Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

Summary

This protocol describes a simple preparation method for gold nanoparticle integrated photo-responsive liposomes with the commercially available materials. It also shows how to measure the microbubble cavitation process of the synthesized liposomes upon the treatment of pulsed laser.

Abstract

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the development process and are unprocurable by clinics. Here, we describe a facile fabrication of these photo-responsive NPs with commercially available gold NPs and thermo-responsive liposomes. Calcein is used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Finally, we show that this photo-triggered release is due to the membrane disruption caused by microbubble cavitation, which can be measured with hydrophone.

Introduction

The possibility to trigger drug release using external stimuli is an attractive way to deliver the drugs in spatial-, temporal- and dosage-controlled fashions with maximized specificity and minimal adverse effects. Among a wide range of exogenous stimuli-responsive systems (light, magnetic field, ultrasound, microwave radiation), light-triggered platforms are attractive, owing to their non-invasiveness, simplicity and adaptability in the clinics.¹ Extensive research in the past decade has provided a variety of platform technologies, such as near-infrared-light responsible gold (Au) nanocages coated with smart polymers,² photo-labile, polymeric nanoparticles (NPs) conjugated with drugs,³ and self-assembled porphysome nanovesicles.⁴ However, these technologies are still in the preclinical stages of development, and require a clear understanding and optimization of parameters involved in the process of initiating and controlling the drug release.

One of the simplest and easily accessible methods for preparing such a system is to integrate Au NPs with thermally-sensitive liposomes^5,6, both of which are widely available in the market and have been extensively investigated in preclinical and even clinical trials. Despite the limitation of deep-tissue activation of Au NPs at their plasmonic wavelength, when compared to near-infrared-activated Au nanostructures (e.g., nanocages), this system still holds great promise when used in small animals or for topical delivery in humans.⁷ There are some early efforts in combining Au NPs with liposomes for light-triggered release.^8-11 While most of them focus on the novelty of materials, accessibility and scalability issues need to be addressed. Moreover, reports on release mechanisms using these nanocarriers are still limited.

Herein, the fabrication of photo-responsive liposomes, simultaneously loaded with drugs and hydrophilic Au NPs has been described. Calcein is used as a model compound to evaluate the encapsulation efficiency and the release profile of the system. In addition, in this system, light absorbed by Au NPs dissipates to the surrounding microenvironment in the form of heat, resulting in an increase in the local temperature. Air microbubbles are generated during the laser heating and cause mechanical disruption of liposomes (Figure 1). The mechanism of microbubble cavitation is confirmed by hydrophone measurements.

Protocol

1. Preparation

1. Clean 100 ml round bottom flasks using aqua regia (1 part of concentrated nitric acid (HNO₃) and 3 parts of concentrated hydrochloric acid (HCl)) and wash the flasks with DI water. Autoclave the flasks and dry them in a hot air oven at 100 °C for 15 min. Wrap and store the sterile flasks until use.
2. Sterilize the hand-held mini-extruder set using 70% ethanol.
3. Turn on the rotary evaporator and set the temperature of the hot water bath and the cooling tower at 37 °C and 4 °C respectively.
4. Prepare 60 mM calcein stock solution by dissolving 374 mg of calcein in 10 ml of 0.1 mM phosphate buffered saline (PBS) (pH 7.4). Adjust the pH to 7.4 using 1 M sodium hydroxide (NaOH) solution.

2. Synthesis of Liposomes

1. Remove the lipids (1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (MPPC) and 1, 2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[carboxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG2000)) from freezer (-20 °C) and thaw them to RT.
2. Weigh 15.9 mg DPPC, 1.3 mg MPPC, and 2.8 mg DSPE-PEG2000. Dissolve them together in 2 ml chloroform.
3. Transfer the chloroform solution to the sterile round bottom flask and evaporate the solvent using the rotary evaporator under reduced pressure, to form a thin, dry lipid layer.
4. Hydrate the lipid layer at 45 °C with 2 ml aqueous solution for 30 min containing 1.95 ml 60 mM calcein prepared in step 1.4 and 50 µl Au NPs (3.36 × 10¹⁶ particles/ml).
5. Pre-heat the heating block to 45 °C. Place a 200 nm polycarbonate membrane filter between the filter supports and assemble the mini-extruder set. Check the potential leakage with DI water.
6. Fill one of the syringes with 1 ml of liposome solution from step 2.4 and extrude the sample by passing the solution to the syringe at the other end of the assemble mini-extruder. Repeat 11 times.
7. Run the synthesized liposomes through a PD-10 desalting column to remove the free Au NPs, lipids and calcein using PBS as the eluent according to manufacturer's protocol.
8. Store the samples in sterile tubes at 4 °C and use in 2 days.

3. Calcein Release from Liposomes with Heating

1. Calculate the lipid molarity of the stock solution by adding up the molarities of DPPC, MPPC, and DSPE-PEG2000 in step 2.2. Dilute the liposome stock solution to 5 mM lipid concentration using 0.1 mM PBS buffer (pH 7.4). Transfer the samples to a centrifuge tube (2 ml).
2. Place the tube in a hot water bath, and raise the temperature gradually from 25 to 70 °C. Increase the temperature at a rate of 1 °C/min here.
3. Collect aliquots (10 µl) at different temperature points (27, 32, 37, 39, 41, 43, 45, 52, 57, 62, 67 and 70 °C).
4. Add 10 µl of 2% Triton X-100 to 2 ml aliquot of liposomal solution to digest the liposomes for 10 min at RT and to achieve complete release of calcein.
5. Transfer 200 µl of the liposomal solution to each well in the 96-well microplate and measure the fluorescence intensity of the collected samples using a fluorescence microplate reader. The excitation and emission wavelengths of calcein are 480 and 515 nm respectively.
6. Taking the fluorescence intensity of the Triton X-100 treated samples as 100% release, calculate the percentage of the released calcein at each time point, using the formula:
   
   Ft is the fluorescence intensity of solution at a given time point. F_i and F_x are the normalized initial and final fluorescence intensities of the solution respectively.

4. Calcein Release from Liposomes with Pulsed Laser

1. Transfer 100 µl liposome solution to a quartz cuvette and place it in a cuvette holder. Use a pulsed Nd:YAG laser with a pulse duration of 6 nsec at 532 nm wavelength as the light source. Use the following Laser Parameters — Repetition rate: 1 Hz; Laser energy density: 1 mJ/cm²; Beam diameter: 0.5 mm.
2. Guide the collimated laser beam through the cuvette such that the light passes through the liposome solution and collect aliquots after various pulses.
3. Add 10 µl of 2% Triton X-100 to 2 ml aliquot of liposomal solution to digest the liposomes for 10 min at RT and to achieve complete release of calcein.
4. Considering calcein is sensitive to light and could be bleached during the laser experiment, pre-measure the bleaching effect of pulsed laser on calcein solution to normalize the data from liposome release.
   1. Specifically, expose calcein solution (60 mM) to pulsed laser with frequency of 1 Hz for varying pulse numbers (0, 25, 50 and 100). Measure the fluorescence intensity of calcein with excitation and emission wavelengths of 480 and 515 nm respectively, before and after laser exposure.
   2. Calculate the amount of bleaching (fluorescence intensity of calcein before laser exposure/ fluorescence intensity of calcein after the specific pulse number). Use this factor to normalize the values obtained from liposome samples by multiplying the fluorescence intensity of the liposomes with the factor.
5. Measure the fluorescence intensity of the collected sample using a fluorescence microplate reader at excitation and emission wavelengths at 480 and 515 nm respectively.
6. Taking the fluorescence intensity of the Triton X-100 treated samples as 100% release, calculate the percentage of the released calcein at each pulse number, using the formula:
   
   Ft is the fluorescence intensity of solution at a given pulse number after normalization. F_i and F_x are the normalized initial and final fluorescence intensities of the solution respectively.

5. Measurement of Pressure Impulses

1. Place 100 µl of the sample on a microscopic slide and set the focus of the laser onto the sample.
2. Immerse a needle hydrophone (1 mm diameter and 450 nV/Pa sensitivity) into the solution.
   Caution: The hydrophone must not be illuminated by the laser light to avoid the damage.
3. Irradiate the sample with the pulsed laser with varying pulse numbers (0-100) and pulse energy (20-160 µJ/pulse).
4. Record the pressure signals using a digital oscilloscope.

Results

Liposomes were prepared using a conventional thin film hydration technique with DPPC, MPPC and DSPE-PEG2000 in a molar ratio of 86:10:4 or 7.95:0.65:1.39 mg/ml.¹² The size of Au NPs is critical to determine the light to heat conversion efficiency during the following laser excitation experiment. Smaller the size of Au NPs, higher is the transducing efficiency.¹³ Thus 5 nm Au NPs, the smallest samples from the vendor, were chosen for encapsulation. During the synthesi...

Discussion

Thin film hydration is the conventional method for preparing liposomes. Organic solvents (chloroform in this case) were first used to dissolve the lipids and then removed in a rotary evaporator at 37 °C to generate a lipid thin film on the flask. This lipid film was hydrated with the aqueous solution containing 60 mM calcein and 5 nm Au NPs. During the hydration process, the temperature was maintained around 50 °C and the flask was constantly agitated by rotating the flask. The key in this step is the choice of...

Materials

Name	Company	Catalog Number	Comments
1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)	Avanti Polar Lipids (Alabama, US)	850355P	Powder, Store at -20 °C
1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (MPPC)	Avanti Polar Lipids (Alabama, US)	855675P	Powder, Store at -20 °C
1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG2000)	Avanti Polar Lipids (Alabama, US)	880120P	Powder, Store at -20 °C
Gold Nanoparticles	Sigma Aldrich	752568-100mL	5 nm particles, stabilized at 0.1 mM PBS
Calcein	Sigma Aldrich	C0875-10g	60 mM, pH 7.4 (adjusted using NaOH)
phosphate buffered saline (PBS)	Sigma Aldrich	P5493	0.1 mM, pH 7.4
Double distilled water	Millipore Milli-DI water purification system
Triton X100	Sigma, Life Sciences	X-100	To disrupt the liposomes to calculate total encapsulation
Rotavapor	Buchi (Switzerland)	R 210	Used for Lipososme preparation
Heating bath	Buchi (Switzerland)	B 491	Used for Lipososme preparation
Vacuum Controller	Buchi (Switzerland)	V-850	Used for Lipososme preparation
Vacuum Pump	Buchi (Switzerland)	V-700	Used for Lipososme preparation
Recirculation bath with temperature controller	Polyscience		Used for Lipososme preparation
Mini-extruder assembly with heating block	Avanti Polar Lipids (Alabama, US)	610000	Used for extrusion of liposomes
Syringes, 1,000 μl	Avanti Polar Lipids (Alabama, US)	610017	Used for extrusion of liposomes
Polycarbonate filter membrane, 200 nm	Whatmann	800281	Used for extrusion of liposomes
Filter Support	Avanti Polar Lipids (Alabama, US)	610014	Used for extrusion of liposomes
PD 10 Desalting coulumns, Sephadex G-25 medium	GE Healthcare, Life sciences	17-0851-01	Used to purify the liposomes
Centrifuge	Sigma Laboratory Centrifuges	3K30	Used to concentrate the liposomal solution
Rotor	Sigma	19777-H	Used to concentrate the liposomal solution
Zetasizer	Nano ZS Malvern		Used for the determination of liposome size and zetapotential
UV-Visible Spectrophotometer	Shimadzu	UV-2450	Used to measure the absorbance of the samples
Fluorescent Spectrofluorometer	Molecular Devices	SpectraMax M5	Used to measure the fluorescence emission of the samples
Nd:YAG Laser	NewWave Research		532 nm; Maximum power: 17 mJ; Width: 406 nsec; Used for sample irradiation
HNR Hydrophone	ONDA	HNR-1000	1 mm diameter and 450 nV/Pa sensitivity, Proper working frequency range: 0.25-10 MHz; Calibration: 50 mV/Bar; Used to measure the acoustic signals
Digital Osciloscope	LECORY - Wave Runner 64Xi-A		Frequency: 600 MHz; Max sample rate: 10 Gs/sec (at two channel); Used to record the measured acoustic signals