPC) and 30% C16 diphytanyl glycerol (GDPE) in 99.9% ethanol
Benzyl-disulfide-tetra-ethyleneglycol-OH	SDx Tethered Membranes Pty. Ltd.	SDx-S2	Spacer molecules
Benzyl-disulfide (tetra-ethyleneglycol) n=2 C20-phytanyl	SDx Tethered Membranes Pty. Ltd.	SDx-S2	Tethered molecules
532 nm green laser continuous light	OBIS LS/OBIS CORE LS, China	ND-1000	The power of this laser was ~135 mW
tethaPod EIS reader	SDx Tethered Membranes Pty. Ltd.	SDx-R1	A reader of conductance and capacitance on six channels simultaneously
tethaPlate cartridge assembly	SDx Tethered Membranes Pty. Ltd.	SDx-BG	Materials to attach the slide with electrodes to the flow cell cartridge
Clamp and slide assembly jig	SDx Tethered Membranes Pty. Ltd.	SDx-A1	Materials to attach the slide with electrodes to the flow cell cartridge
Lipid coated coplanar gold electrodes	SDx Tethered Membranes Pty. Ltd.	SDx-T10	Coplanar  gold electrodes are made from 25 mm x 75 mm x 1 mm polycarbonate base substrate with patterned gold arrays layout, then coated with benzyldisulphide, bis-tetraethylene glycol C16 phytanyl half membrane spanning tethers in a tether ratio of 10%
tethaQuick software	SDx Tethered Membranes Pty. Ltd.	SDx-B1	Software for use with tethaPod to process data and display conductance, impedance and capacitance measurements from the tethaPlate electrodes
99.9% Pure ethanol	Sigma-Aldrich	34963	Absolute,  99.9%
Phosphate buffered saline (PBS)	Sigma-Aldrich	P4417	pH 7