Purpose: Tumor immunology is changing the landscape of modern anticancer therapy and mRNA has emerged as an attractive alternative to DNA for antitumor vaccination, because it has shown to be safer and able to induce higher protein expression upon internalization by Antigen Presenting Cells (APCs). In this work, reversible Addition Fragmentation chain Transfer (RAFT) polymerization has been exploited for the synthesis of a small library of diblock copolymers designed to ideally deliver mRNA encoding tumor-associated antigen (TAA) to APCs and to trigger the immune response and memory against cancer. These novel materials were designed with a poly-cationic Agmatine block to condense oligonucleotides (ONs) and with a glycopolymeric portion to actively target specific lectin receptors expressed on immune cells (e.g. mannose receptor, MR). Importantly, the system is also intended to provide mRNA protection against fast degradation, minimizing its interactions with extracellular nucleases.
Methods: Three cationic block-glycopolymers of increasing molecular weight (Man15-b-Agm12, Man29-b-Agm25 and Man58-b-Agm45) were generated via a 2 step fast RAFT by sequentially polymerizing Mannose and Agmatine monomers at a molar ratio of 1:1. The block copolymers were characterized by 1H NMR and Gel Permeation Chromatography (GPC) analyses. Glycopolyplexes (GPPs) were obtained by simple incubation of the polymers with a 19-base ssDNA, used as model oligonucleotide. Gel retardation assays were performed to select the optimal N/P ratios to achieve complete ssDNA complexation. Glycopolyplexes dimension was characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM) analyses. The zeta-potential of glycoplexes was quantify as an indicator of complexation efficiency. The stability of the glycopolyplexes was investigated by competitive displacement assay with increasing concentration of heparin (0.15-10 IU/mL) and by size analysis of glycopolyplexes incubated at 4°C in phosphate buffer saline (PBS) and at 37°C in PBS added of 10% FBS. Finally, selective recognition and internalization by mannose binding lectin and, in particular, by MR was studied in cellular models non-expressing (CHO) or expressing MR (CHO-MR+). Cells were incubated for 30 minutes with glycopolyplexes loaded with Cy3 labelled ssDNA and the uptake was monitored by flow cytometry.
Results: GPC analysis confirmed a narrow molecular weight distribution for each polymer, with a calculated molecular weight (MW) of 4.4, 8.3 and 16.3 kDa and a polydispersity index (Ð) of 1.13, 1.43 and 1.29 for Man15-b-Agm12, Man29-b-Agm25 and Man58-b-Agm45, respectively.
GPPs were formed by incubating each block co-polymer with a 19-base ssDNA and the optimum N/P ratio to achieve complete ssDNA was found to be of 10, 5 and 3 for Man15-b-Agm12, Man29-b-Agm25 and Man58-b-Agm45, respectively. For the following assays, the N/P ratios of 20, 10 and 5 were used for Man15-b-Agm12/ssDNA, Man29-b-Agm25/ssDNA and Man58-b-Agm45/ssDNA GPPs, respectively. A competitive displacement assay with increasing concentration of heparin demonstrate very high glycopolyplexes stability, with the Man58-b-Agm45/ssDNA being the best performing formulation (complexation preserved until 10 IU/mL). The size of polyplexes by DLS analysis was found to be of 46.3±6.45, 31.1±0.60 and 61.7±3.92 nm for Man15-b-Agm12/ssDNA, Man29-b-Agm25/ssDNA and Man58-b-Agm45/ssDNA GPPs, respectively. Slightly smaller particles were detected by TEM imaging which were ascribed to the technique used that implies dried samples. The zeta-potential of the glycopolyplexes was found to decrease in comparison with the free polymers and more markedly for Man29-b-Agm25/ssDNA and Man58-b-Agm45/ssDNA GPPs, which were formed at lowest N/P ratios in comparison to Man15-b-Agm12/ssDNA GPPs. All glycopolyplexes formulation were stable in physiological medium at 4°C for more than 8 days while at 37°C and in presence of FBS the Man58-b-Agm45/ssDNA GPPs was stable for at least 48 hours. On the contrary, the exposure to serum protein and physiological temperature induces a destabilization of the other formulations at 8 or 24 hours resulting in size increase of the GPPs. The uptake studies on CHO and CHO-MR+ cells showed remarkable and highly specific uptake for Man15-b-Agm12/ssDNA and Man29-b-Agm25/ssDNA glycopolyplexes with negligible internalization by cells non-expressing the receptor. Man58-b-Agm45/ssDNA glycoplexes showed 2-folds higher internalization in CHO-MR+ cells as compared to control CHO cells.
Conclusion: Triggering immune cells response against tumor through the active delivery of mRNA-encoding TAA to APCs have emerged as a new powerful tool in cancer treatment. In here, we successfully synthesized three novel glycopolymers able to efficiently complex a model oligonucleotide as confirmed by gel retardation assays and particle size analysis and which showed high selectivity in delivering model ONs to APCs specific expressing specific receptors. Confocal and FRET studies are ongoing to investigate the glycopolyplexes ability to promote cellular uptake and to facilitate endosomal escape thus favouring rapid expression of protein immediately after being introduced into the cells. The same platform is currently under evaluation for plasmid delivery.
Federica Bellato– Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Padova, Veneto
Silvia Brunato– Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Padova, Veneto
Paolo Caliceti– Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Padova, Veneto
Stefano Salmaso– Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Padova, Veneto