Immune-instructive polymers for modulating macrophage phenotype
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Date
2024-03
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University of Nottingham
Abstract
Background
Medical devices, from implants to gene/drug delivery systems and biosensors,
often elicit immune reactions ranging from transient inflammation to chronic
inflammation and fibrosis, known as the foreign body reaction (FBR). These
immune responses can lead to device failure and tissue damage, highlighting the
need for biomaterials that not only avoid these reactions but actively guide positive
immune interactions for tissue integration and healing. The role of macrophages,
with their highly plastic nature, is critical in the orchestration of these immune
responses, making them a focal point for biomaterial design aimed at minimising
FBR and enhancing implant integration. High-throughput screening using polymer
microarrays has been increasingly utilised to accelerate the discovery of new
biomaterials. Previously, this strategy has identified acrylate and acrylamide
polymers that can modulate the polarisation state of macrophages based on their
expression of surface markers using fluorescent labels and microscopy. Unlike the
macrophage ‘secretome’, surface marker expression only provides a partial insight
into the macrophage functional phenotype. However, since conventional polymer
microarrays do not have defined cell culture chambers for individual polymer spots,
investigating the secretome in this format is not feasible.
Description
Chapter 3: Development of a Novel Screening Platform for Secretome
profiling for the Discovery of Immune-Responsive Polymers
This study presents an innovative screening platform based on the polymer
microarray concept, designed to profile the secretome for each polymer separately.
The aim is to uncover new polymer chemistries that can regulate or modulate
macrophage phenotype, thereby guiding tissue integration and enhancing healing
processes post-implantation. The novel microchamber (7 mm × 7 mm wells) based
polymer array was developed using a contact printing fabrication technique and
ProPlate® multi-well chambers. It comprises 56 polymer spots/wells, with 16 wells
per slide and four slides per plate. It is designed to be compatible with the robotic
printing of polymers and is well-suited for secretome profiling of both adherent and
non-adherent cells. This platform was successfully validated, with its effectiveness
proven through a detailed study assessing the impact of chemically diverse
homopolymers on macrophage viability, attachment, and differentiation into pro inflammatory (M1) or anti-inflammatory (M2) phenotypes. Eighty-one acrylate
and acrylamide polymers were printed into individual wells and cultured with
primary human monocyte-derived macrophages (MDMs) for six days. The
polymers that ensured high MDMs viability (>80%) were then selected for
attachment and secretome analyses, which involved measuring the production of
cytokines TNF-α (Tumor Necrosis Factor-alpha), IL-6 (Interleukin-6), IL-10
(Interleukin-10), TGF-β (Transforming Growth Factor-beta), and CCL-18
(Chemokine (C-C motif) Ligand 18) by cells cultured on them. These cytokines are
key indicators of the macrophages’ immune responses and can signal pro inflammatory or anti-inflammatory activity. Several Polymers that supported cell
attachment and modulated MDM phenotypes were identified. Polymers such as
poly(carbazol-9-yl ethyl acrylate) (pCzEA) and poly(3-hydroxy-2,2-
dimethylpropyl 3-hydroxy-2,2-dimethylpropionate diacrylate) (pHDMPDA) were
categorised as M2-like, while poly(1,1,1-trimethylolpropane trimethacrylate)
(pTMOPTMA) and poly([methylthio]ethyl methacrylate) (pMTEMA) were
categorised as M1-like. The polymer poly(glycerol propoxylate triacrylate)
(pGPOTA) retained macrophages in a naive state, akin to inactivated (M0)
macrophages.
Chapter 4: Evaluating hit Polymers: Exploring the Impact of Seven Promising
Polymers on Macrophage Behaviours and in-vitro Wound Healing
The seven ‘hit’ polymers with most consentient immune modulatory properties in
the screening were synthesised, characterised, and scaled up to larger 13 mm
surfaces. This scale-up was essential for further phenotypic and functional analyses
of these 'hit’ polymers. MDMs showed distinct anti- and pro-inflammatory
characteristics when cultured on these scaled up polymer surfaces. They showed
marked increases/decreases in cytokine production and notable changes in both cell
surface markers and gene expression levels. Furthermore, these polymers indirectly
affected fibroblast migration and proliferation in a 2D wound healing assay using
MDMs-conditioned media. Wounds closed faster when fibroblasts were exposed to media from macrophages cultured on polymers that favoured M2-like
phenotypes like pHDMPDA and pCzEA, and intriguingly pMTEMA, an M1-
polymer, outperforming the tissue culture plastic (TCP) control which is highly
pro-fibroblast proliferation. This suggests that these polymers indirectly influence
stromal cells during tissue healing and regeneration through paracrine signalling,
demonstrating their potential in improving healing outcomes.
The mechanisms behind the polymer-induced changes in MDMs phenotype were
also explored, focusing on the polymers’ stiffness and the thickness of the protein
layer adsorbed from the culture medium. However, unfortunately, there was no
apparent correlation.
Chapter 5: The Impact of Selected Polymers on Fibroblast Behaviours and in vitro Scratch Assay
Having shown the paracrine effect of macrophage conditioned media in previous
chapter, in this chapter I examined the direct impact of selected polymers on
fibroblasts, another key cell type in wound healing, by culturing these cells on
polymer surfaces to evaluate their ability to modify fibroblasts behaviour. All
polymer surfaces supported fibroblast viability, adhesion and proliferation. They
also induced variable secretome profiles and impacted the wound closure rate, with
cells cultured on pHDMPDA and pTMOPTMA showing distinct morphological
characteristics including variations in cell area and perimeter.
Conclusion
This study's innovative approach utilising a microchamber based polymer array
platform has successfully identified novel immune-instructive polymers capable of
steering macrophage activity through secretome analysis, a method surpassing
previous techniques reliant on surface markers. The identified polymers, by
promoting distinct immune and wound healing responses, could pave the way for
the development of next-generation medical devices and regenerative therapies that
are finely tuned to harness and direct the body's own healing mechanisms
Keywords
macrophage phenotypes, Immune-instructive, polymers