Photodegradable hydrogels for dynamic tuning of physical and chemical properties

Abstract

We report a strategy to create photodegradable poly(ethylene glycol)-based hydrogels through rapid polymerization of cytocompatible macromers for remote manipulation of gel properties in situ. Postgelation control of the gel properties was demonstrated to introduce temporal changes, creation of arbitrarily shaped features, and on-demand pendant functionality release. Channels photodegraded within a hydrogel containing encapsulated cells allow cell migration. Temporal variation of the biochemical gel composition was used to influence chondrogenic differentiation of encapsulated stem cells. Photodegradable gels that allow real-time manipulation of material properties or chemistry provide dynamic environments with the scope to answer fundamental questions about material regulation of live cell function and may affect an array of applications from design of drug delivery vehicles to tissue engineering systems.

Figure 1. Photodegradable hydrogel synthesis and degradation for tuning gel properties

(A) The base photodegradable acrylic monomer was used to synthesize (B) the photodegradable crosslinking macromer (Compound 1, M_n ∼ 4070 g/mol), comprised of PEG (black), photolabile moieties (blue), and acrylic end groups (red). (C) Compound 1 was copolymerized with PEGA (M_n ∼ 375 g/mol), creating gels composed of poly(acrylate) chains (red coils) connected by PEG (black lines) with photolabile groups (solid blue boxes) (left). Upon irradiation, the photolabile moiety cleaves (open blue boxes), decreasing ρ_x and releasing modified PEG (right). (D) The physical structure of the hydrogel is degraded via photolysis, decreasing ρ_x and G′. The influence of irradiation on G′, normalized to G′_o, was monitored with rheometry. The degradation rate was precisely controlled with irradiation intensity and wavelength: (a) 365 nm at 20 mW/cm², (b) 365 nm at 10 mW/cm², and (c) 405 nm at 25 mW/cm². (E) The gel degradation was modulated by either (a) continuous or (b) periodic irradiation with 365 nm at 10 mW/cm². The extents of degradation corresponding to the materials used in Fig. 1F and 1G are indicated. (F) hMSCs encapsulated within dense hydrogels exhibit a rounded morphology. (G) Irradiation (480 s, 365 nm at 10 mW/cm²) significantly degrades the gel (ρ_x/ρ_xo ∼ 0.04), promoting hMSC spreading after 3 days in culture. Scale bar, 50 μm.

Figure 2. Two and three-dimensional patterning of photodegradable hydrogels

(A) Thick gels demonstrate surface erosion upon irradiation (left). A gel covalently labeled with fluorescein was eroded spatially via masked flood irradiation (320-500 nm at 40 mW/cm², 400 μm line periodic photomask). Channel depth increased linearly with irradiation, and no changes in hydrogel dimensions due to increased swelling were observed (middle). Feature dimensions were quantified with profilometry: (a) 2.5, (b) 5, (c) 7.5, and (d) 10 minutes irradiation (right). Scale bar, 100 μm. (B) Interconnected three-dimensional channels were fabricated within a photodegradable gel, covalently labeled with rhodamine B, using a two-photon LSM. A thin horizontal channel connected two offset vertical channels of different diameter, which was visualized in brightfield (bottom left) and with confocal LSM (right, intact gel is red and the created feature is black, corresponding cross-sections are noted by blue, green, and orange lines). Scale bar, 100 μm. (C) Channels were eroded within a hydrogel encapsulating fibrosarcoma cells, releasing cells into the degraded channel and enabling migration. Migration of a cell along the edge of a channel is shown in time-lapsed brightfield images (left) and its corresponding position trace (right). Scale bar, 50 μm.

Figure 3. Photolabile RGDS tether synthesis and utilization for dynamic changes in microenvironment chemistry

(A) A photolabile asymmetric biofunctional acrylic monomer (Compound 2) was synthesized containing the adhesion peptide RGDS (black) attached to the PDA (acrylate in red and photolabile moiety in blue). (B) Compound 2 was polymerized (green triangle with closed blue box) into a non-degradable gel (red poly(acrylate) coils connected by black non-degradable PEG crosslinks) whose chemical composition is controlled with light exposure by photolytic release of the tethered biomolecule RGDS (bottom, green triangle with open blue box). hMSCs were encapsulated in non-degradable PEG gels (b) with or (a) without photoreleasable RGDS. The presentation of RGDS was temporally altered by (c) photocleavage of RGDS from the gel on Day 10 in culture. (C) RGDS presentation maintains hMSC viability within PEG-based gels (inset table). RGDS photolytic removal on Day 10 directs hMSC chondrogenesis, (c) increasing GAG production four-fold over (b) persistently-presented RGDS or (a) PEG-only gels by Day 21 (p<0.05), an indicator of hMSC chondrogenesis.

Figure 4. Influence of dynamic microenvironment chemistry on integrin expression and differentiation

(A) Cells (DAPI-labeled nuclei, blue) cultured with persistent RGDS express the cell-surface integrin α_vβ₃ (FITC-labeled, green) on at Day 4 (left) and Day 21 (middle) in culture. Cells with photocleaved RGDS have decreased expression of the α_vβ₃ integrin by Day 21 (right), indicating that the cells have responded to the removal of RGDS. (Representative images shown with average cell percentages noted.) (B) Chondrogenic differentiation of hMSCs was verified by immunostaining for the hMSC marker CD105 (FITC, green) and the chondrocyte marker COLII (TRITC, red). Within error, no cells initially produce COLII (Day 4, left). By Day 21, half of the cells presented persistently with RGDS strongly expressed CD105 and the other half produced COLII (middle). With photolytic removal of RGDS on Day 10, one-fourth of the cells strongly expressed CD105 and three-fourths produced COLII (right), supporting that photolytic removal of RGDS increases chondrogenesis. (Representative images shown with average cell percentages strongly expressing the marker noted.) Scale bars, 100 μm.