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% a...

<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. W., 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=Derangements+of+post-ischemic+cerebral+blood+flow+by+protein+kinase+C+delta.">Derangements of post-ischemic cerebral blood flow by protein kinase C delta.</a> Neuroscience. 171 (2), 566-576 (2010).</li><li>Lin, H. 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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=Nanotechnologies+for+biomolecular+detection+and+medical+diagnostics.">Nanotechnologies for biomolecular detection and medical diagnostics.</a> Current Opinion in Chemical Biology. 10 (1), 11-19 (2006).</li><li>Rosi, N. L., Mirkin, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanostructures+in+biodiagnostics.">Nanostructures in biodiagnostics.</a> Chemical Reviews. 105 (4), 1547-1562 (2005).</li><li>Saha, K., Agasti, S. S., Kim, C., Li, X., Rotello, V. 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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; ...

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 absorptio...

Watch this Scientific Journal Video about Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles at JoVE.com

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 ...

Our unique drug delivery system, which uses 20 nanometer gold particles in two specific short peptides, can stabilize gold nano particles in physiological solution and enhance its cellular uptake, and thus, effectively deliver peptide-based or other drugs into cells. Our GNP peptide hybrid is easy to synthesize and stable. By using gold nanoparticle peptide hybrid as drug carriers, we can transfer peptide drugs into the cells effectively and in abundance.We treated ischemia-reperfusion injury in the lungs with our GNP/PKCi hybrid. GNP brings PKC inhibitor peptides into the cytoplasm. This delivery method was able to reduce more severe lung injury using a lower dosage of PKCi than other delivery methods.To begin this procedure, retrieve the peptides from a freezer at minus 20 degrees Celsius, and thaw them at room temperature. Use a micro scale to weigh 0.01 gram of each thawed peptide, and transfer each peptide into a separate 50 milliliter conical tube. Add 18.74 milliliters of deionized water to the P2 tube, and 16.93 milliliters of deionized water to the P4 tube.Add 8.21 milliliters of 50%acetonitrile diluted in deionized water to the PKC delta inhibitor tube. Vortex the peptide solutions briefly. Then place the tubes into a sonicator at 40 megahertz for five minutes.After this, transfer the solutions to a biosafety cabinet. It is critical to put the 50 mL conical tube in a sonicator for five minutes. If this step is skipped, the GNP/PKCi will form aggregations.Transfer one milliliter of each peptide solution to its own new 50 milliliter conical tube. Add 19 milliliters of deionized water to the tubes containing P2 and P4.And add 19 milliliters of 50%acetonitrile to the tube containing PKC delta inhibitor. Add 475 milliliters of the P2 solution, 25 microliters of the P4 solution, 500 microliters of the PKC delta inhibitor solution to a 15 milliliter tube.Then add nine milliliters of a 20 nanometer GNP solution to the same tube. Remove the combined solution from the biosafety cabinet and wrap the tube in aluminum foil. Place the tube on a shaker overnight.The next day, return the samples to the biosafety cabinet. Transfer one milliliter aliquots of the GNP/PKCi solution into 1.5 milliliter micro tubes. Centrifuge the micro tubes at a micro centrifuge at 15294 times g and at four degrees Celsius for 30 minutes.Transfer the micro tubes back into the biosafety cabinet, and remove the supernatent from each tube, while ensuring the GNP pellet is not disturbed. Resuspend each pellet in the desired solvent according to the concentration required. To assess the GNP/PKCi hybrid solubility, pour 0.5 milliliters of the GNP/PKCi solution into an accrual cuvette.Place the cuvette on a UV-Vis spectrophotometer and test the peak absorption. In this study, protein kinase C-delta inhibitor is conjugated with gold nanoparticles to form a GNP/PKCi hybrid. Care must be taken with evaluating the biophysical properties of the hybrid, as gold nanoparticles tend to aggregate in a solvent.When the nanoparticles aggregate, the color of the solution will change from pink to purple. The samples are analyzed with a UV-Vis spectrophotometer which is able to detect with a high degree of sensitivity. If the hybrid is not aggregated, the peak of an absorption should be at 525 nanometers.If the GNP is aggregated, the peak of absorption will be shifted to the right. As can be seen, when aggregates have formed, the delta optical density decreases. When formulating the GNP peptide hybrids, it is important to remember the ratio of peptides to GNP solution should be one to nine.This GNP/PKCi formula can be used for ischemia-reperfusion injury in organ systems other than the lung. Any peptides can attach to GNP if those peptides have a cysteine residue at the N-terminus. This means GNP peptide hybrid can be used to transport other peptide drugs into the cell.Rationally designed peptides are new technology that can brought specific protein-protein interaction inside those cells. This technique, developed here, can be used for delivery of new peptide drugs or for modified versions of existing drugs.

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Research

JoVE Journal

Bioengineering

5.4K visualizações.  University Health Network. Nosso sistema único de entrega de drogas, que usa partículas de ouro de 20 nanômetros em dois peptídeos curtos específicos, pode estabilizar nanopartículas de ouro em solução fisiológica e melhorar sua absorção celular, e assim, efetivamente fornecer peptídeos ou outras drogas em células. Nosso híbrido de peptídeo GNP é fácil de sintetizar e estável. Usando o híbrido de peptídeo de nanopartículas de ouro como portadores de drogas, podemos transferir drogas de peptídeos p...