Name: synthesis of gold nanoparticle integrated photo-responsive liposomes and measurement of their microbubble cavitation upon pulse laser excitation
Uploaded: 2016-02-24T14:00:00
Description: 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

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

Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and fold into higher order nanostructures. Structurally similar to peptides, peptoids are poly N-substituted glycines, where the side chains are attached to the nitrogen rather than the alpha-carbon. Their ease of synthesis and structural diversity allows testing of basic design principles to drive de novo design and engineering of new biologically-active and nanostructured materials.
Here, a simple manual peptoid synthesis protocol is presented that allows the synthesis of long chain polypeptoids ( up to 50mers) in excellent yields. Only basic equipment, simple techniques (e.g. liquid transfer, filtration), and commercially available reagents are required, making peptoids an accessible addition to many researchers' toolkits. The peptoid backbone is grown one monomer at a time via the submonomer method which consists of a two-step monomer addition cycle: acylation and displacement. First, bromoacetic acid activated in situ with N,N'-diisopropylcarbodiimide acylates a resin-bound secondary amine. Second, nucleophilic displacement of the bromide by a primary amine follows to introduce the side chain. The two-step cycle is iterated until the desired chain length is reached. The coupling efficiency of this two-step cycle routinely exceeds 98% and enables the synthesis of peptoids as long as 50 residues. Highly tunable, precise and chemically diverse sequences are achievable with the submonomer method as hundreds of readily available primary amines can be directly incorporated.
 
Peptoids are emerging as a versatile biomimetic material for nanobioscience research because of their synthetic flexibility, robustness, and ordering at the atomic level. The folding of a single-chain, amphiphilic, information-rich polypeptoid into a highly-ordered nanosheet was recently demonstrated. This peptoid is a 36-mer that consists of only three different commercially available monomers: hydrophobic, cationic and anionic. The hydrophobic phenylethyl side chains are buried in the nanosheet core whereas the ionic amine and carboxyl side chains align on the hydrophilic faces. The peptoid nanosheets serve as a potential platform for membrane mimetics, protein mimetics, device fabrication, and sensors. Methods for peptoid synthesis, sheet formation, and microscopy imaging are described and provide a simple method to enable future peptoid nanosheet designs.

A simple and general manual peptoid synthesis method involving basic equipment and commercially available reagents is outlined, enabling peptoids to be easily synthesized in most laboratories. The synthesis, purification and characterization of an amphiphilic peptoid 36mer is described, as well as its self-assembly into highly-ordered nanosheets.

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and ...

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides1. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously1. This combinatorial platform has been validated with conventional methods2 and the polyanhydride film and nanoparticle libraries have been characterized with 1H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.

This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries.

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect resulting in significant reductions in systemic toxicity. Nonetheless, insufficient release of encapsulated drug from liposomes has limited their clinical efficacy. Temperature-sensitive liposomes have been engineered to provide site-specific release of drug in order to overcome the problem of limited tumor drug bioavailability. Our lab has designed and developed a heat-activated thermosensitive liposome formulation of cisplatin (CDDP), known as HTLC, to provide triggered release of CDDP at solid tumors. Heat-activated delivery in vivo was achieved in murine models using a custom-built laser-based heating apparatus that provides a conformal heating pattern at the tumor site as confirmed by MR thermometry (MRT). A fiber optic temperature monitoring device was used to measure the temperature in real-time during the entire heating period with online adjustment of heat delivery by alternating the laser power. Drug delivery was optimized under magnetic resonance (MR) image guidance by co-encapsulation of an MR contrast agent (i.e.,&#160;gadoteridol) along with CDDP into the thermosensitive liposomes as a means to validate the heating protocol and to assess tumor accumulation. The heating protocol consisted of a preheating period of 5 min prior to administration of HTLC and 20 min heating post-injection. This heating protocol resulted in effective release of the encapsulated agents with the highest MR signal change observed in the heated tumor in comparison to the unheated tumor and muscle. This study demonstrated the successful application of the laser-based heating apparatus for preclinical thermosensitive liposome development and the importance of MR-guided validation of the heating protocol for optimization of drug delivery.

AN MRI-compatible&#160;custom-designed laser-based heating apparatus has been developed to provide local heating of subcutaneous tumors in order to activate release of agents from thermosensitive liposomes specifically at the tumor region.

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect ...

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise regulation of gene activity. In particular, novel stimuli-responsive materials with improved spatiotemporal control over gene expression would unlock translatable platforms in drug discovery and regenerative medicine technologies. Furthermore, an enhanced ability to control nucleic acid release from materials would enable the development of streamlined methods to predict nanocarrier efficacy a priori, leading to expedited screening of delivery vehicles. Herein, we present a protocol for predicting gene silencing efficiencies and achieving spatiotemporal control over gene expression through a modular photo-responsive nanocarrier system. Small interfering RNA (siRNA) is complexed with mPEG-b-poly(5-(3-(amino)propoxy)-2-nitrobenzyl methacrylate) (mPEG-b-P(APNBMA)) polymers to form stable nanocarriers that can be controlled with light to facilitate tunable, on/off siRNA release. We outline two complementary assays employing fluorescence correlation spectroscopy and gel electrophoresis for the accurate quantification of siRNA release from solutions mimicking intracellular environments. Information gained from these assays was incorporated into a simple RNA interference (RNAi) kinetic model to predict the dynamic silencing responses to various photo-stimulus conditions. In turn, these optimized irradiation conditions allowed refinement of a new protocol for spatiotemporally controlling gene silencing. This method can generate cellular patterns in gene expression with cell-to-cell resolution and no detectable off-target effects. Taken together, our approach offers an easy-to-use method for predicting dynamic changes in gene expression and precisely controlling siRNA activity in space and time. This set of assays can be readily adapted to test a wide variety of other stimuli-responsive systems in order to address key challenges pertinent to a multitude of applications in biomedical research and medicine.

We present a novel method that uses photo-responsive block copolymers for more efficient spatiotemporal control of gene silencing with no detectable off-target effects. Additionally, changes in gene expression can be predicted using straightforward siRNA release assays and simple kinetic modeling.

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise ...

Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting and drug delivery are some of the applications of these AuNPs, but endothelial extracellular matrices&#39; defensive properties hamper particle uptake. To address this issue, we describe a synthesis method for ultrasmall gold nanoparticles to improve vascular delivery, with customizable functional groups and polymer lengths for further adjustments. The protocol yields 2.5 nm AuNPs that are capped with tetrakis(hydroxymethyl)phosphonium chloride (THPC). The replacement of THPC with hetero-functional polyethylene glycol (PEG) on the surface of the AuNP increases the hydrodynamic radius to 10.5 nm while providing various functional groups on the surface. The last part of the protocol includes an optional addition of a fluorophore to allow the AuNPs to be visualized under fluorescence to track nanoparticle uptake. Dialysis and lyophilization were used to purify and isolate the AuNPs. These fluorescent nanoparticles can be visualized in both in vitro and in vivo experiments due to the biocompatible PEG coating and fluorescent probes. Additionally, the size range of these nanoparticles render them an ideal candidate for probing the glycocalyx without disrupting normal vasculature function, which may lead to improved delivery and therapeutics.

We describe a method of synthesizing biocompatible 10-nm gold nanoparticles, functionalized by coating poly-ethylene glycol onto the surface. These particles can be used in vitro and in vivo for delivering therapeutics to nanoscale cellular and extracellular spaces that are difficult to access with conventional nanoparticle sizes.

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...