Sustained release of active chemotherapeutics from injectable-solid β-hairpin peptide hydrogel

Abstract

MAX8 β-hairpin peptide hydrogel is a solid, preformed gel that can be syringe injected due to shear-thinning properties and can recover solid gel properties immediately after injection. This behavior makes the hydrogel an excellent candidate as a local drug delivery vehicle. In this study, vincristine, a hydrophobic and commonly used chemotherapeutic, is encapsulated within MAX8 hydrogel and shown to release constantly over the course of one month. Vincristine was observed to be cytotoxic in vitro at picomolar to nanomolar concentrations. The amounts of drug released from the hydrogels over the entire time-course were in this concentration range. After encapsulation, release of vincristine from the hydrogel was observed for four weeks. Further characterization showed the vincristine released during the 28 days remained biologically active, well beyond its half-life in bulk aqueous solution. This study shows that vincristine-loaded MAX8 hydrogels are excellent candidates as drug delivery vehicles, through sustained, low, local and effective release of vincristine to a specific target. Oscillatory rheology was employed to show that the shear-thinning and re-healing, injectable-solid properties that make MAX8 a desirable drug delivery vehicle are unaffected by vincristine encapsulation. Rheology measurements also were used to monitor hydrogel nanostructure before and after drug encapsulation.

Figure 1 -

Triangles correspond to G’ (storage modulus), and squares correspond to G” (loss modulus). (a) A frequency sweep from 0.1–100rad/s with 0.2% strain was run for 0.5 wt% MAX8 hydrogels with 500 μM vincristine (filled symbols) and without vincristine (open symbols). No difference is observed in the viscoelastic properties of the hydrogel with and without vincristine encapsulated. (b) A time sweep at a frequency of 6rad/s with a 0.2% strain was run on a 0.5wt% MAX8 hydrogel with 500μM vincristine encapsulated. Early time shows the initial gelation within 10 minutes. A constant shear at a steady-state shear of 1000/s is applied for 30 seconds at 90 minutes. As soon as the large shear ceases, the time sweep data shows the hydrogel immediately as a solid material and quickly recovering original gel properties.

Figure 2 -

Small-angle neutron scattering from 0.5 wt% MAX8 hydrogels with 500 μM vincristine (yellow) and without (blue) as a function of scattering variable q. Both lines have a similar overall shape and slope throughout the measured q range, implying that the presence of vincristine does not alter the structure of the MAX8 gel or the intramolecular folding of individual MAX8 chains. The cartoon inset shows the possible drug-gel configurations, a.) green fibrils indicating the yellow vincristine bound to the blue MAX8 fibrils, b.) domains of yellow vincristine mostly at the branch and entanglement points, or c.) yellow vincristine evenly scattered throughout the MAX8 network.

Figure 3 -

LDH assays showing percent cell death of DAOY cells (a) after direct treatment with vincristine and (b) vincristine encapsulated and released from a 0.5wt% MAX8 hydrogel. All measurements are taken after 2 days of cell incubation with each listed concentration. * indicates significance (p<0.05). Optical micrographs of DAOY cells treated with (c) 0 nM (d) 1.6 nM (e) 8 nM and (f) 40 nM of vincristine released from a MAX8 hydrogel. Live cells appear elongated and transparent whereas dead cells are rounded and opaque. The scale bar is 200 μm.

Figure 4 -

The 28 day release profile of tritiated vincristine from a 0.5wt% MAX8 peptide hydrogel that initially encapsulated 10 μM vincristine. After 28 days of release, the amount of drug being released was still in nanomolar quantities, as seen in the inset which highlights days 14 through 28 of the release study.

Figure 5 -

To ensure efficacy of the drug released from they hydrogel, 0μM (empty bars), 10μM (lighter gray bars) and 500μM (darker gray bars) of vincristine were encapsulated in 0.5wt% MAX8 hydrogels. There is a noticeable difference in the effectiveness of the hydrophobic drug on cells despite encapsulation in an aqueous. DAOY cell death was measured using LDH assays after cells were exposed to the drug-gel constructs in the listed days of release. Day 0 cells were exposed for an hour to drug-gel transwells, then measured two days later. * indicates noted concentrations are significantly different (p<0.05).