Inkjet-Printed Phospholipid Bilayers on Titanium Oxide Surfaces: Towards Functional Membrane Biointerfaces.

Functional biointerfaces hold broad significance for designing cell-responsive medical implants and sensor devices. Solid-supported phospholipid bilayers are a promising class of biological materials to build bioinspired thin-film coatings, as they can facilitate interactions with cell membranes. However, it remains challenging to fabricate lipid bilayers on medically relevant materials such as titanium oxide surfaces. There are also limitations in existing bilayer printing capabilities since most approaches are restricted to either deposition alone or to fixed microarray patterning. By combining advances in lipid surface chemistry and on-demand inkjet printing, we demonstrate the direct deposition and patterning of covalently tethered lipid bilayer membranes on titanium oxide surfaces, in ambient conditions and without any surface pretreatment process. The deposition conditions were evaluated by quartz crystal microbalance-dissipation (QCM-D) measurements, with corresponding resonance frequency (Δf) and energy dissipation (ΔD) shifts of around −25 Hz and <1 × 10−6, respectively, that indicated successful bilayer printing. The resulting printed phospholipid bilayers are stable in air and do not collapse following dehydration; through rehydration, the bilayers regain their functional properties, such as lateral mobility (>1 µm2/s diffusion coefficient), according to fluorescence recovery after photobleaching (FRAP) measurements. By taking advantage of the lipid bilayer patterned architectures and the unique features of titanium oxide's photoactivity, we further show how patterned cell culture arrays can be fabricated. Looking forward, this work presents new capabilities to achieve stable lipid bilayer patterns that can potentially be translated into implantable biomedical devices.

Amyloid-ß Peptide Triggers Membrane Remodeling in Supported Lipid Bilayers Depending on Their Hydrophobic Thickness.

Amyloid-ß (Aß) peptide has been implicated in Alzheimer's disease, which is a leading cause of death worldwide. The interaction of Aß peptides with the lipid bilayers of neuronal cells is a critical step in disease pathogenesis. Recent evidence indicates that lipid bilayer thickness influences Aß membrane-associated aggregation, while understanding how Aß interacts with lipid bilayers remains elusive. To address this question, we employed supported lipid bilayer (SLB) platforms composed of different-length phosphatidylcholine (PC) lipids (C12:0 DLPC, C18:1 DOPC, C18:1-C16:0 POPC), and characterized the resulting interactions with soluble Aß monomers. Quartz crystal microbalance-dissipation (QCM-D) experiments identified concentration-dependent Aß peptide adsorption onto all tested SLBs, which was corroborated by fluorescence recovery after photobleaching (FRAP) experiments indicating that higher Aß concentrations led to decreased membrane fluidity. These commonalities pointed to strong Aß peptide-membrane interactions in all cases. Notably, time-lapsed fluorescence microscopy revealed major differences in Aß-induced membrane morphological responses depending on SLB hydrophobic thickness. For thicker DOPC and POPC SLBs, membrane remodeling involved the formation of elongated tubule and globular structures as a passive means to regulate membrane stress depending on Aß concentration. In marked contrast, thin DLPC SLBs were not able to accommodate extensive membrane remodeling. Taken together, our findings reveal that membrane thickness influences the membrane morphological response triggered upon Aß adsorption.

Specific Design of Titanium(IV) Phenolato Chelates Yields Stable and Accessible, Effective and Selective Anticancer Agents.

Octahedral titanium(IV) complexes of phenolato hexadentate ligands were developed and showed very high stability for days in water solutions. In vitro cytotoxicity studies showed that, whereas tetrakis(phenolato) systems are generally of low activity presumably due to inaccessibility, smaller bis(phenolato)bis(alkoxo) complexes feature high anticancer activity and accessibility even without formulations, also toward a cisplatin-resistant cell line. An all-aliphatic control complex was unstable and inactive. A leading phenolato complex also revealed: 1) high durability in fully aqueous solutions; accordingly, negligible loss of activity after preincubation for three days in medium or in serum; 2) maximal cellular accumulation and induction of apoptosis following 24-48 h of administration; 3) reduced impact on noncancerous fibroblast cells; 4) in vivo efficacy toward lymphoma cells in murine model; 5) high activity in NCI-60 panel, with average GI50 of 4.6±2 µm. This newly developed family of Ti(IV) complexes is thus of great potential for anticancer therapy.

Highly Stable Tetra-Phenolato Titanium(IV) Agent Formulated into Nanoparticles Demonstrates Anti-Tumoral Activity and Selectivity.

Titanium(IV) complexes exhibit high potential as anti-tumor agents, particularly due to their low intrinsic toxicity and cytotoxicity toward cisplatin resistant cells. Nevertheless, Ti(IV) complexes generally undergo rapid hydrolysis that previously hampered their utilization as anticancer drugs. We recently overcame this difficulty by developing a highly stable Ti(IV) complex that is based on tetra-phenolato, hexadentate ligand, formulated into organic nanoparticles. Herein we investigated the activity of this complex in vitro and in vivo. Although inactive when tested directly due to poor solubility, when formulated, this complex displayed (a) high cytotoxicity toward cisplatin resistant human ovarian cells, A2780-cp, with resistance factor of 1.1; (b) additive behavior in combination with cisplatin toward ovarian and colon cancer cells; (c) selectivity toward cancer cells as implied by its mild activity toward non-cancerous, fibroblast lung cells, MRC-5; (d) high stability and durability as manifested by the ability to maintain cytotoxicity, even following one week of incubation in 100% aquatic medium solution; and (e) in vivo efficacy toward solid tumors of human colon cancer cells, HT-29, in nude mice without any clinical signs of toxicity. These features support the formulated phenolato Ti(IV) complex being an effective and selective anti-tumoral agent.

Anti-proliferative activity of nano-formulated phenolato titanium(IV) complexes against cancer cells.

Nanoparticles of titanium(IV) complexes of phenolato ligands were formed and evaluated for cytotoxicity toward human HT-29 colon cancer, murine T-25 lymphoma, and murine HU-2 multidrug-resistant (MDR) cells. The nano-formulation, besides increasing the complexes' shelf lives, is particularly efficient in overcoming limitations in solubility and cell-penetration, thus enhancing biological accessibility; large complexes that were inactive when measured in a non-formulated form showed marked activity when nano-formulated. For active and accessible small complexes, the effect of the formulation was negligible. Most complexes showed similar activity toward MDR cells and their drug-sensitive analogues, further increasing their therapeutic potential. An exception is a particularly hydrophobic complex, which is presumably more accessible to interaction with the membrane ABCB1 (MDR1) transporter active in the multidrug resistance of HU-2 cells. The most efficient compound is a mononuclear complex of a single hexadentate ligand, combining particularly high activity and hydrolytic stability with accessibility aided by the nano-formulation.

Anticancer metal complexes: synthesis and cytotoxicity evaluation by the MTT assay.

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; therefore their preparation should be conducted under an inert atmosphere. Evaluation of the anticancer activity of these complexes can be achieved by the MTT assay. The MTT assay is a colorimetric viability assay based on enzymatic reduction of the MTT molecule to formazan when it is exposed to viable cells. The outcome of the reduction is a color change of the MTT molecule. Absorbance measurements relative to a control determine the percentage of remaining viable cancer cells following their treatment with varying concentrations of a tested compound, which is translated to the compound anticancer activity and its IC50 values. The MTT assay is widely common in cytotoxicity studies due to its accuracy, rapidity, and relative simplicity. Herein we present a detailed protocol for the synthesis of air sensitive metal based drugs and cell viability measurements, including preparation of the cell plates, incubation of the compounds with the cells, viability measurements using the MTT assay, and determination of IC50 values.

High antitumor activity of highly resistant salan-titanium(IV) complexes in nanoparticles: an identified active species.

A nanoformulated trinuclear hydrolysis product of a bis(alkoxo) salan-Ti(IV) complex shows high antitumor activity, which identifies it as an active species in cells. Additional highly stable mononuclear derivatives also show high activity, when formulated into nanoparticles, thus evincing that biologically friendly Ti(IV) can provide high cytotoxicity with controlled biological function.

Major impact of N-methylation on cytotoxicity and hydrolysis of salan Ti(IV) complexes: sterics and electronics are intertwined.

A series of Ti(IV) complexes containing diamino bis(phenolato) "salan" type ligands with NH coordination were prepared, and their hydrolysis and cytotoxicity were analyzed and compared to the N-methylated analogues. Substituting methyl groups on the coordinative nitrogen donor of highly active and stable Ti(IV) salan complexes with H atoms has two main consequences: the hydrolysis rate increases and the cytotoxic activity diminishes. In addition, the small modification of a single replacement of Me with H leads to a different major hydrolysis product, where a dinuclear Ti(IV) complex with two bridging oxo ligands is obtained, as characterized by X-ray crystallography, rather than a trinuclear cluster. A partial hydrolysis product containing a single oxo bridge was also crystallographically analyzed. Investigation of a series of complexes with NH donors of different steric and electronic effects revealed that cytotoxicity may be restored by fine tuning these parameters even for complexes of low stability.

Different ortho and para electronic effects on hydrolysis and cytotoxicity of diamino bis(phenolato) "salan" Ti(IV) complexes.

Bis(isopropoxo) Ti(IV) complexes of diamino bis(phenolato) "salan" ligands were prepared, their hydrolysis in 1:9 water/THF solutions was investigated, and their cytotoxicity toward colon HT-29 and ovarian OVCAR-1 cells was measured. In particular, electronic effects at positions ortho and para to the binding phenolato unit were analyzed. We found that para substituents of different electronic features, including Me, Cl, OMe, and NO(2), have very little influence on hydrolysis rate, and all para-substituted ortho-H complexes hydrolyze slowly to give O-bridged clusters with a t(1/2) of 1-2 h for isopropoxo release. Consequently, no clear cytotoxicity pattern is observed as well, where the largest influence of para substituents appears to be of a steric nature. These complexes exhibit IC(50) values of 2-18 µM toward the cells analyzed, with activity which is mostly higher than those of Cp(2)TiCl(2), (bzac)(2)Ti(OiPr)(2) and cisplatin. On the contrary, major electronic effects are observed for substituents at the ortho position, with an influence that exceeds even that of steric hindrance. Ortho-chloro or -bromo substituted compounds possess extremely high hydrolytic stability where no major isopropoxo release as isopropanol occurs for days. In accordance, very high cytotoxicity toward colon and ovarian cells is observed for ortho-Cl and -Br complexes, with IC(50) values of 1-8 µM, where the most cytotoxic complexes are the ortho-Cl-para-Me and ortho-Br-para-Me derivatives. In this series of ortho-substituted complexes, the halogen radius is of lesser influence both on hydrolysis and on cytotoxicity, while OMe substituents do not impose similar effect of hydrolytic stability and cytotoxicity enhancement. Therefore, hydrolytic stability and cytotoxic activity are clearly intertwined, and thus this family of readily available Ti(IV) salan complexes exhibiting both features in an enhanced manner is highly attractive for further exploration.

Synthesis, characterization, cytotoxicity, and hydrolytic behavior of C2- and C1-symmetrical Ti(IV) complexes of tetradentate diamine bis(phenolato) ligands: a new class of antitumor agents.

We recently introduced a new class of bis(isopropoxo)-Ti(IV) complexes with diamine bis(phenolato) ligands that possess antitumor activity against colon HT-29 and ovarian OVCAR-1 cells that is higher than that of the known Ti(IV) compounds titanocene dichloride and budotitane as well as that of cisplatin. Herein, we elaborate on this family of compounds; we discuss the effect of structural parameters on the cytotoxic activity and hydrolytic behavior of these complexes, seeking a relationship between the two. Whereas complexes with small steric groups around the metal center possess high activity and lead mostly to formation of O-bridged polynuclear complexes with bound bis(phenolato) ligand upon water addition, bulky complexes hydrolyze to release all free ligands and are inactive. Slightly increasing the size of the N-donor substituents probably weakens the ligand binding in solution, and, thus, rapid hydrolysis is observed, leading to a lack of cytotoxicity, supporting the requirement for ligand inertness. Replacing the two isopropoxo ligands with a single catecholato unit gives a complex with a different geometry that exhibits slower hydrolysis and reduced cytotoxicity, suggesting some participation of labile ligand hydrolysis in the cytotoxicity mechanism. A crystallographically characterized O-bridged polynuclear species obtained from a biologically active bis(isopropoxo) complex upon water addition is inactive, which rules out its participation as the active species, yet suggests some role of the particular steric and electronic requirements allowing its formation in the activity mechanism. Additional measurements support rapid formation of the active species in the presence of cells prior to O-bridged Ti(IV) cluster formation.