p53 Maintains Genomic Stability by Preventing Interference between Transcription and Replication.

p53 tumor suppressor maintains genomic stability, typically acting through cell-cycle arrest, senescence, and apoptosis. We discovered a function of p53 in preventing conflicts between transcription and replication, independent of its canonical roles. p53 deficiency sensitizes cells to Topoisomerase (Topo) II inhibitors, resulting in DNA damage arising spontaneously during replication. Topoisomerase IIα (TOP2A)-DNA complexes preferentially accumulate in isogenic p53 mutant or knockout cells, reflecting an increased recruitment of TOP2A to regulate DNA topology. We propose that p53 acts to prevent DNA topological stress originating from transcription during the S phase and, therefore, promotes normal replication fork progression. Consequently, replication fork progression is impaired in the absence of p53, which is reversed by transcription inhibition. Pharmacologic inhibition of transcription also attenuates DNA damage and decreases Topo-II-DNA complexes, restoring cell viability in p53-deficient cells. Together, our results demonstrate a function of p53 that may underlie its role in tumor suppression.

Delivery of doxorubicin and paclitaxel from double-layered microparticles: The effects of layer thickness and dual-drug vs. single-drug loading.

Double-layered microparticles composed of poly(d,l-lactic-co-glycolic acid, 50:50) (PLGA) and poly(l-lactic acid) (PLLA) were loaded with doxorubicin HCl (DOX) and paclitaxel (PCTX) through a solvent evaporation technique. DOX was localized in the PLGA shell, while PCTX was localized in the PLLA core. The aim of this study was to investigate how altering layer thickness of dual-drug, double-layered microparticles can influence drug release kinetics and their antitumor capabilities, and against single-drug microparticles. PCTX-loaded double-layered microparticles with denser shells retarded the initial release of PCTX, as compared with dual-drug-loaded microparticles. The DOX release from both DOX-loaded and dual-drug-loaded microparticles were observed to be similar with an initial burst. Through specific tailoring of layer thicknesses, a suppressed initial burst of DOX and a sustained co-delivery of two drugs can be achieved over 2months. Viability studies using spheroids of MCF-7 cells showed that controlled co-delivery of PCTX and DOX from dual-drug-loaded double-layered microparticles were better in reducing spheroid growth rate. This study provides mechanistic insights into how by tuning the layer thickness of double-layered microparticles the release kinetics of two drugs can be controlled, and how co-delivery can potentially achieve better anticancer effects. STATEMENT OF SIGNIFICANCE: While the release of multiple drugs has been reported to achieve successful apoptosis and minimize drug resistance, most conventional particulate systems can only deliver a single drug at a time. Recently, although a number of formulations (e.g. micellar nanoparticles, liposomes) have been successful in delivering two or more anticancer agents, sustained co-delivery of these agents remains inadequate due to the complex agent loading processes and rapid release of hydrophilic agents. Therefore, the present work reports the multilayered particulate system that simultaneously hosts different drugs, while being able to tune their individual release over months. We believe that our findings would be of interest to the readers of Acta Biomaterialia because the proposed system could open a new avenue on how two drugs can be released, through rate-controlling carriers, for combination chemotherapy.

Synthesis of small-sized, porous, and low-toxic magnetite nanoparticles by thin POSS silica coating.

In this communication, we report the synthesis of small-sized (<10 nm), water-soluble, magnetic nanoparticles (MNPs) coated with polyhedral oligomeric silsesquioxanes (POSS), which contain either polyethylene glycol (PEG) or octa(tetramethylammonium) (OctaTMA) as functional groups. The POSS-coated MNPs exhibit superparamagnetic behavior with saturation magnetic moments (51-53 emu g(-1)) comparable to silica-coated MNPs. They also provide good colloidal stability at different pH and salt concentrations, and low cytotoxicity to MCF-7 human breast epithelial cells. The relaxivity data and magnetic resonance (MR) phantom images demonstrate the potential application of these MNPs in bioimaging.

Inhibition of 3-D tumor spheroids by timed-released hydrophilic and hydrophobic drugs from multilayered polymeric microparticles.

First-line cancer chemotherapy necessitates high parenteral dosage and repeated dosing of a combination of drugs over a prolonged period. Current commercially available chemotherapeutic agents, such as Doxil and Taxol, are only capable of delivering single drug in a bolus dose. The aim of this study is to develop dual-drug-loaded, multilayered microparticles and to investigate their antitumor efficacy compared with single-drug-loaded particles. Results show hydrophilic doxorubicin HCl (DOX) and hydrophobic paclitaxel (PTX) localized in the poly(dl-lactic-co-glycolic acid, 50:50) (PLGA) shell and in the poly(l-lactic acid) (PLLA) core, respectively. The introduction of poly[(1,6-bis-carboxyphenoxy) hexane] (PCPH) into PLGA/PLLA microparticles causes PTX to be localized in the PLLA and PCPH mid-layers, whereas DOX is found in both the PLGA shell and core. PLGA/PLLA/PCPH microparticles with denser shells allow better control of DOX release. A delayed release of PTX is observed with the addition of PCPH. Three-dimensional MCF-7 spheroid studies demonstrate that controlled co-delivery of DOX and PTX from multilayered microparticles produces a greater reduction in spheroid growth rate compared with single-drug-loaded particles. This study provides mechanistic insights into how distinctive structure of multilayered microparticles can be designed to modulate the release profiles of anticancer drugs, and how co-delivery can potentially provide better antitumor response.

Development of a magnetic 3D spheroid platform with potential application for high-throughput drug screening.

Three-dimensional (3D) cell culture has become increasingly adopted as a more accurate model of the complex in vivo microenvironment compared to conventional two-dimensional (2D) cell culture. Multicellular spheroids are important 3D cell culture models widely used in biological studies and drug screening. To facilitate simple spheroid manipulation, magnetic spheroids were generated from magnetically labeled cells using a scaffold-free approach. This method is applicable to a variety of cell types. The spheroids generated can be targeted and immobilized using magnetic field gradients, allowing media change or dilution to be performed with minimal disruption to the spheroids. Cells in magnetic spheroids showed good viability and displayed typical 3D morphology. Using this platform, a 28 day study was carried out using doxorubicin on magnetic MCF-7 spheroids. The results provided a proof-of-principle for using magnetic tumor spheroids in therapeutic studies. They can offer beneficial insights that help to bridge the gap between in vitro and in vivo models. Furthermore, this platform can be adapted for high-throughput screening in drug discovery.

Design and synthesis of polymer-functionalized NIR fluorescent dyes--magnetic nanoparticles for bioimaging.

The fluorescent probes having complete spectral separation between absorption and emission spectra (large Stokes shift) are highly useful for solar concentrators and bioimaging. In bioimaging application, NIR fluorescent dyes have a greater advantage in tissue penetration depth compared to visible-emitting organic dyes or inorganic quantum dots. Here we report the design, synthesis, and characterization of an amphiphilic polymer, poly(isobutylene-alt-maleic anhyride)-functionalized near-infrared (NIR) IR-820 dye and its conjugates with iron oxide (Fe3O4) magnetic nanoparticles (MNPs) for optical and magnetic resonance (MR) imaging. Our results demonstrate that the Stokes shift of unmodified dye can be tuned (from ~106 to 208 nm) by the functionalization of the dye with polymer and MNPs. The fabrication of bimodal probes involves (i) the synthesis of NIR fluorescent dye (IR-820 cyanine) functionalized with ethylenediamine linker in high yield, >90%, (ii) polymer conjugation to the functionalized NIR fluorescent dye, and (iii) grafting the polymer-conjugated dyes on iron oxide MNPs. The resulting uniform, small-sized (ca. 6 nm) NIR fluorescent dye-magnetic hybrid nanoparticles (NPs) exhibit a wider emissive range (800-1000 nm) and minimal cytotoxicity. Our preliminary studies demonstrate the potential utility of these NPs in bioimaging by means of direct labeling of cancerous HeLa cells via NIR fluorescence microscopy and good negative contrast enhancement in T2-weighted MR imaging of a murine model.

Manipulating magnetic 3D spheroids in hanging drops for applications in tissue engineering and drug screening.

Magnetic spheroid manipulation can be carried out in hanging drops to generate distinctly structured heterotypic microtissues through sequential addition of cells or spheroid to homotypic spheroids. These spheroids can also be incorporated in a droplet-based assay to screen for therapeutic efficacy in prolonged studies. This simple and versatile technique can offer potential benefits in tissue engineering and drug screening applications.

pH-responsive endosomolytic pseudo-peptides for drug delivery to multicellular spheroids tumour models.

Endosomolytic polymers can aid in the endosomal release of therapeutics to improve intracellular drug delivery. pH-responsive biomimetic pseudo-peptides were synthesised by grafting l-phenylalanine onto the pendant carboxylic acids of a polyamide, poly(l-lysine isophthalamide). PP-75 (stoichiometric l-phenylalanine grafting of 75 mol%) was determined to have the best endosomolytic property. The mean hydrodynamic size of PP-75 decreased with lower pH as the polymers adopted a more compact conformation due to protonation of acidic groups and increase in hydrophobicity. PP-75 was demonstrated to deliver model drugs effectively in three dimensional (3D) magnetic HeLa multicellular spheroids used as in vitro tumour models. These spheroids can be isolated easily and quickly by magnetic separation. Due to its relatively small size, PP-75 was able to penetrate from the exterior to the interior of these spheroids and was internalised by the cells in the spheroids. It could retain its pH-mediated membrane-lytic capability in 3D drug delivery by releasing internalised calcein from intracellular endosomes in the tumour models. Furthermore, cell viability results suggest that PP-75 showed no significant cytotoxicity towards cells in the spheroids. The pH-responsive PP-75 can potentially enhance the extracellular and intracellular delivery of therapeutics in tumours.

Effect of magnetite nanoparticle agglomerates on the destruction of tumor spheroids using high intensity focused ultrasound.

Magnetite (Fe(3)O(4)) nanoparticle agglomerates have been shown to enhance the degree of inertial cavitation induced by high-intensity focused ultrasound (HIFU). To investigate the effect of these particles on the destruction of tumor spheroids using HIFU, HeLa spheroids were insonated in the presence and absence of magnetite nanoparticle agglomerates. The HIFU transducer was operated with a frequency of 1.1 MHz, pulse repetition frequency of 1.67 kHz, 5% and 50% duty cycles and peak negative focal pressure of 7.2 MPa for 10 s. The significant increase in the HIFU-induced inertial cavitation caused by the presence of magnetite particles at 50% duty cycle was sufficient to cause cell lysis and disintegrate the whole spheroid (p ≤ 0.001). This suggests that magnetite nanoparticle agglomerates can enhance the efficacy of HIFU in tumor ablation and other related therapies.

Generation and manipulation of magnetic multicellular spheroids.

Multicellular spheroids have important applications in tumour studies, drug screening and tissue engineering. To enable simple manipulation of spheroids, magnetically labelled HeLa cells were cultured in hanging drops to generate magnetic spheroids. HeLa cells were labelled by biotinylating their cell membrane proteins and then binding streptavidin paramagnetic particles onto the biotinylated cell surface. Spheroids of different sizes were obtained by varying the seeding cell concentrations within the hanging drops and the spheroids had good cell viability. Characterisation of the F-actin distribution within the spheroids indicated a three dimensional reorganisation of the cellular cytoskeleton compared to monolayer cultures. The magnetic moment of the spheroids was measured and showed a superparamagnetic response in an applied field. Transmission electron microscopy analysis indicated that the paramagnetic particles were still present in the spheroids even after 21 days of culture. These spheroids could be easily and quickly separated magnetically without the need for centrifugation. The magnetic spheroids were also successfully manipulated and patterned using magnetic fields within a few seconds. The patterned spheroids then fused together to form a larger tissue construct.