Stewart, Chloe L
(2023)
Development of an in vitro model to screen the foreign body response towards hydrogel depot injections.
PhD thesis, University of Nottingham.
Abstract
Background: A wealth of research is directed towards designing long-acting injectable formulations for sustained drug release. However, the foreign body response (FBR) is reported to interfere with sustained release mechanisms and is a major factor in determining the efficacy of depot formulations. In vitro models offer the capacity to screen many test conditions to predict the complex and multifaceted immune response, although no established in vitro FBR model exists. Hence, the overarching aim of this thesis was to develop an in vitro model that recapitulated characteristic elements of the FBR and adapt it to study the potential effect of FBR towards injectable hydrogel materials.
Methods: Eighteen PLGA-PEG-PLGA and PCLA-PEG-PCLA copolymers were synthesised with varying polyester and PEG chain lengths and monomer ratios. The effect of modifying copolymer properties on hydrogel gelation was characterised by rheology. As an essential feature of the FBR, human primary monocyte-derived macrophages were stimulated to fuse in vitro to form foreign body giant cells (FBGCs). Macrophages were cultured on either tissue-culture polystyrene (TCPS) or polyethylene terephthalate (PET) and in cytokine cocktails consisting of M-CSF, GM-CSF, IL-4 and/or IL-13. A diverse selection of cytokines was quantified by ELISA to determine whether markers of innate inflammation were associated with fusion. A range of microenvironmental factors such as growth factors, chemokines, and toll-like receptor (TLR) signals were screened for their effect on FBGC formation. Macrophages were co-cultured with fibroblasts and stimulated to form FBGCs to determine the effect of fibroblasts on FBGC formation and the effect of FBGCs on expression of alpha-smooth muscle actin (α-SMA) and collagen deposition from fibroblasts. To incorporate hydrogels into the model and measure cytotoxicity, a PLGA-PEG-PLGA hydrogel of Mn 1410-1500-1410 g mol-1 and L:G of 13.5 was added to U937 monocyte or human foreskin fibroblast cultures.
Results: Increased copolymer hydrophobicity correlated with increased hydrogel moduli and a lower temperature was required to initiate gelation. However, many of the copolymers formed very weak hydrogels (<100 Pa) at 37 °C and were unable to be incorporated into an in vitro cell model.
The combination of PET, M-CSF and IL-4 induced 90.6% ± 3.2% macrophages to fuse into FBGCs after 28 days, the process of which was associated with a specific programme of soluble mediators characterised by an initial pro-inflammatory response that shifted towards wound healing. No specific molecule was implicated in driving fusion; however, exogenous PDGF enhanced large FBGC formation (14.2 ± 3.1% vs 33.2 ± 5.2% macrophages fused into FBGC containing >10 nuclei/cell). Activation of macrophage TLRs by bacteria induced FBGCs to form on a non-fusogenic surface, whilst the inhibiting TLR4 diminished macrophage fusion. Co-culturing macrophages with fibroblasts enhanced FBGC formation (day 14: <4% vs 52.2% ± 38.6% of FBGCs contained >20 nuclei/cell) and permitted large FBGC formation on non-fusogenic surfaces. Conversely, the addition of FBGCs did not affect the extent of fibroblast α-SMA expression or collagen deposition.
Finally, the PLGA-PEG-PLGA hydrogel demonstrated considerable cell toxicity (<30% viability), which was attributed to be due to the accumulation of acidic degradation products. Adaptations to the model format included an indirect culture set-up that enabled gradual exposure to degradation products and an increased volume ratio of media to hydrogel to better simulate the in vivo clearance.
Conclusion: Macrophage fusion was governed by specific microenvironmental cues that required detection of a foreign antigen and IL-4/IL-13-induced programming. The in vitro co-culture model that recapitulated specific FBGC and fibroblast interactions indicated that large FBGC syncytia may also function as a barrier to drug release in conjunction with the fibrotic capsule. Consequently, both elements of the FBR may dictate sustained drug release kinetics. Enhancing the physiological relevance of the model alleviated cell toxicity and was more reflective of the reported in vivo response. However, further adaptations are required to incorporate weak injectable hydrogels into in vitro models long-term to enable screening of the resultant FBR.
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