Encapsulating a Hydrophilic Chemotherapeutic into Rod-like Nanoparticles of a Genetically Encoded Asymmetric Triblock Polypeptide Improves its Efficacy

Abstract

Encapsulating hydrophilic chemotherapeutics into the core of polymeric nanoparticles can improve their therapeutic efficacy by increasing their plasma half-life, tumor accumulation and intracellular uptake, and by protecting them from premature degradation. To achieve these goals, we designed a recombinant asymmetric triblock polypeptide (ATBP) that self-assembles into rod-shaped nanoparticles, and which can be used to conjugate diverse hydrophilic molecules, including chemotherapeutics, into their core. These ATBPs consist of three segments: a biodegradable elastin-like polypeptide, a hydrophobic Tyrosine-rich segment, and a short Cysteine-rich segment, that spontaneously self-assemble into rod-shaped micelles. Covalent conjugation of a structurally diverse set of hydrophilic small molecules, including a hydrophilic chemotherapeutic -gemcitabine- to the Cysteine residues also leads to formation of nanoparticles over a range of ATBP concentrations. Gemcitabine-loaded ATBP nanoparticles have significantly better tumor regression compared to free drug in a murine cancer model. This simple strategy of encapsulation of hydrophilic small molecules by conjugation to an ATBP can be used to effectively deliver a range of water-soluble drugs and imaging agents in vivo.

Keywords: asymmetric triblock polypeptide; colon cancer; gemcitabine; hydrophilic drug delivery; nanoparticle.

Figure 1. Structure of ATBP, SMM and schematic of the synthesis of ATBP-SMM/GEM nanoparticles

a. Sequence of the asymmetric tri-block polypeptide (ATBP) consists of three segments: an ELP segment that consists of 160 repeats of AGVPG, a self-assembly promoting (YG)₆ segment, and a cysteine-rich (CGG)₈ drug attachment segment that provides reactive Cys residues for the covalent conjugation of maleimide derivatives of model compounds or drug. b. Structure of the small molecule malemide derivatives (SMMs). The circle serves as a visual map of the strcture of model compounds and their hydrophobicity, as measured by their logD; The hydrophobicity increases in clockwise fashion in the diagram. c. Attachment of gemcitabine (GEM) does not disrupt self-assembly of the ATBP into cylindrical nanoparticles with a drug-rich (blue diamonds) core surrounded by a hydrophobic core (red) and hydrophilic polypeptide corona (black chains).

Figure 2. Characterization of ATBP–N-hydroxymaleimide (ATBP-SMM1) conjugates

a. Angular dependence of hydrodynamic radius (R_h) for ATBP–N-hydroxymaleimide conjugate measured by DLS. b. Partial Zimm Plot (Kc/R vs q²) obtained by SLS for ATBP–N-hydroxymaleimide conjugate, c. Cryo-TEM micrograph of ATBP-N-hydroxymaleimide conjugate. d. Determination of transition temperature (T_t) of ATBP-N-hydroxymaleimide conjugate by thermal turbidimetry at 350 nm, e. Determination of CAC of ATBP-N-hydroxymaleimide conjugate by pyrene fluorescence assay.

Figure 3. Characterization of ATBP–GEM conjugate

a-b SDS-PAGE (a) and MALDI-MASS (b) of ATBP and ATBP-GEM conjugate, showing increase in mass from conjugation of GEM. c. Determination of hydrodynamic radius at by single-angle DLS, d. Angular dependence of hydrodynamic radii for ATBP–GEM nanoparticles measured by DLS, e. Partial Zimm Plot (Kc/R vs q²) obtained by SLS for ATBP–GEM conjugate, f. Cryo-TEM micrograph of ATBP-GEM conjugate g. AFM image of ATBP-GEM nanoparticles in air. The scale bar is 200 nm. h. Determination of transition temperature (T_t) of ATBP-GEM conjugate by thermal turbidimetry at 350 nm, i. Determination of CAC of ATBP-GEM conjugate by pyrene fluorescence assay.

Figure 4. In vitro and in vivo activity of ATBP–GEM nanoparticles

a-b Cell viability for ATBP–GEM and free GEM in HCT-116 (a) and Colo 205 (b) cells (mean ± 95%CI). c. Plasma cy5 concentration as a function of time post-administration. A non-compartment model was used to fit the plasma cy5 concentration, which resulted a terminal half-life of 12.8 h for cy5 labelled ATBP–GEM conjugate (mean ± 95% CI, n=5). d. In vivo Tumor uptake. The cy5 concentration in tumor at 1, 6 and 24 h post-administration of cy5 labelled GEM, and cy5-ATBP-GEM nanoparticles. ** and **** indicates p<0.01 and p<0.0001 respectively (Two way ANOVA, Sidak’s test) (mean ± 95% CI, n=4). e-f. Tumor cells (HCT-116) were inoculated on the right flank. When the tumor volume reached ~100 mm³, mice received three doses of PBS (n=6), free GEM (25 mg kg⁻¹ BW, n=6) or ATBP–GEM (25 mg GEM equiv.kg⁻¹ BW, n=6) on day 0, 2 and 4. e, Tumor volume up to day 30 (mean ± 95% CI, n= 8). p < 0.0001 for ATBP–GEM versus GEM and PBS (day 12) respectively (Tukey test). f, Cumulative survival of mice (Kaplan–Meier).