Purpose: The purpose of this study was to design a nanocarrier system capable of enhancing the penetration of doxorubicin (DOX) in an in-vitro 3D lung adenocarcinoma coculture tumor model that expresses the essential components of the extracellular matrix (ECM).
Methods: Coculture spheroids were formed with 200 A549 (lung adenocarcinoma) and 20 NIH-3T3 cells (standard fibroblasts). The spheroids were then grown on 96 well plates coated with poly-HEMA (provide for a non-adhering and curved surface) for 7 days. The expression of collagen and fibronectin (ECM) on cryosections of spheroids was ascertained with immunofluorescent staining. DOX was conjugated to succinamic acid terminated, generation 4, poly (amido-amine) dendrimers (G4SA) through a degradable peptide bond (GFLG) as per Scheme 1. The penetration of the G4SA-GFLG-DOX conjugate into the 3D coculture spheroids was investigated as a function of time by measuring the Mean Fluorescence Intensity (MFI) of DOX in the various Z-stacks (sections) of the spheroids using confocal microscopy. The penetration of the conjugate was compared to free DOX.
Results: Conjugates of DOX to G4SA with (GFLG) peptide bond were synthesized and characterized. An average of 5.5 DOX per dendrimer was observed based on MALDI-TOFF and 1H NMR. The hydrodynamic diameter and zeta potential of the nanoconjugates was 10nm and -17mV, respectively. After one week, single coculture spheroids were formed in each well. They expressed high levels of ECM. DOX in the form of G4SA-GFLG-DOX conjugates was seen to penetrate to a greater rate and extent into the spheroids as compared to free DOX as shown in Figure 1. Insets (equatorial Z-stacks) show that while free DOX largely accumulated intracellularly, the conjugated DOX is found everywhere in the spheroids.
Conclusion: Negatively charged dendrimer-DOX conjugates promoted the penetration of DOX into ECM-expressing 3D in vitro models of lung adenocarcinoma when compared to the free DOX.
Rodrigo Heyder– Virginia Commonwealth University
Elizabeth Bielski– Virginia Commonwealth University
Mikhail Durymanov– South Dakota State University
Joshua Reineke– South Dakota State University
Rashed Almuqbil– Virginia Commonwealth University, Richmond