(L-458) Pro-Inflammatory Peptide-nanogel Conjugates for Macrophage Immunotherapy

Tumor-Associated Macrophages (TAM) are intrinsic components of the tumor microenvironment. They are known to adopt tumor growth-promoting phenotype¹. Pro-inflammatory (M1) phenotype is thought to be tumor suppressive², and therefore targeting TAMs to promote the M1 phenotype is a promising direction in cancer immunotherapy.³ Macrophage polarization in the physiological environment is commonly induced by cytokines; however, they are known to have a short half-life in the body and cytotoxicity to cells when administered in high doses⁴. The objective of this project is to synthesize a nanogel platform with intrinsic pro-inflammatory activity, via delivery of a covalently immobilized M1-inducing peptide. IFN-g (95-133) peptide was selected for this study, as it showed antiviral activity against Encephalomyocarditis Virus Infection in vitro and in vivo⁵ in literature and therefore is used here as a peptide conjugate to induce M1 polarization. We synthesized poly(acrylamide-co-methacrylic acid) (P(AAm-co-MAA)) nanogels covalently conjugated to IFN-g (95-133) peptide and tested the resulting conjugates’ bioactivity on a model macrophage cell line (RAW264.7) using a flow cytometry-based activity assay.

P(AAm-co-MAA) nanogels were synthesized using inverse emulsion polymerization in an inert nitrogen environment from acrylamide (AAm), methacrylic acid (MAA) and Rhodamine-B methacrylate monomers and n.n-bis(acryloyl) cysteamine as a degradable crosslinker. Nanogel hydrodynamic diameter (nm), effective surface charge (mV), and pH response were measured using Dynamic Light Scattering (DLS). IFN-g (95-133) peptide was covalently conjugated to the nanogel at 10% w/w using a two-step conjugation process with a bifunctional amine-PEG-maleimide linker: EDC mediated amidation of carboxylic acid groups and thiol-click reaction of a cysteine residue on the N-terminus of the peptide. Reactions were buffered in 10 mM MES and 1xPBS to compare the conjugation efficiency in different buffers. Micro-BCA assay was used to quantify the amount of peptide conjugated. PEG linker was added in 1.5-fold molar excess relative to methacrylic acid molar concentration and 2-fold molar excess of EDC was used. The bioactivity of IFN-g (95-133) peptide-nanogel conjugates was tested using Flow Cytometry on macrophages incubated with IFN-g (95-133) peptide-nanogel conjugates for 24 h, IFN-g (95-133) peptide, IFN-g protein as a positive control, and whole media as a negative control. The cells were then washed and stained for the co-stimulatory and antigen-presenting markers CD86 and MHC-II, respectively.

P(AAm-co-MAA) nanogels had a hydrodynamic diameter that ranged between 102.1 nm at pH 7.5 and 83.9 nm at pH 3.5 with a 105.7 nm peak at pH 6.5 (Figure 1. a). Zeta potential varied between -27.3 mV at pH 7.5 and -5.9 mV at pH 3.5 (Figure 1. b). IFN-g (95-133)-P(AAm-co-MAA) conjugation efficiency was almost 3-folds greater in MES buffer than in PBS, as it was shown through Micro-BCA assay. The concentration of the peptide in the conjugates was calculated to be 1.04 wt% of peptide mass to nanogel dry mass in PBS buffer and 2.96 wt% in MES buffer (Figure 1. c). EDC-mediated covalent coupling is optimal at pH between 4.5 and 5.0, which is best buffered by MES, while Thiol-click reaction proceeds best at pH 6.5 to 7.5, which is best buffered by PBS. Since EDC-mediated reactions are less efficient than thiol-click reactions, EDC coupling is thought to be the limiting factor in the conjugation reaction. Therefore, the optimization of conditions for EDC-mediated coupling – use of the optimal buffer for pH maintenance – could explain the higher conjugation efficiency in MES buffer. Finally, staining for CD86 and MHC-II on RAW264.7 macrophages after 24 h of incubation with 1.04 wt% IFN-g (95-133)-P(AAm-co-MAA) conjugates showed no significant increase in marker expression via Flow Cytometry (Figure 1. d). To better understand why, we treated the cells with free IFN-g (95-133) at concentrations ranging from 30 to 100 uM. Immune phenotyping showed that even the free peptide did not induce CD86 or MHCII expression.

P(AAm-co-MAA) nanogels have a pH-responsive hydrodynamic diameter and zeta potential around 102.1 nm and – 27.3 mV respectively at physiological pH. Conjugation reaction of IFN-g (95-133) peptide is more effective in MES buffer: 2.96 wt% of the nanogel dry weight vs 1.04 wt% in PBS. Flow Cytometry immune phenotyping showed no significant M1 activation of RAW264.7 macrophages after 24 h incubation with 1.04 wt% IFN-g (95-133)-P(AAm-co-MAA) conjugates.

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3.  Zhao, R. et al., Biomaterials Science, 9(13), 4568–4590.

4.  Lortat-Jacob et al. Journal of Biological Chemistry, 271(27), 16139–16143.

5.  Mujtaba, M. G. et al., Clinical and Vaccine Immunology, 13(8), 944–952.