Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

Bioengineering

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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.

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Konoeda, H., Yang, H., Yang, C., Gower, A., Xu, C., Zhang, W., Liu, M. Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles. J. Vis. Exp. (145), e58741, doi:10.3791/58741 (2019).

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 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 Cδ inhibitor (PKCδi) has been used in the heart, brain and lung4,5,6,7,8. These studies showed that PKCδi decreased inflammation and apoptosis during reperfusion. It has also prevented pulmonary IR injury in rats and in a lung transplant model6. PKCδi 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 PKCδi (CSFNSYELGSL). The optimized molar ratio of P2:P4 to PKCδi is 47.5:2.5:50. GNPs conjugated with PKCδi (GNP/PKCi) are stable in distilled water, 0.9% NaCl, and PBS 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.

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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 MHz) for 5 min.
  7. Bring the peptide solutions to a biosafety cabinet. All peptide solutions prepared should be 1 mM.
  8. 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 PKCδi tube, such that each solution is diluted to 50 µM and stored in its own tube.

2. Formulation of GNP/PKCi

  1. Peptides should be added to the GNP solution while still in the BSC
  2. Add 475 μL of P2, 25μL of P4 and 500 μL of δPKCi solution into a 15 mL tube. Add 9 mL of 20 nm GNP solution (7.0x1011 particle/mL) to the same 15 mL tube.
  3. Exit the biosafety cabinet. Wrap the 15 mL tube with aluminum foil. Leave it on a shaker at RT overnight.
  4. Return the samples to the biosafety cabinet. Aliquot 1 mL of GNP/PKCi into each 1.5 mL microtubes.
  5. Centrifuge the tubes in a micro-centrifuge for 30 min at 15,294 x g at 4 °C.
  6. 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.
  7. 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 µL of 0.9% NaCl, we add 232 µL of 0.9% NaCl to each of three pellets. After pooling them together, we can then collect 500 µL of GNP/PKCi solution.
    NOTE: Mix the desired solvent well before diluting the GNP/PKCi pellet, otherwise the GNP/PKCi will aggregate.

3. Assessment of GNP/PKCi Hybrid Solubility

  1. 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.

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Representative 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 shifted to the right. As an alternate method of analysis, when aggregates have formed the ΔOptical Density (ΔOD = OD at 525 nm - OD at 440 nm) decreases. 

Figure 1
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, ΔOD decreased. Please click here to view a larger version of this figure.

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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
 
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.  Second, GNPs are non-toxic in vitro and in vivo25, making them safe drug carriers.  Third, hydrophobic drugs can be loaded onto the modified GNPs26.  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.  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.  

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.

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Disclosures

The authors have nothing to disclose on this project.

Acknowledgments

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.

Materials

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

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References

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