Evaluation of the feasibility of intrapancreatic delivery of drug-loaded microparticles via EUS-guided fine needle injection using a swine model

Abstract

Background and study aims Patients with pancreatic cancer often have locally advanced or metastatic disease and are not candidates for curative surgery. Polymer-based microparticles (MPs) represent a drug delivery system that offers sustained release of a chemotherapeutic drug after intralesional injection for local tumor management. The aim of this study was to determine the feasibility of endoscopic ultrasound-guided fine-needle injection (EUS-FNI) of drug-loaded MPs tagged with a fluorophore and fiducial markers for locating the injection site. Secondary aims were to determine the tissue-specific effects of MPs. Methods Five pigs underwent EUS with selection of an injection site within the pancreas that was marked by placing fiducial markers prior to the MPs injection. EUS-FNI of either blank microparticles (BMPs), containing no drug, or gemcitabine-loaded microparticles (GMPs) was performed. A saline flush containing Spot Endoscopic Marker was used to expel any residual MPs in the needle shaft and tattoo the injection site. Results A green fluorescent protein flashlight was used to successfully identify the site of MP injection sites in the dissected pancreas. Frozen sections of pig pancreas demonstrated a defined deposit, confirming the delivery of the MPs. Finally, fluorescence microscopy showed activation of caspase-mediated cell death in pancreases of animals that received injections of GMPs. Conclusions This pilot study demonstrated that fiducial marker placement and pancreatic EUS-FNI of MPs was successful in all pigs with no animals demonstrating pancreatitis. Further studies are needed to determine the role for intralesional injection of drug-loaded MPs in borderline resectable or unresectable pancreatic cancer.

Fig. 1

Gross examination of pig pancreas after necropsy. a Pig 3 intact pig pancreas. b Pig pancreas demonstrating fiducial marker (black arrow) and spot ink tattoo (blue arrow) in pig 3. c Pig pancreas demonstrating fiducial marker in pig 5). d Green fluorescent protein flashlight shows FITC excitation in BMP injection site. Pig pancreas demonstrated tattoo (site of Spot ink injection) and green fluorescence indicating site of BMP injection in pig 1.

Fig. 2

EUS images of fiducial and microparticle injections. The pancreas and surrounding structures were well identified prior to each injection. The fiducial markers are evidenced by an echorich focus in pig 1 a and pig 2 b . c This figure demonstrates the pancreas, left kidney and spleen in pig 3. d An anechoic focus denotes the site of the microparticle injection and a hyperechoic strand demonstrates the site of the fiducial marker in pig 3. e The microparticle injection site noted as an anechoic focus in pig 4.

Fig. 3

Gemcitabine-loaded microparticles deposit as a nodule inside the tissue as shown one week after the injection. a The top set of figures show the four pigs with microparticles deposit inside the pancreatic tissue (green). The lower set of figures were used as controls of the injections, an adjacent sample was taken next to the deposit to show the specific location of the microparticles. b Areas close to the center where the nodule was detected for each of the pigs using fluorescence microscopy.

Fig. 4

Activation of apoptosis in pig tissues injected with the drug-loaded microparticles. a One week post-injection, the tail with the 2-mL BMPs deposit with limited caspase-mediated cell death using FAM-FLICA fluorescent reagent (red). The nuclei of the cells were stained with DAPI (blue). The same results are observed in the adjacent control tail tissue. The images were taken close to the center (6,000um depth) of the pancreatic tissue block. b One week post-injection, the neck with the 2-mL GMPs nodule moderately activates caspase-mediated cell death (red) in the surface and the center of the pancreas. The nuclei of the cells were stained with DAPI (blue). As a control, apoptosis was not detected in the adjacent tissue from the neck. The images were taken close the center (~5,000 um) of the tissue block for the site of injection and the adjacent area. c, d The 3-mL and 4-mL deposits, respectively, showed activation of caspase-mediated cell death (red) of groups of cells in the center of the pancreas with the nodule. As a control, apoptosis was not detected in the adjacent tissue from the tail. The images were taken close to the center (~5,000 um) of the tissue block for the site of injection and the adjacent area. All pictures were taken with 10 × magnification.