Multifaceted peptide assisted one-pot synthesis of gold nanoparticles for plectin-1 targeted gemcitabine delivery in pancreatic cancer

Abstract

An astute modification of the plectin-1-targeting peptide KTLLPTP by introducing a C-terminal cysteine preceded by a tyrosine residue imparted a reducing property to the peptide. This novel property is then exploited to fabricate gold nanoparticles (GNP) via an in situ reduction of gold(iii) chloride in a one-pot, green synthesis. The modified peptide KTLLPTPYC also acts as a template to generate highly monodispersed, spherical GNPs with a narrow size distribution and improved stability. Plectin-1 is known to be aberrantly expressed in the surface of pancreatic ductal adenocarcinoma (PDAC) cells while showing cytoplasmic expression in normal cells. The synthesized GNPs are thus in situ surface modified with the peptides via the cysteine residue leaving the N-terminal KTLLPTP sequence free for targeting plectin-1. The visual molecular dynamics based simulations support the experimental observations like particle size, gemcitabine conjugation and architecture of the peptide-grafted nanoassembly. Additionally, GNPs conjugated to gemcitabine demonstrate significantly higher cytotoxicity in vitro in two established PDAC cell lines (AsPC-1 and PANC-1) and an admirable in vivo antitumor efficacy in a PANC-1 orthotopic xenograft model through selective uptake in PDAC tumor tissues. Altogether, this strategy represents a unique method for the fabrication of a GNP based targeted drug delivery platform using a multifaceted peptide that acts as reducing agent, template for GNP synthesis and targeting agent to display remarkable selectivity towards PDAC.

Figure 1. Synthesis, characterization and stability of peptide-templated GNPs

A. Absorption intensity of the reaction solution at SPR peak as a function of time. (inset) UV-vis spectra of the reaction solution obtained at different time intervals. B. TEM image of the synthesized GNPs, scale bar = 20 nm. C. GNP particle size distribution histogram obtained from TEM picture analysis. D. Long-term stability analysis of the synthesized GNPs after 9 months at room temperature (RT), 4 °C and E. at pH 7.4 by measuring respective hydrodynamic size distributions.

Figure 2. Fabrication, characterization and stability of GNP-Gem

A. UV-vis spectra of GNP (Black) and GNP-Gem (Red). (inset) magnified portion of the spectra to highlight the red shift of the SPR peak upon gemcitabine conjugation. B. Saturation curve for gemcitabine conjugation to the GNPs. C. TEM image of the GNP-Gem, scale bar = 20 nm. D. Stability analysis of the GNP-Gem at room temperature by monitoring hydrodynamic diameter for 14 days and E. at 37 °C in presence of 10% FBS for 48 hours. F. In vitro cumulative gemcitabine release curve for GNP-Gem at 37 °C in two different pH environments. Black and red lines represent pH 7.4 and pH 5 respectively.

Figure 3. Computational modeling of nonapeptide bound gold nanoparticle with drug capture

A model for a nonapeptide (KTLLPTPYC) bound to 5.6 nm wide GNP via thiol-gold covalent linkages was generated with capture of Gemcitabine (Gem) drug for study of drug delivery transport mechanism. A. Entire nanoparticle consists of 4,896 Au atoms at the central core shown in yellow VdW spheres. Licorice stick peptides are displayed using conventional color for standard elements (carbon-gray, oxygen-red, nitrogen-blue, sulfur-yellow and hydrogen-white). Thicker stick rendering is shown for the Gemcitabine drug (highlighted ligand carbons colored red and fluorine in pink). Thiol (-SH)-to-gold connected cysteine atoms are shown in olive-yellow. Chlorine counter ions are shown in green spheres. B. Magnification of a representative area on GNP that reveals interaction of peptides and Gem with the GNP surface. N-terminal sequence of the peptides pointing away into solution is available for interacting with target proteins (plectin-1 in this case). C. Physical dimensions of the inner gold nanoparticle (~56 Å or 5.6 nm). D. Binding efficiency of GNP indicated by “% of drug bound”, is increasing over time.

Figure 4. In vitro therapeutic efficacy of GNP-Gem

In vitro cell viability as measured by MTS assay in A. AsPC-1 and B. PANC-1 cells after treating with increasing concentrations of Gem (blue), GNP (red) and GNP-Gem (green) respectively. ** denotes p<0.01.

Figure 5. In vivo therapeutic efficacy of GNP-Gem

Representative in vivo bioluminescence images captured before start and after completion of treatment for each treatment group in A. female and B. male mice. C. In vivo bioluminescence plot against time for female and male separately or in combination. Only (+) error bar was provided to improve clarity. * and ** denote p <0.05 and p<0.01 compared to vehicle-treated group respectively. D. Box plot diagram depicting tumor volume and tumor weight for each treatment group in female and male separately or in combination after completion of the experiment. For comparison, individual data points from each treatment group are also provided side by side. Black = vehicle, Red = GNP, Blue = Gem and Purple = GNP-Gem. * and ** denote p <0.05 and p<0.01 compared to vehicle-treated group respectively.

Figure 6. Immunohistochemical analysis

A. H&E staining of a tissue section from GNP-Gem treated group showing tumor-specific uptake of GNP-Gem without touching adjacent normal pancreas tissue. Scale bar =800 μm. B. Higher magnification showing accumulation of GNPs in tumor tissue. Scale Bar = 200 μm. Accumulated GNPs are shown using black arrows. C. & D. H&E staining of tissue sections obtained from each treatment group showing decrease in cellularity in GNP-Gem treated group in female and male mice respectively. Scale Bar = 200 μm. E. & F. Ki67 staining of tissue sections obtained from each treatment group showing reduction in number of Ki67 positive nuclei in GNP-Gem treated group in female and male mice respectively. Scale bar = 200 μm. G. Box plot diagram depicting percentage of Ki67 positive nuclei for each treatment group in female and male separately or in combination. Three slides were stained for Ki67 per treatment group and ten random areas of equal dimensions from each tissue sections were analyzed in Aperio Imagescope. For comparison, individual data points from each treatment group are also provided side by side. Black = vehicle, Red = GNP, Blue = Gem and Purple = GNP-Gem. * and ** denote p <0.05 and p<0.01 compared to vehicle-treated group respectively.