Self-assembly of gold nanoparticles by chitosan for improved epinephrine detection using a portable surface enhanced Raman scattering device.

Surface enhanced Raman scattering (SERS) has become widely used for identification, quantification and providing structural information about molecular structure in low concentrations as it allows signal Raman enhancement using metallic nanoparticles (NPs). Controlling interaction between analyte and NPs is a major point for the optimization of signal exaltation in SERS analysis. The objective of this study is the improvement and the control of SERS analysis by aggregation/self-assembly optimization of AuNPs using quaternized chitosan. The interest of this approach is to allow stable and reliable measurements with a simple and low cost approach compatible with a massive use in the field. In this work, we used design of experiments by Box-Behnken design to fix optimized conditions to increase signal sensibility of epinephrine water solutions. We also tested SERS signal stability in isotonic sodium chloride 0.9% and glucose 5% matrices. Our results demonstrate that globally neutral AuNPs aggregates were stabilized at a nanometric size by the subsequent addition of polyelectrolyte chains and allows for significant Raman signal enhancement of epinephrine. We succeed to prepare the SERS active material and measure a stable signal of epinephrine at a concentration as down as 0.1 µg mL-1 in less than 5 min. The signal remained stable and exploitable for at least 2 h. Our results reveal a strong correlation between intensity and logarithm of the concentration (concentration before dilution from 0.1 to 10 µg mL-1) suggesting a possible quantification. Furthermore, the signal of epinephrine at 10 µg mL-1 were also exploitable and stable in complex media as isotonic sodium chloride 0.9% and glucose 5%. This represents a particularly interesting application that would allow direct analysis of drugs complex media and open the way to analysis in biological samples.

Discriminative and quantitative analysis of norepinephrine and epinephrine by surface-enhanced Raman spectroscopy with gold nanoparticle suspensions.

Surface-enhanced Raman spectroscopy (SERS) is a powerful analytical technique capable of increasing the Raman signal of an analyte using specific nanostructures. The close contact between those nanostructures, usually a suspension of nanoparticles, and the molecule of interest produces an important exaltation of the intensity of the Raman signal. Even if the exaltation leads to an improvement of Raman spectroscopy sensitivity, the complexity of the SERS signal and the numbers of parameters to be controlled allow the use of SERS for detection rather than quantification. The aim of this study was to develop a robust discriminative and quantitative analysis in accordance with pharmaceutical standards. In this present work, we develop a discriminative and quantitative analysis based on the previous optimized parameters obtained by the design of experiments fixed for norepinephrine (NOR) and extended to epinephrine (EPI) which are two neurotransmitters with very similar structures. Studying the short evolution of the Raman signal intensity over time coupled with chemometric tools allowed the identification of outliers and their removal from the data set. The discriminant analysis showed an excellent separation of EPI and NOR. The comparative analysis of the data showed the superiority of the multivariate analysis after logarithmic transformation. The quantitative analysis allowed the development of robust quantification models from several gold nanoparticle batches with limits of quantification of 32 µg/mL for NOR and below 20 µg/mL for EPI even though no Raman signal is observable for such concentrations. This study improves SERS analysis over ultrasensitive detection for discrimination and quantification using a handheld Raman spectrometer.

Optimization of experimental conditions by surface enhanced Raman Scattering (SERS) spectroscopy with gold nanoparticles suspensions.

Surface Enhanced Raman Scattering (SERS) spectroscopy is a rapid and innovative analysis technique involving metallic nanoparticles (NPs). The interaction between NPs and norepinephrine gives an exaltation of the Raman signal under certain experimental conditions. The control of the signal exaltation, crucial for sensitive analyses, remains one of the main limitations of this technique. The aim of this work is to optimize the exaltation conditions for an optimal SERS signal at two concentrations of norepinephrine (NOR) and spherical gold NPs in suspension. This first work will fix the optimal experimental conditions essential for the development of robust discriminant and quantitative analysis of catecholamine. Two complete 3-factors 3-levels experiment designs were performed at 20 µg.mL-1 and 100 µg.mL-1 norepinephrine concentrations, each experiment being repeated 3 times. The optimization factors were the process of synthesis (variation of the quantity of gold and citrate used for the three synthesis SA, SB and SC) and HCl (0.3 M, 0.5 M, 0.7 M) as well as the volume ratio of NPs and norepinephrine (0.5, 2, 3.5) for SERS acquisition. Spectral acquisitions were performed with a handheld Raman spectrometer with an excitation source at 785 nm. For each sample, 31 acquisitions were realized during 3 s every 8 s. The optimization parameter was the intensity of the characteristic band of norepinephrine at 1280 cm-1. A total of 5,042 spectra were acquired and the pre-treatment selected for all spectra was asymmetric least square combined to a smoothing of Savistsky Golay (ALS - SG). The optimal contact time between norepinephrine and NPs depends on the experimental conditions and was determined for each experiment according to the mean intensity between the three replicates. After interpretation of the experimental designs, the optimal conditions retained were the quantity of gold corresponding to SA and the HCl concentration 0.7 M for the two concentrations of norepinephrine. Indeed, the optimal volume ratio depend on the NOR concentration.

A Case of Phage Therapy against Pandrug-Resistant Achromobacter xylosoxidans in a 12-Year-Old Lung-Transplanted Cystic Fibrosis Patient.

Bacteriophages are a promising therapeutic strategy among cystic fibrosis and lung-transplanted patients, considering the high frequency of colonization/infection caused by pandrug-resistant bacteria. However, little clinical data are available regarding the use of phages for infections with Achromobacter xylosoxidans. A 12-year-old lung-transplanted cystic fibrosis patient received two rounds of phage therapy because of persistent lung infection with pandrug-resistant A. xylosoxidans. Clinical tolerance was perfect, but initial bronchoalveolar lavage (BAL) still grew A. xylosoxidans. The patient's respiratory condition slowly improved and oxygen therapy was stopped. Low-grade airway colonization by A. xylosoxidans persisted for months before samples turned negative. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. Whole genome sequencing indicated that the eight A. xylosoxidans isolates, collected during phage therapy, belonged to four delineated strains, whereby one had a stop mutation in a gene for a phage receptor. The dynamics of lung colonisation were documented by means of strain-specific qPCRs on different BALs. We report the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. The dynamics of airway colonization was more complex than deduced from bacterial culture, involving phage susceptible as well as phage resistant strains.

Pharmaceutical consultation to detect drug interactions in patients treated with oral chemotherapies: A descriptive cross-sectional study.

OBJECTIVE: The development of oral chemotherapy (OC) has led to the recent establishment of multidisciplinary programmes involving pharmacists. We evaluated the utility of our local programme for detecting potential interactions with OCs, particularly drug-drug interactions (DDIs) and herbal-drug interactions (HDIs). METHODS: We performed a single-centre retrospective descriptive study of patients on OC attending a pharmaceutical consultation (PC) during a seven-month period. These consultations included the use of various complementary tools/databases to search for interactions. RESULTS: We analysed 308 treatments taken by 42 consecutive patients. Fifty-four potential interactions with OCs were detected in 26% (n = 79) of the treatments taken by patients: 46 DDIs (32 minor, 12 major, 2 contraindicated) and eight HDIs. Five interventions associated with interactions were suggested by pharmacists during the consultations (4 were taken into account by oncologists). The total mean time spent on each PC for an individual patient was 80 minutes (36 minutes of preparation, 44 minutes with the patient). CONCLUSION: This pilot study highlights the importance of studying interactions in such patients, and of the expertise of pharmacists for detecting interactions, which were found in more than one in four treatment lines. The further development of such activities, which already take up considerable amounts of time, is therefore warranted.

Enhancing digestive fistula healing by the off-label use of a thermoresponsive vessel occluder polymer associated with esophageal stent placement: A case report.

This case report relates to the first-in-man use of a vessel occluder gel medical device as a fistula occluder in a repurposing strategy. A patient with chronic colocutaneous fistula received an off-label treatment with a thermoresponsive Poloxamer 407 gel (20%) via percutaneous administration and injected under endoscopic control. Treatment consisted in the association of esophageal stent placement and gel injection. The product was administered just after the stent placement at<20°C in its liquid form, gelling at body temperature to form a fistula plug. However, the stent was removed at day 26 because of major pain and the fistula was still present. Treatment was continued a total of 14 administrations of thermoresponsive Poloxamer 407 gel during 7 weeks via the external fistula orifice. The treatment reduced fistula orifice diameter from 4.0±0.5 to 1mm and fistula daily output decreased from 425±65 to 23±4mL, when comparing the months before and after treatment. Gel administration was not associated with any toxic effects. The therapeutic outcome remained stable 1 year after treatment. The external fistula diameter and the fistula output were similar to what was observed after the last Poloxamer 407 gel administration.

A mathematical approach to deal with nanoparticle polydispersity in surface enhanced Raman spectroscopy to quantify antineoplastic agents.

Antineoplastic agents are, for most of them, highly toxic drugs prepared at hospital following individualized prescription. To protect patients and healthcare workers, it is important to develop analytical tools able to identify and quantify such drugs on a wide concentration range. In this context, surface enhanced Raman spectroscopy (SERS) has been tested as a specific and sensitive technique. Despite the standardization of the nanoparticle synthesis, a polydispersity of nanoparticles in the suspension and a lack of reproducibility persist. This study focuses on the development of a new mathematical approach to deal with this nanoparticle polydispersity and its consequences on SERS signal variability through the feasibility of 5-fluorouracil (5FU) quantification using silver nanoparticles (AgNPs) and a handled Raman spectrophotometer. Variability has been maximized by synthetizing six different batches of AgNPs for an average size of 24.9 nm determined by transmission electron microscopy, with residual standard deviation of 17.0%. Regarding low performances of the standard multivariate data processing, an alternative approach based on the nearest neighbors were developed to quantify 5FU. By this approach, the predictive performance of the 5FU concentration was significantly improved. The mean absolute relative error (MARE) decreased from 16.8% with the traditional approach based on PLS regression to 6.30% with the nearest neighbors approach (p-value < 0.001). This study highlights the importance of developing mathematics adapted to SERS analysis which could be a step to overcome the spectral variability in SERS and thus participate in the development of this technique as an analytical tool in quality control to quantify molecules with good performances, particularly in the pharmaceutical field.

Forced Degradation of Monoclonal Antibodies After Compounding: Impact on Routine Hospital Quality Control.

Compounded therapeutic mAbs used in a hospital require quality control (QC). In our hospital, analytical QC process intended to mAbs identification and quantification is based on flow injection analysis associated with second-derivative UV spectroscopy and matching method algorithm. We studied the influence of degraded mAbs after compounding on this validated QC. Three forced stress conditions including mechanical, thermal, and freeze-thawing stresses were studied to yield degraded mAbs from 2 model compounds, that is, bevacizumab (IgG1) and nivolumab (IgG4). Different degraded mAbs were generated and were analyzed in terms of turbidity, the percentage of aggregation, size distribution, and changes in tertiary structure. Stresses showed to be mAb-dependent in terms of aggregation. Tertiary structural changes were observed in most of the stressed samples by principal component analysis of the UV second-derivative data. The structural and physicochemical modifications conducted to mismatch depending on the nature of the stress. The mismatch ranged from 17% to 72% for the mAbs, except for freeze-thawed bevacizumab for which a perfect match (100%) was reached. The quantification with an unfulfilled relative error of the concentration (i.e., > ±15%) was detected only for mechanically stressed mAbs. In conclusion, the study revealed that the influence of the mAbs and the type of stress impact on the QC of compounded mAbs.

Pharmaceutical interventions to improve safety of chemotherapy-treated cancer patients: A cross-sectional study.

PURPOSE: Cancer chemotherapy is a high-risk process. To improve patient safety, a systematic pharmaceutical analysis of chemotherapy prescriptions is performed in our institution. The aim of this study was to assess the impact of pharmaceutical interventions (PIs) on the safety of patient chemotherapy prescriptions. METHODS: This prospective cross-sectional study was conducted in an 800-bed university hospital with oncology departments. All chemotherapy prescriptions were included and PIs were collected prospectively during one month. The clinical impact of PIs was scored by an expert panel of oncologists and pharmacists, using the Hatoum scale. Univariate and multivariate analysis were conducted to identify factors associated with a higher frequency of PIs. RESULTS: Of 1346 prescriptions included, 129 required a PI (9.6% (95% CI: 8.1-11.4)). Most PIs were scored as having at least a significant impact for patient safety (69.8% (95% CI: 60.4-76.9)). The frequency of PIs was significantly associated with tumour site (p = 0.04) and weekday of prescription (p = 0.005). Multivariate analysis identified factors independently associated with PI performance, including pancreas and biliary tract cancers (odds ratio = 2.8 (95% CI: 1.4-5.3)), ovary cancers (odds ratio = 2.4 (95% CI: 1.2-4.8)) and head and neck cancers (odds ratio = 2.4 (95% CI: 1.1-5.1)) and the day 1 of the protocol with a cytotoxic agent (odds ratio = 3.7 (95% CI: 1.1-11.1)). CONCLUSIONS: Oncology pharmacists have a critical role in the safety of chemotherapy prescriptions. The coordination between healthcare professionals and access to patient data seem essential to improve the PIs' relevance and their clinical impact on patient safety.

Discriminative and Quantitative Analysis of Antineoplastic Taxane Drugs Using a Handheld Raman Spectrometer.

This study was conducted to evaluate the ability of Raman spectroscopy (RS) to control antineoplastic preparations used for chemotherapy in order to ensure its physical and chemical qualities. Three taxane drugs: cabazitaxel (CBX), docetaxel (DCX) and paclitaxel (PCX) at therapeutic concentration ranges were analyzed using a handheld spectrometer at 785 nm. Qualitative and quantitative models were developed and optimized using a calibration set (n=75 per drug) by partial least square discriminant analysis and regression and validated using a test set (n=27 per drug). All samples were correctly assigned with an accuracy of 100%. Despite optimization, quantitative analysis showed limited performances at the lowest concentrations. The root mean square error of predictions ranged from 0.012 mg/mL for CBX to 0.048 mg/mL for DCX with a minimal coefficient of determination of 0.9598. The linearity range was validated from 0.175 to 0.30 mg/mL for CBX, from 0.40 to 1.00 mg/mL for DCX and from 0.57 to 1.20 mg/mL for PCX. Despite some limitations, this study confirms the potential of RS to control these drugs and also provides substantial advantages to secure the activity for healthcare workers. As a result of its rapidity and the uncomplicated use of a handheld instrument, RS appears to be a promising method to augment security of the medication preparation process in hospitals.

Optimization of classification and regression analysis of four monoclonal antibodies from Raman spectra using collaborative machine learning approach.

The use of monoclonal antibodies (mAbs) constitutes one of the most important strategies to treat patients suffering from cancers such as hematological malignancies and solid tumors. These antibodies are prescribed by the physician and prepared by hospital pharmacists. An analytical control enables the quality of the preparations to be ensured. The aim of this study was to explore the development of a rapid analytical method for quality control. The method used four mAbs (Infliximab, Bevacizumab, Rituximab and Ramucirumab) at various concentrations and was based on recording Raman data and coupling them to a traditional chemometric and machine learning approach for data analysis. Compared to conventional linear approach, prediction errors are reduced with a data-driven approach using statistical machine learning methods. In the latter, preprocessing and predictive models are jointly optimized. An additional original aspect of the work involved on submitting the problem to a collaborative data challenge platform called Rapid Analytics and Model Prototyping (RAMP). This allowed using solutions from about 300 data scientists in collaborative work. Using machine learning, the prediction of the four mAbs samples was considerably improved. The best predictive model showed a combined error of 2.4% versus 14.6% using linear approach. The concentration and classification errors were 5.8% and 0.7%, only three spectra were misclassified over the 429 spectra of the test set. This large improvement obtained with machine learning techniques was uniform for all molecules but maximal for Bevacizumab with an 88.3% reduction on combined errors (2.1% versus 17.9%).

Rapid discrimination and quantification analysis of five antineoplastic drugs in aqueous solutions using Raman spectroscopy.

The aim of this study was to assess the ability of Raman spectroscopy to discriminate and quantify five antineoplastic drugs in an aqueous matrix at low concentrations before patient administration. Five antineoplastic drugs were studied at therapeutic concentrations in aqueous 0.9% sodium chloride: 5-fluorouracil (5FU), gemcitabine (GEM), cyclophophamide (CYCLO), ifosfamide (IFOS) and doxorubicin (DOXO). All samples were packaged in glass vials and analyzed using Raman spectrometry from 400 to 4000cm-1. Discriminant analyses were performed using Partial Least Squares Discriminant Analysis (PLS-DA) and quantitative analyses using PLS regression. The best discrimination model was obtained using hierarchical PLS-DA models including three successive models for concentrations higher than the lower limit of quantification (0% of fitting and cross-validation error rate with an excellent accuracy of 100%). According to these hierarchical discriminative models, 90.8% (n=433) of external validation samples were correctly predicted, 2.5% (n=12) were misclassified and 6.7% (n=32) of the external validation set were not assigned. The quantitative analysis was characterized by the RMSEP that ranged from 0.23mg/mL for DOXO to 3.05mg/mL for 5FU. The determination coefficient (R2) was higher than 0.9994 for all drugs evaluated except for 5FU (R2=0.9986). This study provides additional information about the potential value of Raman spectroscopy for real-time quality control of cytotoxic drugs in hospitals. In some situations, this technique therefore constitutes a powerful alternative to usual methods with ultraviolet (UV) detection to ensure the correct drug and the correct dose in solutions before administration to patients and to limit exposure of healthcare workers during the analytical control process.

Eicosapentaenoic acid membrane incorporation impairs cholesterol efflux from cholesterol-loaded human macrophages by reducing the cholesteryl ester mobilization from lipid droplets.

A diet containing a high n-3/n-6 polyunsaturated fatty acids (PUFA) ratio has cardioprotective properties. PUFAs incorporation into membranes influences the function of membrane proteins. We investigated the impact of the membrane incorporation of PUFAs, especially eicosapentaenoic acid (EPA) (C20:5 n-3), on the anti-atherogenic cholesterol efflux pathways. We used cholesteryl esters (CE)-loaded human monocyte-derived macrophages (HMDM) to mimic foam cells exposed to the FAs for a long period of time to ensure their incorporation into cellular membranes. Phospholipid fraction of EPA cells exhibited high levels of EPA and its elongation product docosapentaenoic acid (DPA) (C22:5 n-3), which was associated with a decreased level of arachidonic acid (AA) (C20:4 n-6). EPA 70µM reduced ABCA1-mediated cholesterol efflux to apolipoprotein (apo) AI by 30% without any alteration in ABCA1 expression. The other tested PUFAs, DPA, docosahexaenoic acid (DHA) (C22:6 n-3), and AA, were also able to reduce ABCA1 functionality while the monounsaturated oleic FA slightly decreased efflux and the saturated palmitic FA had no impact. Moreover, EPA also reduced cholesterol efflux to HDL mediated by the Cla-1 and ABCG1 pathways. EPA incorporation did not hinder efflux in free cholesterol-loaded HMDM and did not promote esterification of cholesterol. Conversely, EPA reduced the neutral hydrolysis of cytoplasmic CE by 24%. The reduced CE hydrolysis was likely attributed to the increase in cellular TG contents and/or the decrease in apo E secretion after EPA treatment. In conclusion, EPA membrane incorporation reduces cholesterol efflux in human foam cells by reducing the cholesteryl ester mobilization from lipid droplets.

Safety analysis of occupational exposure of healthcare workers to residual contaminations of cytotoxic drugs using FMECA security approach.

Handling cytotoxic drugs is associated with chemical contamination of workplace surfaces. The potential mutagenic, teratogenic and oncogenic properties of those drugs create a risk of occupational exposure for healthcare workers, from reception of starting materials to the preparation and administration of cytotoxic therapies. The Security Failure Mode Effects and Criticality Analysis (FMECA) was used as a proactive method to assess the risks involved in the chemotherapy compounding process. FMECA was carried out by a multidisciplinary team from 2011 to 2016. Potential failure modes of the process were identified based on the Risk Priority Number (RPN) that prioritizes corrective actions. Twenty-five potential failure modes were identified. Based on RPN results, the corrective actions plan was revised annually to reduce the risk of exposure and improve practices. Since 2011, 16 specific measures were implemented successively. In six years, a cumulative RPN reduction of 626 was observed, with a decrease from 912 to 286 (-69%) despite an increase of cytotoxic compounding activity of around 23.2%. In order to anticipate and prevent occupational exposure, FMECA is a valuable tool to identify, prioritize and eliminate potential failure modes for operators involved in the cytotoxic drug preparation process before the failures occur.

Rapid discrimination and determination of antibiotics drugs in plastic syringes using near infrared spectroscopy with chemometric analysis: Application to amoxicillin and penicillin.

The aim of this study was to investigate near infrared spectroscopy (NIRS) combined to chemometric analysis to discriminate and quantify three antibiotics by direct measurement in plastic syringes.Solutions of benzylpenicillin (PENI), amoxicillin (AMOX) and amoxicillin/clavulanic acid (AMOX/CLAV) were analyzed at therapeutic concentrations in glass vials and plastic syringes with NIR spectrometer by direct measurement. Chemometric analysis using partial least squares regression and discriminative analysis was conducted to develop qualitative and quantitative calibration models. Discrimination of the three antibiotics was optimal for concentrated solutions with 100% of accuracy. For quantitative analysis, the three antibiotics furnished a linear response (R²>0.9994) for concentrations ranging from 0.05 to 0.2 g/mL for AMOX, 0.1 to 1.0 MUI/mL for PENI and 0.005 to 0.05 g/mL for AMOX/CLAV with excellent repeatability (maximum 1.3%) and intermediate precision (maximum of 3.2%). Based on proposed models, 94.4% of analyzed AMOX syringes, 80.0% of AMOX/CLAV syringes and 85.7% of PENI syringes were compliant with a relative error including the limit of ± 15%.NIRS as rapid, non-invasive and non-destructive analytical method represents a potentially powerful tool to further develop for securing the drug administration circuit of healthcare institutions to ensure that patients receive the correct product at the right dose.

Enzymatic diagnosis of Fabry disease using a fluorometric assay on dried blood spots: An alternative methodology.

Fabry disease (FD, OMIM#301500) is an X-linked lysosomal storage disorder caused by the functional deficiency of α-galactosidase A, a lysosomal enzyme. A method to screen for FD in large populations has been developed using a fluorometric assay of α-galactosidase A activity in dried blood spots (DBS) on filter paper. However, results can be influenced by quenching of fluorescence by haemoglobin which, together with small sample size, may result in a low light emission signal. An alternative, simple and sensitive fluorometric assay was developed for the determination of α-galactosidase A activity in DBS. The assay uses 4-methylumbelliferyl-α-d-galactose as an artificial substrate. To minimize the risk of false-positives, zinc sulfate was used for protein precipitation to stop the enzymatic reaction and eliminate interfering species (hemoglobin). Samples from 209 individuals (60 hemizygotes, 68 heterozygotes, and 81 controls) were tested to establish reference values for the assay. The mean α-galactosidase A activity of the 81 controls was 9.1 ± 3.3 µmol h(-1) L(-1) (mean ± SD). All 60 hemizygotes affected with FD had AGAL activities below 1.7 µmol h(-1) L(-1) (0.2 ± 0.3 µmol h(-1) L(-1)). For the 68 heterozygous females, AGAL activity ranged from 0 to 12.6 µmol h(-1) L(-1) (3.5 ± 2.7 µmol h(-1) L(-1)). Two-thirds of the female patients could be identified using the enzymatic assay and a cut-off level of 40% of the median control value (<3.4 µmol h(-1) L(-1)). Our fluorometric assay using zinc sulfate protein precipitation was shown to have similar sensitivity and robustness while reducing the risk of false positive results due to quenching of 4-MU fluorescence by haemoglobin.