Synthesis of cationic liposome nanoparticles using a thin film dispersed hydration and extrusion method.

Liposome nanoparticles can carry a wide range of therapeutic molecules including small molecules and nucleic acid-based therapeutics. Potential benefits include translocation across physiological barriers, reduced systemic toxicity, and enhanced pharmacokinetic parameters such as absorption, distribution, selective release and optimal elimination kinetics. Liposome nanoparticles can be generated with a wide range of natural and synthetic lipid-based molecules that confer desirable properties depending on the desired therapeutic application Nel et al (2023), Large (2021), Elkhoury (2020). This protocol article seeks to detail the procedures involved in the production of cationic liposomes using thin-film dispersed hydration method with an estimated uniform size of 60-70 nm for targeted drug administration in tumor cells, by modifying the previous one also published by the same authors cited here. The method was carrying out using N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl (DOTAP, 2 mg) as cationic lipid and cholesterol (0.5 mg) in a molar ratio of 7:3 respectively. The liposomal suspension was obtained and its physical, chemical and biological properties were determined. A two-step extrusion process, using 100 nm and 50 nm polycarbonate membranes, was carried. The results demonstrate generation of liposome nanoparticles with a size of 60-70 nm stable for at least 16 weeks and with an encapsulation efficiency of approximately 81% using Doxorubicin.

Surface-Enhanced Raman Analysis of Uric Acid and Hypoxanthine Analysis in Fractionated Bodily Fluids.

In recent years, the disease burden of hyperuricemia has been increasing, especially in high-income countries and the economically developing world with a Western lifestyle. Abnormal levels of uric acid and hypoxanthine are associated with many diseases, and therefore, to demonstrate improved methods of uric acid and hypoxanthine detection, three different bodily fluids were analysed using surface-enhanced Raman spectroscopy (SERS) and high-performance liquid chromatography (HPLC). Gold nanostar suspensions were mixed with series dilutions of uric acid and hypoxanthine, 3 kDa centrifugally filtered human blood serum, urine and saliva. The results show that gold nanostars enable the quantitative detection of the concentration of uric acid and hypoxanthine in the range 5-50 µg/mL and 50-250 ng/mL, respectively. The peak areas of HPLC and maximum peak intensity of SERS have strongly correlated, notably with the peaks of uric acid and hypoxanthine at 1000 and 640 cm-1, respectively. The r2 is 0.975 and 0.959 for uric acid and hypoxanthine, respectively. Each of the three body fluids has a number of spectral features in common with uric acid and hypoxanthine. The large overlap of the spectral bands of the SERS of uric acid against three body fluids at spectra peaks were at 442, 712, 802, 1000, 1086, 1206, 1343, 1436 and 1560 cm-1. The features at 560, 640, 803, 1206, 1290 and 1620 cm-1 from hypoxanthine were common to serum, saliva and urine. There is no statistical difference between HPLC and SERS for determination of the concentration of uric acid and hypoxanthine (p > 0.05). For clinical applications, 3 kDa centrifugal filtration followed by SERS can be used for uric acid and hypoxanthine screening is, which can be used to reveal the subtle abnormalities enhancing the great potential of vibrational spectroscopy as an analytical tool. Our work supports the hypnosis that it is possible to obtain the specific concentration of uric acid and hypoxanthine by comparing the SER signals of serum, saliva and urine. In the future, the analysis of other biofluids can be employed to detect biomarkers for the diagnosis of systemic pathologies.

TiO2-MWCNT nanohybrid: Cytotoxicity, protein corona formation and cellular internalisation in RTG-2 fish cell line.

Titanium dioxide nanoparticles-multiwalled carbon nanotubes (TiO2-MWCNT) nanohydrid has an enhanced photocatalytic activity across the visible light with promising applications in environmental remediation, solar energy devices and antimicrobial technologies. However, it is necessary to evaluate the toxicological effects of TiO2-MWCNT towards safe and sustainable development of nanohybrids. In this work, we studied the cytotoxicity, protein corona formation and cellular internalisation of TiO2-MWCNT on fibroblasts derived from gonadal rainbow trout tissue (RTG-2) for the first time. This nanohydrid did not show any toxicity effect on RTG-2 cells up to 100 mg L-1 after 24 h of exposure as monitored by alamar blue, neutral red and trypan blue assays (in presence or absence of foetal bovine serum, FBS). Futhermore, cryo-transmission electron microscopy analysis demonstrated that TiO2 particles is attached on nanotube surface after FBS-protein corona formation in cell culture medium. Raman spectroscopy imaging showed that TiO2-MWCNT can be internalised by RTG-2 cells. This work is a novel contribution towards better understanding the nanobiointeractions of nanohydrids linked to their in vitro effects on fish cells in aquatic nanoecotoxicology.

Transanal irrigation bowel routine for people with Cauda Equina Syndrome.

OBJECTIVE: Neurologic bowel incontinence and dysfunction are common with Cauda Equina Syndrome (CES). The study objective was to evaluate the efficacy of Peristeen Anal Irrigation System (PAIS)TM in people with CES. DESIGN: Clinical Trial. SETTING: Spinal Cord Rehabilitation outpatient clinic at the Health Sciences Centre in Winnipeg. METHODS: Twelve participants with a mean age of 46.2 years (range 34-72 years, 4 females) with CES used PAISTM bowel routine for 10 weeks. OUTCOME MEASURES: Change in Neurogenic Bowel Dysfunction Score (NBD) over 10 weeks relative to baseline. Secondary outcomes: Change in St. Mark's Fecal Incontinence score (SMFI), Cleveland Clinic Constipation score (CCC), and modified Rectal Surgeons Fecal Incontinence Quality of Life Score (QOL) at week 1, 2, 4, 6, 8 and 10 compared to baseline, and self-rating of bowel function at baseline and 10 weeks. Additionally, colonic transit times were assessed using the radioactive markers (Sitzmarks) method. RESULTS: Ten participants completed the study. Post-intervention primary outcome NBD score improved (p < 0.01). Secondary outcomes also improved significantly, including SMFI (p < 0.01), CCC (p < 0.01), QOL (p < 0.01), self-rating of bowel function (p < 0.01), and transit time improved by 22% (p < 0.05). CONCLUSION: Overall, a significant improvement was observed with the PAISTM for both primary, as well as secondary outcome measures, without any significant adverse effects. As this non-pharmaceutical method of bowel management is effective and has the potential to improve symptoms of bowel dysfunction in people with CES, it should be considered for those in which traditional methods of managing neurogenic bowel fail.

Biochemical impact of solar radiation exposure on human keratinocytes monitored by Raman spectroscopy; effects of cell culture environment.

Understanding and amelioration of the effects of solar radiation exposure are critical in preventing the occurrence of skin cancer. Towards this end, many studies have been conducted in 2D cell culture models under simplified and unrealistic conditions. 3D culture models better capture the complexity of in vivo physiology, although the effects of the 3D extracellular matrix have not been well studied. Monitoring the instantaneous and resultant cellular responses to exposure, and the influence of the 3D environment, could provide an enhanced understanding of the fundamental processes of photocarcinogenesis. This work presents an analysis of the biochemical impacts of simulated solar radiation (SSR) occurring in immortalised human epithelial keratinocytes (HaCaT), in a 3D skin model, compared to 2D culture. Cell viability was monitored using the Alamar Blue colorimetric assay (AB), and the impact of the radiation exposure, at the level of the biomolecular constituents (nucleic acids and proteins), were evaluated through the combination of Raman microspectroscopy and multivariate statistical analysis. The results suggest that SSR exposure induces alterations of the conformational structure of DNA as an immediate impact, whereas changes in the protein signature are primarily seen as a subsequent response.

In vitro cytotoxicity, cellular uptake, reactive oxygen species and cell cycle arrest studies of novel ruthenium(II) polypyridyl complexes towards A549 lung cancer cell line.

Ruthenium(II) polypyridyl complexes have displayed some promising biological responses against a variety of cancers and have emerged as a potential candidate that can show significant antitumor activity. Three ruthenium(II) polypyridyl complexes were biologically evaluated in vitro against the A549 cancer cell line. The complexes were selected based on initial DNA intercalation studies and MTT viability screening and were selected based on the most promising candidates, the [Ru(bpy)2o-CPIP].2PF6 (complex 1), [Ru(phen)2o-CPIP].2PF6 (complex 2) and [Ru(biq)2o-CPIP].2PF6 (complex 3). Confocal cellular uptake studies confirmed the intracellular transport of complexes into A549. Cytoplasmic and the nucleic accumulation of the complex 1 and 2 was seen while no fluorescent microscopy was performed for complex 3 due to instrumental limitations. Cellular cytotoxicity was investigated with the aid of the Alamar blue assay. The complexes displayed concentration and time dependent inhibitory effects yielding IC50 values from 5.00 to 32.75 µM. Complex 1 exhibit highest cytotoxicity with IC50 value of 5.00 ± 1.24 µM. All of the complexes have shown a significant effect in the reduction of intracellular reactive oxygen species (ROS) levels. Finally, the complexes have shown a transient effect on the cell cycle by arresting it at G0/G1 phase except for complex 2 [Ru(phen)2o-CPIP].2PF6 which has shown the significant G0/G1 arrest.

Monitoring the biochemical changes occurring to human keratinocytes exposed to solar radiation by Raman spectroscopy.

Solar radiation exposure is recognised to be a significant contributor to the development of skin cancer. Monitoring the simultaneous and consecutive mechanisms of interaction could provide a greater understanding of the process of photocarcinogenesis. This work presents an analysis of the biochemical and morphological changes occurring to immortalised human epithelial keratinocyte (HaCaT) cell cultures exposed to simulated solar radiation (SSR). Cell viability was monitored with the aid of the Alamar Blue assay, morphological examination was done with haematoxylin and eosin staining (H&E) and changes to the biochemical constituents (nucleic acids and proteins) as a result of the radiation insult were demonstrated through a combination of Raman microspectroscopy and multivariate analysis of spectral patterns. The spectral results suggest that SSR induces changes to the conformational structure of DNA as an immediate result of the radiation, whereas alteration in the protein signature is mostly seen as a later response.

Cold atmospheric plasma induces silver nanoparticle uptake, oxidative dissolution and enhanced cytotoxicity in glioblastoma multiforme cells.

Silver nanoparticles (AgNP) emerged as a promising reagent for cancer therapy with oxidative stress implicated in the toxicity. Meanwhile, studies reported cold atmospheric plasma (CAP) generation of reactive oxygen and nitrogen species has selectivity towards cancer cells. Gold nanoparticles display synergistic cytotoxicity when combined with CAP against cancer cells but there is a paucity of information using AgNP, prompting to investigate the combined effects of CAP using dielectric barrier discharge system (voltage of 75 kV, current is 62.5 mA, duty cycle of 7.5kVA and input frequency of 50-60Hz) and 10 nm PVA-coated AgNP using U373MG Glioblastoma Multiforme cells. Cytotoxicity in U373MG cells was >100-fold greater when treated with both CAP and PVA-AgNP compared with either therapy alone (IC50 of 4.30 µg/mL with PVA-AgNP alone compared with 0.07 µg/mL after 25s CAP and 0.01 µg/mL 40s CAP). Combined cytotoxicity was ROS-dependent and was prevented using N-Acetyl Cysteine. A novel darkfield spectral imaging method investigated and quantified AgNP uptake in cells determining significantly enhanced uptake, aggregation and subcellular accumulation following CAP treatment, which was confirmed and quantified using atomic absorption spectroscopy. The results indicate that CAP decreases nanoparticle size, decreases surface charge distribution of AgNP and induces uptake, aggregation and enhanced cytotoxicity in vitro.

Research priorities for rehabilitation and aging with HIV: a framework from the Canada-International HIV and Rehabilitation Research Collaborative (CIHRRC).

BACKGROUND: People living with HIV are living longer, and can experience physical, mental and social health challenges associated with aging and multimorbidity. Rehabilitation is well positioned to address disability and maximize healthy aging. An international collaborative network, called the Canada-International HIV and Rehabilitation Research Collaborative (CIHRRC), works to guide this emerging field. In this article, we report findings from CIHRRC's aim to identify emerging research priorities in HIV, aging and rehabilitation from the perspectives of people living with HIV, clinicians, researchers, representatives from community organizations and policy stakeholders. METHODS: We conducted a multi-stakeholder multi-method international consultation with people living with HIV, researchers, clinicians and representatives of community-based organizations to identify research priorities in HIV, aging and rehabilitation. Stakeholders identified research priorities during a one-day International Forum comprised of presentations and facilitated discussion. We collated and analyzed data using content analytical techniques, resulting in a framework of research priorities. RESULTS: Sixty-nine stakeholders from countries including Canada (n = 62; 90%), the United Kingdom (n = 5; 7%), United States (n = 1; 1%) and Australia (n = 1; 1%) attended the International Forum on HIV, Aging and Rehabilitation Research. Stakeholders represented community-based organizations (n = 20; 29%), academic institutions (n = 18; 26%), community or institutional healthcare organizations (n = 11; 16%), research or knowledge production organizations (n = 10; 14%), and organizations representing government or industry (n = 10; 14%). The Framework of Research Priorities in HIV, Aging and Rehabilitation includes seven research priorities: (1) nature, extent and impact of disability, concurrent health conditions and chronic inflammation with HIV; (2) prevalence, severity and impact of frailty; (3) community and social participation aging with HIV; (4) strategies for chronic disease management and healthy aging with HIV; (5) facilitators and barriers to access and engagement in, rehabilitation; (6) effectiveness of rehabilitation interventions for healthy aging with HIV; and (7) advancing development and use of patient reported outcome measures in HIV and aging. The Framework highlights methodological considerations to approach the priorities and the importance of knowledge translation and exchange to apply research knowledge into practice, programs and policy. CONCLUSIONS: These priorities offer a foundation for collaboration among international and multidisciplinary teams to advance the field of HIV, aging and rehabilitation in order to promote healthy aging with HIV.

Liposomal encapsulation of silver nanoparticles (AgNP) improved nanoparticle uptake and induced redox imbalance to activate caspase-dependent apoptosis.

Macrophages play a crucial role in several diseases' development and progression, such as in cancer and arthritis through ROS generation and inflammation. This makes macrophages a therapeutic target in these diseases. While silver nanoparticles (AgNP) have been widely used as an antibacterial and investigated as anticancer, its potential against macrophages may be limited due to its inherent oxidative mechanism. Here we encapsulated AgNP in a dipalmitoyl-phosphatidyl choline (DPPC) liposome (forming Lipo-AgNP) to suppress AgNP-induced ROS and enhance its cytotoxicity against THP1-differentiated macrophages (TDM). Our findings showed that while Lipo-AgNP had significantly more of a cytotoxic effect on TDMs (p < 0.01), it also significantly suppressed AgNP induced ROS generation and unexpectedly suppressed reduced glutathione (GSH) levels (p < 0.05) resulting in a redox imbalance in comparison to the unexposed control TDMs. Lipo-AgNP was also found to cause an increase DNA damage through H2AX histone phosphorylation and inhibition of Bcl-2 protein expression. This increased the Bax/Bcl2 ratio causing possible release of cytochrome C and subsequent caspase 3/7-dependent apoptosis. It was found that the difference between the mechanism of AgNP and Lipo-AgNP cytotoxicity may have been through the significantly increased Lipo-AgNP uptake by the TDMs as early as 30 min post-exposure (p < 0.05), changing the nanoparticle pharmacokinetic. In conclusion, the improved uptake of AgNP within the liposome caused ROS-independent caspase activation induced by Lipo-AgNP and this was facilitated by increased DNA damage, the induced redox imbalance and an increased Bax/Bcl-2 ratio.

Cold Atmospheric Plasma induces accumulation of lysosomes and caspase-independent cell death in U373MG glioblastoma multiforme cells.

Room temperature Cold Atmospheric Plasma (CAP) has shown promising efficacy for the treatment of cancer but the exact mechanisms of action remain unclear. Both apoptosis and necrosis have been implicated as the mode of cell death in various cancer cells. We have previously demonstrated a caspase-independent mechanism of cell death in p53-mutated glioblastoma multiforme (GBM) cells exposed to plasma. The purpose of this study was to elucidate the molecular mechanisms involved in caspase-independent cell death induced by plasma treatment. We demonstrate that plasma induces rapid cell death in GBM cells, independent of caspases. Accumulation of vesicles was observed in plasma treated cells that stained positive with acridine orange. Western immunoblotting confirmed that autophagy is not activated following plasma treatment. Acridine orange intensity correlates closely with the lysosomal marker Lyso TrackerTM Deep Red. Further investigation using isosurface visualisation of confocal imaging confirmed that lysosomal accumulation occurs in plasma treated cells. The accumulation of lysosomes was associated with concomitant cell death following plasma treatment. In conclusion, we observed rapid accumulation of acidic vesicles and cell death following CAP treatment in GBM cells. We found no evidence that either apoptosis or autophagy, however, determined that a rapid accumulation of late stage endosomes/lysosomes precedes membrane permeabilisation, mitochondrial membrane depolarisation and caspase independent cell death.

Surface modification of silver nanoparticle (AgNP) by liposomal encapsulation mitigates AgNP-induced inflammation.

Silver nanoparticles (AgNP) are widely used in a variety of consumable products as antibacterial to prevent or treat infection. Unfortunately, evidence exits that AgNP induces inflammation which can worsen with repeated human exposure. However, there is little or no research on how to mitigate these adverse effects due to AgNP induced-toxicity. Here, we investigated if surface modification of AgNP by liposomal encapsulation suppresses AgNP-mediated inflammatory responses in THP1 monocytes and THP1 differentiated macrophages (TDM). AgNP was encapsulated in a dipalmitoyl phosphatidyl choline- (DPPC)/cholesterol-based liposome by extrusion through a 100-nm polycarbonate membrane to form Lipo-AgNP. It was found as expected that AgNP induced significant release of IL-1ß, IL-6, IL-8 and TNF-α in THP1 monocytes more than the basal level. Interestingly, release of these cytokines was suppressed by Lipo-AgNP. In TDMs, AgNP and Lipo-AgNP induced IL-8 release (p < .0001), but Lipo-AgNP maintained IL-8 release at levels significantly lower than that of AgNP (p < .01). However, both AgNP and Lipo-AgNP suppressed IL-1ß and TNF-α release in LPS-stimulated THP1 monocytes and LPS-stimulated or unstimulated TDM respectively. We finally showed that Lipo-AgNP inhibits STAT-3 and this may be responsible for regulating the uncontrolled inflammation induced by AgNP likely mediated STAT-3 protein expression in LPS stimulated THP1 monocytes and TDMs, both LPS-stimulated and unstimulated. This data showed that Lipo-AgNP suppressed AgNP induced inflammation, making Lipo-AgNP particularly useful in treatment of bacteria induced inflammatory diseases and inflammatory cancers.

Design features of a guideline implementation tool designed to increase awareness of a clinical practice guide to HIV rehabilitation: A qualitative process evaluation.

RATIONALE, AIMS, AND OBJECTIVES: A comprehensive electronic guide (e-module) describing an interprofessional, evidence-informed approach to HIV rehabilitation was developed as an education resource for rehabilitation professionals. We developed a guideline implementation tool, consisting of a 10-week, case-based education intervention delivered by email, that was perceived to increase rehabilitation professionals' (occupational therapists (OTs), physical therapists (PTs), and speech language pathologists (SLPs)) knowledge and confidence to apply best practices in HIV rehabilitation. This study aimed to increase understanding of how the design of the guideline implementation tool facilitated increased awareness of and access to the e-module among rehabilitation professionals. METHODS: We conducted a single group intervention study with rehabilitation professionals in Canada and the United Kingdom. Six case studies targeting HIV pathophysiology and associated conditions, an interprofessional approach to rehabilitation assessment and treatment, and psychosocial issues experienced by people living with HIV, were emailed to participants at 2-week intervals. Individual semi-structured interviews were conducted post-intervention. Interview transcripts were analysed using a descriptive qualitative approach. RESULTS: Twenty-six individuals (17 from Canada, and 9 from the UK; 16 PTs, 7 OTs, 3 SLPs) were interviewed. One main theme related to design features of the intervention that facilitated learning and access to the e-module emerged. Subthemes highlighted features of the case-based intervention, including technical feasibility, terminology, formatting and layout, hyperlinks, number and frequency of case studies, and diverse and realistic case scenarios relevant to the learner's practice, that participants described as facilitating access to information and learning. CONCLUSION: Electronically administered case studies were perceived as complementary knowledge tools that increased access to an evidence-informed guide to HIV rehabilitation. Findings provide guidance on using case studies as a guideline implementation tool to facilitate access to information and related resources to optimize learning.

In vitro comparative cytotoxicity study of aminated polystyrene, zinc oxide and silver nanoparticles on a cervical cancer cell line.

Nanoparticles use in nano-biotechnology applications have increased significantly with Aminated polystyrene amine (AmPs NP), Zinc oxide (ZnO NP), and Silver (Ag NP) nanoparticles utilized in wide variety of consumer products. This has presented a number of concerns due to their increased exposure risks and associated toxicity on living systems. Changes in the structural and physicochemical properties of nanoparticles can lead to changes in biological activities. This study investigates, compares, and contrasts the potential toxicity of AmPs, ZnO and Ag NPs on an in vitro model (HeLa cells) and assesses the associated mechanism for their corresponding cytotoxicity relative to the surface material. It was noted that NPs exposure attributed to the reduction in cell viability and high-level induction of oxidative stress. All three test particles were noted to induce ROS to varying degrees which is irrespective of the attached surface group. Cell cycle analysis indicated a G2/M phase cell arrest, with the corresponding reduction in G0/G1 and S phase cells resulting in caspase-mediated apoptotic cell death. These findings suggest that all three NPs resulted in the decrease in cell viability, increase intracellular ROS production, induce cell cycle arrest at the G2/M phase and finally result in cell death by caspase-mediated apoptosis, which is irrespective of their differences in physiochemical properties and attached surface groups.

A randomized double-blind, placebo-controlled, cross-over trial assessing the effect of tadalafil (Cialis) on the cardiovascular response in men with complete spinal cord injury above the sixth thoracic level: A Pilot Study.

STUDY DESIGN: Double-blind, randomized cross-over placebo-controlled pilot study. OBJECTIVES: To determine the effects of tadalafil on systolic blood pressure (SBP), heart rate (HR), and dizziness of men with American Spinal Injury Association Impairment Scale-A (AIS-A) spinal cord injury (SCI) between cervical-4 (C4) and thoracic-5 (T5) levels. SETTING: Outpatient rehabilitation clinic. DESIGN: Double-blind, randomized cross-over placebo-controlled pilot study. METHODS: 20 males with AIS-A SCI, C4-T5 received either tadalafil 20 mg or placebo for the first arm, and then were crossed-over after 1 week to the second arm. SBP, HR, and Visual Analogue Scale (VAS) for dizziness upon sitting up from lying were measured at baseline and again 1, 2, 4, 12, 22, 29, and 36 h post dose administration. The change in each outcome measure (SBP, HR, VAS dizziness) was observed from pre-dose to each time point. A change in VAS dizziness of 2 cm or greater (scale 0-10 cm) was considered positive. RESULTS: SBP did not change significantly in either group. However, HR increased significantly in the tadalafil group at several time points (12 h p < 0.05, 22 h p <0.05, 29 h p <0.01, and 36 h p <0.05), with no change in the placebo group. The VAS dizziness significantly increased (range 2-6 cm changes) at some time point in 1/4 of the subjects after tadalafil, but not in the placebo group; all reports of dizziness were at 12 h or later. CONCLUSIONS: Tadalafil use in people with SCI above T6 is safe with respect to not causing hypotension; hemodynamic changes that occurred 12-36 h post administration were compensated for by elevations in HR. SPONSORSHIP: The Manitoba Medical Services Foundation and the Health Sciences Centre Foundation.

Raman spectroscopy detects biochemical changes due to different cell culture environments in live cells in vitro.

The in vitro cell culture environment can impact on cell biochemistry and cell cycle. The manifestation of such substrate-induced changes in cell cycle in the Raman microspectroscopic profiles of cell cultures is investigated at the level of nucleolus, nucleus and cytoplasm. HeLa immortalised human cervical cells and HaCaT dermal cells were cultured on three different substrates, conventional polystyrene cell culture dishes, CaF2 slides as a commonly used Raman substrate, and glass slides coated with collagen rat tail, as a mimic of the extra-cellular matrix (ECM) environment. A cell cycle study, based on percentage DNA content, as determined using propidium iodide staining and monitored by flow cytometry, was performed on cells of both types, grown on the different substrates, confirming that the in vitro cell culture environment impacts significantly on the cell cycle. Live cell in vitro Raman spectroscopic analysis of cells on the 2D CaF2 and 3D collagen substrates was performed and data was analysed using principal component analysis (PCA). The spectroscopic analysis revealed differences in profiles which reflect the differences in cell cycle for both in vitro culture environments. In particular, the Raman spectra of cells grown on CaF2 show indicators of cell stress, which are also associated with cell cycle arrest at the G0/G1 phase. This work contributes to the field of Raman spectroscopic analysis by providing a fresh look at the significance of the effect of in vitro culture environment to cell cycle and the sensitivity of Raman spectroscopy to such differences in cell metabolism.

Advancing Raman microspectroscopy for cellular and subcellular analysis: towards in vitro high-content spectralomic analysis.

In the confocal mode, Raman microspectroscopy can profile the biochemical content of biological cells at a subcellular level, and any changes to it by exogenous agents, such as therapeutic drugs or toxicants. As an exploration of the potential of the technique as a high-content, label-free analysis technique, this report reviews work to monitor the spectroscopic signatures associated with the uptake and response pathways of commercial chemotherapeutic agents and polymeric nanoparticles by human lung cells. It is demonstrated that the signatures are reproducible and characteristic of the cellular event, and can be used, for example, to identify the mode of action of the agent as well as the subsequent cell death pathway, and even mechanisms of cellular resistance. Data mining approaches are discussed and a spectralomics approach is proposed.

Cold Atmospheric Plasma Induces ATP-Dependent Endocytosis of Nanoparticles and Synergistic U373MG Cancer Cell Death.

Gold nanoparticles (AuNP) have potential as both diagnostic and therapeutic vehicles. However, selective targeting and uptake in cancer cells remains challenging. Cold atmospheric plasma (CAP) can be combined with AuNP to achieve synergistic anti-cancer cytotoxicity. To explore synergistic mechanisms, we demonstrate both rate of AuNP uptake and total amount accumulated in U373MG Glioblastoma multiforme (GBM) cells are significantly increased when exposed to 75 kV CAP generated by dielectric barrier discharge. No significant changes in the physical parameters of AuNP were caused by CAP but active transport mechanisms were stimulated in cells. Unlike many other biological effects of CAP, long-lived reactive species were not involved, and plasma-activated liquids did not replicate the effect. Chemical effects induced by direct and indirect exposure to CAP appears the dominant mediator of enhanced uptake. Transient physical alterations of membrane integrity played a minor role. 3D-reconstruction of deconvoluted confocal images confirmed AuNP accumulation in lysosomes and other acidic vesicles, which will be useful for future drug delivery and diagnostic strategies. Toxicity of AuNP significantly increased by 25-fold when combined with CAP. Our data indicate that direct exposure to CAP activates AuNP-dependent cytotoxicity by increasing AuNP endocytosis and trafficking to lysosomes in U373MG cells.

Synthesis, characterisation and DNA intercalation studies of regioisomers of ruthenium (II) polypyridyl complexes.

Regioisomers of the functional group of the main ligand (L) on a series of [Ru(phen)2L]2+and [Ru(bpy)2L]2+ complexes, where phen is 1,10 phenanthroline and bpy is 2,2'-bipyridine, were synthesised to investigate the interaction with deoxyribonucleic acid (DNA) as potential therapeutics. UV-Vis binding titrations, thermal denaturation and circular dichroism were used to evaluate their interaction with DNA. The conclusions indicated the significance of the auxiliary ligand; especially 1,10-phenanthroline has on the binding constants (Kb). The systematic variation of auxiliary ligand(phen or bpy), and polypyridyl ligand (4-(1H-Imidazo[4,5-f][1,10]phenanthrolin-2-yl)benzonitrile (CPIP), 2-(4-formylphenyl)imidazo[4,5-f] [1,10] phenanthroline (FPIP), 2-(4-bromophenyl)imidazo[4,5-f][1,10]phenanthroline (BPIP) and 2-(4-nitrophenyl)imidazo[4,5-f] [1,10] phenanthroline (NPIP), split in terms of functional group change were investigated for DNA interaction. The CPIP analogues in particular were investigated for the regioisomerism (ortho, meta, para) effect of the nitrile group on the ligand. It was found that both the DNA interaction could be tailored through the systematic variation of the electronic nature of the individual auxiliary ligand and to a lesser extent the functional group and regioisomeric change. Preliminary cell line studies have been carried out to determine the selectivity of the complexes against cell lines such as A375 (Skin Cancer), HeLa (Cervical Cancer), A549 (Lung Cancer), Beas2B (Lung Normal Cell) and MCF-7 (Breast Cancer). Complexes which had strong DNA interactions in the binding studies have proven to be the most efficacious against certain cell lines. Establishing well-defined structure property relationships when looking at trends in spectroscopic properties and DNA binding will aid in the intelligent design of potential therapeutic complexes.

Toxicological assessment of nanomaterials: the role of in vitro Raman microspectroscopic analysis.

The acceleration of nanomaterials research has brought about increased demands for rapid analysis of their bioactivity, in a multi-parametric fashion, to minimize the gap between potential applications and knowledge of their toxicological properties. The potential of Raman microspectroscopy for the analysis of biological systems with the aid of multivariate analysis techniques has been demonstrated. In this study, an overview of recent efforts towards establishing a 'label-free high content nanotoxicological assessment technique' using Raman microspectroscopy is presented. The current state of the art for cellular toxicity assessment and the potential of Raman microspectroscopy are discussed, and the spectral markers of the cellular toxic responses upon exposure to nanoparticles, changes on the identified spectral markers upon exposure to different nanoparticles, cell death mechanisms, and the effects of nanoparticles on different cell lines are summarized. Moreover, 3D toxicity plots of spectral markers, as a function of time and dose, are introduced as new methodology for toxicological analysis based on the intrinsic properties of the biomolecular changes, such as cytoplasmic RNA aberrations, protein and lipid damage associated with the toxic response. The 3D evolution of the spectral markers are correlated with the results obtained by commonly used cytotoxicity assays, and significant similarities are observed between band intensity and percentage viability obtained by the Alamar Blue assay, as an example. Therefore, the developed 3D plots can be used to identify toxicological properties of a nanomaterial and can potentially be used to predict toxicity, which can provide rapid advances in nanomedicine. Graphical Abstract Spectral markers of cytotoxicity as a function of time and dose.