Matrix metalloproteinase responsive hydrogel microplates for programmed killing of invasive tumour cells

Abstract

Interactive materials are an emerging class of systems that can offer control over response and adaptivity in polymer structures towards the meso- and macroscale. Here, we use enzyme regulated cleavage of peptide crosslinkers in polymer hydrogels to release a cytotoxic therapeutic nanoparticle with an adaptable mechanism. Hydrogel microplates were formed through polyethylene glycol/peptide photoinitiated thiol-ene chemistry in a soft-lithography process to give square plates of 20 by 20 μm with a height of 10 μm. The peptide was chosen to be degradable in the presence of matrix metalloproteinase 2/9 (MMP-2/9). The hydrogel material's mechanical properties, swelling, and protease degradation were characterised. The microfabricated hydrogels were loaded with docetaxel (DTXL) containing poly(dl-lactide-co-glycolide) (PLGA) nanoparticles, and characterised for enzyme responsivity, and toxicity to MMP-2/9 overexpressing brain cancer cell line U87-MG. A 5-fold decrease in EC₅₀ was seen compared to free DTXL, and a 20-fold decrease was seen for the MMP responsive microplates versus a non-degradable control microplate. Potential applications of this system in post-resection glioblastoma treatment are envisioned.

Fig. 1. microPlates fabrication and morphological properties. (a) Brief schematic diagram of the sacrificial template soft lithography method used to produce well defined microparticles and chemical structures of polyethylene glycol hydrogels through a radical mediated thiol–ene addition mechanism, using an MMP-2/9 degradable peptide based crosslinker, (b) size distribution of microplates in aqueous solution from multisizer coulter counter, (c) optical profilometry of surface deposited microplates, (d) profile of microplates from optical profilometry, (e) SEM images of hydrogel microplates (scale bar : 10 μm).

Fig. 2. Mechanical characterizations. (a) Compression moduli of macroscopic hydrogels of varying stiffness, from different gel precursor initial concentrations (error bar is S.D. of three measurements), (b) representative stress–strain curves of macroscopic hydrogels of varying stiffness, from different gel precursor initial concentrations, compression moduli taken from linear region and is the average of five samples.

Fig. 3. Biodegradation studies. (a) Macroscopic gel (10 wt% precursors) mass degradation in varying concentrations of MMP-2/9 (collagenase IV) at 37 °C over time, (b) images of macroscale hydrogels (1 cm × 1 cm) at varying points of degradation corresponding to sampling times from panel a (scale bar: 10 mm), (c) hydrogel microplate mass degradation in varying concentrations of MMP-2/9 (collagenase IV) at 37 °C over time, (d) SEM images of hydrogel microplates and their degradation in 10 nM of MMP-2/9 (collagenase IV) at 37 °C, over time (scale bars: 10 μm).

Fig. 4. Drug loading and release. (a) Schematic of the degradation of DTXL containing SPN loaded microplates in MMP-2/9 (collagenase IV), (b) SEM images of surface deposited hydrogel microplates loaded with DTXL-containing PLGA SPNs, (c) release profiles of DTXL from SPN-loaded microplates quantified by HPLC. Scale bars: 10 μm (top) and 2 μm (bottom).

Fig. 5. Cytotoxicity on cancer cells. (a) FRET probe based collagenase IV (MMP-2/9) enzymatic activity assay, conducted with culture media after 24 h culture with cells, compared to 10 nM collagenase IV control solution (inhibitor from supplied kit, Merck InnoZyme), (b) MTT assays of various microplate treatment options (U87-MG cells) at 72 h (non-loaded microplates at the same particle concentration as used in the DTXL containing samples), (c) MTT assays of various microplate treatment options (U87-MG cells) at 72 h, (d) fitted dose response curve EC₅₀ values from various treatment groups, (e) brightfield microscopy images of U87-MG (7500 cells per well), after 48 h of exposure to MMP degradable (top) or non-degradable (bottom) μPL containing DTXL SPNs, EMEM medium, 100 nM total DTXL concentration. Scale bars: 100 μm.