Sustained Small Molecule Delivery from Injectable Hyaluronic Acid Hydrogels through Host-Guest Mediated Retention

Abstract

Self-assembled and injectable hydrogels have many beneficial properties for the local delivery of therapeutics; however, challenges still exist in the sustained release of small molecules from these highly hydrated networks. Host-guest chemistry between cyclodextrin and adamantane has been used to create supramolecular hydrogels from modified polymers. Beyond assembly, this chemistry may also provide increased drug retention and sustained release through the formation of inclusion complexes between drugs and cyclodextrin. Here, we engineered a two-component system from adamantane-modified and β-cyclodextrin (CD)-modified hyaluronic acid (HA), a natural component of the extracellular matrix, to produce hydrogels that are both injectable and able to sustain the release of small molecules. The conjugation of cyclodextrin to HA dramatically altered its affinity for hydrophobic small molecules, such as tryptophan. This interaction led to lower molecule diffusivity and the release of small molecules for up to 21 days with release profiles dependent on CD concentration and drug-CD affinity. There was significant attenuation of release from the supramolecular hydrogels (~20% release in 24h) when compared to hydrogels without CD (~90% release in 24h). The loading of small molecules also had no effect on hydrogel mechanics or self-assembly properties. Finally, to illustrate this controlled delivery approach with clinically used small molecule pharmaceuticals, we sustained the release of two widely used drugs (i.e., doxycycline and doxorubicin) from these hydrogels.

Keywords: cyclodextrin; drug delivery; host-guest chemistry; hydrogel; self-assembly; small molecules; sustained release.

Figure 1

Scheme for tuning release of small molecules from drug loaded supramolecular hydrogels. Ad-HA and CD-HA macromers were synthesized and evaluated for functionalization (x/[x+y]) using ¹H-NMR. Macromers were dissolved in solutions containing varying drugs (green = high affinity for CD, red = low affinity for CD) and combined in varying ratios (Ad:CD) to produce bulk hydrogels. Release kinetics were tuned by varying the CD content available for drug binding or by altering the affinity of the loaded drug for CD.

Figure 2

Isothermal titration calorimetry for HA binding to tryptophan. Solutions of HA (either unmodified or modified with CD) dissolved in MilliQ water were titrated into 40 μM tryptophan in MilliQ water. Baseline isotherms of modified or unmodified HA into water were subtracted to account for heats of dissolution. Representative isotherms and chemical structures of binding components are shown for (A) unmodified HA binding to tryptophan or (B) CD-HA binding to tryptophan.

Figure 3

Comparison of mechanical properties in unloaded and drug loaded (3W peptide, 1mg/mL) hydrogels (1:1 (5 wt%)). (A) Representative frequency sweeps showing G’ (unloaded=blue solid circles, loaded=red solid squares), G” (unloaded=blue open circles, loaded=red open squares), and tan(δ) (unloaded=black open circles, loaded=black crossed squares). (B) Average G’ values for time sweeps for unloaded (blue) and loaded (red) hydrogels for 0.1Hz, 1Hz, and 10Hz frequencies. (C) Representative cyclic strain experiment showing G’ (red solid line), G” (red dashed line), and strain (black dotted line) for loaded hydrogels undergoing cycles of low and high strains.

Figure 4

Fluorescence recovery after photobleaching experiments. Hydrogels loaded with fluorescent peptides were assessed with FRAP analysis to determine payload mobility within the hydrogel. Diffusivity of fluorescent peptide payloads in hydrogels was assessed with changes in (A) CD-HA wt%, (B) Ad:CD ratio at a total polymer concentration of 5 wt%, and (C) payload chemistry (3W versus NoW). (D) Representative FRAP images for changes in payload chemistry, scale bar = 50 μm.

Figure 5

Release profiles of entrapped peptides. Hydrogels containing fluorescent peptides were injected in custom designed erosion cells and peptides released into PBS were quantified. Cumulative release profiles were determined in gels with changes in (A) CD-HA wt%, (B) Ad:CD ratio at total polymer concentration of 5 wt%, and (C) payload chemistry (3W versus NoW). Insets show an expanded view of time points collected during the first 5 days.

Figure 6

Release of doxorubicin (DOX) and doxycycline (DXY) from injectable hydrogels. Cumulative release of (A) DOX or (B) DXY for 1:1 (5 wt%) (red) or 1:2 (7.8 wt%) (blue) formulations over 3 weeks. Insets show an expanded view of time points collected during the first 5 days.