Poster W16 - Investigating nanoparticle shape to overcome the lung tumor microenvironment

Introduction: Lung cancer is responsible for the highest number of deaths among all cancers globally.1 The 5-year survival rate for lung cancer of all stages was 25.4% between 2013 and 2019 in the United States.1 Due to lung cancer’s severe prognosis, there is a need for more effective anticancer treatments. Nanoparticle (NP) encapsulation of chemotherapeutics is a promising method of enhancing efficacy, but NP drug delivery still faces unique challenges associated with structural remodeling in lung cancer.
One of the largest barriers to treating lung cancer is the stiffening and remodeling of the extracellular matrix (ECM) in the tumor microenvironment (TME).2 ECM composition is highly tissue dependent, and studies with materials such as Matrigel or collagen do not accurately reflect the structural differences in ECM due to tissue morphism and disease state. Incorporating decellularized ECM (dECM) derived from native tissues can present a more relevant model of the tumor microenvironment. dECM was used in this study to produce physiologically relevant in vitro results of NP behavior in the lung TME.
The physical properties of NPs, such as shape, impact their ability to diffuse and endocytose in the TME. However, it is not known how the shape of NPs impact their diffusion through ECM or their rate of uptake into tumor cells in a physiologically relevant in vitro model. This study utilizes lung dECM to elucidate the impact of NP shape on behavior in the TME. Namely, NP diffusion and cellular uptake were studied.

Materials and

Methods: To understand the influence of NP shape on ECM diffusion and cellular uptake, we compared rod-shaped NPs (nanorods) and spherical NPs coated in 5kDa polyethylene glycol (PEG). The nanorods were formulated by applying a uniaxial force to 100nm spherical NPs to alter the aspect ratio. Successful stretching was confirmed using transmission electron microscopy (TEM).
Porcine lungs were decellularized using a series of chemical and enzymatic solutions. Decellularization ensured that the presence of native cells did not alter the results, while keeping the structure of the tissue intact. The decellularized lungers were then lyophilized, cryomilled, and digested using pepsin. Lung dECM hydrogels were created from neutralizing the digests and diluting them in phosphate buffered saline at concentrations varied from 4-8 mg/mL, which modeled the increase in ECM components during disease progression.
Diffusion through the ECM was evaluated by incorporating fluorescent NPs into ECM hydrogels and taking videos under a microscope. Multiple Particle Tracking was used to analyze the videos and determine the mean squared displacement (MSD) of each set of NPs. A larger MSD suggested that the nanoparticles were able to move more freely.
Cellular uptake was analyzed in 3D cultures of A549 cells, a human lung adenocarcinoma cell line. 3D cultures were grown in a low-attachment plate to allow for spheroid formation, both with and without ECM gels. Media supplemented with fluorescent NPs was placed in all wells and incubated. Cells were then stained for viability and fixed. Flow cytometry quantified the percentage of cells that had taken up NPs.

Results, Conclusions, and Discussions: TEM images confirmed that nanorods were successfully stretched using the mechanical stretcher. Additional characterization confirmed the qualitative analysis of the success of stretching. A Nanoparticle Tracking Analysis (NTA) machine also gave the size distribution and zeta potential of the nanorods. The size distribution given by the NTA gave both the small and large dimensions of the nanorods because of the two peaks in the distribution. The zeta potential measurement confirmed that the PEG coating on the nanorods was not disturbed by stretching, as the stretched nanorod zeta potential was compared to the zeta potential before stretching.
Preliminary results show that nanorods have a higher MSD than spherical NPs in all dECM hydrogel concentrations, indicating that nanorods are less hindered by ECM remodeling that occurs in lung cancer (Figure 1). MSD is indicative of the NP diffusion rate. A larger MSD means that NPs are diffusing faster. Therefore, a larger percentage of nanorods will be able to navigate through the ECM to reach cells. However, the faster movement of these nanorods will not result in a therapeutic effect if they are not taken up by the cells. Thus, cellular uptake of nanorods was also studied, both with and without ECM incorporated into the cell cultures. These results indicated that the nanorods showed a higher rate of cellular uptake than spherical NPs. This is likely due to the altered aspect ratio of the nanorods, which changes their surface area to volume ratio and allows more efficient entry into cells.

In conclusion, tumors are characterized by both ECM-rich and cell-rich regions. NPs must be able to navigate through both regions of tumors to provide therapeutic benefit and reduce cancer burden. This project looks at the impact of NP shape on behavior in the lung tumor microenvironment. Nanorods were shown to have higher rates of diffusion and cellular uptake in cancerous ECM. This suggests that development of therapeutic NPs formulated in rod shapes may have more success in fighting lung cancer. With more effective strategies to combat lung cancer, patient health will increase.

Acknowledgements (Optional): [1] Nat Cancer Inst, 2024
[2] Rosenblum, D. Nature Commun, 2018