Sharon M. Sagnella

Effective delivery of siRNA into cancer cells and tumors using well-defined biodegradable cationic star polymers.

Cancer is one of the most common causes of death worldwide. Two types of cancer that have high mortality rates are pancreatic and lung cancer. Despite improvements in treatment strategies, resistance to chemotherapy and the presence of metastases are common. Therefore, novel therapies which target and silence genes involved in regulating these processes are required. Short-interfering RNA (siRNA) holds great promise as a therapeutic to silence disease-causing genes. However, siRNA requires a delivery vehicle to enter the cell to allow it to silence its target gene. Herein, we report on the design and synthesis of cationic star polymers as novel delivery vehicles for siRNA to silence genes in pancreatic and lung cancer cells. Dimethylaminoethyl methacrylate (DMAEMA) was polymerized via reversible addition-fragmentation transfer polymerization (RAFT) and then chain extended in the presence of both cross-linkers N,N-bis(acryloyl)cistamine and DMAEMA, yielding biodegradable well-defined star polymers. The star polymers were characterized by transmission electron microscopy, dynamic light scattering, ζ potential, and gel permeation chromatography. Importantly, the star polymers were able to self-assemble with siRNA and form small uniform nanoparticle complexes. Moreover, the ratios of star polymer required to complex siRNA were nontoxic in both pancreatic and lung cancer cells. Treatment with star polymer-siRNA complexes resulted in uptake of siRNA into both cell lines and a significant decrease in target gene mRNA and protein levels. In addition, delivery of clinically relevant amounts of siRNA complexed to the star polymer were able to silence target gene expression by 50% in an in vivo tumor setting. Collectively, these results provide the first evidence of well-defined small cationic star polymers to deliver active siRNA to both pancreatic and lung cancer cells and may be a valuable tool to inhibit key genes involved in promoting chemotherapy drug resistance and metastases.

Dextran-Based Doxorubicin Nanocarriers with Improved Tumor Penetration

Drug delivery systems with improved tumor penetration are valuable assets as anticancer agents. A dextran-based nanocarrier system with aldehyde functionalities capable of forming an acid labile linkage with the chemotherapy drug doxorubicin was developed. Aldehyde dextran nanocarriers (ald-dex-dox) demonstrated efficacy as delivery vehicles with an IC50 of ∼300 nM against two-dimensional (2D) SK-N-BE(2) monolayers. Confocal imaging showed that the ald-dex-dox nanocarriers were rapidly internalized by SK-N-BE(2) cells. Fluorescence lifetime imaging microscopy (FLIM) analysis indicated that ald-dex-dox particles were internalized as intact complexes with the majority of the doxorubicin released from the particle four hours post uptake. Accumulation of the ald-dex-dox particles was significantly enhanced by ∼30% in the absence of glucose indicating a role for glucose and its receptors in their endocytosis. However, inhibition of clathrin dependent and independent endocytosis and macropinocytosis as well as membrane cholesterol depletion had no effect on ald-dex-dox particle accumulation. In three-dimensional (3D) SK-N-BE(2) tumor spheroids, which more closely resemble a solid tumor, the ald-dex-dox nanoparticles showed a significant improvement in efficacy over free doxorubicin, as evidenced by decreased spheroid outgrowth. Drug penetration studies in 3D demonstrated the ability of the ald-dex-dox nanocarriers to fully penetrate into a SK-N-BE(2) tumor spheroids, while doxorubicin only penetrates to a maximum distance of 50 μM. The ald-dex-dox nanocarriers represent a promising therapeutic delivery system for the treatment of solid tumors due to their unique enhanced penetration ability combined with their improved efficacy over the parent drug in 3D.

Stimuli-responsive functionalized mesoporous silica nanoparticles for drug release in response to various biological stimuli

A silica-based mesoporous nanosphere (MSN) controlled-release drug delivery system has been synthesized and characterized. The system uses l-cysteine derivatized gold nanoparticles (AuNPs), bound to the MSNs using Cu2+ as a bridging ion. The AuNPs serve as removable caps that hinder the release of drug molecules inside the amino functionalized MSN mesoporous framework. The modified MSNs themselves exhibit negligible cytotoxicity to living cells, as revealed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The drug delivery system requires one of two biological stimuli to trigger drug release. These stimuli are either: low pH (pH < 5); or elevated levels of adenosine triphosphate (ATP) (concentration > 4 mM). The feasibility of biologically controlled release was demonstrated through the stimuli-induced removal of the AuNP caps over the MSN releasing the anticancer drug doxorubicin. We envisage that this MSN system could play a significant role in developing new generations of controlled-release delivery vehicles.

Functionalizing Biodegradable Dextran Scaffolds Using Living Radical Polymerization: New Versatile Nanoparticles for the Delivery of Therapeutic Molecules

Conferring biodegradability to nanoparticles is vitally important when nanomedicine applications are being targeted, as this prevents potential problems with bioaccumulation of byproducts after delivery. In this work, dextran has been modified (and rendered hydrophobic) by partial acetalation. A solid state NMR method was first developed to fully characterize the acetalated polymers. In a subsequent synthetic step, RAFT functionality was attached via residual unmodified hydroxyl groups. The RAFT groups were then used in a living free radical polymerization reaction to control the growth of hydrophilic PEG-methacrylate chains, thereby generating amphiphilic comblike polymers. The amphiphilic polymers were then self-assembled in water to form various morphologies, including small vesicles, wormlike rods, and micellar structures, with PEG at the periphery acting as a nonfouling biocompatible polymer layer. The acetalated dextran nanoparticles were designed for potential doxorubicin (DOX) delivery application based on the premise that in the cell compartments (endosome, lysozome) the acetalated dextran would hydrolyze, destroying the nanoparticle structure, releasing the encapsulated DOX. In-vitro studies confirmed minimal cytotoxicity of the (unloaded) nanoparticles, even after 3 days, proving that the hydrolysis products from the acetal groups (methanol and acetone) had no observable cytotoxic effect. An intriguing initial result is reported that in vitro studies of DOX-loaded dextran-nanoparticles (compared to free DOX) revealed an increased differential toxicity toward a cancer cell line when compared to a normal cell line. Efficient accumulation of DOX in a human neuroblastoma cell line (SY-5Y) was confirmed by both confocal microscopy and flow cytometry measurements. Furthermore, the time dependent release of DOX was monitored using fluorescence lifetime imaging microscopy (FLIM) in SY-5Y live cells. FLIM revealed bimodal lifetime distributions, showing the accumulation of both DOX-loaded dextran-nanoparticles and subsequent release of DOX in the living cells. From FLIM data analysis, the amount of DOX released in SY-5Y cells was found to increase from 35% to 55% when the incubation time increased from 3 h to 24 h.

Drug delivery: Beyond active tumour targeting

UNLABELLED Despite improvements in our understanding of cancer and the concept of personalised medicine, cancer is still a major cause of death. It is established that solid tumours are highly heterogeneous, with a complex tumour microenvironment. Indeed, the tumour microenvironment is made up of a collection of immune cells, cancer-activated fibroblasts, and endothelial cells and in some cases a dense extracellular matrix. Accumulating evidence shows that the tumour microenvironment is a major barrier for the effective delivery of therapeutic drugs to tumour cells. Importantly, nanotechnology has come to the forefront as highly effective delivery vehicles for therapeutic agents. This perspective will discuss how nanomedicine can be used to target and deliver therapeutic drugs specifically to tumour cells. Moreover, emerging opportunities to modulate the tumour microenvironment and increase the delivery and efficacy of chemotherapy agents to solid tumours will be highlighted. FROM THE CLINICAL EDITOR Improving drug delivery to treatment resistant tumors is a major target of many nanomedicine-based applications. This comprehensive review discusses the currently available and emerging opportunities, in addition to discussing tumor microenvironment modulation to facilitate efficient delivery.

Soft ordered mesoporous materials from nonionic isoprenoid-type monoethanolamide amphiphiles self-assembled in water

The thermal and lyotropic liquid crystalline phase behaviour of a series of amide and monoethanolamide amphiphiles with isoprenoid-type hydrocarbon chains has been investigated. The amphiphilic nature of these molecules combined with their ability to form nanostructured self-assemblies makes them ideal candidates as delivery vehicles of bioactive molecules. For both families of molecules, increased branching in the hydrophobic chain, associated with increasing chain length, results in a decreased melting point. The melting points of the amides are significantly higher than their monoethanolamide homologues. Interestingly, both hexahydrofarnesoyl (H-farnesoyl) and phytanoyl monoethanolamide exhibit a glass transition temperature at around −72 °C to −74 °C. H-Farnesoyl and phytanoyl monoethanolamide form lyotropic liquid crystalline phases in water, whilst H-farnesoyl and phytanoyl amide form a spontaneous emulsion at the amphiphile–water interface. In particular, at room temperature H-farnesoyl and phytanoyl monoethanolamide form the Schwarz diamond (QIID) and the Schoen gyroid (QIIG) bicontinuous cubic phases which are retained down to temperatures as low as 1 °C. Furthermore, phytanoyl monoethanolamide displays a QIID, QIIG and inverse hexagonal phase (HII) at physiological temperature. Both phytanoyl and H-farnesoyl monoethanolamide form mesoporous cubic phases at room temperature that are easily dispersed into cubosomes. The robust nature of the nanostructured phase formation of these two monoethanolamides over a wide range of temperatures makes them ideal candidates for a variety of applications.

Ordered Nanostructured Amphiphile Self-Assembly Materials from Endogenous Nonionic Unsaturated Monoethanolamide Lipids in Water

The self-assembly, solid state and lyotropic liquid crystalline phase behavior of a series of endogenous n-acylethanolamides (NAEs) with differing degrees of unsaturation, viz., oleoyl monoethanolamide, linoleoyl monoethanolamide, and linolenoyl monoethanolamide, have been examined. The studied molecules are known to possess inherent biological function. Both the monoethanolamide headgroup and the unsaturated hydrophobe are found to be important in dictating the self-assembly behavior of these molecules. In addition, all three molecules form lyotropic liquid crystalline phases in water, including the inverse bicontinuous cubic diamond (Q(II)(D)) and gyroid (Q(II)(G)) phases. The ability of the NAEs to form inverse cubic phases and to be dispersed into ordered nanostructured colloidal particles, cubosomes, in excess water, combined with their endogenous nature and natural medicinal properties, makes this new class of soft mesoporous amphiphile self-assembly materials suitable candidates for investigation in a variety of advanced multifunctional applications, including encapsulation and controlled release of therapeutic agents and incorporation of medical imaging agents.

Enhanced uptake of an integral membrane protein, the dopamine D2L receptor, by cubic nanostructured lipid nanoparticles doped with Ni(II) chelated EDTA amphiphiles

Intrinsic difficulties in characterizing the structure of combined membrane protein–lyotropic liquid crystalline lipidic cubic systems have hampered the development of techniques such as membrane protein (MP) crystallization, which remain largely empirical with consequently low success rates. Here we have incorporated an integral membrane protein and important neurological drug target, the dopamine D2L receptor, within nanostructured nanoparticles of lipidic cubic phase, known as Cubosomes. We show that MPs are incorporated within Cubosomes and that they exert a structural effect which is qualitatively similar to that seen in bulk cubic phase for some systems, exemplifying the potential of Cubosomes to characterize MP incorporation. In addition we have shown, for Cubosomes doped with Ni(II) chelated EDTA amphiphiles, that the strong affinity interaction between the bio-engineered histidine(His)-tag on the protein and the Ni(II) chelated EDTA headgroup of the doped amphiphile leads to enhanced interaction between the membrane protein and the nanostructured cubic nanoparticle. This indicates that protein loading within a cubic phase can be increased as required either to facilitate crystal growth within cubic mesophases or for drug loading. In addition it exemplifies the potential of Cubosome nanostructured nanoparticles to be targeted to specific sites in the body.

Monodisperse nonionic phytanyl ethylene oxide surfactants: high throughput lyotropic liquid crystalline phase determination and the formation of liposomes, hexosomes and cubosomes

The phase behaviour (both neat and lyotropic) and toxicity of eight new ethylene oxide amphiphiles (EO = 1 to 8) with a single phytanyl chain (3,7,11,15 tetramethylhexadecyl) is reported. There is a discontinuity at EO > 4 where the neat and lyotropic behaviour exhibit a tipping point which is qualitatively rationalised in terms of the molecular geometry of the surfactant. Below four EO units the behaviour of the neat surfactants show only a glass transition, Tg ∼ −90 °C. Above four EO units crystallisation (Tcrys) and crystal-isotropic liquid (Tm) transitions are also observed. These increase monotonically with the hydrophilicity of the surfactant; consistent with the greater cohesiveness of the molecules due to van der Waals interactions. The increase in hydrophilicity corresponds to a decrease in curvature of the surfactant layer towards water. However, the exaggerated splay of the phytanyl chain is effective in promoting various self-assembled structures with inverse cubic and hexagonal phases preferred below ambient temperatures for EO < 4, and these are stable to dilution. Variation of the EO head group length promotes an interesting diversity of cubic phases, with an inverse micellar cubic phase (Fd3m) present for EO = 2 and the bicontinuous gyroid cubic (Ia3d) and double diamond cubic (Pn3m) phases present at higher ethoxylation. DIT-NIR microspectroscopy provided a high throughput, low volume, fast equilibrating method for obtaining the approximate partial temperature-composition phase diagrams of the binary systems with water. The toxicity of colloidal dispersions of these amphiphiles was assayed against normal breast epithelial (HMEpiC) and breast cancer (MCF7) cell lines. The IC50 of the EO amphiphiles was similar in both cell lines with moderate toxicity ranging from ∼80–110 μM in an in vitro cell viability assay.

Synthesis, self-assembly and stimuli responsive properties of cholesterol conjugated polymers

Reversible addition–fragmentation chain transfer (RAFT) polymerization was used to generate well-defined pH-responsive biofunctional polymers as potential ‘smart’ gene delivery systems. A series of five poly(dimethylamino ethyl methacrylate-co-cholesteryl methacrylate) P(DMAEMA-co-CMA) statistical copolymers, with similar molecular weights and varying cholesterol content, were prepared. The syntheses, compositions and molecular weight distributions for P(DMAEMA-co-CMA) were monitored by nuclear magnetic resonance (NMR), solid-state NMR and gel permeation chromatography (GPC) evidencing well-defined polymeric structures with narrow polydispersities. Aqueous solution properties of the copolymers were investigated using turbidimetry and light scattering to determine hydrodynamic diameters and zeta potentials associated with the phase transition behaviour of P(DMAEMA-co-CMA) copolymers. UV-Visible spectroscopy was used to investigate the pH-responsive behaviour of copolymers. Hydrodynamic radii were measured in the range 10–30 nm (pH, temperature dependent) by dynamic light scattering (DLS). Charge studies indicated that P(DMAEMA-co-CMA) polymers have an overall cationic charge, mediated by pH. Potentiometric studies revealed that the buffering capacity and pKa values of polymers were dependent on cholesterol content as well as on cationic charge. The buffering capacity increased with increasing charge ratio, overall demonstrating transitions in the pH endosomal region for all five copolymeric structures. Cell viability assay showed that the copolymers displayed increasing cytotoxicity with decreasing number of cholesterol moieties. These preliminary results show the potential of these well-defined P(DMAEMA-co-CMA) polymers as in vitro siRNA delivery agents.