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. Preparation
<ol>
	<li>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 &#176;C for 15 min. Wrap and store the sterile flasks until use.</li>
	<li>Sterilize the hand-held mini-extruder set using 70% ethanol.</li>
	<li>Turn on the rotary evaporator and set the temperature of the hot water bath and the cooling tower at 3...

<ol>
	<li>McCoy, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triggered+drug+delivery+from+biomaterials.">Triggered drug delivery from biomaterials.</a> Expert Opin. Drug Deliv. 7 (5), 605-616 (2010).</li><li>Yavuz, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+nanocages+covered+by+smart+polymers+for+controlled+release+with+near-infrared+light.">Gold nanocages covered by smart polymers for controlled release with near-infrared light.</a> Nat. Mater. 8 (12), 935-939 (2009).</li><li>Gohy, J. F., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photo-responsive+block+copolymer+micelles:+design+and+behavior.">Photo-responsive block copolymer micelles: design and behavior.</a> Chem. Soc. Rev. 42 (17), 7117-7129 (2013).</li><li>Lovell, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porphysome+nanovesicles+generated+by+porphyrin+bilayers+for+use+as+multimodal+biophotonic+contrast+agents.">Porphysome nanovesicles generated by porphyrin bilayers for use as multimodal biophotonic contrast agents.</a> Nat. Mater. 10 (4), 324-332 (2011).</li><li>Needham, D., Dewhirst, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+and+testing+of+a+new+temperature-sensitive+drug+delivery+system+for+the+treatment+of+solid+tumors.">The development and testing of a new temperature-sensitive drug delivery system for the treatment of solid tumors.</a> Adv. Drug Deliv. Rev. 53 (3), 285-305 (2001).</li><li>Landon, C. D., Park, J. Y., Needham, D., Dewhirst, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+drug+delivery+and+hyperthermia:+the+materials+design+and+preclinical+and+clinical+testing+of+low+temperature-sensitive+liposomes+used+in+combination+with+mild+hyperthermia+in+the+treatment+of+local+cancer.">Nanoscale drug delivery and hyperthermia: the materials design and preclinical and clinical testing of low temperature-sensitive liposomes used in combination with mild hyperthermia in the treatment of local cancer.</a> Open Nanomed. J. 3, 38-64 (2011).</li><li>Sykes, E. A., Dai, Q., Tsoi, K. M., Hwang, D. M., Chan, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+exposure+in+animals+can+be+visualized+in+the+skin+and+analysed+via+skin+biopsy.">Nanoparticle exposure in animals can be visualized in the skin and analysed via skin biopsy.</a> Nat. Commun. 5, 3796 (2014).</li><li>Paasonen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+nanoparticles+enable+selective+light-induced+contents+release+from+liposomes.">Gold nanoparticles enable selective light-induced contents release from liposomes.</a> J. Control. Release. 122 (1), 86-93 (2007).</li><li>Wu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remotely+Triggered+Liposome+Release+by+Near-Infrared+Light+Absorption+via+Hollow+Gold+Nanoshells.">Remotely Triggered Liposome Release by Near-Infrared Light Absorption via Hollow Gold Nanoshells.</a> J. Am. Chem. Soc. 130 (26), 8175-8177 (2008).</li><li>Leung, S. J., Kachur, X. M., Bobnick, M. C., Romanowski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wavelength-Selective+Light-Induced+Release+from+Plasmon+Resonant+Liposomes.">Wavelength-Selective Light-Induced Release from Plasmon Resonant Liposomes.</a> Adv. Funct. Mater. 21 (6), 1113-1121 (2011).</li><li>Volodkin, D. V., Skirtach, A. G., M&#246;hwald, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-IR+Remote+Release+from+Assemblies+of+Liposomes+and+Nanoparticles.">Near-IR Remote Release from Assemblies of Liposomes and Nanoparticles.</a> Angew. Chem. Int. Ed. 48 (10), 1807-1809 (2009).</li><li>Mills, J. K., Needham, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysolipid+incorporation+in+dipalmitoylphosphatidylcholine+bilayer+membranes+enhances+the+ion+permeability+and+drug+release+rates+at+the+membrane+phase+transition.">Lysolipid incorporation in dipalmitoylphosphatidylcholine bilayer membranes enhances the ion permeability and drug release rates at the membrane phase transition.</a> Biochim. Biophys. Acta. 1716 (2), 77-96 (2005).</li><li>Jiang, K., Smith, D. A., Pinchuk, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size-dependent+Photothermal+Conversion+Efficiencies+of+Plasmonically+Heated+Gold+Nanoparticles.">Size-dependent Photothermal Conversion Efficiencies of Plasmonically Heated Gold Nanoparticles.</a> J. Phys. Chem. C. 117 (51), 27073-27080 (2013).</li><li>Chongsiriwatana, N., Barron, A., Giuliani, A., Rinaldi, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+bacterial+membrane+interaction+of+antimicrobial+peptides+and+their+mimics.">Comparing bacterial membrane interaction of antimicrobial peptides and their mimics.</a> Antimicrobial Peptides. 618, 171-182 (2010).</li><li>Egerev, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acoustic+signals+generated+by+laser-irradiated+metal+nanoparticles.">Acoustic signals generated by laser-irradiated metal nanoparticles.</a> Appl. Opt. 48 (7), C38-C45 (2009).</li><li>Gonz&#225;lez, M. G., Liu, X., Niessner, R., Haisch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strong+size-dependent+photoacoustic+effect+on+gold+nanoparticles+by+laser-induced+nanobubbles.">Strong size-dependent photoacoustic effect on gold nanoparticles by laser-induced nanobubbles.</a> Appl. Phys. Lett. 96, 174104 (2010).</li></ol>

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 &#176;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 &#176;C and the flask was constantly agitated by rotating the flask. The key in this step is the choice of...

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.

<table border="0" cellpadding="0" cellspacing="0" width="943">
	<tbody>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)</td>
			<td dir="LTR" style="width:140px;">Avanti Polar Lipids (Alabama, US)</td>
			<td dir="LTR" style="width:119px;">850355P</td>
			<td dir="LTR" style="width:213px;">Powder, Store at -20 &#176;C</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (MPPC)</td>
			<td dir="LTR" style="width:140px;">Avanti Polar Lipids (Alabama, US)</td>
			<td dir="LTR" style="width:119px;">855675P</td>
			<td dir="LTR" style="width:213px;">Powder, Store at -20 &#176;C</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">1, 2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG2000)&#160;</td>
			<td dir="LTR" style="width:140px;">Avanti Polar Lipids (Alabama, US)</td>
			<td dir="LTR" style="width:119px;">880120P</td>
			<td dir="LTR" style="width:213px;">Powder, Store at -20 &#176;C</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">Gold Nanoparticles</td>
			<td dir="LTR" style="width:140px;">Sigma Aldrich</td>
			<td dir="LTR" style="width:119px;">752568-100mL</td>
			<td dir="LTR" style="width:213px;">5nm particles, stabilized at 0.1mM PBS</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">Calcein</td>
			<td dir="LTR" style="width:140px;">Sigma Aldrich</td>
			<td dir="LTR" style="width:119px;">C0875-10g</td>
			<td dir="LTR" style="width:213px;">60mM, pH 7.4 - adjusted using NaOH</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:471px;">phosphate buffered saline (PBS)</td>
			<td dir="LTR" style="width:140px;">Sigma Aldrich</td>
			<td dir="LTR" style="width:119px;">P5493</td>
			<td dir="LTR" style="width:213px;">0.1 mM, pH 7.4</td>
		</tr>
		<tr height="63">
			<td dir="LTR" height="63" style="height:63px;width:471px;">Double distilled water</td>
			<td dir="LTR" style="width:140px;">Millipore Milli-DI water purification system</td>
			<td dir="LTR" style="width:119px;"></td>
			<td dir="LTR" style="width:213px;"></td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">Triton X100&#160;&#160;</td>
			<td dir="LTR" style="width:140px;">Sigma, Life Sciences</td>
			<td dir="LTR" style="width:119px;">X-100</td>
			<td dir="LTR" style="width:213px;">To disrupt the liposomes to calculate total encapsulation</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">Rotavapor&#160;&#160;</td>
			<td dir="LTR" style="width:140px;">Buchi (Switzerland)</td>
			<td dir="LTR" style="width:119px;">R 210</td>
			<td dir="LTR" style="width:213px;">Used for Lipososme preparation</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">Heating bath</td>
			<td dir="LTR" style="width:140px;">Buchi (Switzerland)</td>
			<td dir="LTR" style="width:119px;">B 491</td>
			<td dir="LTR" style="width:213px;">Used for Lipososme preparation</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">Vacuum Controller&#160;&#160;</td>
			<td dir="LTR" style="width:140px;">Buchi (Switzerland)</td>
			<td dir="LTR" style="width:119px;">V-850</td>
			<td dir="LTR" style="width:213px;">Used for Lipososme preparation</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">Vacuum Pump</td>
			<td dir="LTR" style="width:140px;">Buchi (Switzerland)</td>
			<td dir="LTR" style="width:119px;">V-700</td>
			<td dir="LTR" style="width:213px;">Used for Lipososme preparation</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">Recirculation bath with temperature controller</td>
			<td dir="LTR" style="width:140px;">Polyscience</td>
			<td dir="LTR" style="width:119px;"></td>
			<td dir="LTR" style="width:213px;">Used for Lipososme preparation</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">&#160;Mini-extruder assembly with heating block&#160;</td>
			<td dir="LTR" style="width:140px;">Avanti Polar Lipids (Alabama, US)</td>
			<td dir="LTR" style="width:119px;">610000</td>
			<td dir="LTR" style="width:213px;">Used for extrusion of liposomes</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">Syringes, 1000 uL</td>
			<td dir="LTR" style="width:140px;">Avanti Polar Lipids (Alabama, US)</td>
			<td dir="LTR" style="width:119px;">610017</td>
			<td dir="LTR" style="width:213px;">Used for extrusion of liposomes</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">Polycarbonate filter membrane, 200nm&#160;&#160;</td>
			<td dir="LTR" style="width:140px;">Whatmann</td>
			<td dir="LTR" style="width:119px;">800281</td>
			<td dir="LTR" style="width:213px;">Used for extrusion of liposomes</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">Filter Support</td>
			<td dir="LTR" style="width:140px;">Avanti Polar Lipids (Alabama, US)</td>
			<td dir="LTR" style="width:119px;">610014</td>
			<td dir="LTR" style="width:213px;">Used for extrusion of liposomes</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">PD 10 Desalting coulumns, Sephadex G-25 medium</td>
			<td dir="LTR" style="width:140px;">GE Healthcare, Life sciences</td>
			<td dir="LTR" style="width:119px;">17-0851-01</td>
			<td dir="LTR" style="width:213px;">Used to purify the liposomes</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">Centrifuge&#160;&#160;</td>
			<td dir="LTR" style="width:140px;">Sigma Laboratory Centrifuges</td>
			<td dir="LTR" style="width:119px;">3K30</td>
			<td dir="LTR" style="width:213px;">Used to concentrate the liposomal solution&#160;</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:471px;">Rotor</td>
			<td dir="LTR" style="width:140px;">Sigma</td>
			<td dir="LTR" style="width:119px;">19777-H</td>
			<td dir="LTR" style="width:213px;">Used to concentrate the liposomal solution&#160;</td>
		</tr>
		<tr height="63">
			<td dir="LTR" height="63" style="height:63px;width:471px;">Zetasizer&#160;&#160;</td>
			<td dir="LTR" style="width:140px;">Nano ZS Malvern</td>
			<td dir="LTR" style="width:119px;"></td>
			<td dir="LTR" style="width:213px;">Used for the determination of liposome size and zetapotential</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">UV- Visible Spectrophotometer</td>
			<td dir="LTR" style="width:140px;">Shimadzu</td>
			<td dir="LTR" style="width:119px;">UV-2450</td>
			<td dir="LTR" style="width:213px;">Used to measure the absorbance of the samples</td>
		</tr>
		<tr height="63">
			<td dir="LTR" height="63" style="height:63px;width:471px;">Fluorescent Spectrofluorometer&#160;&#160;</td>
			<td dir="LTR" style="width:140px;">Molecular Devices</td>
			<td dir="LTR" style="width:119px;">SpectraMax M5</td>
			<td dir="LTR" style="width:213px;">Used to measure the fluorescence emission of the samples</td>
		</tr>
		<tr height="63">
			<td dir="LTR" height="63" style="height:63px;width:471px;">Nd:YAG Laser</td>
			<td dir="LTR" style="width:140px;">NewWave Research</td>
			<td dir="LTR" style="width:119px;"></td>
			<td dir="LTR" style="width:213px;">532 nm; Maximum power: 17mJ; Width: 406 ns; Used for sample irradiation</td>
		</tr>
		<tr height="126">
			<td dir="LTR" height="126" style="height:126px;width:471px;">HNR Hydrophone</td>
			<td>ONDA</td>
			<td dir="LTR" style="width:119px;">HNR-1000</td>
			<td dir="LTR" style="width:213px;">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</td>
		</tr>
		<tr height="84">
			<td dir="LTR" height="84" style="height:84px;width:471px;">Digital Osciloscope</td>
			<td dir="LTR" style="width:140px;">LECORY - Wave Runner 64Xi-A</td>
			<td dir="LTR" style="width:119px;"></td>
			<td dir="LTR" style="width:213px;">Frequency: 600 MHz; Max sample rate : 10 Gs/s (at two channel); Used to record the measured acoustic signals</td>
		</tr>
	</tbody>
</table>

synthesis of gold nanoparticle integrated photo-responsive liposomes and measurement of their microbubble cavitation upon pulse laser excitation

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.

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...

Watch this Scientific Journal Video about Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation at JoVE.com

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

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 ...

The overall goal of this protocol is to demonstrate the synthesis of a light responsive Liposomes and reveal the mechanism for the light controlled drug release from these Liposomes. This method helps answer the key questions in a figurative light response of Liposomes for drug delivery, where the key challenges are to synthesize the Liposomes and enhance the magnesium of controlled release. The main advantage of this technique is the cell synthesis of light responsive Liposomes with the commercially available materials.The implication of this technology extends towards the superficial tissue therapy. The use of minimal evasive procedures as well as a part of the laser device, will make the tech platform more accessible to the patients. Besides providing insights into the top care and accurate drug delivery, our system can also be applied to other systems such as small ani-ma-mo-rose as well as mucus system through guided lasers.First clean a 100 milliliter round bottom flask using Aqua regia. After washing with deionized water and autoclaving, dry the flask in a hot air oven at 100 degrees Celsius for 15 minutes. Then wrap and store the sterile flask until use.Sterilize a hand-held mini-extruder set using 70%Ethanol. Next turn on the rotary evaporator and set the temperature of the hot water bath and the cooling tower to 37 and 4 degree Celsius, respectively. Prepare a 60 millimolar Calcium stock solution by dissolving 374 milligrams of Calcein and 10 milliliters of 0.1 millimolar Phosphate buffered saline or PBS.Adjust the pH to 7.4 using one mollar Sodium Hydroxide solution. Remove the lipids from the freezer and thaw them to room temperature. Once the lipids have been thawed dissolve 15.9 milligrams of DPPC, 1.3 milligrams of MPPC and 2.8 milligrams of DSPE-PEG2000 and two milliliters of Chloroform.After transferring the Chloroform solution to a sterile round bottom flask, evaporate the solvent using the rotary evaporator under reduced pressure to form a thin dry lipid layer. Following this, hydrate the lipid layer at 45 degree Celsius with two milliliters of Aqueous solution containing 1.95 milliliters of the previously prepared 60 millimolar Calcium solution and 50 microliters of Gold nanoparticles for 30 minutes. Pre-heat a heating block to 45 degree Celsius.Place a 200 nanometer polycarbonate membrane filter between the filters supports and assemble the mini extruder set. Then check for potential leakage with deionized water. Fill one of the syringes with one milliliter of the previously prepared Liposome solution and extrude the sample by passing the solution through the syringe at the other end of the assembled mini extruder.After repeating the previous step 11 times run the synthesized Liposomes through a PD-10 desalting Column to remove the free gold nano particles, lipids and Calcein, using PBS as the eluent according to the manufacturers protocol. When finished store the samples in sterile tubes at four degree Celsius. At this point, calculate the lipid molarity of the stock solution by adding up the molarities of DPPC, MPPC and DSPE-PEG2000.Dilute the Liposome stock solution to a 5 millimolar lipid concentration using 0.1 millimolar PBS, then transfer the samples to 2 milliliters centrifuge tubes. Next, place the tubes in a hot water bath and raise the temperature gradually from 25 to 70 degrees Celsius. Collect 10 microliter aliquots at different temperature points.At 10 microliters of two percent triton x-100 to a two milliliter aliquot of Liposomal solution to digest the Liposomes for 10 minutes at room temperature and to achieve complete release of Calcein. Following this, transfer 200 microliters of the Liposomal solution to each well in a 96 well microplate. Measure the fluorescence intensity of the collected samples using a fluorescence micro plate reader.Taking the fluorescence intensity of the triton x-100 treated samples as 100 percent release, calculate the percentage of the released Calcein at each time point using the following formula:Transfer 100 microliters of the Liposome solution to a Quartz cuvette and place the cuvette in a cuvette holder. Guide the collimated laser beam through the cuvette such that the light passes through the Liposme solution. Then collect aliquots after various pulses.Following this add 10 microliters of 2%triton x-100, to a 2 milliliter aliquot of Liposomal solution to digest the Liposomes for 10 minutes at room temperature and to achieve complete release of Calcein. To pre-measure the bleaching effect of the laser on the Calcein solution expose the solution to a post laser with a frequency of 1 Hertz for varying pulse numbers. Measure the fluorescence intensity of Calcein with excitation and emission wavelengths of 480 and 515 Nanometers respectively before and after laser exposure.After calculating the amounts of bleaching use this factor to normalize the values obtained from Liposomes samples by multiplying the fluorescence intensity of the Liposomes with the factor. Next measure the fluorescence intensity of the collected sample using a fluorescence micro plate reader at excitation and emission wavelengths of 480 and 515 nanometers respectively. Taking the fluorescence intensity of the triton x-100 treated samples as 100 percent release, calculate the percentage of the released Calcein at each pulse number.Place 100 microliters of the sample on a microscope slide then set the focus of the laser on to the sample. The laser is guided through the sample manually hence it is important to make sure that the focal point of the beam is well within the sample and not on the microscopic slide to avoid experiments later. Following this immerse a needle hydrophone into the solution.The tip of the hydrophone is emerged into the sample at a sufficient distance away from the focal point of the laser. This is done to prevent any laser induced damages with the hydrophone. Irradiate the sample with the pulse laser with varying pulse numbers and pulse energy.Finally record the pressure signals using a digital oscilloscope. No significant difference in the release percentage is observed for the Liposomes with and without gold nano-particles. The Liposomes were almost intact at physiological temperature with a leakage percentage of less than 10 percent with or without gold nano-particles.When the temperature was increased to 42 degrees Celsius, 60 to 80 percent of the encapsulated Calcein was released from the Liposomes within two minutes. Liposomes with gold nano-particles released the encapsulated Calcein upon excitation in which the amount of released Calcein increased as the pulse numbers increased. The acoustic signals recorded for the gold nano-particles, the Liposomes with gold nano-particles and the Liposomes without gold nano-particles are shown here:The pressure impulse, contains both positive and negative phases and suggests that microbubbles form and disrupt in the solution.As expected the acoustic signal amplitude gradually increased with the increasing laser energy and should be due to the increased intensity of the oscillation of the particles attributing to the subsequent thermoelastic expansion and contraction. Once mastered the fabrication of Liposomes can be done within 3 hours provided all glassware and equipment is prepared in advanced. While attempting the laser controlled release it is important to remember that the laser beam is focused completely unto the sample to avoid loss of laser energy.Following this procedure other experiments like in vitro and in viol laser controlled release can be done to answer additional questions like how the physiological conditions will impact the laser controlled release. After this development, this technique will be resourceful in the field of reactivity to explore, localize and control the release to skin, eyes and mucus membrane.

synthesis-gold-nanoparticle-integrated-photo-responsive-liposomes

Research

JoVE Journal

Bioengineering

8.0K visualizações.  Nanyang Technological University. The overall goal of this protocol is to demonstrate the synthesis of a light responsive Liposomes and reveal the mechanism for the light controlled drug release from these Liposomes. This method helps answer the key questions in a figurative light response of Liposomes for drug delivery, where the key challenges are to synthesize the Liposomes and enhance the magnesium of controlled release. The main advantage of this technique is the cell synthesis of light responsive Liposomes with the comm...

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