Purpose: The hyper-permeable vasculature and dysfunctional lymphatic system of tumors result in elevated interstitial fluid pressure of the tumor extracellular matrix (ECM). This reduces the convective transport through the ECM, thus leaving behind diffusion as the primary mode of transport for therapeutic agents. Diffusivity of therapeutic macromolecules depends on their size, charge, and configuration and their interactions with ECM. We aim to design fast diffusing nano-sized peptides as drug carriers for improved drug delivery through a tumor microenvironment. We hypothesize that by using phage display and Next Generation DNA sequencing (NGS) method, we can identify and optimize desired physicochemical properties (i.e. Surface charge and hydrophilicity) that improve diffusivity of macromolecules through the tumor ECM. Therefore, we are shortlisting potential peptides with the fastest diffusivity through an in vitro model of tumor ECM from a random phage-presenting peptide library, which possesses a collection of peptides with diverse physical and chemical properties.
Methods: We constructed a phage library, where random seven amino acid peptide sequences are displayed on the surface of T7 phage. Here, each phage expresses a different peptide sequence and each phage effectively serves as a formulation with unique surface chemistry. Then, we screen the potential sequences with fast diffusivity by making the library to diffuse through the tumor ECM. Repeated rounds of selection are performed to narrow down the diversity and screen possible peptides with the specific desired physicochemical characteristics. The peptide clones are identified by NGS method, as the phage genome genetically encodes the displayed peptides. Further, few clones are selected for validation based on their reproducibility and frequency of appearance during screening, and motif analysis. Additionally, the specific physicochemical properties of the selected clones are obtained. The selected clones are validated by comparing the diffusivity of the peptides displayed on the T7 phage using a transwell diffusion assay. T7 phage presenting peptides with each type of physicochemical characteristics are considered as positive and negative controls. We also calculated and compared the diffusivity of the selected sequences in their peptide level using same transwell diffusion assay.
Results: We obtained enrichment of the randomly designed phage-presenting peptide library after four rounds of selection. NGS analysis showed the average net charge and hydrophilicity of the top-20 frequent sequences to approach neutral through the subsequent rounds of selection. Transwell diffusion assay of the peptide presenting on phage, displayed higher diffusion of hydrophilic and neutral surface charge clones through the in vitro tumor ECM with above ten times improved diffusivity in comparison to the negative control (negative surface charge). Interestingly, the positive control (positive surface charge) exhibit 500 times stronger diffusing behavior in comparison to the negative control possibly because of the Donnan effect. However, the diffusion trend observed in phage level (60 nm) is found not to be conserved in peptide level (~3 nm) possibly because of the dominance of the steric interaction on electrostatic interaction. In peptide level, the neutral charged and hydrophilic peptide showed significantly higher diffusivity in comparison to the positively charged control (p<0.05). Surprisingly, it was found that weakly positive surface charged peptides diffuse faster (nearly two-times) through tumor ECM than their unhindered diffusion through saline. This reveals the possible action of Donnan partitioning because of the weak interaction of the positively surface charged peptides with net negative tumor ECM, which will be further investigated in the study.
Conclusion: In this study, we have selected two neutral charged hydrophilic peptides with improved diffusivity through an in vitro tumor ECM model. Also, we observed the possible action of Donnan partitioning enabling weakly positive surface charge to diffuse faster through tumor ECM than that of their unhindered diffusivity. We will further investigate the ability of the peptides to improve diffusivity of nanoparticle chemotherapeutic drug carriers through the tumor ECM.
Debadyuti Ghosh– Assistant Professor, University of Texas at Austin