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 na....

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 (PB....

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

List of materials used in this article

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