Identification of peptide coatings that enhance diffusive transport of nanoparticles through the tumor microenvironment

Rashmi Mohanty; Xinquan Liu; Jae You Kim; Xiujuan Peng; Sahil Bhandari; Jasmim Leal; Dhivya Arasappan; Dennis C. Wylie; Tony Dong; Debadyuti Ghosh

Iterative screening allows the tumor-like ECM to serve as a selection pressure to filter away phage unable to consistently penetrate through the ECM, irrespective of the heterogeneity of its mesh network. In each replicate, we observed an increased amount of phage that permeated through the tumor-like ECM after four rounds (Figure 1b) . The average library titer (i.e. amount of phage) of both replicates increased 200-fold and 400-fold in the third and fourth round compared to the second round. In the third and fourth rounds, the increase of permeating phage suggests there is enrichment of the library for phage that may possess desired functionalities (i.e. unhindered transport) through the tumor-like ECM. There was no increase in the first two rounds, but the lack of initial enrichment is typically observed in phage selections 70 . The enrichment of phage during selection is expected, as observed with affinity-based panning against specific targets [70] [71] [72] . The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/659524 doi: bioRxiv preprint round 1 (R1) to round 2 (R2) and had moderate increase in rounds 3 and 4 (R3 and R4); these results have been observed by others and is indicative of their enrichment [69] [70] [71] 73 . From NGS data, we selected one peptide sequence from the original, naïve library as a negative control for our subsequent experiments. This control peptide sequence is present in the original library, but its frequency decreased with successive rounds of selection, which suggests it was unable to withstand the selection pressure of the tumor-like ECM and cannot permeate through the barrier. Two additional sequences, P4 and P5-peptides, were selected from multiple sequence alignment using Seq2Logo as described elsewhere 69, 74 . Here, multiple sequence alignment of the top thirty frequent peptides from the fourth round of both replicates was done to identify the most abundant amino acid at each position of the 7-mer 75 . From this analysis, we generated and selected The selected peptides along with their physicochemical properties (net charge at pH 7.0 and hydropathy/hydrophobicity) are listed in Table 1 . Three of the five sequences selected are hydrophilic, one is hydrophobic, and one of the sequences is neither hydrophilic nor hydrophobic, which we term as hydro-neutral. The net charge of two of the selected sequences is negative, two are positive, and one of them has a net neutral surface charge at pH 7.0. The net charge of all the selected peptides are near neutral, ranging from -1.0 to +1.1. Table 1 . Peptides selected for validation based on frequency, reproducibility and multiple sequence alignment of the thirty most frequent sequences from both replicates of phage library screening, along with their physicochemical properties.