Hyperbranched Double Hydrophilic Block Copolymer Micelles of Poly(ethylene oxide) and Polyglycerol for pH-Responsive Drug Delivery

We report the synthesis of a well-defined hyperbranched double hydrophilic block copolymer of poly­(ethylene oxide)-<i>hyperbranched</i>-polyglycerol (PEO-<i>hb</i>-PG) to develop an efficient drug delivery system. In specific, we demonstrate the hyperbranched PEO-<i>hb</i>-PG can form a self-assembled micellar structure on conjugation with the hydrophobic anticancer agent doxorubicin, which is linked to the polymer by pH-sensitive hydrazone bonds, resulting in a pH-responsive controlled release of doxorubicin. Dynamic light scattering, atomic force microscopy, and transmission electron microscopy demonstrated successful formation of the spherical core–shell type micelles with an average size of about 200 nm. Moreover, the pH-responsive release of doxorubicin and in vitro cytotoxicity studies revealed the controlled stimuli-responsive drug delivery system desirable for enhanced efficiency. Benefiting from many desirable features of hyperbranched double hydrophilic block copolymers such as enhanced biocompatibility, increased water solubility, and drug loading efficiency as well as improved clearance of the polymer after drug release, we believe that double hydrophilic block copolymer will provide a versatile platform to develop excellent drug delivery systems for effective treatment of cancer

<i>De novo</i> chromatinization of the KSHV genome following infection.

<p>(<b>A</b>) FAIRE assay showing the degree of chromatinization of the indicated viral and cellular promoters in SLK cells infected for 1, 8 or 24 hours or in latently infected SLK cells. (<b>B</b>) and (<b>C</b>) The enrichment of histones H3 and H2A was calculated as ChIP/input (% Input or occupancy) at the indicated viral and cellular promoters following <i>de novo</i> infection at 1, 8 and 24 hpi and in latently infected SLK cells. (<b>D</b>) Real time RT-PCR analysis was performed to determine the expression of the indicated viral genes in SLK cells infected by KSHV for 8 and 24 hours and in latently infected SLK cells. Expression levels of viral genes are shown relative to those of 18S. A 2-tailed student's t-test was performed between 8 hpi and latency for each tested lytic gene and all p values were less than 0.05.</p

Both PRC2 and PRC1 are involved in the inhibition of lytic gene expression following <i>de novo</i> infection.

<p>(<b>A</b>) Immunoblot analysis of EZH2 and RING1B expression in shRNA-treated SLK cells. (<b>B</b>) RT-qPCR analysis of viral gene expression of KSHV infected cells upon depletion of EZH2 and RING1B expression. The expression of viral genes and MYT1 cellular gene is shown relative to that of KSHV infected SLK cells treated with scrambled shRNA. (<b>C</b>) ChIP assays show that depletion of the EZH2 expression during <i>de novo</i> infection altered the deposition profiles of histone modifications and led to the reduction of RING1B recruitment on the KSHV genome. The asterisk denotes p<0.02. (<b>D</b>) Following 2 days of treatment with either DMSO or the EZH2 inhibitor, GSK343, SLK cell lysates were used for immunoblot analysis with the indicated antibodies. (<b>E</b>) ChIP analysis of the recruitment of PcG proteins and the deposition of histone modifications on KSHV promoters in KSHV infected cells pretreated with GSK343. (<b>F</b>) GSK343-treated TIME, SLK and 293T cells were infected by KSHV for 72 hours and RT-PCR was performed to test the expression of viral genes and the cellular gene MYT1. The fold change represents the induction of viral gene expression in GSK343-treated cells compared to DMSO-treated cells.</p

Biphasic euchromatin-to-heterochromatin transition on the KSHV genome following <i>de novo</i> infection.

<p>As soon as the viral genome enters the nucleus, histones are recruited on the viral DNA, resulting in the chromatinization of the KSHV genome. The viral genome initially adopts a transcriptionally permissive chromatin, characterized by high levels of the H3K27ac and H3K4me3 and this is accompanied by the transient expression of lytic genes. Subsequently, the PRC2 and PRC1 are recruited to viral genome, where they are responsible for the enrichment of H3K27me3 and H2Ak119ub on the viral chromatin as well as the repression of lytic genes. During latency, both PRC2 and PRC1 remain on the KSHV genome for the maintenance of the repression of lytic genes.</p

Analysis of the deposition of histone modifications on KSHV promoters during <i>de novo</i> infection using time-course ChIP assays.

<p>ChIPs were performed using latently infected SLK cells or SLK cells following <i>de novo</i> infection for the indicated hours post infection (hpi) and the enrichment of histone modifications was measured by qPCR using primers specific for the indicated viral and cellular promoters. ChIPs for each histone modification were normalized for the amount of the relevant histones at each promoter. T-test was applied to compare the values between the indicated time points (*). (<b>A</b>) H3K4me3 ChIP (for LANA, RTA and K2 p<0.05, for ORF25 p = 0.1 between 1 and 24 hpi). (<b>B</b>) H3K27ac ChIP (for LANA p = 0.6, RTA and K2 p<0.05, for ORF25 p = 0.18 between 1 and 72 hpi). (<b>C</b>) H3K27me1 ChIP (for LANA p = 0.063, for RTA, K2 and ORF25 p<0.05 between 1 and 8 hpi). (<b>D</b>) H3K27me3 ChIP (for LANA p = 0.33, for RTA, K2 and ORF25 p<0.005 between 1 and 72 hpi). (<b>E</b>) H2AK119ub ChIP (for LANA p = 0.3, for RTA, K2 and ORF25 p<0.02 between 1 and 72 hpi). (<b>F</b>) Sequential deposition of H3K27ac and H3K27me1 on viral promoters during infection was confirmed by sequential ChIP assays. The first ChIP was performed with H3K27ac-specific antibody at 1 hpi and 8 hpi, followed by elution of the immunoprecipitated DNA, The eluted DNA was used as the input for a second ChIP performed with H3K27me1 antibody. (<b>G</b>) Sequential deposition and colocalization of H3K4me3 and H3K27me3 on the RTA promoter following <i>de novo</i> infection were confirmed by sequential ChIP assays. The first ChIP was performed with H3K4me3-specific antibody at 24 hpi and 72 hpi followed by the second ChIP for H3K27me3 using the eluted first ChIPs as the input.</p

Euchromatinization of the KSHV genome in gingival oral epithelial cells following <i>de novo</i> infection.

<p>(<b>A</b>) Measurement of viral DNA replication in SLK, OEPI, SCC15 and NOK cells infected by KSHV for 4, 24, 48 and 72 hours. The viral DNA polymerase inhibitor, PAA, was applied to block the replication of KSHV. (<b>B</b>) FAIRE assay showing the degree of chromatinization of the indicated viral and cellular promoters in SLK and OEPI cells infected for 8, 24 or 72 hours. (<b>C</b>) SLK and OEPI cells were infected with KSHV for 1, 2 and 3 days and immunoblots were performed to test the expression of RTA and K3 viral proteins. Actin served as a loading control. The “C” indicates immunoblot analysis of uninfected cells. (<b>D</b>) Quantitative RT-PCR analysis of viral gene expression in KSHV infected SLK and OPEI cells. (<b>E</b>) and (<b>F</b>) ChIP analysis of the indicated histone modifications on a selection of KSHV promoters in OEPI cells at 8, 24 and 72 hpi. The cellular promoters (ACT and MYT1) were used as controls. (<b>G</b>) Comparative immunoblot analysis of the indicated cellular proteins between SLK and oral epithelial cells.</p

RTA is involved in the deposition of activating histone marks on the KSHV genome following <i>de novo</i> infection.

<p>(<b>A</b>) Comparative ChIP analysis of H3K4me3, H3K27ac and H3K27me3 at the indicated viral promoters of wild type (wt) or RTA knockout (RTAstop) KSHV-infected SLK cells at 8, 24 and 72 hpi. The promoters of the cellular actin (ACT) and MYT1 genes were used as controls. ChIPs were normalized for the amount of histone H3 at each promoter. The asterisk denotes p<0.05 between wt and RTAstop at 8 hpi. (<b>B</b>) RT-qPCR analysis of viral gene expression in RTAstop KSHV-infected SLK cells at 24 hpi. The expression of the indicated viral genes was calculated relative to wt KSHV-infected cells. (<b>C</b>) ChIP experiments showing the binding of RTA and CBP on the RTA-responsive RTA and K2 promoters as well as the LANA promoter in latently-infected cells and naïve cells infected for 8 or 24 hours by wild type KSHV. ORF25 promoter was used as a negative control. (<b>D</b>) RTA and CBP binding on viral promoters in RTAstop KSHV-infected SLK cells.</p

Recruitment of components of the PRC2 and PRC1 complexes onto the KSHV genome during <i>de novo</i> infection.

<p>(<b>A</b>) Time-course ChIP analysis of the binding of EZH2 (PRC2), RING1B and RYBP (PRC1) onto KSHV promoters at 4, 24, and 72 hpi and during latency. (<b>B</b>) Genome-wide recruitment of EZH2 and RING1B to the KSHV genome at 4 and 72 hpi. The Pearson correlation between the binding of EZH2 and RING1B is 0.6 at 72 hpi. Labels are the same as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003813#ppat-1003813-g003" target="_blank">Figure 3</a>.</p

Genome-wide view of the deposition of histone modifications on the KSHV genome following <i>de novo</i> infection.

<p>(<b>A</b>) ChIP-on-chip was performed for H3K27ac, H3K4me3 and H3K27me3 at 4, 24 and 72 hpi using SLK infected cells. The alternating dark and light blue squares represent viral ORFs. (<b>B</b>) Heat map representation of changes in histone modifications at the gene regulatory regions of KSHV genes grouped by expression class (latent, La, immediate-early, IE, early, E, late, L). The rows display the relative abundance of the indicated histone modification within the −1 kb to +1 kb genomic regions flanking the translational start site (TSS) of each viral gene. The blue and yellow colors denote lower-than-average and higher-than-average enrichment, respectively, whereas gray represents missing values for enrichment due to lack of probes in those genomic regions.</p