Non-Covalent Carrier Hydrophobicity as a Universal Predictor of Intracellular Protein Activity

Abstract

Over the past decade, extensive optimization of polymeric cell-penetrating peptide (CPP) mimics (CPPMs) by our group has generated a substantial library of broadly effective carriers which circumvent the need for covalent conjugation often required by CPPs. In this study, design rules learned from CPPM development were applied to reverse-engineer the first library of simple amphiphilic block copolypeptides for non-covalent protein delivery, namely, poly­(alanine-block-arginine), poly­(phenylalanine-block-arginine), and poly­(tryptophan-block-arginine). This new CPP library was screened for enhanced green fluorescent protein and Cre recombinase delivery alongside a library of CPPMs featuring equivalent side-chain configurations. Due to the added hydrophobicity imparted by the polymer backbone as compared to the polypeptide backbone, side-chain functionality was not a universal predictor of carrier performance. Rather, overall carrier hydrophobicity predicted the top performers for both internalization and activity of protein cargoes, regardless of backbone identity. Furthermore, comparison of protein uptake and function revealed carriers which facilitated high gene recombination despite remarkably low Cre internalization, leading us to formalize the concept of intracellular availability (IA) of the delivered cargo. IA, a measure of cargo activity per quantity of cargo internalized, provides valuable insight into the physical relationship between cellular internalization and bioavailability, which can be affected by bottlenecks such as endosomal escape and cargo release. Importantly, carriers with maximal IA existed within a narrow hydrophobicity window, more hydrophilic than those exhibiting maximal cargo uptake. Hydrophobicity may be used as a scaffold-independent predictor of protein uptake, function, and IA, enabling identification of new, effective carriers which would be overlooked by uptake-based screening methods.

Red Light-Emitting Water-Soluble Luminescent Iridium-Containing Polynorbornenes: Synthesis, Characterization and Oxygen Sensing Properties in Biological Tissues In Vivo

“…Among the abovementioned polymerization methods, ROMP is the most suitable polymerization route because ROMP reactions proceed rapidly in the presence of air-stable and functional groups of tolerant ruthenium Grubbs catalysts in mild conditions and allow the acquisition of different functional polymeric materials with desired structure and composition [10][11][12][13]. The employment of ROMP for the preparation of polymeric materials for different biomedical applications is presented in recent articles [14][15][16]. Hango et al developed oxanorbornene-based block copolymers capable of effective intracellularly protein delivery [14].…”

“…The employment of ROMP for the preparation of polymeric materials for different biomedical applications is presented in recent articles [14][15][16]. Hango et al developed oxanorbornene-based block copolymers capable of effective intracellularly protein delivery [14]. In the paper by Kockelmann et al, 2020, it is reported that poly(norbornene)-based nanogels can be utilized as drug delivery agents in anticancer immunotherapy [15].…”

Increased block copolymer length improves intracellular availability of protein cargo

“…S26†). 29,40,48,49 Cargo binding appeared to be driven by the polymer repeat unit to protein ratio ( i.e. , mass concentration of polymer in the system) rather than PTDM DP.…”

“…In a recent study using the same reporter Jurkat T cell line, the polymers MePh 10 - b -dG 5 , dMe 10 - b -dG 5 , and dPh 5 - b -dG 5 yielded recombination rates of 25%, 50%, and 45%, respectively (560 nM, 5 : 1). 29 In a different human T cell line, MePh 9 - b -dG 5 reached 70% using Cre and 90% using a Tat-Cre conjugate, which facilitates some cellular entry on its own (125 nM, 10 : 1). 42 Two additional reports in this cell line demonstrated the abilities of dPh 5 - b -dG 5 and dPh 5 - grad -dG 5 (gradient architecture) to edit up to 70% (Cre) and 90% (Tat-Cre) of cells (100–150 nM, 5 : 1–10 : 1).…”

“…This is consistent with previous findings that the simple addition of a hydrophobic block dramatically improves protein internalization. 29,40,41 Block copolymer degree of polymerization was found to affect protein delivery in competing ways. Although shortening these PTDMs maximized protein internalization, arranging the same number of monomers into fewer, longer polymer chains improved functional Cre delivery.…”