Distribution of PLGA-modified nanoparticles in 3D cell culture models of hypo-vascularized tumor tissue

Abstract

Background: Advanced stage cancer treatments are often invasive and painful-typically comprised of surgery, chemotherapy, and/or radiation treatment. Low transport efficiency during systemic chemotherapy may require high chemotherapeutic doses to effectively target cancerous tissue, resulting in systemic toxicity. Nanotherapeutic platforms have been proposed as an alternative to more safely and effectively deliver therapeutic agents directly to tumor sites. However, cellular internalization and tumor penetration are often diametrically opposed, with limited access to tumor regions distal from vasculature, due to irregular tissue morphologies. To address these transport challenges, nanoparticles (NPs) are often surface-modified with ligands to enhance transport and longevity after localized or systemic administration. Here, we evaluate stealth polyethylene-glycol (PEG), cell-penetrating (MPG), and CPP-stealth (MPG/PEG) poly(lactic-co-glycolic-acid) (PLGA) NP co-treatment strategies in 3D cell culture representing hypo-vascularized tissue.

Results: Smaller, more regularly-shaped avascular tissue was generated using the hanging drop (HD) method, while more irregularly-shaped masses were formed with the liquid overlay (LO) technique. To compare NP distribution differences within the same type of tissue as a function of different cancer types, we selected HeLa, cervical epithelial adenocarcinoma cells; CaSki, cervical epidermoid carcinoma cells; and SiHa, grade II cervical squamous cell carcinoma cells. In HD tumors, enhanced distribution relative to unmodified NPs was measured for MPG and PEG NPs in HeLa, and for all modified NPs in SiHa spheroids. In LO tumors, the greatest distribution was observed for MPG and MPG/PEG NPs in HeLa, and for PEG and MPG/PEG NPs in SiHa spheroids.

Conclusions: Pre-clinical evaluation of PLGA-modified NP distribution into hypo-vascularized tumor tissue may benefit from considering tissue morphology in addition to cancer type.

Keywords: 3D cell culture; Cell penetrating peptide (CPP); Cervical cancer; Nanoparticle transport; Nanoparticles; Tumor spheroid; Tumor vascularization.

Fig. 1

Schematic representing NP formulations used in this study. From left to right: unmodified, MPG, and PEG formulations

Fig. 2

NP distribution through liquid overlay (LO) spheroids in mid-plane cross-sections (top three rows) and 3D composite (bottom three rows) confocal images. Nuclei are blue (Hoechst) and NPs are green (Coumarin 6). Scale bar: 50 μm

Fig. 3

NP distribution through hanging drop (HD) spheroids in mid-plane cross-sections (top three rows) and 3D composite (bottom three rows) confocal images. Nuclei are blue (Hoechst) and NPs are green (Coumarin 6). Scale bar: 50 μm

Fig. 4

NP distribution profiles quantifying the mean fluorescence intensity (MFI) vs. penetration distance through liquid overlay (LO) spheroids. Distribution profiles are shown as a function of NP treatment and tumor cell type. Average of the values along distance is denoted by the dark lines

Fig. 5

NP distribution profiles quantifying the mean fluorescence intensity (MFI) vs. penetration distance through hanging drop (HD) spheroids. Distribution profiles are shown as a function of NP treatment and tumor cell type. Average of the values along distance is denoted by the dark lines

Fig. 6

NP distribution represented as AUC for each tumor cell type (HeLa, SiHa, or CaSki) as a function of NP treatment group, relative to spheroid type (LO, black and HD, gray). Values of all significant correlations, including each treatment group relative to unmodified NPs, relative to other treatment groups, or relative to the same treatment group in a different spheroid type are given with degree of significance indicated (* p < 0.01, ** p < 0.001, *** p < 0.0001, **** p < 0.00001). Error bars: average ± standard deviation (n = 3)

Fig. 7

Schematic of spheroid formation techniques for a liquid overlay (LO) and b hanging drop (HD) spheroids, respectively representing larger, more irregularly-shaped and smaller, regularly-shaped avascular tissue

Fig. 8

Typical morphologies of liquid overlay (LO) and hanging drop (HD) tumor spheroids, evaluated via bright field microscopy. Scale bar: 200 μm