Enzyme-triggered- and tumor-targeted delivery with tunable, methacrylated poly(ethylene glycols) and hyaluronic acid hybrid nanogels

Abstract

Enzyme-responsive polymeric-based nanostructures are potential candidates for serving as key materials in targeted drug delivery carriers. However, the major risk in their prolonged application is fast disassembling of the short-lived polymeric-based structures. Another disadvantage is the limited accessibility of the enzyme to the moieties that are located inside the network. Here, we report on a modified environmentally responsive and enzymatically cleavable nanogel carrier that contains a hybrid network. A properly adjusted volume phase transition (VPT) temperature allowed independent shrinking of a) poly(ethylene glycol) methyl ether methacrylate (OEGMA) with di(ethylene glycol) and b) methyl ether methacrylate (MEO₂MA) part of the network, and the exposition of hyaluronic acid methacrylate (MeHa) network based carboxylic groups for its targeted action with the cellular based receptors. This effect was substantial after raising temperature in typical hyperthermia-based treatment therapies. Additionally, novel tunable NGs gained an opportunity to store- and to efficient-enzyme-triggered release relatively low but highly therapeutic doses of doxorubicin (DOX) and mitoxantrone (MTX). The controlled enzymatic degradation of NGs could be enhanced by introducing more hyaluronidase enzyme (HAdase), that is usually overexpressed in cancer environments. MTT assay results revealed effective cytotoxic activity of the NGs against the human MCF-7 breast cancer cells, the A278 ovarian cancer cells and also cytocompatibility against the MCF-10A and HOF healthy cells. The obtained tunable, hybrid network NGs might be used as a useful platform for programmed delivery of other pharmaceuticals and diagnostics in therapeutic applications.

Keywords: Targeted drug delivery; controlled release; enzymatic degradation; hybrid network nanogel; methacrylated hyaluronic acid; poly(ethylene glycol).

Figure 1.

Overview of strategy in synthesis of hybrid network MEO₂MA-OEGMA-MeHa-based NGs with enzymatically sensitive DEGDA crosslinker.

Figure 2.

(a) Comparison of ¹H NMR spectra of MEO₂MA-OEGMA30%-DEGDA-, MEO₂MA-OEGMA30%-MeHa-DEGDA NGs and MeHa polymer (in D₂O). (b) TEM micrograph exhibiting surface activity of carboxylic groups from MeHa chains in MEO₂MA-OEGMA30% -MeHa-DEGDA NGs obtained after application of 1% uranyl acetate (UA) staining protocol.

Figure 3.

(a) Plots of hydrodynamic diameter, D_h, vs temperature, obtained for MEO₂MA-OEGDA30%-MeHa-DEGDA (blue line), MEO₂MA-OEGDA15%-MeHa-DEGDA (red line) and MEO₂MA-OEGDA30%-DEGDA (dark line). (b) Dependences of D_h on temperature obtained at various pH and referred to an aggregation process. MEO₂MA-OEGDA- MeHa-DEGDA at pH 7.4 (green line), 5.0 (pink line), 9.0 (blue line), and MEO₂MA-OEGDA-DEGDA at pH 5.0 (dark line). (c) Plots of zeta potential vs. temperature for increased carboxylic content in the MeHa-based network. (d) Quasi-reversible cycles of polydispersity index (PDI) changes seen at physiological- and hyperthermia-used temperatures and low acidic environment (pH 5); state of minimal tendency to NGs aggregation. All measurements were repeated three times. For clarity of the figure a single mean standard error was shown.

Figure 4.

TEM micrographs of A) MEO₂MA-OEGMA30%-MeHa-DEGDA NGs (pH 7.4), (B) MEO₂MA-OEGMA30%-MeHa-DEGDA NGs taken 1-day after their treatment with 2 mg·mL⁻¹ hyaluronidase enzyme (HAdase, pH 5.0). (C) MEO₂MA-OEGMA30%-MeHa-DEGDA NGs reveal their HAdase-based enzymatic degradation at pH 5.0 after 1-week treatment.

Figure 5.

(a) Dependencies of light scattering intensity ratio, I_t/I_o, on degradation time of MEO₂MA-OEGMA30%-MeHa-DEGDA NGs at 37 °C in presence and absence of HAdase. Subscripts “0” and “t” represent time t = 0 and t = t, respectively. (b) Distribution of MEO₂MA-OEGMA30%-MeHa-DEGDA NGs size presented as changes in scattered light intensity after various treatment times by HAdase enzyme. (c) Time dependencies of scattered light intensity (derived count rate, DCR) and hydrodynamic diameter, for MEO₂MA-OEGMA30%-MeHa-DEGDA NGs after treatment with HAdase (2 mg/ml) and at pH 7.4 and 5.0. All measurements were repeated three times. For clarity of the figure a single mean standard error was shown.

Figure 6.

Profiles of DOX and MTX release from MEO₂MA-OEGMA30%-MeHa-DEGDA NGs during enzymatic degradation at buffered solutions at 37 °C; HAdase concentration - 2 mg/ml. All measurements were repeated three times. For clarity of the figure a single mean standard error was shown.

Figure 7.

Results of MTT assay for MCF-10A and HOF healthy cell lines after 72-h treatment with free DOX, DOX-loaded and drug-free NGs. The red line depicts the threshold of 70% of cell viability in accordance with ISO 10993–5 (ISO 10993–5:2009, 2009). One way ANOVA was used to test for statistical significance. Differences from control sample were marked with *, whereas ** marked differences between groups. The difference was considered significant for P values <05.

Figure 8.

Results of MTT assay with MCF-7 cancer cell line and with and without HAdase after 72-h treatment with free DOX, DOX-loaded and drug-free NGs. One way ANOVA was used to test for statistical significance. Differences from control sample were marked with *, whereas ** marked differences between groups. The difference was considered significant for P values <05.

Figure 9.

Results of MTT assay with A2780 cancer cell line and with and without HAdase after 72-h treatment with free DOX, DOX-loaded and drug-free NGs. One way ANOVA was used to test for statistical significance. Differences from control sample were marked with *, whereas ** marked differences between groups. The difference was considered significant for P values <05.

Figure 10.

(a) Confocal images of cancer- (MCF-7) and health- (MCF-10A) cells obtained after 72-h incubation with DOX-loaded MEO₂MA-OEGMA30%-MeHa-DEGDA NGs. Cell nuclei were stained with Hoechst fluorescent dye (blue color). Red color was emitted by DOX absorbed by cells. In case of cancer cells overlapping fluorescence signals of DOX and Hoechst dye were separated. (b) 3 D projections of MCF-7 and MCF-10A cell nuclei recorded after 72-h treatment with DOX-loaded MEO₂MA-OEGMA30%-MeHa-DEGDA NGs. For each cell line separated fluorescent signals and merged signal are presented. Blue color marks cell nuclei stained with Hoechst dye. Red color indicates presence of DOX. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Figure 11.

Representative flow cytometry traces characterizing A278 and MCF-7 cell lines after 24 h of incubation of NGs loaded with 0.005 µM DOX.