(M1130-05-27) Novel Materials and Formulations for Driving Intracellular Delivery of Macrocyclic Peptides

Purpose: Peptides have become increasingly popular therapeutic prospects for the treatment of various diseases. They have high potency, high specificity, and can often access unique pathways and desired biological outcomes where other modalities fall short. To date, however, the success of therapeutic peptide drugs has been mostly limited to extracellular targets accessed via parenteral routes of administration. These limitations are driven by the physical and chemical properties of peptides which renders them almost entirely impermeable. Improving delivery of these molecules across cell membranes would greatly expand the therapeutic indications for which peptides could be developed as drugs. To this end, we have pursued novel approaches for achieving intracellular delivery of therapeutic macrocyclic peptides. Novel materials identified through an exhaustive literature search and assessed via internal formulation development were profiled for their ability to functionally deliver peptides intracellularly.

Methods: An extensive literature search was performed to identify novel fit-for-purpose formulation-based approaches for improving intracellular delivery [Figure 1]. Prioritized polymer carriers were synthesized as previously described^1-5 and characterized by nuclear magnetic resonance (NMR) spectroscopy. Polymeric carriers and protein/peptide cargos were incubated at room temperature to form complexes. Complex size and charge were determined by dynamic light scattering (DLS) and zeta potential measurements. The cargo loading efficiency of the individual carriers was assessed by a fluorescence plate-based assay using fluorescently tagged protein or peptide cargo. Cells were incubated with complexes, and microscopy and cell viability assays were employed to assess intracellular uptake, trafficking, and cytotoxicity.

Results: The selected polymer carriers have predominantly been tested with protein cargo in the literature. Therefore, initial formulation assessments of guanidinobenzoic acid (GBA) and biguanide benzoic acid (BGBA) functionalized PAMAM dendrimers [Figure 1] were performed with a model BSA-FITC protein. Dendrimer:BSA-FITC complexes were recapitulated with similar sizes and zeta potentials as previously reported^{1, 2} [Figure 2]. Increased BSA-FITC loading was observed with increasing dendrimer functionality (BGBA >GBA >unfunctionalized). A selection of macrocyclic peptides was then tested with these dendrimers. Successful complexation was achieved with peptides of varying charge and solubility [Figure 3]. Cellular imaging assays were used to assess the intracellular delivery efficiency of dendrimer:peptide complexes. Peptide internalization to the cytosol was achieved, but a decrease in cell viability was also observed, likely due to the high cationic charge of the dendrimers.

Conclusion: Novel formulation strategies represent a promising approach for improving intracellular delivery of non-permeable peptides. We determined that GBA- and BGBA-dendrimers can mediate peptide loading and intracellular delivery but have undesired cytotoxic effects. Incorporating the novel guanidine functionalities onto innately less charged materials or testing other less charged carriers may provide a window for improved delivery with minimized cytotoxicity.

References: 1. Chang, H.; Lv, J.; Gao, X.; Wang, X.; Wang, H.; Chen, H.; He, X.; Li, L.; Cheng, Y., Rational design of a polymer with robust efficacy for intracellular protein and peptide delivery. Nano letters 2017, 17 (3), 1678-1684.
2. Yu, C.; Tan, E.; Xu, Y.; Lv, J.; Cheng, Y., A guanidinium-rich polymer for efficient cytosolic delivery of native proteins. Bioconjugate chemistry 2018, 30 (2), 413-417.
3. Ren, L.; Lv, J.; Wang, H.; Cheng, Y., A coordinative dendrimer achieves excellent efficiency in cytosolic protein and peptide delivery. Angewandte Chemie International Edition 2020, 59 (12), 4711-4719.
4. Backlund, C. M.; Hango, C. R.; Minter, L. M.; Tew, G. N., Protein and antibody delivery into difficult-to-transfect cells by polymeric peptide mimics. ACS Applied Bio Materials 2019, 3 (1), 180-185.
5. Lin, J.-L.; Wang, Z.-K.; Xu, Z.-Y.; Wei, L.; Zhang, Y.-C.; Wang, H.; Zhang, D.-W.; Zhou, W.; Zhang, Y.-B.; Liu, Y., Water-soluble flexible organic frameworks that include and deliver proteins. Journal of the American Chemical Society 2020, 142 (7), 3577-3582.

Acknowledgements:The authors have declared no conflict of interest


Summary of reviewed literature concepts and prioritized Tier 1 carriers.


Characterization of dendrimer complexation with BSA-FITC protein.


Properties of selected macrocyclic peptides and characterization of dendrimer:peptide complexes.