Long circulating genetically encoded intrinsically disordered zwitterionic polypeptides for drug delivery

Abstract

The clinical utility of many peptide and protein drugs is limited by their short in-vivo half-life. To address this limitation, we report a new class of polypeptide-based materials that have a long plasma circulation time. The design of these polypeptides is motivated by the hypothesis that incorporating a zwitterionic sequence, within an intrinsically disordered polypeptide motif, would impart "stealth" behavior to the polypeptide and increase its plasma residence time, a behavior akin to that of synthetic stealth polymers. We designed these zwitterionic polypeptides (ZIPPs) with a repetitive (VPX₁X₂G)_n motif, where X₁ and X₂ are cationic and anionic amino acids, respectively, and n is the number of repeats. To test this hypothesis, we synthesized a set of ZIPPs with different pairs of cationic and anionic residues with varied chain length. We show that a combination of lysine and glutamic acid in the ZIPP confer superior pharmacokinetics, for both intravenous and subcutaneous administration, compared to uncharged control polypeptides. Finally, to demonstrate their clinical utility, we fused the best performing ZIPP sequence to glucagon-like peptide-1 (GLP1), a peptide drug used for treatment of type-2 diabetes and show that the ZIPP-GLP1 fusion outperforms an uncharged polypeptide of the same molecular weight in a mouse model of type-2 diabetes.

Keywords: Bioinspired materials; Recombinant proteins; Unstructured polypeptides; Zwitterionic polypeptides.

Figure 1.. Design and synthesis of zwitterionic polypeptides.

A. Schematic of the zwitterionic polypeptides (ZIPPs). ZIPPs are homopolymers of a VPX₁X₂G repeat unit, wherein X₁ and X₂ are positively and negatively charged amino acids respectively. B. SDS-PAGE of various ZIPPs sequences and the two ELP controls, (VPGAG)₁₂₀ (control for equal number of repeat units) and (VPGAG)₁₆₀ (MW controls). The polypeptide bands were compared to a standard MW ladder as a reference.

Figure 2.. Physiochemical characterization of ZIPPs.

A. ZIPPs and ELPs show a high degree of disorder as seen from their CD spectra. The presence of a negative ellipticity peak ~197nm is characteristic of an intrinsically disordered polypeptide. B. Hydrodynamic radius (R_h) measured by Dynamic Light Scattering (DLS) shows that ZIPPs have anti-poly-electrolyte behavior, as they assume an extended conformation in a high ionic strength buffer such as PBS but have a collapsed structure in water. Uncharged ELPs, used as a control, do not display this behavior, as seen by the negligible change in R_h between water and PBS. (ns-not significant, ****p<0.0001 2-way repeated measures ANOVA, Sidak post-hoc test). C. Native PAGE shows that ZIPPs do not have any non-specific interaction with mouse serum albumin as well as human serum albumin, as seen by the similar mobility of ZIPPs in the presence or absence of albumin. An Albumin binding peptide (ABP) fused to an ELP was used as a positive control.

Figure 3.. Pharmacokinetics and biodistribution of ZIPPs.

A. Circulating ZIPPs and ELPs in plasma as a function of time following a single i.v. injection. The data represent mean ± SE (n=5). The plasma concentration was fit to a two-compartment model, which yielded the pharmacokinetic parameters in Table 2. B. Plasma pharmacokinetics upon s.c. administration, shown as mean ± SE (n=3-4). Data from the s.c. administration study was fit to a non-compartmental model to determine the parameters in Table 3. C. Plasma pharmacokinetics upon i.v. administration for a shorter ZIPP, (VPKEG)₈₀. Data represent mean ± SE (n=4).

Figure 4.. Biodistribution of ZIPPs.

A. Biodistribution of ELP and ZIPP after i.v. injection at 2 h, 6 h, 24 h and 72 h revealed that there is not a large difference in organ distribution between ZIPP and ELP, with a few exceptions at several time points that are marked in the figure. Data shown as mean ± SE (n=3-4). (*p<0.05, ** p<0.01, ***p<0.0001, unpaired t-test). B. Retention of ELP and ZIPP in the organs of the reticuloendothelial system (RES) expressed as organ to blood ratio, calculated by normalizing the amount present in each organ to that in the blood. Each bar is the mean ± SE (n=3-4). (*p<0.05, **p<0.01, ***p<0.0001, unpaired t-test).

Figure 5.. GLP1-ZIPP fusions are active in vitro and in vivo.

A. cAMP response of native GLP1 and GLPl-(VPKEG)₁₂₀, GLPl-(VPGAG)₁₆₀ was tested in BHK cells expressing the GLP-1R (n=3). B. Blood glucose was monitored for 3 days after treating 7-week-old DIO mice (n=5) with a single s.c. injection of GLP1-(VPKEG)₁₂₀. GLP1-(VPGAG)₁₆₀ or PBS. C. Glucose AUC was calculated for each mouse up to 72 h and normalized to the mean of the PBS control group. (^#p<0.0001, one-way ANOVA and Dunnett’s test). D. Percent body weight change relative to weight at time of injection, t=0 h. For B and D (*p<0.05, **p<0.01, ***p<0.001, ****p< 0.0001 2-way repeated measures ANOVA, Tukey’s post-hoc test).