Multiresponsive Nanogels for Targeted Anticancer Drug Delivery

Nanogels with a biomolecular coating (biocoating) were shown to be capable of triggered delivery of anticancer drug Doxorubicin. The biocoating was formed utilizing binding between glycogen and the tetra-functional lectin Concanavalin A, which can be triggered to disassemble (and release) upon exposure to glucose and changes in solution pH. We also show the nanogel’s thermoresponsivity can be used to accelerate Doxorubicin release. Moreover, we showed that transferrin immobilized on the nanogel surface could accelerate nanogel uptake by cancer cells. In these experiments, we showed that Doxorubicin was able to be released to the nucleus of human liver cancer cell line (HepG2) within 3 h. Doxorubicin-loaded nanogels exhibit a strong growth inhibition ability toward HepG2. This investigation showcases how nanogel design and chemistry can be tuned to achieve useful biomedical applications

Tuning Hsf1 levels drives distinct fungal morphogenetic programs with depletion impairing Hsp90 function and overexpression expanding the target space

<div><p>The capacity to respond to temperature fluctuations is critical for microorganisms to survive within mammalian hosts, and temperature modulates virulence traits of diverse pathogens. One key temperature-dependent virulence trait of the fungal pathogen <i>Candida albicans</i> is its ability to transition from yeast to filamentous growth, which is induced by environmental cues at host physiological temperature. A key regulator of temperature-dependent morphogenesis is the molecular chaperone Hsp90, which has complex functional relationships with the transcription factor Hsf1. Although Hsf1 controls global transcriptional remodeling in response to heat shock, its impact on morphogenesis remains unknown. Here, we establish an intriguing paradigm whereby overexpression or depletion of <i>C</i>. <i>albicans HSF1</i> induces morphogenesis in the absence of external cues. <i>HSF1</i> depletion compromises Hsp90 function, thereby driving filamentation. <i>HSF1</i> overexpression does not impact Hsp90 function, but rather induces a dose-dependent expansion of Hsf1 direct targets that drives overexpression of positive regulators of filamentation, including Brg1 and Ume6, thereby bypassing the requirement for elevated temperature during morphogenesis. This work provides new insight into Hsf1-mediated environmentally contingent transcriptional control, implicates Hsf1 in regulation of a key virulence trait, and highlights fascinating biology whereby either overexpression or depletion of a single cellular regulator induces a profound developmental transition.</p></div

<i>HSF1</i> depletion induces filamentation independently of changes in <i>HSP90</i> expression.

<p>Strains were grown in the absence or presence of 20 μg/mL DOX in rich medium at 30°C. a) A strain was engineered where <i>HSP90</i> is under the control of a constitutive promoter, <i>ACT1p</i>, in the <i>tetO-HSF1/hsf1Δ</i> background. In the <i>ACT1p-HSP90</i> strain, <i>HSF1</i> levels were depleted (left panel) with DOX without altering <i>HSP90</i> expression (right panel). <i>HSF1</i> and <i>HSP90</i> transcript levels were normalized to <i>ACT1</i> and <i>GPD1</i>. Data are means +/- standard error of the means for triplicate samples. ** indicates P value <0.01, ns indicates no significant difference, unpaired t test. b) <i>HSF1</i> depletion induces filamentation independently of changes in <i>HSP90</i> expression.</p

Hsf1 binds to an expanded set of taget genes upon overexpression, inducing the expression of positive regulators of filamentation.

<p>a) ChIP-seq was performed to determine the targets of Hsf1 in strains expressing basal (<i>HSF1-TAP/HSF1</i>) and overexpressed (<i>tetO-HSF1-TAP/tetO-HSF1</i>) levels of Hsf1, which identified an expansion of Hsf1 targets upon overexpression. Genes previously identified as Hsf1 targets and genes involved in <i>C</i>. <i>albicans</i> filamenation are indicated. b) Hsf1 signals at the summit of Hsf1 ChIP-seq peaks in wild-type (basal conditions, top) and <i>HSF1</i> overexpression (bottom) strains. Weak Hsf1 ChIP-seq signal can be seen at the overexpression-specific target sites under basal conditions, suggesting that Hsf1 also binds these sites under basal conditions albeit at a lower level. Legend shows Hsf1 ChIP-seq signal across a 2 kb window spanning Hsf1 binding summits identified by MACS analysis. c) Both basal and <i>HSF1</i> overexpression-specific targets contain the conserved heat shock element (HSE) binding sites. The percentage of ChIP-seq peaks with the HSE motifs TTCnnGAA, TTCn⁷TTC, TTCnnGAAnnTTC, or variations of the motifs termed triple cis motifs (GAA n^0-3 GAA n^0-3 GAA), triple trans motifs (GAA n^0-3 TTC n^0-3 GAA) or triple trans/cis motifs (TTC n^0-3 GAA n^0-3 GAA, GAA n^0-3 GAA n^0-3 TTC, TTC n^0-3 TTC n^0-3 GAA, GAA n^0-3 TTC n^0-3 TTC) are shown for basal and overexpression-specific targets. d) MA plot showing difference in gene expression between wild-type and <i>HSF1</i> overexpression strains. Genes more than 1.5-fold up-regulated in the <i>HSF1</i> overexpression strain as compared to the wild-type strain are shown in red, whereas genes more than 1.5-fold down-regulated are shown in blue. Genes of interest based on their roles in filamentation are indicated. <i>HSF1</i> is highlighted in purple. e) A Euler diagram depicting the overlap between differentially regulated genes upon <i>HSF1</i> overexpression determined by RNA-seq and Hsf1 bound targets determined by ChIP-seq. Hsf1 targets includes those promoters bound in either the wild-type or overexpression strain. Selected genes associated with filamentation are indicated in the diagram. f) A heat map showing gene expression changes upon <i>HSF1</i> overexpression for filamentation regulators. Colored dots are included to indicate whether the respective gene is bound by Hsf1 in the wild-type strain (in red) or only when Hsf1 is overexpressed (in orange). The colour bar depicts the change in expression as determined by RNA-seq.</p

<i>HSF1</i> overexpression induces filamentation independently of changes in <i>HSP90</i> transcript or protein levels, and function.

<p>a) To monitor the effects of <i>HSF1</i> overexpression on filamentation independently of changes in <i>HSP90</i> expression, we engineered strains where <i>HSP90</i> is under the control of a constitutive promoter, <i>ACT1p</i>, in the <i>tetO-HSF1/tetO-HSF1</i> strain. <i>HSF1</i> and <i>HSP90</i> transcript levels were normalized to <i>ACT1</i> and <i>GPD1</i>. Data are means +/- standard error of the means for triplicate samples. In the <i>ACT1p-HSP90</i> strain, <i>HSF1</i> levels are overexpressed (left panel) but <i>HSP90</i> levels do not differ substantially from the wild-type levels (right panel). * indicates P value <0.05, ns indicates no significant difference, unpaired t test. b) Western blot analysis shows that overexpression of <i>HSF1</i> does not impact the levels of Hsp90 protein. Tubulin serves as loading control. c) <i>HSF1</i> overexpression induces filamentation independently of changes in <i>HSP90</i> expression. Strains were grown in the absence of DOX at 30°C. d) Western blot analysis was performed to assay if <i>HSF1</i> overexpression compromises Hsp90 function by monitoring the Hsp90 client protein Hog1. Cells were treated with 5 mM hydrogen peroxide (H₂O₂) for 10 minutes to induce oxidative stress before protein extraction. Overexpression of <i>HSF1</i> does not affect the stability of Hog1, nor does it block its activation. Tubulin serves as a loading control. e) <i>HSF1</i> overexpression does not cause hypersensitivity to the Hsp90 inhibitor geldanamycin (Gda). Growth curves were generated by measuring the optical density of cells grown in the absence and presence of 20 μg/mL DOX in the presence of 3.13 μM geldanamycin. Optical density measurements at 595 nm were taken every 15 minutes with a TECAN plate reader. Experiment was performed in biological quadruplicate, with one representative graph shown.</p

<i>HSF1</i> overexpression and depletion induce filaments that differ in their morphology, nuclear content and dependence on Ras1.

<p>a) Reduced temperature blocks filamentation in response to <i>HSF1</i> overexpression but not in response to <i>HSF1</i> depletion. Strains were grown in the absence or presence of high levels of DOX (20 μg/mL) at the indicated temperature. b) Filaments induced by <i>HSF1</i> overexpression and depletion have distinct features. Cell walls and septa were visualized using calcofluor white and the nuclei were visualized using a strain with the nucleolar protein, Nop1, GFP tagged. c) Filaments induced by <i>HSF1</i> depletion have a significant increase in the percentage of cells that are multinucleate compared to filaments induced by <i>HSF1</i> overexpression. The number of nuclei in at least 300 cells were counted for each condition, for two biological replicates. Means are graphed with the error bars displaying the standard error of means. Unpaired t-test indicates a significant difference in percentage of multinucleate cells, P value is 0.0056. d) Filaments induced by <i>HSF1</i> depletion, but not overexpression, are dependent on the GTPase Ras1. Strains were grown in rich medium with 80 mg/L uridine added, in the presence of no DOX, 0.1 μg/mL DOX (Low DOX), or 20 μg/mL DOX (High DOX) at 30°C.</p

<i>HSF1</i> depletion induces filamentation by compromising Hsp90 function, independently of changes in <i>HSP90</i> expression.

<p>a) Western blot analysis was performed to assay if <i>HSF1</i> depletion compromises Hsp90 function by monitoring the Hsp90 client protein Hog1. Strains were grown in the absence or presence of 20 μg/mL DOX. Cells were treated with 5 mM hydrogen peroxide (H₂O₂) for 10 minutes to induce oxidative stress before protein extraction. Depletion of Hsf1 reduces the levels of phosphorylated Hog1 (pHog1) but not total Hog1 levels, even in the isogenic strain with constitutive <i>HSP90</i> expression. Tubulin levels serve as loading control. WT indicates the wild type control. Experiment was performed in biological quadruplicate and a representative image is shown, with the western blots for the additional replicates shown in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007270#pgen.1007270.s015" target="_blank">S8 Fig</a>.</b> b) <i>HSF1</i> depletion causes a hypersensitivity to the Hsp90 inhibitor geldanamycin, even when <i>HSP90</i> expression is constitutive and independent of Hsf1. Growth curves were generated by measuring the optical density of cells grown in the absence or presence of 20 μg/mL DOX in the presence of a concentration of geldanamycin that does not inhibit the growth of the wild-type strain (3.13 μM). Optical density at 595 nm was measured every 15 minutes with a TECAN plate reader grown with high orbital shaking at 30°C. Experiment was performed in biological quadruplicate, with one representative graph shown. c) <i>HSF1</i> depletion causes filamentation at lower concentrations of geldanamycin (Gda) than is necessary to induce filamentation of the wild-type strain. Strains were grown in static conditions in the presence of no DOX or 20 μg/mL DOX, and in the presence of no geldanamycin (Gda) or 3.13 μM geldanamycin (Gda) at 30°C. In static growth conditions, 3.13 μM geldanamycin does not inhibit growth of the strains.</p

Overexpression of <i>HSF1</i> induces filamentation through overexpression of positive regulators of filamentation.

<p>a) Overexpression of <i>HSF1</i> drives the overexpression of <i>UME6</i> and <i>BRG1</i>. Strains were grown in the presence of no DOX, 0.1 μg/mL DOX (Low DOX), or 20 μg/mL DOX (High DOX) at 30°C. <i>UME6</i> and <i>BRG1</i> transcript levels were normalized to <i>ACT1</i> and <i>GPD1</i>. Data are means +/- standard error of the means for triplicate samples. b) Overexpression of <i>UME6</i> or <i>BRG1</i> induces filamentation. Strains with a tetracycline-inducible promoter (<i>tetON</i>) driving the expression of target genes were grown in the presence of 50 μg/mL DOX to induce overexpression at 30°C. Overexpression of <i>LEU3</i> acts as a negative control. c) Loss of Ume6 or Brg1 blocks filamentation induced by <i>HSF1</i> overexpression. Strains were grown in the absence of DOX at 30°C.</p

Model for how <i>HSF1</i> overexpression and depletion induce filamentation.

<p>Under basal conditions, <i>HSF1</i> levels are moderate and <i>C</i>. <i>albicans</i> exists in the yeast form. A reduction in <i>HSF1</i> levels leads to filamentous growth both by compromising Hsp90 function and through circuitry that is independent of Hsp90 but dependent on Efg1. An increase in <i>HSF1</i> levels induces a dose-dependent expansion of Hsf1 direct targets that drives overexpression of positive regulators of morphogenesis, including Brg1 and Ume6, and decreased expression of negative regulators of morphogenesis, such as Nrg1, resulting in filamentous growth. Filaments induced by <i>HSF1</i> overexpression and depletion are structurally distinct, require different genetic circuitry and are induced through distinct mechanisms.</p

The genetic circuitry through which Hsf1 and Hsp90 regulate filamentation are distinct.

<p>a) Filaments induced by compromised Hsp90 function are not dependent on the transcription factors Rob1 or Efg1, but are dependent on the protease Kex2 and the cell cycle checkpoint protein Bub2. Cultures were grown in the absence or presence of 10 μM of geldanamycin (Gda) for 6.5 hours to inhibit Hsp90 function. b) Unlike filaments induced by compromised Hsp90 function, filaments induced by <i>HSF1</i> depletion are not dependent on Bub2 but are dependent on Efg1. Cultures were treated with no DOX, 0.1 μg/mL DOX (Low DOX), or 20 μg/mL DOX (High DOX) at 30°C. For both a) and b), blue outlines indicate that the homozygous deletion mutant of the morphogenetic regulator filaments to a comparable level as the non-mutant control and red outlines indicates that the mutant blocks filamentation.</p