Utilizing surface plasmon resonance as a novel method for monitoring in-vitro P-glycoprotein efflux.

P-glycoprotein (Pgp) is known for its dichotomous roles as both a safeguarding efflux transporter against xenobiotics and as a catalyst for multidrug resistance. Given the susceptibility of numerous therapeutic compounds to Pgp-mediated resistance, compliance with Food and Drug Administration (FDA) guidelines mandates an in-depth in vitro transport assay during drug development. This study introduces an innovative transport assay that aligns with these regulatory imperatives but also addresses limitations in the currently established techniques. Using Pgp-reconstituted liposomes and employing surface plasmon resonance (SPR), this study developed a distinct method of measuring the relative transport rates of Pgp substrates in a controlled microenvironment. The Pgp substrates selected for this study-quinidine, methadone, and desipramine-resulted in transport ratios that corroborate with trends previously observed. To assess the kinetics of Pgp-mediated transport, the results were analyzed by fitting the data to both currently proposed Pgp substrate translocation models-the vacuum cleaner and flippase models. While the resulting kinetic analysis in this study lends support predominantly to the vacuum cleaner model, this study most notably developed a novel method of assessing Pgp-mediated transport rates and real-time kinetics using surface plasmon resonance.

Bile Acid-Targeted Hyaluronic Acid Nanoparticles for Enhanced Oral Absorption of Deferoxamine.

Patients with ß-thalassemia and sickle cell disease often rely on blood transfusions which can lead to hemochromatosis and chronic oxidative stress in cells and tissues. Deferoxamine (DFO) is clinically approved to treat hemochromatosis but is suboptimal to patients due to its poor pharmacokinetics which requires long-term infusion regimens. Although the oral route is preferable, DFO has limited oral bioavailability. Studies have shown that hyaluronic acid (HA) and bile acid (BA) can enhance the oral absorption of poorly absorbed drugs. To improve upon the oral delivery of DFO, we report on the synthesis and characterization of HA (MW 15 kD) conjugated to two types of BA, deoxycholic acid (DOCA) and taurocholic acid (TCA), and DFO. The resulting seven polymeric conjugates all formed self-assembled nanoparticles. The degree of BA and DFO conjugation to the HA polymer was confirmed at each step through nuclear magnetic resonance, Fourier transform infrared spectroscopy, and UV-Vis spectroscopy. The best formulations for further in vitro testing were determined based on physicochemical characterizations and included HA-DFO, TCA9-HA-DFO, and DOCA9-HA-DFO. Results from in vitro assays revealed that TCA9-HA-DFO enhanced the permeation of DFO the most and was also less cytotoxic to cells compared to the free drug DFO. In addition, ferritin reduction studies indicated that the conjugation of DFO to TCA9-HA did not compromise its chelation efficiency at equivalent free DFO concentrations. This research provides supportive data for the idea that TCA conjugated to HA may enhance the oral absorption of DFO, improve its cytocompatibility, and maintain its iron chelation efficiency.

Oral Non-absorbable Polymer-Deferoxamine Conjugates for Reducing Dietary Iron Absorption.

Hereditary hemochromatosis (HH) is a non-transfusional genetic iron overload (IO) disease wherein patients are not able to regulate dietary iron absorption, which ultimately leads to excess cellular iron accumulation. Preventative measures for HH mainly include phlebotomy and asking patients to minimize dietary iron intake. To investigate alternative iron reduction strategies, we report on prophylactic non-absorbable polymer-deferoxamine (DFO) conjugates capable of chelating and reducing excessive gut uptake of dietary iron. Three different sizes of the conjugates (56 nm, 256 nm, and 7.4 µm) were prepared, and their physicochemical properties, transit times in the gut under fed/fasted conditions, acute safety, and efficacy at reducing iron absorption in a dietary iron-overload mouse model were investigated. The conjugates were synthesized through reverse phase water-in-oil (w/o) emulsions, followed by conjugation of DFO to the resulting polymer scaffolds. In vitro studies using Caco-2 transwell assays showed that the conjugates could not permeate across the monolayer, were poorly endocytosed, and did not induce cellular toxicity. In vivo mouse studies via oral gavage demonstrated that polymer-DFO conjugates remained in the gastrointestinal (GI) tract for up to 12 h and significantly prevented escalation of serum ferritin levels and excess liver iron accumulation. Ex vivo images of the duodenum suggest that nanometer-sized conjugates (56 and 246 nm) perform better at chelating dietary iron based on longer retention times (i.e., entrapment in the villi of the duodenum) and an overall slower transit from the GI tract compared to larger micron-sized (7.4 µm) conjugates. Overall, nanometer-sized polymer-DFO conjugates were orally non-absorbable, appeared safe, and were more efficacious at reducing dietary iron absorption when taken with non-heme containing food.

Antibacterial and Biofilm-Eradicating Activities of pH-Responsive Vesicles against Pseudomonas aeruginosa.

The formation of biofilms by a microcolony of bacteria is a significant burden on the healthcare industry due to difficulty eradicating it. In this study, pH-responsive vesicles capable of releasing apramycin (APR), a model aminoglycoside antibiotic, in response to the low pH typical of establishedPseudomonas aeruginosa biofilms resulted in improved eradication of existing biofilms in comparison to the free drug. The amphiphilic polymeric vesicle (PV) comprised of block polymer poly (ethylene glycol)-block-poly 2-(dimethylamino) ethyl methacrylate (mPEG-b-pDEAEMA) averaged 128 nm. The drug encapsulation content of APR in PV/APR was confirmed to be 28.2%, and the drug encapsulation efficiency was confirmed to be 51.2%. At pH 5.5, PV/APR released >90% APR after 24 h compared to <20% at pH 7.4. At pH 5.5, protonation of the pDEAEMA block results in a zeta potential of +23 mV compared to a neutral zeta potential of +2.2 mV at pH 7.4. Confocal microscopy, flow cytometry, and scanning electron microscopy reveal that the positively charged vesicles can compromise the integrity of the planktonic bacterial membrane in a pH-dependent manner. In addition, PV/APR is able to diffuse into mature biofilms to release APR in the acidic milieu of biofilm bacteria, and PV/APR was more efficient at eliminating preexisting biofilms compared to free APR at 128 and 256 µg/mL. This study reveals that dynamic charge density in response to pH can lead to differential levels of interactions with the biofilm and bacterial membrane. This effectively results in enhanced antibacterial and antibiofilm properties against both planktonic and difficult-to-treat biofilm bacteria at concentrations significantly lower than those of the free drug. Overall, this pH-responsive vesicle could be especially promising for treating biofilm-associated infectious diseases.

ROS-sensitive micelles for controlled delivery of antibiotics to combat intracellular Staphylococcus aureus-associated infections.

Bacteria can evade the immune system once they are engulfed by phagocytic host cells. This protects them against the bactericidal action of antibiotics and allows the infection to remain latent or to recur. Reactive oxygen species (ROS)-related stress has been implicated in various pathological conditions such as inflammatory diseases involving infections of host cells and can serve as a useful trigger for intracellular controlled drug delivery. We herein report on a fluorescent ROS-sensitive intracellular antibiotic delivery nanoparticle for encapsulation of rifampin (RIF) based on the principles of Förster Resonance Energy Transfer (FRET) that is capable of ratiometrically sensing H2O2 levels and monitoring the drug release process. The fluorescent micelles (MFs) are formed through the self-assembly of amphiphilic diblock copolymers consisting of a poly(ethylene glycol) (PEG) segment and a fluorescent oxidation-responsive hydrophobic phenylboronic pinacol ester (PBA) block. Specifically, MFs could encapsulate the model antibiotic RIF (MF/RIF) and ROS-triggered controlled release of RIF within infected macrophages (where ROS levels are elevated) improved the elimination of intracellular bacteria compared to MF or RIF alone. This antibiotic delivery system may be especially effective at fighting intracellular pathogens that have managed to evade the immune system and could minimize exposure of normal cells and tissues to high drug concentrations.

Synthesis and evaluation of an amphiphilic deferoxamine:gallium-conjugated cationic random copolymer against a murine wound healing infection model of Pseudomonas aeruginosa.

Multidrug resistant (MDR) Gram-negative bacteria are an urgent global health threat. We report on the design and evaluation of a xenosiderophore-conjugated cationic random copolymer (pGQ-DG) which exhibits selective antibacterial activity against Pseudomonas aeruginosa (P. aeruginosa) by targeting select outer membrane (OM) receptors for scavenging xenosiderophores such as deferoxamine (DFO), while possessing favorable cytocompatibility and exhibiting low hemolysis, to enhance and safely damage the bacterial OM. pGQ-DG demonstrated synergistic properties in combination with vancomycin (VAN) when evaluated in vitro against P. aeruginosa. In addition, pGQ-DG plus VAN cleared the P. aeruginosa infection and efficiently accelerated healing in a murine wound healing model as effectively as colistin, suggesting that this strategy could serve as an alternative to colistin against MDR bacteria. STATEMENT OF SIGNIFICANCE: P. aeruginosa exhibits intrinsic antibiotic resistance due to limited permeability of its outer membrane (OM). A triple combination antipseudomonal approach was investigated by 1) selectively targeting P. aeruginosa through the complex DFO:gallium, 2) disrupting the OM through a cationic random copolymer, and 3) enhancing bacteria sensitivity to VAN as a result of the OM disruption. Synthesis and characterization of the lead polymer pGQ-DG, mechanism of action, antimicrobial activity, and biocompatibility were investigated in vitro and in vivo. Overall pGQ-DG plus VAN cleared the P. aeruginosa infection and accelerated wound healing in mice as effectively as colistin, suggesting that this strategy could serve as an alternative to colistin against multidrug resistant P. aeruginosa.

Challenges and Opportunities of Deferoxamine Delivery for Treatment of Alzheimer's Disease, Parkinson's Disease, and Intracerebral Hemorrhage.

Deferoxamine mesylate (DFO) is an FDA-approved, hexadentate iron chelator routinely used to alleviate systemic iron burden in thalassemia major and sickle cell patients. Iron accumulation in these disease states results from the repeated blood transfusions required to manage these conditions. Iron accumulation has also been implicated in the pathogenesis of Alzheimer's disease (AD), Parkinson's disease (PD), and secondary injury following intracerebral hemorrhage (ICH). Chelation of brain iron is thus a promising therapeutic strategy for improving behavioral outcomes and slowing neurodegeneration in the aforementioned disease states, though the effectiveness of DFO treatment is limited on several accounts. Systemically administered DFO results in nonspecific toxicity at high doses, and the drug's short half-life leads to low patient compliance. Mixed reports of DFO's ability to cross the blood-brain barrier (BBB) also appear in literature. These limitations necessitate novel DFO formulations prior to the drug's widespread use in managing neurodegeneration. Herein, we discuss the various dosing regimens and formulations employed in intranasal (IN) or systemic DFO treatment, as well as the physiological and behavioral outcomes observed in animal models of AD, PD, and ICH. The clinical progress of chelation therapy with DFO in managing neurodegeneration is also evaluated. Finally, the elimination of intranasally administered particles via the glymphatic system and efflux transporters is discussed. Abundant preclinical evidence suggests that intranasal DFO treatment improves memory retention and behavioral outcome in rodent models of AD, PD, and ICH. Several other biochemical and physiological metrics, such as tau phosphorylation, the survival of tyrosine hydroxylase-positive neurons, and infarct volume, are also positively affected by intranasal DFO treatment. However, dosing regimens are inconsistent across studies, and little is known about brain DFO concentration following treatment. Systemic DFO treatment yields similar results, and some complex formulations have been developed to improve permeability across the BBB. However, despite the success in preclinical models, clinical translation is limited with most clinical evidence investigating DFO treatment in ICH patients, where high-dose treatment has proven dangerous and dosing regimens are not consistent across studies. DFO is a strong drug candidate for managing neurodegeneration in the aging population, but before it can be routinely implemented as a therapeutic agent, dosing regimens must be standardized, and brain DFO content following drug administration must be understood and controlled via novel formulations.

Effects of Polyethyelene Glycol-Desferrioxamine:Gallium Conjugates on Pseudomonas aeruginosa Outer Membrane Permeability and Vancomycin Potentiation.

Pseudomonas aeruginosa exhibits a broad spectrum of intrinsic antibiotic resistance because of the limited permeability of its outer membrane. Given this situation, molecules that could make Gram-negative bacteria more permeable and more susceptible to large-scaffold Gram-positive antibiotics may be advantageous. Herein, we evaluate the antimicrobial activity of a series of targeted poly(ethylene glycol)-desferrioxamine/gallium (PEG-DG) conjugates that can improve the sensitivity of P. aeruginosa to the glycopeptide vancomycin (VAN). We observed that single-ended mPEG-DG and double-ended PEG-DG2 conjugates characterized by PEG MW ≥2000 synergistically enhanced the sensitivity of VAN against P. aeruginosa reference strains PAO1 and ATCC 27853 and three clinically isolated carbapenem-resistant strains, but not Escherichia coli strain ATCC 25922. Although the exact mechanism of this phenomenon is currently under investigation, PEG-DG conjugates enhanced nitrocefin (NCF), hexidium iodide (HI), and VAN permeability only when PEG and DG were directly conjugated. The two most important physicochemical factors contributing to the synergistic activity observed with VAN relate to (1) the final concentration of DG ligands conjugated to the polymer and (2) the polymer length, wherein MW ≥2000 yielded a similar fractional inhibitory concentration.

Reactive Oxygen Species-Triggered Dissociation of a Polyrotaxane-Based Nanochelator for Enhanced Clearance of Systemic and Hepatic Iron.

Chronic blood transfusions are used to alleviate anemic symptoms in thalassemia and sickle cell anemia patients but can eventually result in iron overload (IO) and subsequently lead to severe oxidative stress in cells and tissues. Deferoxamine (DFO) is clinically approved to treat transfusional IO, but the use of the iron chelator is hindered by nonspecific toxicity and poor pharmacokinetic (PK) properties in humans, resulting in the need to administer the drug via long-term infusion regimens that can often lead to poor patient compliance. Herein, a nanochelator system that uses the characteristic IO physiological environment to dissociate was prepared through the incorporation of DFO and reactive oxygen species (ROS)-sensitive thioketal groups into an α-cyclodextrin-based polyrotaxane platform (rPR-DFO). ROS-induced dissociation of this nanochelator (ca. 10 nm) into constructs averaging 2 nm in diameter significantly increased urine and fecal elimination of excess iron in vivo. In addition to significantly improved PK properties, rPR-DFO was well-tolerated in mice and no adverse side effects were noted in single high dose or multiple dose acute toxicity studies. The overall features of rPR-DFO as a promising system for iron chelation therapy can be attributed to a combination of the nanochelator's improved PK, favorable distribution to the liver, and ROS-induced dissociation properties into constructs <6 nm for faster renal elimination. This ROS-responsive nanochelator design may serve as a promising alternative for safely prolonging the circulation of DFO and more rapidly eliminating iron chelates from the body in iron chelation therapy regimens requiring repeated dosing of nanochelators.

Antibacterial and Potentiation Properties of Charge-Optimized Polyrotaxanes for Combating Opportunistic Bacteria.

Bacteria are now becoming more resistant to most conventional antibiotics. Approaches for the treatment of multidrug-resistant bacterial infections are urgently required. Cationic polymers have broad-spectrum antibacterial activity but can also induce non-specific damage to mammalian cells. Herein, we report on the design of cationic polyrotaxanes (cPRs) with variable charge densities. cPRs were prepared by conjugating neutral ethanolamine and cationic ethylenediamine at various ratios onto threaded alpha-cyclodextrins and their antimicrobial and cytocompatible properties were investigated in vitro. In contact with Gram-negative bacteria, cPRs can disrupt the bacterial outer membrane integrity via electrostatic interactions and penetrate into the cytosol. The ability of cPRs to serve as potentiators at sub-MIC concentrations, to enhance the permeability and activity of poorly permeable antibiotics such as vancomycin, erythromycin and rifampicin, was also investigated against Gram-negative P. aeruginosa PAO1 and E. coli ATCC 25922.

Desferrioxamine:gallium-pluronic micelles increase outer membrane permeability and potentiate antibiotic activity against Pseudomonas aeruginosa.

The outer membrane of Pseudomonas aeruginosa functions primarily as a permeability barrier and imparts a broad spectrum of intrinsic antibiotic resistance. Herein, we describe the synthesis, characterization, and antimicrobial evaluation of a targeted polymeric micelle that specifically permeabilizes the outer membrane and potentiates antibiotic activity against P. aeruginosa.

Terpyridine-Micelles for Inhibiting Bacterial Biofilm Development.

Iron plays a critical role in bacterial infections and is especially critical for supporting biofilm formation. Until recently, Fe(III) was assumed to be the most relevant form of iron to chelate in therapeutic antimicrobial strategies due to its natural abundance under normal oxygen and physiologic conditions. Recent clinical data obtained from cystic fibrosis (CF) patients found that there is actually quite an abundance of Fe(II) present in sputum and that there exists a significant relationship between sputum Fe(II) concentration and severity of the disease. A biocompatible mixed micelle formed from the self-assembly of poly(lactic- co-glycolic acid)- block-methoxy poly(ethylene glycol) (PLGA- b-mPEG) and poly(lactic- co-glycolic acid)- block-poly(terpyridine)5 [PLGA- b-p(Tpy)5] polymers was prepared to chelate Fe(II) (Tpy-micelle). Tpy-micelles showed high selectivity for Fe(II) over Fe(III), decreased biofilm mass more effectively under anaerobic conditions at >4 µM Tpy-micelles, reduced bacteria growth in biofilms by >99.9% at 128 µM Tpy-micelles, effectively penetrated throughout a 1-day old biofilm, and inhibited biofilm development in a concentration-dependent manner. This study reveals that Fe(II) chelating Tpy-micelles are a promising addition to Fe(III) chelating strategies to inhibit biofilm formation in CF lung infections.

ROS-triggered degradable iron-chelating nanogels: Safely improving iron elimination in vivo.

Iron-mediated generation of highly toxic Reactive Oxygen Species (ROS) plays a major role in the process leading to iron overload-related diseases. The long-term subcutaneous administration of Deferoxamine (DFO) is currently clinically-approved to improve patient symptoms and survival. However, non-specific toxicity and short circulation times of the drug in humans often leads to poor patient compliance. Herein, thioketal-based ROS-responsive polymeric nanogels containing DFO moieties (rNG-DFO) were designed to chelate iron and to degrade under oxidative stimuli into fragments <10 nm to enhance excretion of iron-bound chelates. Serum ferritin levels and iron concentrations in major organs of IO mice decreased following treatment with rNG-DFO, and fecal elimination of iron-bound chelates increased compared to free DFO. Furthermore, rNG-DFO decreased iron mediated oxidative stress levels in vitro and reduced iron-mediated inflammation in the liver of IO mice. The study confirms that ROS-responsive nanogels may serve as a promising alternative to DFO for safer and more efficient iron chelation therapy.

A bioorthogonal turn-on fluorescent strategy for the detection of lysine acetyltransferase activity.

Lysine acetylation plays vital roles in the regulation of fundamental cellular processes, which is mediated by lysine acetyltransferases (KATs). Developing chemical biology probes for KAT activity detection is of important value in providing improved understanding of their biological functions. We reported a panel of "turn-on" fluorescent probes for sensitive and selective detection of KAT enzymatic activity through a simple mix-and-read format. Combined with bioorthogonal substrate labelling and click chemistry, these probes produced strong "turn-on" fluorescent signals in response to KAT-mediated acylation process. This chemical biology strategy diversifies the assay toolboxes to investigate functions and mechanisms of acetyltransferase enzymes.

Nanogel-DFO conjugates as a model to investigate pharmacokinetics, biodistribution, and iron chelation in vivo.

Deferoxamine (DFO) to treat iron overload (IO) has been limited by toxicity issues and short circulation times and it would be desirable to prolong circulation to improve non-transferrin bound iron (NTBI) chelation. In addition, DFO is currently unable to efficiently target the large pool of iron in the liver and spleen. Nanogel-Deferoxamine conjugates (NG-DFO) can prove useful as a model to investigate the pharmacokinetic (PK) properties and biodistribution (BD) behavior of iron-chelating macromolecules and their overall effect on serum ferritin levels. NG-DFO reduced the cytotoxicity of DFO and significantly reduced cellular ferritin levels in IO macrophages in vitro. PK/BD studies in normal rats revealed that NG-DFO displayed prolonged circulation and preferential accumulation into the liver and spleen. IO mice treated with NG1-DFO presented significantly lower levels of serum ferritin compared to DFO. Total renal and fecal elimination data point to the need to balance prolonged circulation with controlled degradation to accelerate clearance of iron-chelating macromolecules.

Multifunctional Polymeric Micelles for Combining Chelation and Detection of Iron in Living Cells.

Multifunctional self-assembled micelles composed of Pluronics F127 polymer chains are developed and investigated for chelation and selective detection of iron(III) in vitro and in iron-overloaded cells. Tetraphenylethene (TPE) is encapsulated into the micelle core and the iron chelate drug deferoxamine (DFO) is conjugated to micelles to generate a fluorescence quenching detection system termed DFO-TFM for short, where T stands for TPE, F for F127, and M for micelle. The key to the successful formation of this fluorescence quenching system is due to the near-ideal overlap between the absorption spectrum of the DFO:iron(III) complex and fluorescence spectrum of TPE. DFO-TFM can retain the iron-chelation properties of DFO and exhibits negligible cytotoxicity compared to free DFO. Furthermore, this fluorescence "turn-off" system can be utilized to detect the presence of iron and to monitor the chelation process in an iron overload cell model. This study serves as an effective proof-of-concept model for designing future in vivo systems capable of combining the features of iron chelation with iron detection and efforts toward the development of such detection systems are currently underway.

Enzymatically Biodegradable Polyrotaxane-Deferoxamine Conjugates for Iron Chelation.

Chelation therapy is frequently used to help reduce excess iron in the body, but current chelators such as deferoxamine (DFO) are plagued by short blood circulation times, which necessitates infusions and can cause undesirable toxic side effects in patients. To address these issues, polyrotaxanes (PR) were synthesized by threading α-cyclodextrin (α-CD) onto poly(ethylene glycol) bis(amine) (PEG-BA, MW 3400 g/mol) capped with enzymatically cleavable bulky Z-L phenylalanine (Z-L Phe) moieties. The resulting PR was conjugated to DFO and hydroxypropylated to generate the final polyrotaxane-DFO (hPR-DFO). The iron chelating capability of hPR-DFO was verified by UV-vis absorption spectroscopy and the ability of materials to degrade into smaller CD-conjugated DFO fragments (hCD-DFO) in the presence of the protease was confirmed via gel permeation chromatography. In vitro studies in iron-overloaded macrophages reveal that hPR-DFO can significantly reduce the cytotoxicity of the drug while maintaining its chelation efficacy, and that it is more rapidly endocytosed and trafficked to lysosomes of iron-overloaded cells in comparison to non-iron-overloaded macrophages. In vivo studies indicate that iron-overloaded mice treated with hPR-DFO displayed lower serum ferritin levels (a measure of iron burden in the body) and could eliminate excess iron by both the renal and fecal routes. Moreover, there was no gross evidence of acute toxicological damage to the liver or spleen.

Modulation of oxidative stress and subsequent induction of apoptosis and endoplasmic reticulum stress allows citral to decrease cancer cell proliferation.

The monoterpenoid, citral, when delivered through PEG-b-PCL nanoparticles inhibits in vivo growth of 4T1 breast tumors. Here, we show that citral inhibits proliferation of multiple human cancer cell lines. In p53 expressing ECC-1 and OVCAR-3 but not in p53-deficient SKOV-3 cells, citral induces G1/S cell cycle arrest and apoptosis as determined by Annexin V staining and increased cleaved caspase3 and Bax and decreased Bcl-2. In SKOV-3 cells, citral induces the ER stress markers CHOP, GADD45, EDEM, ATF4, Hsp90, ATG5, and phospho-eIF2α. The molecular chaperone 4-phenylbutyric acid attenuates citral activity in SKOV-3 but not in ECC-1 and OVCAR-3 cells. In p53-expressing cells, citral increases phosphorylation of serine-15 of p53. Activation of p53 increases Bax, PUMA, and NOXA expression. Inhibition of p53 by pifithrin-α, attenuates citral-mediated apoptosis. Citral increases intracellular oxygen radicals and this leads to activation of p53. Inhibition of glutathione synthesis by L-buthionine sulfoxamine increases potency of citral. Pretreatment with N-acetylcysteine decreases phosphorylation of p53 in citral-treated ECC-1 and OVCAR-3. These results define a p53-dependent, and in the absence of p53, ER stress-dependent mode of action of citral. This study indicates that citral in PEG-b-PCL nanoparticle formulation should be considered for treatment of breast and other tumors.