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.
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.
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 liposomes5,6, 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.
1. Preparation
2. Synthesis of Liposomes
3. Calcein Release from Liposomes with Heating
4. Calcein Release from Liposomes with Pulsed Laser
5. Measurement of Pressure Impulses
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 synthesis, hydrating medium containing Au NPs and calcein was added to generate multi-lamellar vesicles, which were then subject to size extrusion and gel filtration chromatography to remove the free Au NPs and calcein.
Calcein is a hydrophilic fluorescent dye is encapsulated within the aqueous core of the liposomes at a concentration of 60 mM. At such high concentrations, the fluorescence of calcein self-quenches. However, calcein is diluted as it is released from the liposomes, leading to fluorescence re-gain, indicating triggered drug release.14 Relying on this property of calcein liposomes were diluted to 5 mM and the response of the liposomal system to the temperature change was observed. As indicated in Figure 2A, regardless of the presence of Au NPs, the liposomes were almost intact at the physiological temperature (i.e., 37 °C) with a leakage percentage of less than 10%. However, when the temperature was raised to 42 °C (slightly above the transition temperature of lipids), 60%-80% of the encapsulated calcein is released from liposomes within 2 min. No significant difference in the release percentage is observed within the liposomes with and without Au NPs, when the release is triggered using heat. This is due to the phase transition temperature of the system. Moreover, the quick and efficient release is due to the presence of 10% MPPC, which enhances the permeability of liposome bilayer at the transition temperature.
Next, the calcein release from liposomes upon laser irradiation was examined. The liposomal solution was taken in a quartz cuvette which was placed in a cuvette holder. 532 nm Nd:YAG pulse laser with a pulse duration of 6 nsec and an instantaneous power density of 166.67 kW/cm2 was focused onto the liposomal solution. Aliquots of samples were collected at varying pulse numbers (0, 25, 50, and 100) in which the pulse frequency was fixed at 1 Hz. The samples were immediately transferred to a microcentrifuge tube and placed in an ice bath. This is to prevent any further release of calcein. The laser was turned off while collecting the samples. As shown in Figure 2B, liposomes with Au NPs released the encapsulated calcein upon excitation, in which the amount of released calcein increased as the pulse numbers increased.
The last step was to study the mechanism of calcein release from the liposomes with hydrophone. Figure 3A is the acoustic signal recorded for Au NPs (red), liposomes with Au NPs (black) and liposomes without Au NPs (blue). While Au NPs and liposomes with Au NPs showed characteristic pressure wave signals, no signal was observed from liposomes without Au NPs. The pressure impulse contains both positive and negative phases (inset in Figure 3A). It suggests that microbubbles form (positive phase) and disrupt (negative phase) in the solution. The maximum and the minimum values of the recorded peak in the Au NP-containing liposomes were 0.00093 and -0.00074 V respectively. Finally, the average maxima and minima of the pressure impulses as a function of increasing laser energy were calculated. As expected, the acoustics signal amplitude gradually increased with the increasing laser energy (Figure 3B). This should be due to the increased intensity of the oscillation of the particles attributing to the subsequent thermo-elastic expansion and contraction.15,16
Figure 1. The proposed drug release mechanism of photo-responsive liposomes: Au NPs (red dots) when being irradiated, generate microbubbles that disrupt the liposome membrane and thus trigger calcein release. Calcein, self-quenching within liposomes (as shown), fluoresces as being released to the surrounding. Please click here to view a larger version of this figure.
Figure 2. Heat and pulsed laser triggered release of calcein from liposomes. (A) Percentage of released calcein from liposomes with or without Au NPs, which were placed in water bath with various temperatures for 2 min; (B) Percentage of released calcein from liposomes with Au NPs, which were treated with pulsed laser (pulse duration = 6 nsec; power density ~12 mW/cm2). Error bars represent the mean ± standard deviation (SD) of three independent experiments performed in triplicate. Please click here to view a larger version of this figure.
Figure 3. (A) Acoustic signals measured using a hydrophone system for Au NPs (red), liposomes with Au NPs (black) and liposomes without Au NPs (blue). While Au NPs and liposomes with Au NPs showed characteristic pressure wave signals, no signal was observed from liposomes without Au NPs. The inset shows an enlarged view of acoustic signals of photo-responsive liposomes. (B) Maximum and minimum peak value of the pressure impulses as a function of increasing laser power. The acoustics signal amplitude gradually increases with the increasing laser energy. Error bars represent the mean ± SD of three independent experiments performed in ten runs. Please click here to view a larger version of this figure.
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 temperatures at which the evaporation and hydration were carried out respectively. The phase transition temperature (Tm) of DPPC, the major constituent of the liposome, is 41 °C. During the formation of the lipid think film, the temperature should be below 41 °C to prevent the formation of aggregated clumps of dried lipids. During the hydration, a higher temperature than Tm was chosen to drive the phospholipids to self-assemble into spherical multilamellar structures. Although thin film hydration method is easy to perform, it is limited by poor encapsulation efficiency (<1%) of the calcein and Au NPs in the aqueous solution. In the future, the encapsulation might be improved through freeze-thaw cycles.
Subsequent size-extrusion was done using a hand held mini extruder, placed on a heating block. A maximum of 1 ml solution can be extruded using this set-up, which is ideal for a small scale preparation in the laboratory. The key in this step is still the temperature, which should be above the phase transition temperature of liposomes. By handing the extrusion at 50 °C, the size of liposomes became uniform after 10 cycles of extrusion through the membrane with 200 nm pores. The motion in this step should be slow and gentle as the pressure within the extruder is big while the membrane is fragile.
One often-overlooked but important step during the synthesis of drug containing liposomes is the purification or removal of the un-encapsulated drugs (i.e., calcein and Au NPs here). This was done by gel filtration chromatography. It is critical for the researcher to notice that the PD-10 column used in this study is only suitable for processing <2 ml solution. Samples with larger volume could be processed by using multiple columns.
The study of calcein release from the liposome relies on the self-quenching of calcein at higher concentrations and re-fluorescence as diluted. When the samples were exposed to heat or light for thermal or laser treatment respectively, aliquots of samples were constantly withdrawn at varying temperature points or pulse numbers and transferred to separate vials. The vials should be immediately placed in an ice bath to prevent the potential further release of calcein, for accurate measurements. Another thing to notice is that calcein is sensitive to light and hence could be bleached during the laser experiment. To overcome this, the bleaching effect of a pulsed laser on the calcein solution should be measured and the data from liposome release normalized accordingly.
The protocol herein describes a facile preparation method for light sensitive liposomes. Thermal release is first demonstrated by exploiting the temperature responsive of the phospholipids. The laser set up is illustrated and light triggered release is achieved by pulsed laser. The mechanism of the light triggered release is also explored and is found to be due to membrane disruption by microbubble cavitation.
Different from all reported protocols, this protocol chooses materials (i.e., lipids and Au NPs) that have been frequently used in clinical trials and widely available in the market. The use of readily accessible materials allows anyone to prepare such light-sensitive system by themselves, without the need to seek technical help. In addition, this protocol makes use of thin film hydration to prepare liposomes, which is both cost-effective and easily scalable.
This simple but powerful protocol will benefit researchers and clinicians who are interested in achieving controlled drug delivery to superficial tissues like skin. One potential application would be to deliver anti-sunburn agents by incorporating this system within sunscreen.
The authors have nothing to disclose.
This work was partially supported by the Tier-1 Academic Research Funds by Singapore Ministry of Education (RG 64/12 to CX) and NTU-Northwestern Institute of Nanomedicine.
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 | 5nm particles, stabilized at 0.1mM PBS |
Calcein | Sigma Aldrich | C0875-10g | 60mM, 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, 1000 uL | Avanti Polar Lipids (Alabama, US) | 610017 | Used for extrusion of liposomes |
Polycarbonate filter membrane, 200nm | 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: 17mJ; Width: 406 ns; Used for sample irradiation | |
HNR Hydrophone | ONDA | HNR-1000 | 1000 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/s (at two channel); Used to record the measured acoustic signals |