(W1130-11-73) IR-820 Dye-Based Layer-by-Layer Inhalation System as a Theranostic Agent for Non-invasive NIRF Imaging of Tumor Margins in Lung Cancers

Purpose: We report the development and pre-clinical evaluation of IR820-LBL particles as an inhalable and biocompatible theranostic carrier system to facilitate real-time imaging of lung cancer progression and treatment under visible light. Layer-by-layer particles (LBLs) have been identified as promising candidates for inhalation applications owing to their tunable size, composition, and surface properties. The IR820-LBL inhalation is composed of a Layer-By-Layer (LBL) particle system incorporating a biodegradable near-infrared absorbing component (i.e., IR-820 dye) to demonstrate effective deep lung NIRF (Near Infrared Fluorescence) imaging under NIR/visible light over a wavelength range of 400nm to 900nm. While imaging modalities such as X-rays, CT scans, PET scans, and MRIs are valuable tools for diagnosing and monitoring lung diseases, their use may cause health problems with repeated exposure. Certain imaging techniques, such as bronchoscopy or biopsy, are invasive and carry a risk of complications such as bleeding, infection, or lung collapse. To address these issues, innovative modalities such as IR820-LBL-based inhalable NIRF imaging probes offer a non-invasive, safe, and effective solution for lung tumor imaging. Our objective is to expand the potential applications of previously designed Layer by layer (LBL) particles by our lab group for new applications of operative NIRF imaging and theranostic potential.

Methods: IR820-LbL assembled particles were prepared by coating successive layers of, Glycol chitosan, and IR820 dye loaded Bovine serum albumin on 0.5ml polystyrenes particles of size = 0.5micron (with 3 layers each), starting with a layer of glycol chitosan. For IR820 loading, it was dissolved in BSA (Bovine Serum Albumin, 5mg/ml), and coated on the particles. The resultant particles were washed 3 times by centrifugation to ensure removal of free polyelectrolytes. Particle size, zeta potential, and polydispersity index were measured using Zetasizer Nano (Malvern Instruments, Malvern, UK). Direct as well as indirect methods were adopted to determine the loading % and entrapment % of IR820 dye, The in-vitro deposition behavior of LbL coated 0.5-µm particles was assessed using a Copley-170 Next Generation Impactor™ (NGI, MSP corporation, MN, USA), using a PARI LC PLUS® nebulizer connected to the Pari FAST-NEB compressor system.

Results: The IR820-LbL assembly of polyelectrolytes was confirmed by the reversible change in zeta potential as shown in Fig. 1A and sequential increase in the particle size as shown in Fig. 1B, after accumulation of successive layers and the homogeneity of the formulation was confirmed by measuring the polydispersity index as shown in Fig.1C. As mentioned in Fig. 2, three formulation trials were performed with varying amounts of IR820 dye concentrations (0.1mg/ml, 0.3mg/ml, and 0.9mg/ml) based on literature survey, in order to achieve optimum entrapment for imaging applications. The entrapment efficiency of IR820-LBL microparticles was evaluated to be 75.3% by the indirect method as mentioned in Fig. 2. The in-vitro aerosolization study performed using the next-generation impactor coupled with Pari LC Plus nebulizer showed that IR820-LbL assembled 0.5µm particles have suitable aerodynamic properties for non-invasive lung delivery as in Fig. 3. The deposition pattern shows maximum particles deposited in stages 4 and 5 as in Fig. 3A, which suggests that 0.5-micron-coated particles are capable of efficiently reaching the lungs using a Pari LC nebulizer. The in-vitro cytotoxicity assay of the IR820-LbL-coated particles is being performed on normal lung epithelial and fibroblast cell lines to assess the safety of the formulation for clinical translation.

Conclusion: The IR820-LBL inhalable particle system is aimed to perform a multifunctional activity, offering the potential to perform targeted delivery of chemotherapeutic drugs to the lung tissues along with real-time monitoring of lung tissues. We explore theranostic applications of the IR820-LBL-based inhalation system to monitor lung cancer pathology and provide dynamic information about changes in lung tissue over time. Currently, the formulation development is in process, to be followed by an evaluation of safety and efficacy in vitro as well as in vivo.

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Acknowledgements: I acknowledge Gupta Lab, St. John's University to help with the facilities and provide funding for my research.

Figure.1: Preliminary characterization of IR820-LBL microparticle formulation, (a) Shows the layer-wise reversible zeta potential measured, (b) shows the increasing particle size with deposition of each layer, (c) shows the change in polydispersity index of the formulation with deposition of each layer

Figure.2: Entrapment efficiency of IR820-LBL microparticles calculated for different formulation trial

Figure.3: Shows deposition pattern of IR820-LBL microparticles over stage1 to 8 of NGI on evaluation of in-vitro pulmonary deposition behavior (a) shows % retention of IR820 – LBL microparticles at each stage of NGI while (b) shows cumulative deposition of the IR820-LBL microparticles at each stage of NGI.