If strong cation exchange chromatography (SCX) is combined with ion-pair chromatography, then the solute could be retained selectively with the power of mixed separation modes. This combination is termed selectivity enhanced strong cation exchange chromatography (SE-SCX). Macroporous polystyrene-divinylbenzene (PS/DVB) resin with sulfonate coating that conveys ion exchange and reversed phase characteristics was employed. Sodium dodecyl sulfate (SDS) was utilized as a selectivity modifier and an ion-pair reagent. This separation strategy is exploited for a challenging simultaneous separation of peptide variants having similar isoelectric points (pIs) and comparable retention behaviour. Insulin variants were used as a model in this study. The selective separation of insulin and five structurally-related analogues namely; ASPART, LISPRO, GLULISIN, GLARGIN, and DETEMIR was conducted using gradient elution mode. Three eluents were used for the separation of the target compounds. Eluent A was a mixture of acetonitrile and 10 mmol L-1 SDSat ratio (1:1) and was kept at 20% through the run. Eluent B was 20 mmol L-1 KH2PO4 adjusted at pH=4.0 and eluent C was eluent B plus 1 mol L-1 NaCl that was increased linearly till 80% at 20 min. It was found that the retention of the tested variants can be modeled mainly by electrostatic interaction that might be hydrophobically-assisted. The developed method was validated in accordance with ICH guidelines and was appropriate for the intended purposes. Finally, this study introduces SE-SCX as a new selective separation strategy for peptides and proteins that may open the door for novel mixed mode perspectives in protein analysis.

If strong cation exchange chromatography (SCX) is combined with ion-pair chromatography, then the solute could be retained selectively with the power of mixed separation modes. This combination is termed selectivity enhanced strong cation exchange chromatography (SE-SCX). Macroporous polystyrene-divinylbenzene (PS/DVB) resin with sulfonate coating that conveys ion exchange and reversed phase characteristics was employed. Sodium dodecyl sulfate (SDS) was utilized as a selectivity modifier and an ion-pair reagent. This separation strategy is exploited for a challenging simultaneous separation of peptide variants having similar isoelectric points (pIs) and comparable retention behaviour. Insulin variants were used as a model in this study. The selective separation of insulin and five structurally-related analogues namely; ASPART, LISPRO, GLULISIN, GLARGIN, and DETEMIR was conducted using gradient elution mode. Three eluents were used for the separation of the target compounds. Eluent A was a mixture of acetonitrile and 10 mmol L-1 SDSat ratio (1:1) and was kept at 20% through the run. Eluent B was 20 mmol L-1 KH2PO4 adjusted at pH=4.0 and eluent C was eluent B plus 1 mol L-1 NaCl that was increased linearly till 80% at 20 min. It was found that the retention of the tested variants can be modeled mainly by electrostatic interaction that might be hydrophobically-assisted. The developed method was validated in accordance with ICH guidelines and was appropriate for the intended purposes. Finally, this study introduces SE-SCX as a new selective separation strategy for peptides and proteins that may open the door for novel mixed mode perspectives in protein analysis.

There is a need to develop asymmetric routes to functionalised β-lactams, which remain the most important group of antibacterials. Here we describe biocatalytic and protein engineering studies concerning carbapenem biosynthesis enzymes, aiming to enable stereoselective production of functionalised carbapenams with three contiguous chiral centres. Structurally-guided substitutions of wildtype carboxymethylproline synthases enable tuning of their C-N and C-C bond forming capacity to produce 5-carboxymethylproline derivatives substituted at C-4 and C-6, from amino acid aldehyde and malonyl-CoA derivatives. Use of tandem enzyme incubations comprising an engineered carboxymethylproline synthase and an alkylmalonyl-CoA forming enzyme (i.e. malonyl-CoA synthetase or crotonyl-CoA carboxylase reductase) can improve stereocontrol and expand the product range. Some of the prepared 4,6-disubstituted-5-carboxymethylproline derivatives are converted to bicyclic β-lactams by carbapenam synthetase catalysis. The results illustrate the utility of tandem enzyme systems involving engineered crotonases for asymmetric bicyclic β-lactam synthesis.

In situ polymerization is a simple and efficient technique for modification and fabrication of modified electrodes in voltammetry. An efficient and highly sensitive square wave voltammetric (SWV) method was developed for analysis of a 5-HT3 antagonist granisetron (GRN) using in situ polymerized glycine on pencil graphite electrode surface. It was found that the fabricated polymer enhanced the sensitivity by more than two times and enhanced the surface activity by more than three times. Surface area measurements showed that poly-Gly/PGE have large surface area of 44.3 mm2, when compared to that of bare PEG (12.1 mm2). Several methods as cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM) techniques were used to characterize the modified surface. Further, FT-IR spectroscopic study was used to predict the way of glycine polymerization on electrode surface and the possible interaction mechanism with GRN. After optimization, the proposed method showed a linear response of GRN concentrations in the range from 0.08 to 3.00 mmol L1 with a limit of detection (LOD) of 26.2 nmol L1 (9.14 ng mL1). The method was utilized for GRN determination in ampoules and in real human plasma samples.

In situ polymerization is a simple and efficient technique for modification and fabrication of modified electrodes in voltammetry. An efficient and highly sensitive square wave voltammetric (SWV) method was developed for analysis of a 5-HT3 antagonist granisetron (GRN) using in situ polymerized glycine on pencil graphite electrode surface. It was found that the fabricated polymer enhanced the sensitivity by more than two times and enhanced the surface activity by more than three times. Surface area measurements showed that poly-Gly/PGE have large surface area of 44.3 mm2, when compared to that of bare PEG (12.1 mm2). Several methods as cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM) techniques were used to characterize the modified surface. Further, FT-IR spectroscopic study was used to predict the way of glycine polymerization on electrode surface and the possible interaction mechanism with GRN. After optimization, the proposed method showed a linear response of GRN concentrations in the range from 0.08 to 3.00 mmol L1 with a limit of detection (LOD) of 26.2 nmol L1 (9.14 ng mL1). The method was utilized for GRN determination in ampoules and in real human plasma samples.

Intravenous delivery of poorly water-soluble anticancer drugs such as docetaxel (DTX) is challenging due to the low bioavailability and the toxicity related to solubilizing excipients. Colloidal nanoparticles are used as alternative carriers, but low drug loading capacity and circulation instability limit their clinical translation. To address these challenges, DTX nanocrystals (NCs) were prepared using Pluronic F127 as an intermediate stabilizer and albumin as a functional surface modifier, which were previously found to be effective in producing small and stable NCs. We hypothesize that the albumin-coated DTX NCs (DTX-F-alb) will remain stable in serum-containing medium so as to effectively leverage the enhanced permeability and retention effect. In addition, the surface-bound albumin, in its native form, may contribute to cellular transport of NCs through interactions with albumin-binding proteins such as secreted protein acidic and rich in cysteine (SPARC). DTX-F-alb NCs showed sheet-like structure with an average length, width, and thickness of 284 ± 96, 173 ± 56, and 40 ± 8 nm and remained stable in 50% serum solution at a concentration greater than 10 μg/mL. Cytotoxicity and cellular uptake of DTX-F-alb and unformulated (free) DTX were compared on three cell lines with different levels of SPARC expression and DTX sensitivity. While the uptake of free DTX was highly dependent on DTX sensitivity, DTX-F-alb treatment resulted in relatively consistent cellular levels of DTX. Free DTX was more efficient in entering drug-sensitive B16F10 and SKOV-3 cells than DTX-F-alb, with consistent cytotoxic effects. In contrast, multidrug-resistant NCI/ADR-RES cells took up DTX-F-alb more than free DTX with time and responded better to the former. This difference was reduced by SPARC knockdown. The high SPARC expression level of NCI/ADR-RES cells, the known affinity of albumin for SPARC, and the opposing effect of SPARC knockdown support that DTX-F-alb have exploited the surface-bound albumin-SPARC interaction in entering NCI/ADR-RES cells. Albumin-coated NC system is a promising formulation for the delivery of hydrophobic anticancer drugs to multidrug-resistant tumors.

Intravenous delivery of poorly water-soluble anticancer drugs such as docetaxel (DTX) is challenging due to the low bioavailability and the toxicity related to solubilizing excipients. Colloidal nanoparticles are used as alternative carriers, but low drug loading capacity and circulation instability limit their clinical translation. To address these challenges, DTX nanocrystals (NCs) were prepared using Pluronic F127 as an intermediate stabilizer and albumin as a functional surface modifier, which were previously found to be effective in producing small and stable NCs. We hypothesize that the albumin-coated DTX NCs (DTX-F-alb) will remain stable in serum-containing medium so as to effectively leverage the enhanced permeability and retention effect. In addition, the surface-bound albumin, in its native form, may contribute to cellular transport of NCs through interactions with albumin-binding proteins such as secreted protein acidic and rich in cysteine (SPARC). DTX-F-alb NCs showed sheet-like structure with an average length, width, and thickness of 284 ± 96, 173 ± 56, and 40 ± 8 nm and remained stable in 50% serum solution at a concentration greater than 10 μg/mL. Cytotoxicity and cellular uptake of DTX-F-alb and unformulated (free) DTX were compared on three cell lines with different levels of SPARC expression and DTX sensitivity. While the uptake of free DTX was highly dependent on DTX sensitivity, DTX-F-alb treatment resulted in relatively consistent cellular levels of DTX. Free DTX was more efficient in entering drug-sensitive B16F10 and SKOV-3 cells than DTX-F-alb, with consistent cytotoxic effects. In contrast, multidrug-resistant NCI/ADR-RES cells took up DTX-F-alb more than free DTX with time and responded better to the former. This difference was reduced by SPARC knockdown. The high SPARC expression level of NCI/ADR-RES cells, the known affinity of albumin for SPARC, and the opposing effect of SPARC knockdown support that DTX-F-alb have exploited the surface-bound albumin-SPARC interaction in entering NCI/ADR-RES cells. Albumin-coated NC system is a promising formulation for the delivery of hydrophobic anticancer drugs to multidrug-resistant tumors.

Intravenous delivery of poorly water-soluble anticancer drugs such as docetaxel (DTX) is challenging due to the low bioavailability and the toxicity related to solubilizing excipients. Colloidal nanoparticles are used as alternative carriers, but low drug loading capacity and circulation instability limit their clinical translation. To address these challenges, DTX nanocrystals (NCs) were prepared using Pluronic F127 as an intermediate stabilizer and albumin as a functional surface modifier, which were previously found to be effective in producing small and stable NCs. We hypothesize that the albumin-coated DTX NCs (DTX-F-alb) will remain stable in serum-containing medium so as to effectively leverage the enhanced permeability and retention effect. In addition, the surface-bound albumin, in its native form, may contribute to cellular transport of NCs through interactions with albumin-binding proteins such as secreted protein acidic and rich in cysteine (SPARC). DTX-F-alb NCs showed sheet-like structure with an average length, width, and thickness of 284 ± 96, 173 ± 56, and 40 ± 8 nm and remained stable in 50% serum solution at a concentration greater than 10 μg/mL. Cytotoxicity and cellular uptake of DTX-F-alb and unformulated (free) DTX were compared on three cell lines with different levels of SPARC expression and DTX sensitivity. While the uptake of free DTX was highly dependent on DTX sensitivity, DTX-F-alb treatment resulted in relatively consistent cellular levels of DTX. Free DTX was more efficient in entering drug-sensitive B16F10 and SKOV-3 cells than DTX-F-alb, with consistent cytotoxic effects. In contrast, multidrug-resistant NCI/ADR-RES cells took up DTX-F-alb more than free DTX with time and responded better to the former. This difference was reduced by SPARC knockdown. The high SPARC expression level of NCI/ADR-RES cells, the known affinity of albumin for SPARC, and the opposing effect of SPARC knockdown support that DTX-F-alb have exploited the surface-bound albumin-SPARC interaction in entering NCI/ADR-RES cells. Albumin-coated NC system is a promising formulation for the delivery of hydrophobic anticancer drugs to multidrug-resistant tumors.

The most effective and safe contraceptive method, intrauterine devices (IUDs), is still underutilized due to the pain barrier during IUD insertion. Lidocaine, a well-known local anesthetic, can be used to relieve IUD insertion pain. This study aimed at formulation, in vitro, in vivo and clinical evaluation of a novel lidocaine dual-responsive in situ gel. Pluronic and Gelrite® were used as thermosenstive and ion-activated polymers, respectively. In situ gels containing 2% lidocaine, pluronics and/or Gelrite® were prepared. The optimized dual-responsive formula (F 5) was clear, with 95% drug content, free flowing at room temperature and gel at vaginal temperature (T gel of 28 C). This optimized dual-responsive in situ gel was found to be superior to single-responsive one due to presence of Gelrite®, imparting resistance to dilution effect of simulated vaginal fluids. DSC thermograms revealed no interaction between …

The most effective and safe contraceptive method, intrauterine devices (IUDs), is still underutilized due to the pain barrier during IUD insertion. Lidocaine, a well-known local anesthetic, can be used to relieve IUD insertion pain. This study aimed at formulation, in vitro, in vivo and clinical evaluation of a novel lidocaine dual-responsive in situ gel. Pluronic and Gelrite® were used as thermosenstive and ion-activated polymers, respectively. In situ gels containing 2% lidocaine, pluronics and/or Gelrite® were prepared. The optimized dual-responsive formula (F 5) was clear, with 95% drug content, free flowing at room temperature and gel at vaginal temperature (T gel of 28 C). This optimized dual-responsive in situ gel was found to be superior to single-responsive one due to presence of Gelrite®, imparting resistance to dilution effect of simulated vaginal fluids. DSC thermograms revealed no interaction between …