Robin L. McCarley

Redox-Triggered Contents Release from Liposomes

An exciting new direction in responsive liposome research is endogenous triggering of liposomal payload release by overexpressed enzyme activity in affected tissues and offers the unique possibility of active and site-specific release. Bringing to fruition the fully expected capabilities of this new class of triggered liposomal delivery system requires a collection of liposome systems that respond to different upregulated enzymes; however, a relatively small number currently exist. Here we show that stable, approximately 100 nm diameter liposomes can be made from previously unreported quinone-dioleoyl phosphatidylethanolamine (Q-DOPE) lipids, and complete payload release (quenched fluorescent dye) from Q-DOPE liposomes occurs upon their redox activation when the quinone headgroup possesses specific substituents. The key component of the triggerable, contents-releasing Q-DOPE liposomes is a trimethyl-locked quinone redox switch attached to the N-terminus of DOPE lipids that undergoes a cleavage event upon two-electron reduction. Payload release by aggregation and leakage of uncapped Q-DOPE liposomes is supported by results from liposomes wherein deliberate alteration of the trimethyl-locked switch completely deactivates the redox-destructible phenomena (liposome opening). We expect that Q-DOPE liposomes and their variants will be important in treatment of diseases with associated tissues that overexpress quinone reductases, such as cancers and inflammatory diseases, because the quinone redox switch is a known substrate for this group of reductases.

Detection and Cellular Imaging of Human Cancer Enzyme Using a Turn-On, Wavelength-Shiftable, Self-Immolative Profluorophore

A frontier area in the development of activatable (turn-on) fluorescence-based probes is that concerned with rapid and selective stimulus triggering of probe activation so as to allow for biomarker identification and cellular imaging. The work here is concerned with a cloaked fluorophore composed of a reporter whose fluorescence is efficiently quenched by it being bound to an activatable trigger group through a novel self-immolative linker. Highly selective and rapid activation of the trigger group is achieved by chemical and enzymatic means that result in activated trigger group detachment from the self-immolative linker, with the latter subsequently cleaved from the reporter autonomously, thereby unmasking intense, red-shifted fluorescence emission. To achieve this success, we used a trimethyl-locked quinone propionic acid trigger group and an N-methyl-p-aminobenzyl alcohol self-immolative linker attached to the reporter. Delineated here are the synthesis and characterization of this cloaked fluorophore and the evaluation of its triggered turning on in the presence of an up-regulated enzyme in human cancer cells, NAD(P)H:quinone oxidoreductase-1 (NQO1, DT-diaphorase, EC 1.6.99.2).

Profluorogenic Reductase Substrate for Rapid, Selective, and Sensitive Visualization and Detection of Human Cancer Cells that Overexpress NQO1

Achieving the vision of identifying and quantifying cancer-related events and targets for future personalized oncology is predicated on the existence of synthetically accessible and economically viable probe molecules fully able to report the presence of these events and targets in a rapid and highly selective and sensitive fashion. Delineated here are the design and evaluation of a newly synthesized turn-on probe whose intense fluorescent reporter signature is revealed only through probe activation by a specific intracellular enzyme present in tumor cells of multiple origins. Quenching of molecular probe fluorescence is achieved through unique photoinduced electron transfer between the naphthalimide dye reporter and a covalently attached, quinone-based enzyme substrate. Fluorescence of the reporter dye is turned on by rapid removal of the quinone quencher, an event that immediately occurs only after highly selective, two-electron reduction of the sterically and conformationally restricted quinone substrate by the cancer-associated human NAD(P)H:quinone oxidoreductase isozyme 1 (hNQO1). Successes of the approach include rapid differentiation of NQO1-expressing and -nonexpressing cancer cell lines via the unaided eye, flow cytometry, fluorescence imaging, and two-photon microscopy. The potential for use of the turn-on probe in longer-term cellular studies is indicated by its lack of influence on cell viability and its in vitro stability.

Rapid, Photoinduced Electron Transfer-Modulated, Turn-on Fluorescent Probe for Detection and Cellular Imaging of Biologically Significant Thiols

There is a very limited number of existing probes whose fluorescence is turned on in the presence of the class of biological thiols made up of glutathione, cysteine, and homocysteine. The extant probes for this class of biological thiols commonly have poor aqueous solubility and long analyte response times, and they demand a very high probe/thiol ratio for decreased time of significant reporter signal generation; knowledge regarding their selectivity with respect to other sulfur-based analytes is unclear. Described here is a previously unreported photoinduced electron-transfer-quenched probe (HMBQ-Nap 1) that offers highly selective and rapid in vitro detection of this class of biologically important thiols at low concentrations and low probe/thiol ratio, and importantly, very rapid imaging of these biological thiols in human cells.

Structure−Activity Relationships in Peptide Modulators of β-Amyloid Protein Aggregation: Variation in α,α-Disubstitution Results in Altered Aggregate Size and Morphology

Neuronal cytotoxicity observed in Alzheimers disease (AD) is linked to the aggregation of β-amyloid peptide (Aβ) into toxic forms. Increasing evidence points to oligomeric materials as the neurotoxic species, not Aβ fibrils; disruption or inhibition of Aβ self-assembly into oligomeric or fibrillar forms remains a viable therapeutic strategy to reduce Aβ neurotoxicity. We describe the synthesis and characterization of amyloid aggregation mitigating peptides (AAMPs) whose structure is based on the Aβ hydrophobic core Aβ(17-20), with α,α-disubstituted amino acids (ααAAs) added into this core as potential disrupting agents of fibril self-assembly. The number, positional distribution, and side-chain functionality of ααAAs incorporated into the AAMP sequence were found to influence the resultant aggregate morphology as indicated by ex situ experiments using atomic force microscopy (AFM) and transmission electron microscopy (TEM). For instance, AAMP-5, incorporating a sterically hindered ααAA with a diisobutyl side chain in the core sequence, disrupted Aβ(1-40) fibril formation. However, AAMP-6, with a less sterically hindered ααAA with a dipropyl side chain, altered fibril morphology, producing shorter and larger sized fibrils (compared with those of Aβ(1-40)). Remarkably, ααAA-AAMPs caused disassembly of existing Aβ fibrils to produce either spherical aggregates or protofibrillar structures, suggesting the existence of equilibrium between fibrils and prefibrillar structures.

Environmentally Robust Rhodamine Reporters for Probe-based Cellular Detection of the Cancer-linked Oxidoreductase hNQO1

UNLABELLEDnWe successfully synthesized a fluorescent probe capable of detecting the cancer-associatednnnNAD(P)Hnquinoneoxidoreductase isozyme-1 within human cells, based on results from an investigation of the stability of various rhodamines and seminaphthorhodamines toward the biological reductant NADH, present at ∼100-200 μM within cells. While rhodamines are generally known for their chemical stability, we observe that NADH causes significant and sometimes rapid modification of numerous rhodamine analogues, including those oftentimes used in imaging applications. Results from mechanistic studies lead us to rule out a radical-based reduction pathway, suggesting rhodamine reduction by NADH proceeds by a hydride transfer process to yield the reduced leuco form of the rhodamine and oxidized NAD(+). A relationship between the structural features of the rhodamines and their reactivity with NADH is observed. Rhodamines with increased alkylation on the N3- and N6-nitrogens, as well as the xanthene core, react the least with NADH; whereas, nonalkylated variants or analogues with electron-withdrawing substituents have the fastest rates of reaction. These outcomes allowed us to judiciously construct a seminaphthorhodamine-based, turn-on fluorescent probe that is capable of selectively detecting the cancer-associated, NADH-dependent enzymennnNAD(P)Hnquinoneoxidoreductase isozyme-1 in human cancer cells, without the issue of NADH-induced deactivation of the seminaphthorhodamine reporter.

Cascade Reaction-Based, Near-Infrared Multiphoton Fluorescent Probe for the Selective Detection of Cysteine

The ability to detect and visualize cellular events and their associated target biological analytes through use of cell-permeable profluorogenic probes is dependent on the availability of activatable probes that respond rapidly and selectively to target analytes by production of fluorescent reporting molecules whose excitation and emission energies span a broad range. Herein is described a new probe, DCM-Cys, that preferentially reacts with cysteine to form a dicyanomethylene-4H-pyran (DCM) reporter whose red-energy fluorescence can be stimulated by two-photon, near-infrared excitation so as to provide visualization of cysteine presence inside living human cells with a high signal-to-background ratio. These aforementioned characteristics and the ability of DCM-Cys to provide selective, nanomolar-level in vitro cysteine detection, as demonstrated by its lack of significant response to other thiols and potential interfering agents from biological environments, are attributed to the molecular designs of the DCM-Cys probe and DCM reporter. Attachment of an acryl moiety to the DCM reporter via a self-eliminating, electron-withdrawing benzyl alcohol-carbamate linker offers a probe having selective, sensitive reaction with cysteine to rapidly produce a reporter whose energies of excitation and emission (λabsreport = 480 nm, λemisreport = 640 nm) are red-shifted from those of the DCM-Cys probe (λabsprobe = 440 nm, λemisprobe = 550 nm), thereby leading to low background signal from abundant probe and a large signal from the resulting reporter of cysteine presence.

Oxidoreductase-Facilitated Visualization and Detection of Human Cancer Cells

UNLABELLEDnAchieving highly selective and sensitive detection/visualization of intracellular biological events through the use of cell-penetrable, bioanalyte-activatable, turn-on probes is dependent on the presence of specific event-linked cellular biomarkers, if and only if there exist activatable probes that appropriately respond to the biomarker analyte. Here is described the evaluation of, and use in cellular imaging studies, a previously undisclosed naphthalimide probe QMeNN, whose fluorescence is deactivated by photoinduced electron transfer (PeT) quenching that results from the presence of a covalently linked biomarker-specific quinone trigger group. Highly selective and rapid activation of the quinone group by the human cancer tumor-linkednnnNAD(P)Hnquinone oxido-reductase isozyme 1 (hNQO1) results in fast trigger group removal to yield a highly fluorescent green-energy-range reporter that possesses a high molar absorptivity; there is a 136-fold increase in brightness for the enzymatically produced reporter versus probe precursor, a value 4 times greater than previously reported for the hNQO1 analyte. The novel probe is taken up and activated rapidly within only hNQO1-positive human cancer cells; addition of an hNQO1 inhibitor prevents the selective activation of the probe. Comparison of cytosolic fluorescence intensity in positive cells versus background in negative cells yields a quantitative metric (positive-to-negative ratio, PNR) for judging hNQO1 activity. We show it is possible to determine hNQO1 presence in previously studied colorectal cancer cells and the unexplored ovarian cancer cell line NIH:OVCAR-3, with respective PNR values of 926 and 34 being obtained. Even with 10 min probe incubation, ready discrimination of positive cells from negative cells is achieved. Cell viability is unaffected by probe presence, thereby highlighting the practicality of probe use in live-cell imaging applications.

Aqueous-Based Initiator Attachment and ATRP Grafting of Polymer Brushes from Poly(methyl methacrylate) Substrates

Many polymers, such as PMMA, are very susceptible to swelling or dissolution by organic solvents. Growing covalently attached polymer brushes from these surfaces by atom-transfer radical polymerization (ATRP) is challenging because of the typical requirement of organic solvent for initiator immobilization. We report an unprecedented, aqueous-based route to graft poly(N-isopropylacrylamide), PNIPAAm, from poly(methyl methacrylate), PMMA, surfaces by ATRP, wherein the underlying PMMA is unaffected. Successful attachment of the ATRP initiator, N-hydroxysuccinimidyl-2-bromo-2-methylpropionate, on amine-bearing PMMA surfaces was confirmed by XPS. From this surface-immobilized initiator, thermoresponsive PNIPAAm brushes were grown by aqueous ATRP to yield optically transparent PNIPAAm-grafted PMMA surfaces. This procedure is valuable, as it can be applied for the aqueous-based covalent attachment of ATRP initiator on any amine-functionalized surface, with subsequent polymerization of a variety of monomers.

Lipid nature and their influence on opening of redox-active liposomes.

The pathway for content release from reduction-sensitive liposomes based on a quinone-dioleoylphosphatidylethanolamine lipid conjugate (Q-DOPE) is outlined using results from fluorescent dye content release assays as well as single- and multiple-angle light scattering. Experimental observations are consistent with a shape/size change of the reduced liposomes prior to their aggregation, with subsequent near-quantitative content release achieved only when the lipid membrane experiences conditions favorable to a lamellar to an inverted hexagonal phase transition. Addition of poly(ethyleneglycol)-modified DOPE (PEG-DOPE) to the Q-DOPE liposomal formulation results in stabilization of the lipid bilayer, whereas incorporation of DOPE yields faster content release. At high DOPE concentrations, DOPE/PEG-DOPE/Q-DOPE liposomes exhibit larger content release, indicating a change in pathway for content release. The outcomes here provide a better understanding of the underlying principles of triggered liposomal content release and the potential utility of specific lipid properties for the rational design of drug delivery systems based on the novel Q-DOPE lipid.