Aram Omer Saeed

Modular Construction of Multifunctional Bioresponsive Cell-Targeted Nanoparticles for Gene Delivery

Multifunctional and modular block copolymers prepared from biocompatible monomers and linked by a bioreducible disulfide linkage have been prepared using a combination of ring-opening and atom-transfer radical polymerizations (ATRP). The presence of terminal functionality via ATRP allowed cell-targeting folic acid groups to be attached in a controllable manner, while the block copolymer architecture enabled well-defined nanoparticles to be prepared by a water-oil-water double emulsion procedure to encapsulate DNA with high efficiency. Gene delivery assays in a Calu-3 cell line indicated specific folate-receptor-mediated uptake of the nanoparticles, and triggered release of the DNA payload via cleavage of the disulfide link resulted in enhanced transgene expression compared to nonbioreducible analogues. These materials offer a promising and generic means to deliver a wide variety of therapeutic payloads to cells in a selective and tunable way.

Synthetic polymers for biopharmaceutical delivery

The increasing complexity of biological molecules being developed for medical applications requires more sophisticated carrier materials to take these potent but delicate therapeutics to in vivo targets. Recent advances in polymer synthesis chemistries are enabling carrier vehicles of greater sophistication in architecture and function to be prepared. In this outline review, the application of polymer chemistries to protein and nucleic acid therapeutics are considered.

One-pot controlled synthesis of biodegradable and biocompatible co-polymer micelles

A facile route to biocompatible poly(lactic acid-co-glycolic acid)-co-poly(ethyleneglycolmethacrylate) (PLGA–PEGMA) block co-polymers is described utilising a combination of ring-opening polymerisation (ROP) and reversible addition fragmentation transfer (RAFT) methods. A series of PLGA–PEGMA polymers varying in co-monomer content and block length were synthesised with low polydispersities. All the block co-polymers formed micelles in aqueous solution as shown by dynamic light scattering, while critical micelle concentrations were found to be in the micromolar range. The polymer micelles were able to encapsulate model drugs (carboxyfluorescein and fluorescein isothiocyanate) and selected co-polymer micelles incubated with 3T3 fibroblasts as a model cell line were rapidly taken up as indicated by fluorescence microscopy assays. The combination of the polymer chemistries opens the way to highly flexible syntheses of micellar drug carrier systems .

Facile synthesis of responsive nanoparticles with reversible, tunable and rapid thermal transitions from biocompatible constituents

Responsive polymeric nanoparticles composed of hybrid block co-polymers were prepared from biocompatible components that displayed rapid, tunable and multiply reversible transitions in response to change of temperature.

Programmable polymer-DNA hydrogels with dual input and multiscale responses

Combination switchable polymer-DNA hydrogels have been synthesized to respond to both a specific oligonucleotide recognition signal and a non-specific but biorelevant environmental trigger. The hydrogels exhibit rheological properties that can be modulated through interaction with complementary DNA strands and/or reduction. Furthermore, individual and combined oligonucleotide recognition and reduction responses allow control over pore sizes in the gel, enabling programmable release and transport of objects ranging from the nano- to micro-scale.

‘Isothermal’ phase transitions and supramolecular architecture changes in thermoresponsive polymers via acid-labile side-chains

Polymers designed to change their conformation via a phase transition triggered by acidic cleavage of a hydrophobic side-chain have been synthesized and characterised. The new materials were prepared by co-polymerising N-isopropylacrylamide with an acetal-containing pH-sensitive monomer N-(2-(2,4,6-trimethoxyphenyl)-1,3-dioxan-5-yl)acrylamide (TMPDA) and then grafting the resultant linear co-polymers to branched poly(ethyleneimine). The final three-component polycations exhibited Lower Critical Solution Temperature (LCST) behaviour. The structures of these polymers, their solution behaviour and their self-association were characterized by DLS and TEM in water and buffer solutions. The acid-triggered hydrolysis of trimethoxybenzeneacetal side-chains on the poly(N-isopropylacrylamide-co-TMPDA) grafts resulted in changes in lower critical solution temperatures and in solution self-assembly; thus in effect creating an ‘isothermal’ phase transition. The changes in polymer conformation, at acidity levels corresponding to those in cell endosomes, offer promise for these polymers to act as controlled release materials.

Mannan binding lectin-associated serine protease 1 is induced by hepatitis C virus infection and activates human hepatic stellate cells

Mannan binding lectin (MBL)‐associated serine protease type 1 (MASP‐1) has a central role in the lectin pathway of complement activation and is required for the formation of C3 convertase. The activity of MASP‐1 in the peripheral blood has been identified previously as a highly significant predictor of the severity of liver fibrosis in hepatitis C virus (HCV) infection, but not in liver disease of other aetiologies. In this study we tested the hypotheses that expression of MASP‐1 may promote disease progression in HCV disease by direct activation of hepatic stellate cells (HSCs) and may additionally be up‐regulated by HCV. In order to do so, we utilized a model for the maintenance of primary human HSC in the quiescent state by culture on basement membrane substrate prior to stimulation. In comparison to controls, recombinant MASP‐1 stimulated quiescent human HSCs to differentiate to the activated state as assessed by both morphology and up‐regulation of HSC activation markers α‐smooth muscle actin and tissue inhibitor of metalloproteinase 1. Further, the expression of MASP‐1 was up‐regulated significantly by HCV infection in hepatocyte cell lines. These observations suggest a new role for MASP‐1 and provide a possible mechanistic link between high levels of MASP‐1 and the severity of disease in HCV infection. Taken together with previous clinical observations, our new findings suggest that the balance of MASP‐1 activity may be proinflammatory and act to accelerate fibrosis progression in HCV liver disease.

A thermoresponsive and magnetic colloid for 3D cell expansion and reconfiguration

A dual thermoresponsive and magnetic colloidal gel matrix is described for enhanced stem-cell culture. The combined properties of the material allow enzyme-free passaging and expansion of mesenchymal stem cells, as well as isolation of cells postculture by the simple process of lowering the temperature and applying an external magnetic field. The colloidal gel can be reconfigured with thermal and magnetic stimuli to allow patterning of cells in discrete zones and to control movement of cells within the porous matrix during culture.

Gelatin embedding for the preparation of thermoreversible or delicate scaffolds for histological analysis

Thermoreversible hydrogels for tissue engineering (TE) purposes have gained increased attention in recent years as they can be combined with cells and drugs and directly injected into the body. Following the fate of transplanted cells in situ is essential in characterizing their distribution and survival, as well as the expression of specific markers or cell-matrix interactions. Existing histological embedding methods, such as paraffin wax embedding, can mechanically damage some biomaterials during processing. In this study, we describe a broadly applicable preparation protocol that allows the handling of delicate, thermoreversible scaffolds for histological sectioning. The gelatin solution permits the embedding of samples at 37 °C, which suits the solid phase of most TE scaffolds. A thermoreversible scaffold of polycaprolactone microparticles, combined with poly(polyethylene glycol methacrylate ethyl ether) and containing human adipose-derived stem cells, was prepared for histology by an initial gelatin embedding step in addition to the standard cryosectioning and paraffin processing protocols. Sections were evaluated by hematoxylin eosin staining and immunostaining for human vimentin. The gelatin embedding retained the scaffold particles and permitted the complete transfer of the construct. After rapid cooling, the solid gelatin blocks could be cryosectioned and paraffin infiltrated. In contrast to direct cryosectioning or paraffin infiltration, the extended protocol preserved the scaffold structure as well as the relevant cell epitopes, which subsequently allowed for immunostaining of human cells within the material. The gelatin embedding method proposed is a generalizable alternative to standard preparations for histological examination of a variety of delicate samples.