Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

Summary

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.

Abstract

Protein kinase C-delta inhibitor (PKCδi) 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 PKCδi. 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 PKCδi (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.

Introduction

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 transplantat....

Protocol

1. Preparation of Peptide Solutions

1. Retrieve the peptides (P2: CAAAAE, P4: CAAAAW, PKCδi: CSFNSYELGSL) from the -20 °C freezer and thaw at room temperature (RT).
   NOTE: Keep the bottle closed to prevent moisture from condensing on the peptides.
2. Weigh 0.01 g of each peptide on a microscale. Put each peptide into a separate 50 mL conical tube.
3. Add 18.74 mL of deionized (DI) water to the P2 tube.
4. Add 16.93 mL of DI water to the P4 tube.
5. Add 8.21 mL of 50% acetonitrile diluted in DI water to the PKCδi tube.
6. Vortex the peptide solutions briefly. Put the 50 mL conical tubes in a sonicator (40....

Results

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 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 1b). If the GNP is aggregated, the peak of absorption will be .......

Discussion

To ensure proper formulation, it is crucial that the δPKCi solution undergoes the sonication step outlined in 1.6. δPKCi 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.  The GNP/PKCi hybrid will not be well formulated if these steps are not done properly, due to the aggregation of the δPKCi peptide²³. 
.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
negatively charged glutamic acid peptide (P2)	CanPeptide		Sequence: CAAAAE-NH2 Length: 6aa Modification: C-terminal amidation Quantity: 50mg Purity: >95%
hydrophobic tryptophan peptide (P4)	CanPeptide		Sequence: CAAAAW-NH2 Length: 6aa Modification: C-terminal amidation Quantity: 50mg Purity: >95%
δPKCi peptide	CanPeptide		Seqeuence: CSFNSYELGSL-NH2 Length: 11aa Modification: C-terminal amidation Quantity: 50mg Purity: >95%
Conical tube(50ml)	Corning Life Sciences	3582070
Conical tube(15ml)	Corning Life Sciences	3582096
Acetonitrile	Sigma-Aldrich	271004-100ML
Sonicator	Branson Ultrasonics Corp.	Branson 2510MTH
Microtube	Diamed.ca	AD 150-N
Gold nanoparticle solution	Ted Pella	15705-5	A particle size is 20nm
Rocking Platform shaker	VWR international	40000-304
Microcentrifuge	Eppendorf	5417R
Acryl cuvette	SARSREDT	67.758
UV-Vis spectrophotometer	Agilent	Caty 60 UV-Vis