Highly Efficient and Safe Delivery of VEGF siRNA by Bioreducible Fluorinated Peptide Dendrimers for Cancer Therapy

RNA interference (RNAi) has a great promise in treating various acquired and hereditary diseases. However, it remains highly desirable to develop new delivery system to circumvent complex extra- and intracellular barriers for successful clinical translation. Here, we report on a versatile polymeric vector, bioreducible fluorinated peptide dendrimers (BFPD), for efficient and safe small interfering RNA (siRNA) delivery. In virtue of skillfully integrating all of the unique advantages of reversible cross-linking, fluorination, and peptide dendrimers, this novel vector can surmount almost all extra- and intracellular barriers associated with local siRNA delivery through highly improved physiological stability and serum resistance, significantly increased intratumoral enrichment, cellular internalization, successful facilitation of endosomal escape, and cytosolic siRNA release. BFPD polyplexes, carrying small interfering vascular endothelial growth factor (siVEGF), demonstrated excellent VEGF silencing efficacy (∼65%) and a strong capability for inhibiting HeLa cell proliferation. More importantly, these polyplexes showed superior performance in long-term enrichment in the tumor sites and had a high level of tumor growth inhibition. Furthermore, these polyplexes not only exhibited excellent in vivo antitumor efficacy but also demonstrated superior biocompatibility, compared with LPF2000, both in vivo and in vitro. These findings indicate that BFPD is an efficient and safe siRNA delivery system and has remarkable potential for RNAi-based cancer treatment

TrRas1 regulates filamentation and sporulation in <i>T. reesei</i>.

<p>The colonial phenotypes (A) and hyphal morphology (B) of strains TU-6, Δ<i>TrRas1</i> and <i>cbh1-TrRas1</i> cultured on PDA plates (glucose) or MM plates (cellulose) for 5 days. The microscopic images were captured using DIC. Scale bars are shown in the figure.</p

Growth of TU-6, Δ<i>TrRas1</i> and Δ<i>TrRas2</i> on different carbon sources.

<p>Strains were grown on plates containing minimal medium supplemented with 1% glucose, glycerol, lactose or cellulose at 30°C for 4 days.</p

Ras GTPases Modulate Morphogenesis, Sporulation and Cellulase Gene Expression in the Cellulolytic Fungus <em>Trichoderma reesei</em>

<div><h3>Background</h3><p>The model cellulolytic fungus <em>Trichoderma reesei</em> (teleomorph <em>Hypocrea jecorina</em>) is capable of responding to environmental cues to compete for nutrients in its natural saprophytic habitat despite its genome encodes fewer degradative enzymes. Efficient signalling pathways in perception and interpretation of environmental signals are indispensable in this process. Ras GTPases represent a kind of critical signal proteins involved in signal transduction and regulation of gene expression. In <em>T. reesei</em> the genome contains two Ras subfamily small GTPases TrRas1 and TrRas2 homologous to Ras1 and Ras2 from <em>S. cerevisiae</em>, but their functions remain unknown.</p> <h3>Methodology/Principal Findings</h3><p>Here, we have investigated the roles of GTPases TrRas1 and TrRas2 during fungal morphogenesis and cellulase gene expression. We show that both TrRas1 and TrRas2 play important roles in some cellular processes such as polarized apical growth, hyphal branch formation, sporulation and cAMP level adjustment, while TrRas1 is more dominant in these processes. Strikingly, we find that TrRas2 is involved in modulation of cellulase gene expression. Deletion of <em>TrRas2</em> results in considerably decreased transcription of cellulolytic genes upon growth on cellulose. Although the strain carrying a constitutively activated <em>TrRas2^G16V</em> allele exhibits increased cellulase gene transcription, the <em>cbh1</em> and <em>cbh2</em> expression in this mutant still strictly depends on cellulose, indicating TrRas2 does not directly mediate the transmission of the cellulose signal. In addition, our data suggest that the effect of TrRas2 on cellulase gene is exerted through regulation of transcript abundance of cellulase transcription factors such as Xyr1, but the influence is independent of cAMP signalling pathway.</p> <h3>Conclusions/Significance</h3><p>Together, these findings elucidate the functions for Ras signalling of <em>T. reesei</em> in cellular morphogenesis, especially in cellulase gene expression, which contribute to deciphering the powerful competitive ability of plant cell wall degrading fungi in nature.</p> </div

TrRas1 and TrRas2 play similar roles in increasing cAMP content.

<p>(A) The cAMP levels of TU-6 and Δ<i>TrRas1</i> on PDA plates. (B) Comparison of the cAMP levels of strains TU-6-Z, Δ<i>TrRas2</i> and <i>PAnigpdA</i>-<i>TrRas2^G16V</i>. Cultivation of respective strains and cAMP level determination were performed as described in the methods. The cAMP content is given in relation to parental strain.(C) Phenotypes of wild-type and Δ<i>TrRas1</i> upon growth on PDA plates in the presence or absence of 2 mM cAMP in the medium. Strains were grown for 7 days at 30°C. Scale bars are shown in the figure.</p

Schematic model depicting TrRas1/2 signalling in regulating morphogenesis and cellulase gene expression in <i>T. reesei</i>.

<p>The network described in this work is indicated by black lines, while regulatory relationships described previously are indicated by gray lines. Solid lines indicate genetically determined steps and dashed lines represent hypothesized steps. (A) Activation of GNA3 results in increased cAMP levels, which regulates the PKAC1 to control vegetative growth <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048786#pone.0048786-Schuster1" target="_blank">[31]</a>. Morphogenesis is also regulated by GTPases TrRas1 and TrRas2, which probably act through cAMP-PKAC1 pathway and MAP kinase pathway to regulate filamentation, vegetative growth and sporulation. TrRas1 and TrRas2 possess similar roles in controlling these cellular processes. (B) In the presence of cellulose, TrRas2 senses extracellular signals (e.g., light) and acts through an unidentified pathway to modulate the transcript abundance of transcriptional regulators Xyr1, Ace1 and Cre1, which directly or indirectly regulate the cellulase gene expression. TrRas2 positively influences the transcription of <i>xyr1</i> and <i>cre1</i> but negatively influences <i>ace1</i> expression. Xyr1 (activator) and Ace1 (repressor) subsequently regulate cellulase genes (<i>cbh1</i> and <i>cbh2</i>) expression. Meanwhile, Ace1 negatively influences the <i>xyr1</i> expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048786#pone.0048786-MachAigner1" target="_blank">[41]</a>, while Cre1 has a positive effect on the transcription of <i>xyr1 </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048786#pone.0048786-Portnoy1" target="_blank">[40]</a>. In addition, Xyr1 may also be repressed by an unknown element in the presence of glucose; while under cellulose-inducing conditions, the repressor departs from Xyr1 thus making this activator being unrepressing status.</p

TrRas2 influences cellulase formation in <i>T. reesei</i>.

<p>Comparison of Cellobiohydrolase activity (A) and extracellular protein concentration (B) of stains TU-6-Z, Δ<i>TrRas2</i> and <i>PAnigpdA</i>-<i>TrRas2^G16V</i> grown on 1% Avicel cellulose. (C) SDS-PAGE of secreted proteins in culture supernatants from the TU-6-Z, Δ<i>TrRas2</i> and <i>PAnigpdA</i>-<i>TrRas2^G16V</i> strains upon growth on 1% Avicel cellulose. Transcript levels of the major cellulase genes <i>cbh1</i> (E) and <i>cbh2</i> (F) were detected just before inducing (BF, 0 h) and 12 h, 20 h, 28 h, 36 h, 44 h after the beginning of the cultivation on cellulose and 10 h, 20 h on glucose. The relative <i>cbh1</i> and <i>cbh2</i> mRNA levels were present by setting the amount of <i>cbh1</i> and <i>cbh2</i> mRNA obtained from BF as 1 respectively. Each reaction was done in triplicate.</p

Analysis of transcript levels of <i>TrRas1</i> and <i>TrRas2</i> mRNA by qRT-PCR.

<p>(A) Transcript abundances of <i>TrRas1</i> and <i>TrRas2</i> on different carbon sources. The ratios of the expression of <i>TrRas1</i> and <i>TrRas2</i> to that of the actin reference gene were calculated in <i>T. reesei</i> QM9414 cultured on glycerol, glucose or cellulose. The relative mRNA levels were presented by setting the amount of <i>TrRas2</i> mRNA detected on glucose at 6 h as 1. (B) Quantitative real-time PCR analysis of <i>TrRas1</i> or <i>TrRas2</i> mRNA levels in the Δ<i>TrRas2</i> or Δ<i>TrRas1</i> mutant. The ratios of the expression of <i>TrRas1</i> and <i>TrRas2</i> to that of the actin reference gene were calculated. The relative mRNA levels were presented by setting the amount of <i>TrRas2</i> mRNA in <i>T. reesei</i> TU-6 detected at 12 h as 1. Values are means of three independent experiments. Error bars represent standard deviations.</p

Phylogenetic analysis of Ras proteins.

<p>The Ras-related Rho orthologues were used as an outgroup. The analysis was performed using Neighbor-joining method in the MEGA4.0 software and 1000 Bootstrap replications as test of phylogeny. GenBank accession numbers for the proteins are as follows: S. cerevisiae-Ras1, AAA34958; S. cerevisiae-Ras2, AAA34959; C. albicans-Ras1, AAF03566; U. maydis-Ras1, AAO19640; U. maydis-Ras2, AAO19639; S. sclerotiorum-Ras, AAT75139; P. marneffei-RasA, AAO64439; P. marneffei-RasB, EEA25931; A. fumigatus-RasA, EAL91488; A. fumigatus-RasB, EAL93074; A. nidulans-RasA,; N. crassa-Ras1, CAA37612; N. crassa-Ras2, BAA03708; H. sapiens-HRas, AAM12630; H. sapiens-KRas, AAB41942; H. sapiens-NRas, AAA60255; C. elegans-Ras2, CAA84796; T. reesei-Ras1, EGR51722; T. reesei-Ras2, EGR45548; S. cerevisiae-Rho1, AAA34977; N. crassa-Rho1, ACD01425; C. albicans-Rho1, XP_715825; A. nidulans-RhoA, AAK08118; P. marneffei-RhoA, XP_002144340.</p

TrRas2 modulates polarized apical growth, branch formation and sporulation in <i>T. reesei</i>.

<p>Morphological phenotypes of strains TU-6-Z, Δ<i>TrRas2</i>, Re<i>TrRas2</i> and <i>PAnigpdA</i>-<i>TrRas2^G16V</i> on PDA plates (A) and in liquid minimal medium (LMM) (B). Strains were cultured on PDA plates for 5 days at 30°C or were grown in LMM supplemented with 2% glucose as the carbon source for 48 h. (C) Hyphal phenotypes of <i>TrRas2</i> strains in LMM. The microscopic images were captured using DIC. Scale bars are shown in the figure.</p