Cholecystokinin Receptor-Targeted Polyplex Nanoparticle Inhibits Growth and Metastasis of Pancreatic Cancer

Abstract

Background & aims: Pancreatic ductal adenocarcinoma (PDAC) remains the most aggressive malignancy with the lowest 5-year survival rate of all cancers in part owing to the lack of tumor-specific therapy and the rapid metastatic nature of this cancer. The gastrointestinal peptide gastrin is a trophic peptide that stimulates growth of PDAC in an autocrine fashion by interaction with the cholecystokinin receptor that is overexpressed in this malignancy.

Methods: We developed a therapeutic novel polyplex nanoparticle (NP) that selectively targets the cholecystokinin receptor on PDAC. The NP was characterized in vitro and stability testing was performed in human blood. The effects of the target-specific NP loaded with gastrin small interfering RNA (siRNA) was compared with an untargeted NP and with an NP loaded with a scrambled siRNA in vitro and in 2 orthotopic models of PDAC. A polymerase chain reaction metastasis array examined differentially expressed genes from control tumors compared with tumors of mice treated with the targeted polyplex NP.

Results: The polyplex NP forms a micelle that safely delivers specific gastrin siRNA to the tumor without off-target toxicity. Consistent with these findings, cellular uptake was confirmed only with the targeted fluorescently labeled NP by confocal microscopy in vitro and by IVIS fluorescent based imaging in mice bearing orthotopic pancreatic cancers but not found with untargeted NPs. Tumor uptake and release of the gastrin siRNA NP was verified by decreased cellular gastrin gene expression by quantitative reverse-transcription polymerase chain reaction and peptide expression by immunohistochemistry. Growth of PDAC was inhibited in a dose-related fashion in cell culture and in vivo. The targeted NP therapy completely blocked tumor metastasis and altered tumor-specific genes.

Conclusions: Our polyplex nanoparticle platform establishes both a strong foundation for the development of receptor-targeted therapeutics and a unique approach for the delivery of siRNA in vivo, thus warranting further exploration of this approach in other types of cancers.

Keywords: CCK Receptor; CCK, cholecystokinin; Ex/Em, maximal excitation and emission wavelengths; Ga-10, gastrin 10 peptide; Gastrin; Gene Therapy; MW, molecular weight; N/P, ratio of “amines” of poly (L-lysine) unit and “phosphates” of siRNA complexed in the polyplex; NMR, nuclear magnetic resonance; NP, nanoparticle; Nanotechnology; Orthotopic; PBS, phosphate-buffered saline; PDAC, pancreatic ductal adenocarcinoma; PEG, polyethylene glycol; PanIN, pancreatic intraepithelial neoplasia; mRNA, messenger RNA; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; siRNA, small interfering RNA.

Graphical abstract

Figure 1

Synthesis of the gastrin-targeted polyplex. (A) Synthesis of thiol-functionalized polyplex forming block copolymer SH-PEG-PLL. SH-PEG-PLL block copolymer was synthesized from Tr-S-PEG-PLL (Tr-S-PEG-PLL) by reducing with trifluoroacetic acid and triethylsilane (98:2 vol/vol). (B) ¹H NMR of trityl protected polymer and thiol-functionalized polymer SH-PEG-PLL (absence of aromatic peaks at δ, 7.35 ppm). (C) Synthesis of CCK-B–receptor–targeted PEG-PLL block copolymer (Ga-PEG-PLL). A CCK-receptor–specific peptide, Ga-10, was conjugated to the SH-PEG-PLL block copolymer via a thiol-maleimide–coupling reaction. ¹H NMR of purified Ga-PEG-PLL indicates the presence of an aromatic proton (δ, 7.5–6.8 ppm) from the Ga-10 peptide. (D) Both Ga-PEG-PLL and SH-PEG-PLL were assessed by gel permeation chromatography to confirm the Ga-10 conjugation to the polymer backbone using a Shodex protein KW 403-4F column using both fluorescence (Ex/Em: λ= 280/350 nm) and UV detector (220 nm). Because of the presence of fluorescent tryptophan (Ex/Em: λ= 280/350 nm) in the Ga-10 peptide, the target-specific polymer, Ga-PEG-PLL elution was strongly detected via fluorescent detector (D1), whereas the untargeted precursor, SH-PEG-PLL elution was barely detected (D2) at an identical polymer concentration. On the other hand, both targeted and untargeted polymers showed an identical elution profile and intensity via UV detector at 220 nm (D3 and D4). (E) Finally, the targeted polyplex was prepared by mixing the Ga-PEG-PLL with selective gastrin siRNA to form the polyplex micelle.

Figure 2

Characterization of the targeted polyplex. (A) Hydrodynamic size of both targeted (Ga-PEG-PLL) and untargeted (PEG-PLL) polyplex measured by dynamic light scattering technique. The targeted polyplex was synthesized from Ga-PEG5K-PLL27 and the untargeted version from PEG5K-PLL30 block copolymer by complexing with gastrin siRNA at the N/P ratio of 5. Attachment of the targeting ligand, Ga-10, on the targeted polyplex surface did not change the polyplex size significantly. (B) A series of untargeted PEG-PLL polyplex formulations from varied PEG (5k–20k) and PLL block lengths (30–100 lysine units) encapsulating gastrin siRNA (19 bp) at the N/P ratio of 5 showed remarkable enhancement in serum stability compared with free siRNA. No siRNA degradation was observed for at least 7 hours in 90% fresh human serum. PEG5K-PLL30 was chosen as an untargeted polyplex for all in vitro and in vivo investigations. (C) Unencapsulated (free) siRNA degrades very fast in human serum (serum half-life, <10 min).

Figure 3

Effects of gastrin siRNA polyplex on growth of pancreatic cancer in vitro. (A) Effects of siRNA-loaded NPs on growth of pancreatic cancer in vitro. Growth of pancreatic cancer cells PANC-1 (A1) and AsPC-1 (A2) was decreased significantly with the addition of polyplex loaded with the gastrin siRNA compared with scrambled control RNA. (B) Localization of polyplex in cancer cells after uptake. Confocal microscopy (Zeiss) images of BxPC-3 human pancreatic cancer cells in culture treated with PBS (control, B1) or polyplex loaded with fluorescently labeled Cy3 siRNA shows uptake and localization of the siRNA in the cancer cell cytoplasm when treated with 240 nmol/L (B2) or 480 nmol/L (B3) of siRNA. (C) Measurement of target mRNA after treatment with siRNA-loaded polyplex. Quantitative RT-PCR showed a dose-dependent decrease in gastrin mRNA in AsPC-1 (C1) and BxPC-3 (C2) cells treated with polyplex carrying gastrin siRNA at 120, 240, or 480 nmol/L, but not in vehicle controls. (D) Analysis of gastrin peptide expression by immunofluorescence in AsPC-1 (D1) and PANC-1 (D2) cells after treatment with siRNA polyplex confirms that the polyplex loaded with gastrin siRNA also decreased peptide expression.

Figure 4

Selective uptake of targeted gastrin siRNA polyplex decreases orthotopic pancreatic tumor growth in vivo. (A) Final BxPC-3 tumor weights tended to be smaller in mice treated with 240 nmol/L siRNA, but this was not statistically significant. (B) Mice bearing human PANC-1 tumors and treated with 480 nmol/L targeted siRNA had significantly smaller tumors compared with controls. (C) Targeted siRNA are taken up into BxPC-3 orthotopic pancreatic tumors in mice. Polyplex that were either targeted to the CCK-B receptor or untargeted were loaded with fluorescently labeled Cy3 gastrin siRNA and injected intraperitoneally and imaged by fluorescent microscopy. Only the mice receiving the targeted siRNA showed fluorescent uptake within the tumors after 5 hours (arrows showing fluorescence). There was no uptake of fluorescent particles detected in the mice treated with untargeted polyplex circles show area of tumor and magnified view. (D) qRT-PCR for gastrin mRNA. Treatment with targeted siRNA had significantly less gastrin mRNA in PANC-1 tumors. (E1-5) Immunohistochemistry for gastrin peptide is shown from a representative BxPC-3 tumor from each treatment group. (E6) Densitometry analysis of immunostaining for gastrin reactivity showed significantly less gastrin (P = .02) in the tumors of mice treated with targeted siRNA. (F) Ki67 staining proliferation index was markedly reduced in PANC-1 tumors of mice treated with targeted siRNA (F1). PBS control Ki67 immunoreactivity (F2). Tumors treated with targeted NP with gastrin siRNA have reduced Ki67 immunoreactivity (F3). ∗P < .05, ∗∗∗P < .005.

Figure 5

CCK-receptor–targeted polyplex gastrin siRNA blocks metastases in a pancreatic cancer orthotopic model. (A) Percentage of mice with metastases in various treatment groups is shown. No metastases were found in mice treated with targeted gastrin siRNA polyplex. (B) Representative H&E image of a metastatic lesion in the liver of a PBS-treated control mouse. Scale bar: 200 μm. (C) Heat map showing absolute expression levels of metastatic genes altered with targeted siRNA treatment. (D) Fold change in genes from tumors treated with targeted siRNA compare to control tumors. Names of genes from excised tumors that were increased (left) or decreased (right) with targeted siRNA therapy and the fold change. (E) Graphic representation of differentially expressed genes from tumors of mice treated with targeted siRNA treatment. Masson’s trichrome stain of representative PBS-treated control tumor (left) showing extensive intratumoral fibrosis. Tumor on right shows marked decreased fibrosis in tumors of mice treated with targeted siRNA nanoparticles. Quantitative analysis with of fibrosis shows significantly less fibrosis (∗P = .031) in tumors of mice treated with the NPs. (F) IHC stain of tumors with anti-SMAa staining shows statistically less fibrosis (∗∗P = .003) in the tumors of mice treated with the NP (right) compared to tumors of PBS-treated control mice (left).