Tumor-activated prodrug nanoparticles sensing endogenous levels of matrix metallo-proteinase 2 in 3D tumor spheroids for the on-demand release of doxorubicin

Matrix Metalloproteinases (MMPs) are proteolytic enzymes that play a key role in tumor metastasis progression so a novel approach in nanoparticles (NPs) design may be the one that benefits from MMPs over-expression, in order... [ view full abstract ]

Matrix Metalloproteinases (MMPs) are proteolytic enzymes that play a key role in tumor metastasis progression so a novel approach in nanoparticles (NPs) design may be the one that benefits from MMPs over-expression, in order to realize a stimuli-responsive nanocarrier.

To this aim we designed biocompatible and biodegradable NPs made up of a poly(D,L-lactic-co-glycolic acid) (PLGA)–block–polyethylene glycol (PEG) copolymer (namely PELGA), blended with a tumor activated prodrug (TAP) composed by an MMP2-sensitive peptide bound to doxorubicin (Dox) and to a PLGA molecule. The same prodrug was produced without the MMP-sensitive linker as negative control.

PELGA-TAP and PELGA-Dox NPs were produced and characterized. DLS results show that NPs diameter is below 100 nm with a PDI< 0.2 and a negative z-potential for both NPs formulations. SEM and Cryo-TEM images show that NPs are spherical and monodispersed. In vitro Dox release from NPs, in presence and absence of free MMP2 enzyme, was tested performing a cleavage assay on both NPs. Release profiles show that PELGA-TAP NPs allow Dox controlled release specifically upon MMP2 cleavage of the TAP while PELGA-Dox NPs drug release is comparable to PELGA-TAP NPs release in absence of the enzyme.

To demonstrate PELGA-TAP NPs ability to sense the differences in expression levels of endogenous MMP2 enzyme, U87 and HDF, tumor and healthy cell lines respectively, were used to built up an in vitro spheroidal model. Spheroids were treated with PELGA-TAP and PELGA-Dox NPs. Confocal microscopy images show the presence of a fluorescence signal within U87 spheroids after 24h exposure to PELGA-TAP NPs, inducing spheroids’ disaggregation after 48h, while a very low fluorescence signal was observed for U87 spheroids treated with PELGA-Dox after 24h and 48h of incubation. Interestingly, HDF spheroids show a lower fluorescence signal than U87, with little differences between PELGA-TAP and PELGA-Dox NPs treatment.

The results presented above suggest that PELGA-TAP NPs can deliver Dox specifically upon enzymatic cleavage and promote drug penetration within 3D tumor spheroids so, starting from these promising results, NPs could be tested on more complex in vitro tissue equivalents to further validate their selective drug release and their efficacy as drug delivery systems.