Self-assembly of thermally responsive nanoparticles of a genetically encoded peptide polymer by drug conjugation

Abstract

Chimeric polypeptides (CPs) that are derived from elastin-like polypeptides (ELPs) can self-assemble to form nanoparticles by site-specific covalent attachment of hydrophobic molecules to one end of the biopolymer backbone. Molecules with a distribution coefficient greater than 1.5 impart sufficient amphiphilicity to drive self-assembly into sub-100 nm nanoparticles.

Figure 1. A) Sequence of the chimeric polypeptide

The 62 kDa ELP segment of the CP consists of 160 repeats of VPGXG with the guest residue X having the composition Val₁Gly₇Ala₈. The 1.6 kDa cysteine-rich sequence at the C-terminus provides sites for covalent conjugation of maleimide derivatives of model compounds, shown in (B). B) Structure of the model compounds. The circle represents a visual map of the model compounds and their hydrophobicity as measured by the distribution coefficient at pH 7.4. The attachment of compounds with a Log(D) ≤ 1.5 (shown in blue) did not trigger self-assembly of the CP, whereas compounds with a Log(D) > 1.5 (shown in pink) triggered the CP to self-assemble into nanoparticles.

Figure 2. Physical properties of CP nanoparticles

(A) CP thermal characterization. Transition temperature (T_t) as a function of CP concentration of CPs conjugated to hydrophilic compounds (blue; unimer; compounds 1–5), and hydrophobic compounds (red; nanoparticle; compounds 6–10, 14) compared with an unconjugated control (black; unimer). The thermal behavior of all 6 CP-small molecule conjugates that formed nanoparticles was identical, and is hence plotted together as the mean of the T_t of each of the CP-small molecule conjugate, and the error bars are the standard deviation. The lines are linear fits to the data. (B) DLS results of the CP-conjugate of compound 8 with a Log(D) of 2.1, which shows the increase in R_h from ~6 nm corresponding to unimers prior to conjugation to the formation of nanoparticles with a R_h of ~33 nm after conjugation. (C) Relationship between the T_t (left Y-axis, data in red) and R_h (right Y-axis, data in black) as a function of Log(D) for all 14 conjugates. As the Log(D) increases to greater than 1.5, the particle R_h increases from the unimer size of 6 nm to nanoparticles with an R_h of 30 – 55 nm for different conjugates. The concentration dependence of the T_t (slope from A) decreases from an average value of −5.5 to −1.0 °C/Log(concentration). The curves in (C) are solely a guide to the eye.

Figure 3. Cryo-TEM

CP conjugates were imaged via cryo-TEM in phosphate buffered saline. A) N-methoxycarbonylmaleimide (Compound 1) did not form nanoparticles and is displayed as a negative control. The remaining conjugates spontaneously formed nanoparticles: B) n-benzylmaleimide (Compound 7); C) n-[4-(2-benzimidazolyl)phenyl]maleimide (Compound 9); D) 2-maleimido fluorene (Compound 12); E) n-(1-pyrenyl)maleimide (Compound 14); and F) paclitaxel. Scale bars represent 100 nm.

Figure 4

The apparent coordination number (#CPs per NP) versus the Log(D) of the small molecules conjugated to each CP. Above the threshold of Log(D) = ~1.5, the number of CPs per nanoparticle (#CPs/NP) increases with hydrophobicity of the conjugated small molecule [Log(D)]. The blue diamond, green square, and red triangle markers indicate gemcitabine, oxycodone, and paclitaxel, respectively. The lines are drawn solely as a guide to the eye.

Figure 5. Design of thermally sensitive CP nanoparticles

The transition temperature (T_t) as a function of CP₁ and CP₂ concentration. The CP₁ data represents an average of conjugates 6–10 and 14, whereas the CP₂ data represents an average of conjugates 6, 7, and 14. The black lines represent the targeted range of hyperthermia (38–42°C). The error bars are the standard deviation. The lines are linear fits to the data.