Curvature-Coupled Hydration of Semicrystalline Polymer Amphiphiles Yields flexible Worm Micelles but Favors Rigid Vesicles: Polycaprolactone-Based Block Copolymers

Crystallization processes are in general sensitive to detailed conditions, but the present understanding of underlying mechanisms is insufficient. A crystallizable chain within a diblock copolymer assembly, for example, is expected to couple curvature to crystallization and thereby impact rigidity as well as preferred morphology, and yet the effects on dispersed phases have remained unclear. The hydrophobic polymer polycaprolactone (PCL) is semicrystalline in bulk (Tm = 60 °C) and is shown here to generate flexible worm micelles or rigid vesicles in water from several dozen poly(ethylene oxide)-based diblocks (PEO−PCL). Despite the fact that “worms” have a mean curvature between that of vesicles and spherical micelles, “worms” are seen only within a narrow, process-dependent wedge of morphological phase space that is deep within the vesicle phase. Fluorescence imaging shows worms are predominantly in one of two states − either entirely flexible with dynamic thermal undulations or fully rigid; only a few worms appear rigid at room temperature (T ≪ Tm), indicating suppression of crystallization by both curvature and PCL hydration. Worm rigidification, which depends on molecular weight, is also prevented by copolymerization of caprolactone with just 10% racemic lactide that otherwise has little impact on bulk crystallinity. In contrast to worms, vesicles of PEO−PCL are always rigid and typically leaky. Defects between crystallite domains induce dislocation-roughening with focal leakiness although select PEO−PCLwhich classical surfactant arguments would predict make wormsyield vesicles that retain encapsulant and appear smooth, suggesting a gel or glassy state. Hydration in dispersion thus tends to selectively soften high curvature microphases

Curvature-Coupled Hydration of Semicrystalline Polymer Amphiphiles Yields flexible Worm Micelles but Favors Rigid Vesicles: Polycaprolactone-Based Block Copolymers

Crystallization processes are in general sensitive to detailed conditions, but the present understanding of underlying mechanisms is insufficient. A crystallizable chain within a diblock copolymer assembly, for example, is expected to couple curvature to crystallization and thereby impact rigidity as well as preferred morphology, and yet the effects on dispersed phases have remained unclear. The hydrophobic polymer polycaprolactone (PCL) is semicrystalline in bulk (Tm = 60 °C) and is shown here to generate flexible worm micelles or rigid vesicles in water from several dozen poly(ethylene oxide)-based diblocks (PEO−PCL). Despite the fact that “worms” have a mean curvature between that of vesicles and spherical micelles, “worms” are seen only within a narrow, process-dependent wedge of morphological phase space that is deep within the vesicle phase. Fluorescence imaging shows worms are predominantly in one of two states − either entirely flexible with dynamic thermal undulations or fully rigid; only a few worms appear rigid at room temperature (T ≪ Tm), indicating suppression of crystallization by both curvature and PCL hydration. Worm rigidification, which depends on molecular weight, is also prevented by copolymerization of caprolactone with just 10% racemic lactide that otherwise has little impact on bulk crystallinity. In contrast to worms, vesicles of PEO−PCL are always rigid and typically leaky. Defects between crystallite domains induce dislocation-roughening with focal leakiness although select PEO−PCLwhich classical surfactant arguments would predict make wormsyield vesicles that retain encapsulant and appear smooth, suggesting a gel or glassy state. Hydration in dispersion thus tends to selectively soften high curvature microphases

Curvature-Coupled Hydration of Semicrystalline Polymer Amphiphiles Yields flexible Worm Micelles but Favors Rigid Vesicles: Polycaprolactone-Based Block Copolymers

Crystallization processes are in general sensitive to detailed conditions, but the present understanding of underlying mechanisms is insufficient. A crystallizable chain within a diblock copolymer assembly, for example, is expected to couple curvature to crystallization and thereby impact rigidity as well as preferred morphology, and yet the effects on dispersed phases have remained unclear. The hydrophobic polymer polycaprolactone (PCL) is semicrystalline in bulk (Tm = 60 °C) and is shown here to generate flexible worm micelles or rigid vesicles in water from several dozen poly(ethylene oxide)-based diblocks (PEO−PCL). Despite the fact that “worms” have a mean curvature between that of vesicles and spherical micelles, “worms” are seen only within a narrow, process-dependent wedge of morphological phase space that is deep within the vesicle phase. Fluorescence imaging shows worms are predominantly in one of two states − either entirely flexible with dynamic thermal undulations or fully rigid; only a few worms appear rigid at room temperature (T ≪ Tm), indicating suppression of crystallization by both curvature and PCL hydration. Worm rigidification, which depends on molecular weight, is also prevented by copolymerization of caprolactone with just 10% racemic lactide that otherwise has little impact on bulk crystallinity. In contrast to worms, vesicles of PEO−PCL are always rigid and typically leaky. Defects between crystallite domains induce dislocation-roughening with focal leakiness although select PEO−PCLwhich classical surfactant arguments would predict make wormsyield vesicles that retain encapsulant and appear smooth, suggesting a gel or glassy state. Hydration in dispersion thus tends to selectively soften high curvature microphases

Curvature-Coupled Hydration of Semicrystalline Polymer Amphiphiles Yields flexible Worm Micelles but Favors Rigid Vesicles: Polycaprolactone-Based Block Copolymers

Crystallization processes are in general sensitive to detailed conditions, but the present understanding of underlying mechanisms is insufficient. A crystallizable chain within a diblock copolymer assembly, for example, is expected to couple curvature to crystallization and thereby impact rigidity as well as preferred morphology, and yet the effects on dispersed phases have remained unclear. The hydrophobic polymer polycaprolactone (PCL) is semicrystalline in bulk (Tm = 60 °C) and is shown here to generate flexible worm micelles or rigid vesicles in water from several dozen poly(ethylene oxide)-based diblocks (PEO−PCL). Despite the fact that “worms” have a mean curvature between that of vesicles and spherical micelles, “worms” are seen only within a narrow, process-dependent wedge of morphological phase space that is deep within the vesicle phase. Fluorescence imaging shows worms are predominantly in one of two states − either entirely flexible with dynamic thermal undulations or fully rigid; only a few worms appear rigid at room temperature (T ≪ Tm), indicating suppression of crystallization by both curvature and PCL hydration. Worm rigidification, which depends on molecular weight, is also prevented by copolymerization of caprolactone with just 10% racemic lactide that otherwise has little impact on bulk crystallinity. In contrast to worms, vesicles of PEO−PCL are always rigid and typically leaky. Defects between crystallite domains induce dislocation-roughening with focal leakiness although select PEO−PCLwhich classical surfactant arguments would predict make wormsyield vesicles that retain encapsulant and appear smooth, suggesting a gel or glassy state. Hydration in dispersion thus tends to selectively soften high curvature microphases

Volumetric CHIKV VLP yield enhancement resulting from adaptation of <i>Sf</i>21 to elevated culture pH.

<p>(<b>A</b>) CHIKV VLP concentration of culture supernatants from <i>Sf</i>21 and <i>Sf</i>Basic small-scale shake flask cultures. Error bars represent 95% confidence intervals (N = 10 independent infections). (<b>B</b>) CHIKV VLP concentration of culture supernatants from scale-up of <i>Sf</i>Basic from shake flasks (SF) into a PID controlled 3-L stirred tank bioreactor (STBR).</p

Immunogenicity assay titers from guinea pig sera after vaccination with adjuvanted <i>Sf</i>Basic-derived and HEK293-derived CHIKV VLPs.

<p>(<b>A</b>) Anti-CHIKV IgG geomean titer and CHIKV 181/25 geomean neutralizing titer (NT100) at study day 14 (14 days after first dose). (<b>B</b>) Anti-CHIKV IgG geomean titer and CHIKV 181/25 geomean neutralizing titer (NT100) at study day 21 (7 days after second dose). Geomean IgG ELISA background from pre-vaccination sera is indicated by a dashed line. Error bars represent 95% confidence intervals (N = 4 animals per group), and asterisks indicate a statistically significant increase in titer over background (p<0.05).</p

Biophysical characterization of CHIKV VLPs derived from SfBasic cells and comparison to a VLP standard derived from HEK293 cells.

<p>(<b>A</b>) Western blot of density gradient ultracentrifugation fractions containing CHIKV VLPs, using E1, E2, and capsid peptide-specific antibodies. (<b>B</b>) Dynamic light scattering (DLS) distribution of purified CHIKV VLP diameters (Ø). (<b>C</b>) Raw/unprocessed and 2D class average transmission electron microscopy images of purified CHIKV VLPs.</p

CHIKV structural polyprotein expression and processing in AcMNPV-CHIKV37997 infected <i>Sf</i>21 cells and pV1JNS-CHIKV37997 transfected HEK293 cells.

<p>Cell lysate Western blots depicting (<b>A</b>) E2 expression and processing, detected by an E2 peptide-specific antibody. (<b>B</b>) E1 expression, detected by an E1 peptide-specific antibody. (<b>C</b>) E1/E2 (co-migrating) and capsid expression and processing, detected by an anti-CHIKV polyclonal antibody. AcMNPV-GFP infected <i>Sf</i>21 lysate was included as a negative control for insect cells and baculovirus vector.</p

Transmission electron microscopy (TEM) images of thin-sections of AcMNPV-CHIKV37997 infected <i>Sf</i>21 cells and pV1JNS-CHIKV37997 transfected HEK293 cells.

<p>(<b>A</b>) Putative CHIKV capsids formed in the cytoplasm of HEK293 cells. Capsid diameters are approximately 30–35 nm. (<b>B</b>) Baculovirus showing hallmark multiple nucleocapsids per envelope, with very electron dense nucleocapsids. Nucleocapsid diameter is approximately 40 nm. (<b>C</b>) Putative CHIKV capsids formed in the cytoplasm of <i>Sf</i>21 cells. Capsid diameters are approximately 30–35 nm. Scale bar is equivalent for all images and represents 500 nm.</p

Characterization of the <i>Sf</i>Basic cell line and comparison to the parental <i>Sf</i>21 cell line.

<p>(<b>A</b>) Cell diameter (Ø) distribution histogram with a representative bright field image. (<b>B</b>) Fluorescence microscopy images of propidium iodide stained cells. Mean fluorescence intensity (MFI) of the G1-phase cell population from flow cytometry cell cycle analysis of the same sample is indicated below the corresponding image. (<b>C</b>) Cell line population doubling time (PDT) as a function of growth medium pH. The growth medium was Sf-900II-BES-MISS, adjusted to various pH levels by titration with 1 N NaOH.</p