Temperature triggered self-assembly of polypeptides into multivalent spherical micelles

Abstract

We report herein thermally responsive elastin-like polypeptides (ELPs) in a linear AB diblock architecture with an N-terminal peptide ligand that self-assemble into spherical micelles when heated slightly above body temperature. A series of 10 ELP block copolymers (ELP(BC)'s ) with different molecular weights and hydrophilic-to-hydrophobic block ratios were genetically synthesized by recursive directional ligation. The self-assembly of these polymers from unimers into micelles was investigated by light scattering, fluorescence spectroscopy, and cryo-TEM. These ELP(BC)'s undergo two phase transitions as a function of solution temperature: a unimer-to-spherical micelle transition at an intermediate temperature and a micelle-to-bulk aggregate transition at a higher temperature when the hydrophilic-to-hydrophobic block ratio is between 1:2 and 2:1. The critical micelle temperature is controlled by the length of the hydrophobic block, and the size of the micelle is controlled by both the total ELP(BC) length and hydrophilic-to-hydrophobic block ratio. These polypeptide micelles display a critical micelle concentration in the range 4-8 microM demonstrating the high stability of these structures. These studies have also identified a subset of ELP(BC)'s bearing terminal peptide ligands that are capable of forming multivalent spherical micelles that present multiple copies of the ligand on their corona in the clinically relevant temperature range 37-42 degrees C and target cancer cells. These ELP(BC)'s may be useful for drug targeting by thermally triggered multivalency. More broadly, the design rules uncovered by this study should be applicable to the design of other thermally reversible nanoparticles for diverse applications in medicine and biology.

Figure 1

Temperature triggered self-assembly of an ELPBC to form multivalent spherical micelles. An N-terminal ELP[V₁A₈G₇-n] gene (hydrophilic, high T_t) and C-terminal ELP[V₅-n] gene (hydrophobic, low T_t) are seamlessly fused together to create a gene that encodes an ELP_BC. When the size and ratio of the blocks are correctly selected, the ELP_BC self-assembles into a spherical micelle at ~40 °C. In the cartoon shown, upon self-assembly the spherical micelles present multiple copies of an affinity targeting moiety (green triangle) and sequester a drug or imaging agent (lightning bolt) within the core of the micelle.

Figure 2

Turbidity profile of ELP-64/60 at various concentrations as a function of temperature. Turbidity profiles were obtained by monitoring optical density (OD) at 350 nm in PBS as the solution was heated at a rate of 1 °C/min. The increase in OD indicates formation of ELP particles larger than an ELP unimer. The sharpness of the micelle-to-aggregate transition can be obtained from dOD/dT as shown in the top portion of this figure.

Figure 3

Dependence of transition temperature (T_t) on ELP_BC concentration for the 6 ELP_BCs that formed nanoparticles in PBS. The first T_t is defined by the temperature at the first apparent increase in turbidity (filled circle). The second T_t is defined by the temperature at the maximum in dOD/dT (filled hourglass). Both the first and second T_t had a decreasing logarithmic dependence on ELP concentration. The dependence of T_t on ELP concentration was quantified by fitting these data with the following equation, T_t=m*ln([ELP]) + b, shown by a solid line. The parent ELP[V₅-n]'s thermal response is shown by the dashed line.

Figure 4

Hydrodynamic radius (R_h) and turbidity of A) ELP-96/60 and B) ELP-64/90 versus temperature. The DLS data were acquired at a 50 degree angle in PBS at a 25 μM ELP concentration. The R_h is displayed as the fast mode of a biexponential fit at temperatures below the first T_t and from a monoexponential fit at temperatures above the first T_t.

Figure 5

A) dynamic light scattering (DLS) and B) static light scattering (SLS) collected at multiple angles for ELP64/90 in PBS at 37 °C and an ELP_BC concentration of 25 μM. Angle is expressed as the absolute value of the scattering vector (q) squared. A) DLS reveals no angular dependence of apparent diffusion coefficient (D_app) indicated by the solid line plotted with a slope of zero. The graph is displayed with ± 10% of the mean D_app value. The z-averaged diffusion coefficient (D_z) is the average of all six measurements and the hydrodynamic radius (R_h) is calculated from the Stokes-Einstein equation. B) The radius of gyration (R_g) and apparent molecular weight (MW_app) were determined with SLS by calculating the slope and intercept of the solid line relating the inverse of the scattered intensity to q².

Figure 6

Dependence of pyrene fluorescence on A) temperature and B) concentration for 25 uM ELP-64/90 in PBS. A) I₁/I₃ of pyrene decreased from 20-60 °C indicating a reduction in the polarity of the ELP-64/90 solution. There was a pronounced decrease in I₁/I₃ at temperatures above the CMT and below the second transition. B) I₁/I₃ at the inflection point of each temperature scan (as shown in A) was plotted as a function of ELP-64/90 concentration. The inflection point of a sigmoid fit (solid line) was defined as the CMC. Data are the mean ± SD in B (n = 3).

Figure 7

PC₃P I_E/I_M ratio as a function of temperature for ELP-64/90 in PBS at 25 μM. This ratio increased at the CMT of ELP-64/90 (~40 °C) and at the bulk aggregation temperature for the control ELP[V₅A₂G₃-150] (~42 °C), indicating a greater microviscosity in the microenvironment of PC₃P. The I_E/I_M ratio decreased at the second T_t of ELP-64/90 (~50 °C). Date are mean ± SD (n = 3).

Figure 8

Cryo-TEM of A) ELP-64/60, B) ELP-96/90, C) ELP-64/90, and D) ELP-96/60 vitrified at a temperature that induced micelle formation in PBS at 25 μM. The bar is 20 nm in all images.

Figure 9

Hydrodynamic radius (R_h) and turbidity of ELP-96/60 (open symbols and dashed line) and NGR-ELP-96/60 (closed symbols and solid line) versus temperature in PBS at a 25 μm ELP concentration. The turbidity profile is shown from an upward thermal ramp (1 °C/min). DLS data were acquired at fixed angle of 90 degrees and a 25 μM ELP_BC concentration using a DynaPro LSR instrument. The autocorrelation function was analyzed using a cumulant and regularization algorithm for Rayleigh spheres provided by the manufacturer to determine the hydrodynamic radii. Data are reported as are mean ± polydispersity.

Figure 10

Confocal images illustrating the effects of heat and ligand presentation on cellular uptake of NGR-ELP- and ELP-96/60-Alexa488. Size bars represent 20 μM. NGR-ELP-96/60-Alexa488 (green) demonstrated limited accumulation in HT-1080 cells (red) in monovalent form (T < Body T, A) but accumulated in polyvalent micellar form (T >Body T, B). ELP-96/60 accumulated in the cell at low amounts both in monovalent (T < Body T, C) and polyvalent (T > Body T, D).