Physiologically relevant oxidative degradation of oligo(proline) cross-linked polymeric scaffolds

Abstract

Chronic inflammation-mediated oxidative stress is a common mechanism of implant rejection and failure. Therefore, polymer scaffolds that can degrade slowly in response to this environment may provide a viable platform for implant site-specific, sustained release of immunomodulatory agents over a long time period. In this work, proline oligomers of varying lengths (P(n)) were synthesized and exposed to oxidative environments, and their accelerated degradation under oxidative conditions was verified via high performance liquid chromatography and gel permeation chromatography. Next, diblock copolymers of poly(ethylene glycol) (PEG) and poly(ε-caprolactone) (PCL) were carboxylated to form 100 kDa terpolymers of 4%PEG-86%PCL-10%cPCL (cPCL = poly(carboxyl-ε-caprolactone); i% indicates molar ratio). The polymers were then cross-linked with biaminated PEG-P(n)-PEG chains, where P(n) indicates the length of the proline oligomer flanked by PEG chains. Salt-leaching of the polymeric matrices created scaffolds of macroporous and microporous architecture, as observed by scanning electron microscopy. The degradation of scaffolds was accelerated under oxidative conditions, as evidenced by mass loss and differential scanning calorimetry measurements. Immortalized murine bone-marrow-derived macrophages were then seeded on the scaffolds and activated through the addition of γ-interferon and lipopolysaccharide throughout the 9-day study period. This treatment promoted the release of H(2)O(2) by the macrophages and the degradation of proline-containing scaffolds compared to the control scaffolds. The accelerated degradation was evidenced by increased scaffold porosity, as visualized through scanning electron microscopy and X-ray microtomography imaging. The current study provides insight into the development of scaffolds that respond to oxidative environments through gradual degradation for the controlled release of therapeutics targeted to diseases that feature chronic inflammation and oxidative stress.

Figure 1

GPC chromatograms of the synthesized scaffold components. (A) PEG-P_n-PEG crosslinkers (10 mg/mL in DMF + 0.1M LiBr) prior to final Fmoc deprotection are detectable via a UV detector set at 310 nm. The presence of a small, earlier-eluting hump at ~27 min represents the tri-PEGylated peptides due to incomplete acetylation of the peptide sequences. The major peak at 29–30 min is the major, bi-PEGylated product. (B) The 4%PEG-86%PCL-10%cPCL backbone (10 mg/mL in THF) is relatively monodisperse, as evidenced by the near-complete overlap of the MALS and differential refractive index (dRI) chromatograms. With the dn/dc of the terpolymer measured at 0.0663 mL/g in THF, the molecular weights were calculated at M_n = 99.4 kDa and M_w = 115 kDa (PDI = 1.16).

Figure 2

Metal-catalyzed oxidation of proline oligomers. (A) P₁₀ was incubated at 37°C for 4 days in PBS only or PBS containing H₂O₂ and Cu(II), then analyzed via HPLC-MS. The latter treatment resulted in the disappearance of chromatograms and mass spectra characteristic of the intact peptide. To further confirm oxidative degradation of the peptide, PEG-P_n-PEG was incubated under the same conditions prior to analysis via GPC (B-E). In all cases, these molecules eluted at later times following only 2 d in the oxidative environment. (E) Peak molecular weights were calculated based on elution time, relative to monodisperse PEG standards. Within the first 2 d of treatment, degradation rate was proportional to the length of the proline oligomers. Further, all crosslinkers degraded to form a 550 Da product within 6 d, which is consistent with the molecular weight of the PEG reagent that was coupled to both ends of the peptides used in the study, to form the PEG-P_n-PEG crosslinkers for the scaffolds.

Figure 3

SEM images of the scaffolds of 4%PEG-86%PCL-10%cPCL by crosslinker type. Two different magnifications showcase macropores of > 100 μm diameter (top row) and micropores of < 10 μm in diameter (bottom row). Only the PEG-dihydrazide-crosslinked scaffolds failed to show any widespread microporous architecture. Macropores were templated into the polymer network through a salt-leaching procedure, while micropores were generated through the phase separation of the water generated during the crosslinking reaction from the hydrophobic solvent used to dissolve the pre-polymer.

Figure 4

Box-and-whisker plots representing swelling ratios of 4%PEG-86%PCL-10%cPCL scaffolds by crosslinker type. Upper and lower ends of boxes represent the 25th and 75th percentiles, respectively. Solid lines represent the median swelling ratios. Whiskers indicate 90th and 10th percentiles, and dots indicate outliers. The ability of the scaffolds to retain water was somewhat related to the length of the proline oligomer used as a crosslinker. PEG-P₁₀-PEG-crosslinked scaffolds retained significantly less water than PEG-dihydrazide-crosslinked scaffolds (*p < 0.05, n = 24). Differences in swelling ratios versus the other two scaffold types were not statistically significant.

Figure 5

Accelerated degradation of terpolymer scaffolds crosslinked with PEG-P₇-PEG crosslinkers. Scaffolds crosslinked with PEG-dihydrazide or PEG-P₇-PEG were soaked in PBS or PBS + 1 mM SIN-1 for 28 days. (A) At each time point, scaffolds were dried and massed. The average remaining mass fraction of each scaffold is calculated by dividing dry mass following treatment, by dry mass at the beginning of the study. Scaffolds containing both crosslinker types experienced some degree of oxidative degradation, but PEG-P₇-PEG-crosslinked scaffolds lost more mass under oxidative conditions (#,* p < 0.05, n = 3). (B) Heat capacity of scaffolds for the melting point transition following 14 d treatment was measured via DSC. Error bars represent standard deviation of 3 independent experiments (# p < 0.01, n = 3; * p < 0.05; § p < 0.05).

Figure 6

LPS/IFNγ-activated BMDMs exhibited H₂O₂-dependent degradation of PEG-P₇-PEG-crosslinked scaffolds. (A) Immortalized murine BMDMs cultured in tissue culture plates for 24 h in the presence of 50 ng/mL IFNγ and 10 μg/mL LPS produced higher levels of H₂O₂ per cell (H₂O₂ production normalized to cell number indirectly via protein assay), relative to untreated BMDMs (*p < 0.05, n = 3). (B) SEM images (40x and 900x) of scaffolds incubated with untreated or activated BMDMs for 9 d. Only PEG-P₇-PEG-crosslinked scaffolds incubated with activated BMDMs exhibited the appearance of widespread pitting and < 10 μm pores in the polymer network.

Figure 7

μCT imaging of scaffolds incubated with NGL-BMDMs (Mϕ). Isotropic voxel size = 1 μm. (A) 3D pore diameter heat maps of scaffolds following incubation with untreated or activated (LPS/IFNγ-treated) BMDMs for 9 d. (B-C) Pore diameter histograms for scaffolds by crosslinker and treatment (average of n = 3 independent experiments). (D) From these histograms, a range of pore diameters (0–10 μm) was gated as an ROI, and the cumulative percentage of voxels containing pores of diameters within this range was plotted versus crosslinker type and treatment method. Consistent with the intended drug delivery function of this scaffold, the PEG-P₇-PEG-crosslinked scaffolds experienced an increase in the appearance of small pores ≤ 10 μm in diameter. These results are consistent with SEM observations demonstrating the appearance of micropores and pits in these polymer matrices, as well as the widespread disintegration of the macroporous scaffold structure as shown in (A).

Scheme 1

Synthesis of Biaminated PEG-P_n-PEG Crosslinkers

Scheme 2

Synthesis of x%PEG-y%PCL-z%cPCL Backbone Polymers