This work is supported by research grants from Canadian Institutes of Health Research (PJT-148847), Ministry of Research and Innovation of Ontario (RE-08-029), and Canada First Research of Excellence Program, Medicine by Design at University of Toronto. Dr. Mingyao Liu is James and Mary Davie Chair in Lung Injury, Repair, and Regeneration.

1. Preparation of Peptide Solutions
<ol>
	<li>Retrieve the peptides (P2: CAAAAE, P4: CAAAAW, PKC&#948;i: CSFNSYELGSL) from the -20 &#176;C freezer and thaw at room temperature (RT). 
	NOTE: Keep the bottle closed to prevent moisture from condensing on the peptides.</li>
	<li>Weigh 0.01 g of each peptide on a microscale. Put each peptide into a separate 50 mL conical tube.</li>
	<li>Add 18.74 mL of deionized (DI) water to the P2 tube.</li>
	<li>Add 16.93 mL of DI water to the P4 tube.</li>
	<li>Add 8.21 mL of 50% acetonitrile diluted in DI water to the PKC&#948;i tube.</li>
	<li>Vortex the peptide solutions briefly. Put the 50 mL conical tubes in a sonicator (40 MHz) for 5 min.</li>
	<li>Bring the peptide solutions to a biosafety cabinet. All peptide solutions prepared should be 1 mM.</li>
	<li>Transfer 1 mL of each peptide solution to a new 50 mL conical tube. Add 19 mL of DI water to the tubes of P2 and P4 and add 19 mL of 50% acetonitrile to the PKC&#948;i tube, such that each solution is diluted to 50 &#181;M and stored in its own tube.</li>
</ol>
2. Formulation of GNP/PKCi
<ol>
	<li>Peptides should be added to the GNP solution while still in the BSC</li>
	<li>Add 475 &#956;L of P2, 25&#956;L of P4 and 500 &#956;L of &#948;PKCi solution into a 15 mL tube. Add 9 mL of 20 nm GNP solution (7.0x1011 particle/mL) to the same 15 mL tube.</li>
	<li>Exit the biosafety cabinet. Wrap the 15 mL tube with aluminum foil. Leave it on a shaker at RT overnight.</li>
	<li>Return the samples to the biosafety cabinet. Aliquot 1 mL of GNP/PKCi into each 1.5 mL microtubes.</li>
	<li>Centrifuge the tubes in a micro-centrifuge for 30 min at 15,294 x g at 4 &#176;C.</li>
	<li>Remove the supernatant from each tube under a biosafety cabinet. 
	NOTE: Be careful to remove the supernatant while ensuring that the GNP pellet remains intact and is not aspirated.</li>
	<li>Re-suspend the pellet in the desired solvent according to the concentration required. Applicable solvents can be DI water, PBS, and 0.9% NaCl. 
	NOTE: Starting from 1 mL of GNP/PKCi, the GNP pellet contains 6.3 x 1011 particles, based on the GNP concentration provided by the manufacturer. To administer 1.3 x 1012 particles in 500 &#181;L of 0.9% NaCl, we add 232 &#181;L of 0.9% NaCl to each of three pellets. After pooling them together, we can then collect 500 &#181;L of GNP/PKCi solution. 
	NOTE: Mix the desired solvent well before diluting the GNP/PKCi pellet, otherwise the GNP/PKCi will aggregate.</li>
</ol>
3. Assessment of GNP/PKCi Hybrid Solubility
<ol>
	<li>Pour 0.5 mL of GNP/PKCi solution into an acryl cuvette. Place the acryl cuvette on a UV-Vis spectrophotometer and test the peak absorption15.</li>
</ol>

<ol>
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D., Keshavjee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+graft+dysfunction:+definition,+risk+factors,+short-+and+long-term+outcomes.">Primary graft dysfunction: definition, risk factors, short- and long-term outcomes.</a> Seminars in Respiratory and Critical. 31 (2), 161-171 (2010).</li><li>Chatterjee, S., Nieman, G. F., Christie, J. D., Fisher, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear+stress-related+mechanosignaling+with+lung+ischemia:+lessons+from+basic+research+can+inform+lung+transplantation.">Shear stress-related mechanosignaling with lung ischemia: lessons from basic research can inform lung transplantation.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 307 (9), 668-680 (2014).</li><li>Inagaki, K., 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=Inhibition+of+delta-protein+kinase+C+protects+against+reperfusion+injury+of+the+ischemic+heart+in+vivo.">Inhibition of delta-protein kinase C protects against reperfusion injury of the ischemic heart in vivo.</a> Circulation. 108 (19), 2304-2307 (2003).</li><li>Lincoff, A. M., 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=Inhibition+of+delta-protein+kinase+C+by+delcasertib+as+an+adjunct+to+primary+percutaneous+coronary+intervention+for+acute+anterior+ST-segment+elevation+myocardial+infarction:+results+of+the+PROTECTION+AMI+Randomized+Controlled+Trial.">Inhibition of delta-protein kinase C by delcasertib as an adjunct to primary percutaneous coronary intervention for acute anterior ST-segment elevation myocardial infarction: results of the PROTECTION AMI Randomized Controlled Trial.</a> European Heart Journal. 35 (37), 2516-2523 (2014).</li><li>Kim, H., 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=deltaV1-1+Reduces+Pulmonary+Ischemia+Reperfusion-Induced+Lung+Injury+by+Inhibiting+Necrosis+and+Mitochondrial+Localization+of+PKCdelta+and+p53.">deltaV1-1 Reduces Pulmonary Ischemia Reperfusion-Induced Lung Injury by Inhibiting Necrosis and Mitochondrial Localization of PKCdelta and p53.</a> American Journal of Transplantation. 16 (1), 83-98 (2016).</li><li>Lin, H. 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The authors have nothing to disclose on this project.

To ensure proper formulation, it is crucial that the &#948;PKCi solution undergoes the sonication step outlined in 1.6. &#948;PKCi peptide sequence contains hydrophobic moieties, so a sonicator assists in dissolving PKCi in the 50% acetonitrile solution. In addition, it is very important to mix the solvent meticulously, as outlined in step 2.7.&#160; The GNP/PKCi hybrid will not be well formulated if these steps are not done properly, due to the aggregation of the &#948;PKCi peptide23.&#160; 
&#160; 
GNP-based drug formulation provides several advantages. First, GNPs can be easily synthesized in well controlled sizes, ranging from a few nanometers to ~100 nm. Usually, the smaller sized GNPs can deliver drugs into cells more efficiently than the larger ones as the smaller ones can easily diffuse into their target region24.&#160; Second, GNPs are non-toxic in vitro and in vivo25, making them safe drug carriers.&#160; Third, hydrophobic drugs can be loaded onto the modified GNPs26.&#160; Forth, the surface chemistry of GNPs is readily modified for specific applications. In our studies, two short peptides are used to modify the GNP surface, to stabilize them in physiological conditions and impart new bioactivities. The peptides were thoughtfully designed with three regions including gold binding, spacing, and functional regions.&#160; Specifically, the N terminus of the peptide has cysteine (C) residue containing thiol group that can bind with gold. The middle part has four hydrophobic alanine residues to promote peptide assembly into a densely packed monolayer on the GNPs. The amino acid at the C terminus is a functional amino acid pointing outward, which can be used to manipulate the surface properties of the GNPs. The 95:5 ratio of these two peptides was systemically selected in a previous study14. The ratio between PKCi and P2/P4 peptides was also systemically tested and selected15.&#160;&#160;
On the other hand, the GNP drug delivery system has limitations. GNPs are not cell type or tissue specific. GNPs are mainly accumulated in the lung, liver, and spleen after intravenous administration27,28,29. So far, this formula has only been tested in cell culture and small animal models. To translate it to clinical application, further studies on large animal models and its pharmacokinetics, tissue distribution and potential toxicity need to be determined.

Lung transplantation saves patients with end-stage lung disease1. However, serious complications after lung transplantation remain an obstacle. In the early stages following lung transplantation, primary graft dysfunction is the most harmful complication1, and its primary cause is ischemia-reperfusion (IR)-induced acute lung injury2.
Under cold preservation, metabolism in a donor lung is restricted to a very low level. However, reactive oxygen species and nitric oxide synthesis are activated due to the cessation of blood flow3. After transplantation, blood circulation is restored, and reactive oxygen species and nitric oxide generated during cold ischemia enhance inflammation and cell death, resulting in tissue injury.
To prevent IR injury, a protein kinase C&#948; inhibitor (PKC&#948;i) has been used in the heart, brain and lung4,5,6,7,8. These studies showed that PKC&#948;i decreased inflammation and apoptosis during reperfusion. It has also prevented pulmonary IR injury in rats and in a lung transplant model6. PKC&#948;i is usually conjugated with a cell-penetrating peptide, TAT, for intracellular delivery. However, it has been shown that the TAT peptide alone has non-specific biological effects, including promotion of angiogenesis, apoptosis, and inhibition of multiple cytokines9,10,11. Nanoparticles, small particles ranging from 1 to 100 nm in diameter12, have been explored as candidates in facilitating drug delivery13. In particular, gold nanoparticles (GNPs) are regarded as noninvasive and nontoxic. Therefore, we have developed GNPs as drug delivery carriers for peptide-based drugs14,15.
The surface of GNPs can be manipulated for specific applications such as molecular recognition16,17, chemical sensing18, imaging19, and drug delivery. A GNP/peptide hybrid system has been developed, containing 20 nm GNPs and two short peptides (P2: CAAAAE and P4: CAAAAW) at a 95:5 ratio, to modify the surface properties of GNPs. The P2 peptide, with the negatively charged glutamic acid (E) at the end, stabilizes GNPs in an aqueous solution, and the P4 peptide, with the hydrophobic tryptophan (W) at the end, helps GNPs entrance into cells14. The cysteine (C) residue at the N terminus of these peptides contains a thiol group that can conjugate to the gold surfaces14. This peptide/GNP hybrid was further used to deliver PKC&#948;i (CSFNSYELGSL). The optimized molar ratio of P2:P4 to PKC&#948;i is 47.5:2.5:50. GNPs conjugated with PKC&#948;i (GNP/PKCi) are stable in distilled water, 0.9% NaCl, and PBS&#160;containing bovine albumin or fetal bovine serum14. Intravenous injection of GNP/PKCi has been shown to prevent ischemia-reperfusion injury of the lung15. This article outlines a method to formulate GNP/PKCi and describes how to evaluate the physicochemical properties of GNP/PKCi. We have used similar methods to formulate other peptide-based drugs conjugated to GNP20,21,22. We hope this article will draw more attention to this novel formulation for intracellular drug delivery.

<table border="0" cellpadding="0" cellspacing="0" style="width:603px;" width="603">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">negatively charged glutamic acid peptide (P2)</td>
			<td style="width:189px;">CanPeptide</td>
			<td></td>
			<td style="width:197px;">Sequence: CAAAAE-NH2 
			Length: 6aa 
			Modification: C-terminal amidation 
			Quantity: 50mg 
			Purity: &#62;95%</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">hydrophobic tryptophan peptide (P4)</td>
			<td>CanPeptide</td>
			<td></td>
			<td style="width:197px;">Sequence: CAAAAW-NH2 
			Length: 6aa 
			Modification: C-terminal amidation 
			Quantity: 50mg 
			Purity: &#62;95%</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">&#948;PKCi peptide</td>
			<td>CanPeptide</td>
			<td></td>
			<td style="width:197px;">Seqeuence: CSFNSYELGSL-NH2 
			Length: 11aa 
			Modification: C-terminal amidation 
			Quantity: 50mg 
			Purity: &#62;95%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Conical tube(50ml)</td>
			<td>Corning Life Sciences</td>
			<td align="right" style="width:119px;">3582070</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Conical tube(15ml)</td>
			<td>Corning Life Sciences</td>
			<td align="right" style="width:119px;">3582096</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Acetonitrile</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">271004-100ML</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Sonicator</td>
			<td style="width:71px;">Branson Ultrasonics Corp.</td>
			<td style="width:119px;">Branson 2510MTH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microtube</td>
			<td>Diamed.ca</td>
			<td style="width:119px;">AD 150-N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gold nanoparticle solution</td>
			<td>Ted Pella</td>
			<td style="width:119px;">15705-5</td>
			<td>A particle size is 20nm</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Rocking Platform shaker</td>
			<td style="width:71px;">VWR international</td>
			<td style="width:119px;">40000-304&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microcentrifuge</td>
			<td style="width:71px;">Eppendorf</td>
			<td style="width:119px;">5417R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Acryl cuvette</td>
			<td style="width:71px;">SARSREDT</td>
			<td align="right" style="width:119px;">67.758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UV-Vis spectrophotometer</td>
			<td style="width:71px;">Agilent</td>
			<td style="width:119px;">Caty 60 UV-Vis</td>
			<td></td>
		</tr>
	</tbody>
</table>

protein kinase c-delta inhibitor peptide formulation using gold nanoparticles

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a cell-penetrating peptide, TAT, for intracellular delivery. However, TAT has shown non-specific biological activities. Gold nanoparticles (GNPs) can be used as drug delivery carriers without recognized toxicity. Therefore, we have used a GNP/peptide hybrid to deliver PKC&#948;i. Two short peptides (P2: CAAAAE and P4: CAAAAW), at a 95:5 ratio, were used to modify the surface properties of GNP. GNPs conjugated with PKC&#948;i (GNP/PKCi) are stable in distilled water, 0.9% NaCl, and phosphate-buffered saline (PBS) containing bovine serum albumin or fetal bovine serum. Intravenous injection of GNP-PKCi was previously shown to prevent ischemia-reperfusion injury of the lung. This article outlines a protocol to formulate GNP/PKCi and assess the physiochemical properties of GNP/PKCi. We have used similar methods to formulate other peptide-based drugs with GNP. This article will hopefully draw more attention to this novel intracellular drug delivery technology and its applications in vivo.

Care should be taken to evaluate the biophysical properties of the GNP/PKCi hybrid, as GNP tends to aggregate in solvent. When GNP is aggregated, the color of the solution changes from pink to purple (Figure&#160;1a). UV-Vis spectrophotometer is able to detect the changes more sensitively. If the GNP/PKCi is not aggregated the peak of absorption should be at 525 nm (Figure&#160;1b). If the GNP is aggregated, the peak of absorption will be shifted to the right. As an alternate method of analysis, when aggregates have formed the &#916;Optical Density (&#916;OD = OD at 525 nm - OD at 440 nm) decreases.&#160;
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58741/58741fig1v2.jpg" /> 
Figure 1: Quality of GNP/PKCi. (A) Properly prepared GNP/PKCi is pink in color (left). Aggregated GNP/PKCi appears light purple (right). (B) Good GNP/PKCi preparation is stable in water, PBS, or in 0.9% NaCl solution. Readings on a UV-Vis spectrometer indicated that the peak of absorption was at 525 nm in all solutions. (C) An example of good and bad GNP/PKCi preparations. When the GNP was aggregated, the peak of absorption was shifted to the right. Moreover, &#916;OD decreased. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58741/58741fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

We have previously used a gold nanoparticle peptide hybrid to intravenously deliver a synthetic peptide, protein kinase C-delta inhibitor, which reduced ischemia-reperfusion-induced acute lung injury. Here we show the detailed protocol of the drug formulation. Other intracellular peptides can be formulated similarly.

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a ...

protein-kinase-c-delta-inhibitor-peptide-formulation-using-gold

Nanyang Technological University

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JoVE Journal

Bioengineering

5.4K Views.  University Health Network. We have previously used a gold nanoparticle peptide hybrid to intravenously deliver a synthetic peptide, protein kinase C-delta inhibitor, which reduced ischemia-reperfusion-induced acute lung injury. Here we show the detailed protocol of the drug formulation. Other intracellular peptides can be formulated similarly.

Video: Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. 
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5 (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10.

This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

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 ...

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. HerDox is formed by non-covalent self-assembly of a tumor targeted cell penetration protein with the chemotherapy agent, doxorubicin, via a small nucleic acid linker. A combination of electrophilic, intercalation, and oligomerization interactions facilitate self-assembly into round 10-20 nm particles. HerDox exhibits stability in blood as well as in extended storage at different temperatures. Systemic delivery of HerDox in tumor-bearing mice results in tumor-cell death with no detectable adverse effects to non-tumor tissue, including the heart and liver (which undergo marked damage by untargeted doxorubicin). HER2 elevation facilitates targeting to cells expressing the human epidermal growth factor receptor, hence tumors displaying elevated HER2 levels exhibit greater accumulation of HerDox compared to cells expressing lower levels, both in vitro and in vivo. Fluorescence intensity imaging combined with in situ confocal and spectral analysis has allowed us to verify in vivo tumor targeting and tumor cell penetration of HerDox after systemic delivery. Here we detail our methods for assessing tumor targeting via multimode imaging after systemic delivery.

This article details the procedures for optical imaging analysis of the tumor-targeted nanoparticle, HerDox. In particular, detailed use of the multimode imaging device for detecting tumor targeting and assessing tumor penetration is described here.

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. ...

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, biomedical device coatings, and drug delivery tools. Nanoporous gold (np-Au), produced by a nano-scale self-assembly process, is a relatively new material that exhibits large effective surface area, high electrical conductivity, and catalytic activity. These properties have made np-Au an attractive material to scientific community. Most studies on np-Au employ macro-scale specimens and focus on fundamental science of the material and its catalytic and sensor applications. The macro-scale specimens limit np-Au's potential in miniaturized systems, including biomedical devices. In order to address these issues, we initially describe two different methods to micropattern np-Au thin films on rigid substrates. The first method employs manually-produced stencil masks for creating millimeter-scale np-Au patterns, while the second method uses lift-off photolithography to pattern sub-millimeter-scale patterns. As the np-Au thin films are obtained by sputter-deposition process, they are compatible with conventional microfabrication techniques, thereby amenable to facile integration into microsystems. These systems include electrically-addressable biosensor platforms that benefit from high effective surface area, electrical conductivity, and gold-thiol-based surface bioconjugation. We describe cell culture, immunostaining, and image processing techniques to quantify np-Au's interaction with mammalian cells, which is an important performance parameter for some biosensors. We expect that the techniques illustrated here will assist the integration of np-Au in platforms at various length-scales and in numerous applications, including biosensors, energy storage systems, and catalysts.

We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.

We describe the method of programming stem cells to overexpress therapeutic factors for angiogenesis using biodegradable polymeric nanoparticles. Processes described include polymer synthesis, transfecting adipose-derived stem cells in vitro, and validating the efficacy of programmed stem cells to promote angiogenesis in a murine hindlimb ischemia model.

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly.
Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

We have engineered the capsid protein of hepatitis E virus as a theranostic nanoparticle (HEVNP). HEVNP self-assembles into a stable icosahedral cage in mucosal delivery. Here, we describe the modification of HEVNPs for tumor targeting by mutating surface-exposed residues to cysteines, which conjugate synthetic ligands that specifically bind tumor cells.

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...

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 ...

Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

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Chapters in this video

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

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

Viral Nanoparticles for In vivo Tumor Imaging

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Harmonic Nanoparticles for Regenerative Research

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth