Carbon Dots-Biomembrane Interactions and Their Implications for Cellular Drug Delivery.

The effect of carbon dots (CDs) on a model blayer membrane was studied as a means of comprehending their ability to affect cell membranes. Initially, the interaction of N-doped carbon dots with a biophysical liposomal cell membrane model was investigated by dynamic light scattering, z-potential, temperature-modulated differential scanning calorimetry, and membrane permeability. CDs with a slightly positive charge interacted with the surface of the negative-charged liposomes and evidence indicated that the association of CDs with the membrane affects the structural and thermodynamic properties of the bilayer; most importantly, it enhances the bilayer's permeability against doxorubicin, a well-known anticancer drug. The results, like those of similar studies that surveyed the interaction of proteins with lipid membranes, suggest that carbon dots are partially embedded in the bilayer. In vitro experiments employing breast cancer cell lines and human healthy dermal cells corroborated the findings, as it was shown that the presence of CDs in the culture medium selectively enhanced cell internalization of doxorubicin and, subsequently, increased its cytotoxicity, acting as a drug sensitizer.

A Triphenylphosphonium-Functionalized Delivery System for an ATM Kinase Inhibitor That Ameliorates Doxorubicin Resistance in Breast Carcinoma Mammospheres.

The enzyme ataxia-telangiectasia mutated (ATM) kinase is a pluripotent signaling mediator which activates cellular responses to genotoxic and metabolic stress. It has been shown that ATM enables the growth of mammalian adenocarcinoma stem cells, and therefore the potential benefits in cancer chemotherapy of a number of ATM inhibitors, such as KU-55933 (KU), are currently being investigated. We assayed the effects of utilizing a triphenylphosphonium-functionalized nanocarrier delivery system for KU on breast cancer cells grown either as a monolayer or in three-dimensional mammospheres. We observed that the encapsulated KU was effective against chemotherapy-resistant mammospheres of breast cancer cells, while having comparably lower cytotoxicity against adherent cells grown as monolayers. We also noted that the encapsulated KU sensitized the mammospheres to the anthracycline drug doxorubicin significantly, while having only a weak effect on adherent breast cancer cells. Our results suggest that triphenylphosphonium-functionalized drug delivery systems that contain encapsulated KU, or compounds with a similar impact, are a useful addition to chemotherapeutic treatment schemes that target proliferating cancers.

Development of stimuli-responsive lyotropic liquid crystalline nanoparticles targeting lysosomes: Physicochemical, morphological and drug release studies.

The abilities of sub-cellular targeting and stimuli-responsiveness are critical challenges in pharmaceutical nanotechnology. In the present study, glyceryl monooleate (GMO)-based non-lamellar lyotropic liquid crystalline nanoparticles were stabilized by the poly(2-(dimethylamino)ethyl methacrylate)-b-poly(lauryl methacrylate) block copolymer carrying tri-phenyl-phosphine cations (TPP-QPDMAEMA-b-PLMA), either used alone or in combination with other polymers as co-stabilizers. The systems were designed to perform simultaneously sub-cellular targeting, stimuli-responsiveness and to exhibit stealthiness. The physicochemical characteristics and fractal dimensions of the resultant nanosystems were obtained from light scattering techniques, while their micropolarity and microfluidity from fluorescence spectroscopy. Their morphology was assessed by cryo-TEM, while their thermal behavior by microcalorimetry and high-resolution ultrasound spectroscopy. The analyzed properties, including the responsiveness to pH and temperature, were found to be dependent on the combination of the polymeric stabilizers. The subcellular localization was monitored by confocal microscopy, revealing targeting to lysosomes. Subsequently, resveratrol was loaded into the nanosystems, the entrapment efficiency was investigated and in vitro release studies were carried out at different conditions, in which a stimuli-triggered drug release profile was achieved. In conclusion, the proposed multi-functional nanosystems can be considered as potentially stealth, stimuli-responsive drug delivery nanocarriers, with targeting ability to lysosomes and presenting a stimuli-triggered drug release profile.

The Prophylactic and Multimodal Activity of Two Isatin Thiosemicarbazones against Alzheimer's Disease In Vitro.

Alzheimer's disease (AD) is a multifactorial disorder strongly involving the formation of amyloid-ß (Aß) oligomers, which subsequently aggregate into the disease characteristic insoluble amyloid plaques, in addition to oxidative stress, inflammation and increased acetylcholinesterase activity. Moreover, Aß oligomers interfere with the expression and activity of Glycogen synthase kinase-3 (GSK3) and Protein kinase B (PKB), also known as AKT. In the present study, the potential multimodal effect of two synthetic isatin thiosemicarbazones (ITSCs), which have been previously shown to prevent Aß aggregation was evaluated. Both compounds resulted in fully reversing the Aß-mediated toxicity in SK-NS-H cells treated with exogenous Aß peptides at various pre-incubation time points and at 1 µM. Cell survival was not recovered when compounds were applied after Aß cell treatment. The ITSCs were non-toxic against wild type and 5xFAD primary hippocampal cells. They reversed the inhibition of Akt and GSK-3ß phosphorylation in 5xFAD cells. Finally, they exhibited good antioxidant potential and moderate lipoxygenase and acetylcholinesterase inhibition activity. Overall, these results suggest that isatin thiosemicarbazone is a suitable scaffold for the development of multimodal anti-AD agents.

Engineering Mitochondriotropic Carbon Dots for Targeting Cancer Cells.

Aiming to understand and enhance the capacity of carbon dots (CDs) to transport through cell membranes and target subcellular organelles-in particular, mitochondria-a series of nitrogen-doped CDs were prepared by the one-step microwave-assisted pyrolysis of citric acid and ethylenediamine. Following optimization of the reaction conditions for maximum fluorescence, functionalization at various degrees with alkylated triphenylphosphonium functional groups of two different alkyl chain lengths afforded a series of functionalized CDs that exhibited either lysosome or mitochondria subcellular localization. Further functionalization with rhodamine B enabled enhanced fluorescence imaging capabilities in the visible spectrum and allowed the use of low quantities of CDs in relevant experiments. It was thus possible, by the appropriate selection of the alkyl chain length and degree of functionalization, to attain successful mitochondrial targeting, while preserving non-toxicity and biocompatibility. In vitro cell experiments performed on normal as well as cancer cell lines proved their non-cytotoxic character and imaging potential, even at very low concentrations, by fluorescence microscopy. Precise targeting of mitochondria is feasible with carefully designed CDs that, furthermore, are specifically internalized in cells and cell mitochondria of high transmembrane potential and thus exhibit selective uptake in malignant cells compared to normal cells.

Multi-Walled Carbon Nanotubes Decorated with Guanidinylated Dendritic Molecular Transporters: An Efficient Platform for the Selective Anticancer Activity of Doxorubicin.

An efficient doxorubicin (DOX) drug delivery system with specificity against tumor cells was developed, based on multi-walled carbon nanotubes (MWCNTs) functionalized with guanidinylated dendritic molecular transporters. Acid-treated MWCNTs (oxCNTs) interacted both electrostatically and through hydrogen bonding and van der Waals attraction forces with guanidinylated derivatives of 5000 and 25,000 Da molecular weight hyperbranched polyethyleneimine (GPEI5K and GPEI25K). Chemical characterization of these GPEI-functionalized oxCNTs revealed successful decoration with GPEIs all over the oxCNTs sidewalls, which, due to the presence of guanidinium groups, gave them aqueous compatibility and, thus, exceptional colloidal stability. These GPEI-functionalized CNTs were subsequently loaded with DOX for selective anticancer activity, yielding systems of high DOX loading, up to 99.5% encapsulation efficiency, while the DOX-loaded systems exhibited pH-triggered release and higher therapeutic efficacy compared to that of free DOX. Most importantly, the oxCNTs@GPEI5K-DOX system caused high and selective toxicity against cancer cells in a non-apoptotic, fast and catastrophic manner that cancer cells cannot recover from. Therefore, the oxCNTs@GPEI5K nanocarrier was found to be a potent and efficient nanoscale DOX delivery system, exhibiting high selectivity against cancerous cells, thus constituting a promising candidate for cancer therapy.

Novel Isatin Thiosemicarbazone Derivatives as Potent Inhibitors of ß-Amyloid Peptide Aggregation and Toxicity.

Inhibition of ß-amyloid peptide (Αß) aggregation in Alzheimer's disease (AD) is among the therapeutic approaches against AD which still attracts scientific research interest. In the search for compounds that interact with Aß and disrupt its typical aggregation course toward oligomeric or polymeric toxic assemblies, small organic molecules of natural origin, combining low molecular weight (necessary blood-brain barrier penetration) and low toxicity (necessary for pharmacological application), are greatly sought after. Isatin (1H-indoline-2,3-dione), a natural endogenous indole, and many of its derivatives exhibit a wide spectrum of neuropharmacological and chemotherapeutic properties. The synthesis and biological evaluation of four new isatins as inhibitors of Aß aggregation is presented herein. In these derivatives, the N-phenyl thiosemicarbazide moiety is joined at the 3-oxo position of isatin through Schiff base formation, and substitutions are present at the indole nitrogen and position 5 of the isatin core. Biophysical studies employing circular dichroism, thioflavin T fluorescence assay, and transmission electron microscopy reveal the potential of the isatin thiosemicarbazones (ITSCs) to alter the course of Αß aggregation, with two of the derivatives exhibiting outstanding inhibition of the aggregation process, preventing completely the formation of amyloid fibrils. Furthermore, in in vitro studies in primary neuronal cell cultures, the ITSCs were found to inhibit the Aß-induced neurotoxicity and reactive oxygen species production at concentrations as low as 1 µM. Taken all together, the novel ITSCs can be considered as privileged structures for further development as potential AD therapeutics.

Targeting breast cancer stem-like cells using chloroquine encapsulated by a triphenylphosphonium-functionalized hyperbranched polymer.

Cancer stem cells (CSCs) have garnered increasing attention over the past decade, as they are believed to play a crucial role in tumor progression and drug resistance. Accumulating evidence provides insight into the function of autophagy in maintenance and survival of CSCs. Here, we studied the impact of a mitochondriotropic triphenylphosphonium-functionalized dendrimeric nanocarrier on cultured breast cancer cell lines, grown either as adherent cells or as mammospheres that mimic a stem-like phenotype. The nanocarrier manifested a substantial cytotoxicity both alone as well as after encapsulation of chloroquine, a well-known autophagy inhibitor. The cytotoxic effects of the nanocarrier could be ascribed to interference with mitochondrial function. Importantly, mammospheres were selectively sensitive to encapsulated chloroquine and this depends on the expression of the gene encoding ATM kinase. Ataxia-telangiectasia mutated (ATM) kinase is an enzyme that functions as an essential signaling mediator that enables growth of cancer stem cells through the regulation of autophagy. We noted that this ATM-dependent sensitivity of mammospheres to encapsulated chloroquine was independent of the status of the tumor suppressor gene p53. Our study suggests that breast cancer stem cells, as they are modeled by mammospheres, are sensitive to encapsulated chloroquine, depending on the expression of the ATM kinase, which is thereby characterized as a potential biomarker for sensitivity to this type of treatment.

A combination drug delivery system employing thermosensitive liposomes for enhanced cell penetration and improved in vitro efficacy.

Drug-loaded thermosensitive liposomes are investigated as drug delivery systems in combination with local mild hyperthermia therapy due to their capacity to release their cargo at a specific temperature range (40-42 °C). Additional benefit can be achieved by the development of such systems that combine two different anticancer drugs, have cell penetration properties and, when heated, release their drug payload in a controlled fashion. To this end, liposomes were developed incorporating at low concentration (5 mol%) a number of monoalkylether phosphatidylcholine lipids, encompassing the platelet activating factor, PAF, and its analogues that induce thermoresponsiveness and have anticancer biological activity. These thermoresponsive liposomes were efficiently (>90%) loaded with doxorubicin (DOX), and their thermal properties, stability and drug release were investigated both at 37 ◦C and at elevated temperatures. In vitro studies of the most advantageous liposomal formulation containing the methylated PAF derivative (methyl-PAF, edelfosine), an established antitumor agent, were performed on human prostate cancer cell lines. This system exhibits controlled release of DOX at 40-42 °C, enhanced cell uptake due to the presence of methyl-PAF, and improved cell viability inhibition due to the combined action of both medications.

A New Perspective in Utilizing MMP-9 as a Therapeutic Target for Alzheimer's Disease and Type 2 Diabetes Mellitus.

Matrix metalloprotease 9 (MMP-9) is a 92 kDa type IV collagenase and a member of the family of endopeptidases. MMP-9 is involved in the degradation of extracellular matrix components, tissue remodeling, cellular receptor stripping, and processing of various signaling molecules. In the CNS, the effects of MMP-9 are quite complex, since it exerts beneficial effects including neurogenesis, angiogenesis, myelogenesis, axonal growth, and inhibition of apoptosis, or destructive effects including apoptosis, blood-brain barrier disorder, and demyelination. Likewise, in the periphery, physiological events, as the involvement of MMP-9 in angiogenesis, for instance in wound healing, can be turned into pathological, such as in tumor metastasis, depending on the state of the organism. Alzheimer's disease is a neurodegenerative disorder, characterized by amyloid accumulation and deposition in the brain. Amyloidogenesis, however, also occurs in diseases of the periphery, such as type II diabetes mellitus, where an analogous type of amyloid, is deposited in the pancreas. Interestingly, both diseases exhibit similar pathology and disease progression, with insulin resistance being a major common denominator. Hence, combinatorial strategies searching new or existing molecules to apply for therapeutic use for both diseases are gaining momentum. MMP-9 is extensively studied due to its association with a variety of physiological and pathological processes. Consequently, meticulous design could render MMP-9 into a potential therapeutic target for Alzheimer's disease and type 2 diabetes mellitus; two seemingly unrelated diseases.

Synthesis of Hydroxyapatite, ß-Tricalcium Phosphate and Biphasic Calcium Phosphate Particles to Act as Local Delivery Carriers of Curcumin: Loading, Release and In Vitro Studies.

The successful synthesis of hydroxyapatite (HA), ß-Tricalcium phosphate (ß-TCP) and two biphasic mixtures (BCPs) of the two was performed by means of wet precipitation. The resulting crystals were characterized and the BCP composition was analyzed and identified as 13% HA-87% TCP and 41% HA-59% TCP. All samples were treated with curcumin solutions, and the degree of curcumin loading and release was found to be proportional to the TCP content of the ceramic. No further cytotoxicity was observed upon MG-63 treatment with the curcumin-loaded ceramics. Finally, the alkaline phosphatase activity of the cells was found to increase with increasing content of TCP, which provides an encouraging proof of concept for the use of curcumin-loaded synthetic biomaterials in bone remodeling.

A randomized controlled clinical trial on the effectiveness of three different mouthrinses (chlorhexidine with or without alcohol and C31G), adjunct to periodontal surgery, in early wound healing.

OBJECTIVES: The use of chlorhexidine (CHX) with or without alcohol has been recommended for a number of clinical applications. On the other hand, there is a plethora of widely subscribed antiseptics, such as agent C31G (alkyl dimethyl glycine/alkyl dimethyl amine oxide), which has not yet been evaluated postsurgically. The effectiveness of three different mouthrinses (CHX with and without alcohol, C31G) in plaque control and early wound healing was compared postoperatively. MATERIALS AND METHODS: In this, randomized, double-blind, controlled clinical trial 42 patients were allocated to three groups assigned to 2 weeks rinsing after non-regenerative periodontal flap surgery with or without osseous surgery with C31G (group A), alcohol-free CHX 0.12% (group B) or alcohol-based CHX 0.12% (group C). At days 7 and 14, plaque and early wound healing indices were recorded. At day 14, total bacterial counts were estimated utilizing real-time quantitative polymerase chain reaction (qPCR). Statistics included linear and generalized linear mixed models. RESULTS: At day 7, healing response was not significantly different among groups. At day 14, group A revealed the highest while group C demonstrated the lowest plaque index values (B vs A, odds ratio-OR = 0.18, p = 0.012; C vs A, OR = 0.01, p < 0.001; C vs B, OR = 0.06, p < 0.001). Group C demonstrated the lowest bacterial counts levels at day 14 (38.470 × 106, 48.190 × 106, and 3.020 × 106 for groups A, B, and C, respectively). At day 14, healing was significantly better in group C compared to B (p = 0.007). Group A showed no significant differences compared to other groups. CONCLUSIONS: (1) The presence of alcohol may increase the effectiveness of CHX in early wound healing, (2) C31G might be an alternative solution prescribed during early postoperative period after non-regenerative periodontal flap surgery. CLINICAL RELEVANCE: The present study found that active agent C31G displayed no significant differences to CHX formulations regarding periodontal wound healing improvement and might be used alternatively after non-regenerative periodontal flap surgery. In addition, an alcohol based 0.12% CHX mouthwash was more effective than an alcohol-free 0.12% CHX and C31G mouthrinse on plaque control in the absence of mechanical oral hygiene.

Overexpression of matrix metalloproteinase-9 (MMP-9) rescues insulin-mediated impairment in the 5XFAD model of Alzheimer's disease.

A hallmark of Alzheimer's disease (AD) is the accumulation of oligomeric amyloid-ß (Aß) peptide, which may be primarily responsible for neuronal dysfunction. Insulin signaling provides a defense mechanism against oligomer-induced neuronal loss. We previously described the neuroprotective role of matrix metalloproteinase 9 (MMP-9) in decreasing the formation of Aß oligomers. In the present study, we examined the role of MMP-9 on the insulin survival pathway in primary hippocampal cultures and hippocampal cell extracts from 3 month-old wild type, AD (5XFAD), MMP-9-overexpressing (TgMMP-9), and double transgenic mice (5XFAD/TgMMP-9). The data demonstrate that the insulin pathway was compromised in samples from 5XFAD mice, when compared to the wild type and TgMMP-9. This was due to enhanced phosphorylation of IRS1 at Serine 636 (pIRS1-Ser636), which renders IRS1 inactive and prevents insulin-mediated signaling. In 5XFAD/TgMMP-9 samples, the insulin survival pathway was rescued through enhanced activation by phosphorylation of IRS1 at Tyrosine 465 (pIRS1-Tyr465), downstream increased phosphorylation of Akt and GSK-3ß, and decreased phosphorylation of JNK kinase. Oligomeric Aß levels decreased and BDNF levels increased in 5XFAD/TgMMP-9 mice, compared to 5XFAD mice. Our findings indicate that overexpression of MMP-9 rescued insulin survival signaling in vitro and in early stages in the 5XFAD model of AD.

Pharmacogenetic inhibition of eIF4E-dependent Mmp9 mRNA translation reverses fragile X syndrome-like phenotypes.

Fragile X syndrome (FXS) is the leading genetic cause of autism. Mutations in Fmr1 (fragile X mental retardation 1 gene) engender exaggerated translation resulting in dendritic spine dysmorphogenesis, synaptic plasticity alterations, and behavioral deficits in mice, which are reminiscent of FXS phenotypes. Using postmortem brains from FXS patients and Fmr1 knockout mice (Fmr1(-/y)), we show that phosphorylation of the mRNA 5' cap binding protein, eukaryotic initiation factor 4E (eIF4E), is elevated concomitant with increased expression of matrix metalloproteinase 9 (MMP-9) protein. Genetic or pharmacological reduction of eIF4E phosphorylation rescued core behavioral deficits, synaptic plasticity alterations, and dendritic spine morphology defects via reducing exaggerated translation of Mmp9 mRNA in Fmr1(-/y) mice, whereas MMP-9 overexpression produced several FXS-like phenotypes. These results uncover a mechanism of regulation of synaptic function by translational control of Mmp-9 in FXS, which opens the possibility of new treatment avenues for the diverse neurological and psychiatric aspects of FXS.

Intracellular accumulation of aggregated pyroglutamate amyloid beta: convergence of aging and Aß pathology at the lysosome.

Deposition of aggregated amyloid beta (Aß) is a major hallmark of Alzheimer's disease (AD)-a common age-related neurodegenerative disorder. Typically, Aß is generated as a peptide of varying lengths. However, a major fraction of Aß peptides in the brains of AD patients has undergone posttranslational modifications, which often radically change the properties of the peptides. Aß3(pE)-42 is an N-truncated, pyroglutamate-modified variant that is abundantly present in AD brain and was suggested to play a role early in the pathogenesis. Here we show that intracellular accumulation of oligomeric aggregates of Aß3(pE)-42 results in loss of lysosomal integrity. Using a novel antibody specific for aggregates of AßpE3, we show that in postmortem human brain tissue, aggregated AßpE3 is predominantly found in the lysosomes of both neurons and glial cells. Our data further demonstrate that AßpE3 is relatively resistant to lysosomal degradation, which may explain its accumulation in the lysosomes. The intracellular AßpE3 aggregates increase in an age-dependent manner. The results presented in this study support a model where Aß pathology and aging converge, leading to accumulation of the degradation-resistant pE-modified Aß in the lysosomes, lysosomal dysfunction, and neurodegeneration.

A novel G3337A mitochondrial ND1 mutation related to cardiomyopathy co-segregates with tRNALeu(CUN) A12308G and tRNAThr C15946T mutations.

We describe a novel mutation in human mitochondrial NADH dehydrogenase 1 gene (ND1), a G to A transition at nucleotide position 3337, which is co-segregated with two known mutations in tRNALeu(CUN) A12308G and tRNAThr C15946T. These mutations were detected in two unrelated patients with different clinical phenotypes, exhibiting cardiomyopathy as the common symptom. The ND1 G3337A mutation that was detected was found almost homoplasmic in the two patients and it was absent in 150 individuals that were tested as control group. Mitochondrial respiratory chain complex I activity of the patients platelets was also tested and found decreased compared to those of controls. We suggest that the co-existence of mutations in tRNA and ND1 genes may act synergistically affecting the clinical phenotype. Our study highlights the enormous phenotypic diversity that exists among pathogenic mtDNA mutations and re-emphasizes the need for a more careful clinical approach.