Developing a robust in vitro release method for a polymeric nanoparticle: Challenges and learnings.

Nanomedicines have emerged as a promising approach for targeted therapeutic delivery and specifically as a beneficial alternative to conventional cancer therapies as they can deliver higher concentrations of chemotherapeutic agents at the tumour site compared to healthy tissue, thus providing improved drug efficacy and lower systemic toxicity. Long acting injectables are increasingly becoming the focus of pharmaceutical research, as they can reduce dosing frequency and improve the life quality of patients. Development of an in vitro release (IVR) method for modified release nanomedicines is challenging because of the uniqueness and range of different formulation design approaches, as well as the complex nature of drug release mechanisms which may result in inherent variability. Regulatory guidance on the development of dissolution or release methods for parenteral products is limited relative to oral products. This article details the extensive in vitro release method development work conducted on a polymeric nanoparticle to develop the release media composition and selection of suitable apparatus and sampling technique to separate the released drug from the formulation. The aim was to develop a suitably robust analytical method that generated clinically relevant in vitro release data.

Selecting Counterions to Improve Ionized Hydrophilic Drug Encapsulation in Polymeric Nanoparticles.

Hydrophobic ion pairing (HIP) can successfully increase the drug loading and control the release kinetics of ionizable hydrophilic drugs, addressing challenges that prevent these molecules from reaching the clinic. Nevertheless, polymeric nanoparticle (PNP) formulation development requires trial-and-error experimentation to meet the target product profile, which is laborious and costly. Herein, we design a preformulation framework (solid-state screening, computational approach, and solubility in PNP-forming emulsion) to understand counterion-drug-polymer interactions and accelerate the PNP formulation development for HIP systems. The HIP interactions between a small hydrophilic molecule, AZD2811, and counterions with different molecular structures were investigated. Cyclic counterions formed amorphous ion pairs with AZD2811; the 0.7 pamoic acid/1.0 AZD2811 complex had the highest glass transition temperature (Tg; 162 °C) and the greatest drug loading (22%) and remained as phase-separated amorphous nanosized domains inside the polymer matrix. Palmitic acid (linear counterion) showed negligible interactions with AZD2811 (crystalline-free drug/counterion forms), leading to a significantly lower drug loading despite having similar log P and pKa with pamoic acid. Computational calculations illustrated that cyclic counterions interact more strongly with AZD2811 than linear counterions through dispersive interactions (offset π-π interactions). Solubility data indicated that the pamoic acid/AZD2811 complex has a lower organic phase solubility than AZD2811-free base; hence, it may be expected to precipitate more rapidly in the nanodroplets, thus increasing drug loading. Our work provides a generalizable preformulation framework, complementing traditional performance-indicating parameters, to identify optimal counterions rapidly and accelerate the development of hydrophilic drug PNP formulations while achieving high drug loading without laborious trial-and-error experimentation.

Introduction of a Hexalysine (6 K) Tag Can Protect from N-Terminal Cleavage and Increase Yield of Recombinant Proteins Expressed in the Periplasm of E. coli.

Recombinant expression of proteins in the periplasm of E. coli is frequently used for proteins containing disulfide bonds that are essential for protein folding and activity, as the cytosol of E. coli constitutes a reducing environment. The periplasm in contrast is an oxidative environment which supports proper protein folding. However, yields can be limited compared with cytoplasmic expression, and protocols must be adjusted to avoid overloading the periplasmic transportation machinery. Another less-appreciated issue with periplasmic expression is the potential generation of unwanted N-terminal cleavage products, a persistent issue which we encountered when expressing the disulfide bond containing extracellular regions of several Helicobacter pylori adhesins (BabA, BabB, BabC, and LabA) in the periplasm of E. coli XL10 GOLD, a strain traditionally not used for proteins expression. Here, we describe how introducing a C-terminal hexa-lysine (6 K) tag enhanced solubility and protected BabA from N-terminal proteolytic degradation (BabA), enabling crystallization and subsequent X-ray structural analysis. However. the same strategy had no advantageous effect for LabA, which using this protocol could be retrieved from the periplasm in relatively high yields (20-40 mg/L).

Structural and binding characterization of the LacdiNAc-specific adhesin (LabA; HopD) exodomain from Helicobacter pylori.

Helicobacter pylori (H. pylori) uses several outer membrane proteins for adhering to its host's gastric mucosa, an important step in establishing and preserving colonization. Several adhesins (SabA, BabA, HopQ) have been characterized in terms of their three-dimensional structure. A recent addition to the growing list of outer membrane porins is LabA (LacdiNAc-binding adhesin), which is thought to bind specifically to GalNAcß1-4GlcNAc, occurring in the gastric mucosa. LabA47-496 protein expressed as His-tagged protein in the periplasm of E. coli and purified via subtractive IMAC after TEV cleavage and subsequent size exclusion chromatography, resulted in bipyramidal crystals with good diffraction properties. Here, we describe the 2.06 â€‹Å resolution structure of the exodomain of LabA from H. pylori strain J99 (PDB ID: 6GMM). Strikingly, despite the relatively low levels of sequence identity with the other three structurally characterized adhesins (20-49%), LabA shares an L-shaped fold with SabA and BabA. The 'head' region contains a 4 â€‹+ â€‹3 α-helix bundle, with a small insertion domain consisting of a short antiparallel beta sheet and an unstructured region, not resolved in the crystal structure. Sequence alignment of LabA from different strains shows a high level of conservation in the N- and C-termini, and identifies two main types based on the length of the insertion domain ('crown' region), the 'J99-type' (insertion ~31 â€‹amino acids), and the H. pylori '26695 type' (insertion ~46 â€‹amino acids). Analysis of ligand binding using Native Electrospray Ionization Mass Spectrometry (ESI-MS) together with solid phase-bound, ELISA-type assays could not confirm the originally described binding of GalNAcß1-4GlcNAc-containing oligosaccharides, in line with other recent reports, which also failed to confirm LacdiNAc binding.

Metal and metallothionein levels in zooplankton in relation to environmental exposure: spatial and temporal variability (Saronikos Gulf, Greece).

Metal and metallothionein (MT) in mixed zooplankton were investigated as means of monitoring metal availability regarding environmental exposure. Spatial and temporal variability of Cd, Cu, Ni, Zn, Fe, Mn and Pb in zooplankton and seawater were studied in Saronikos Gulf (Aegean Sea, Eastern Mediterranean), once every second month during an annual cycle (2011-2012). Particulate organic carbon and chlorophyll α were also measured in seawater samples. Median zooplankton metal concentrations were 0.65, 32.4, 7.1, 864, 1420, 40.2 and 26.8 µg g-1 dw for Cd, Cu, Ni, Zn, Fe, Mn and Pb, respectively, and 109 µg g-1 ww for MTs. Metal levels in zooplankton and MTs were higher at sites influenced by human-derived pressures. Additionally, metal concentrations in pelagic fish flesh from the Greek MED-POL data base were used for bioconcentration and biomagnification factors calculation. Bioconcentration from water to zooplankton was higher than metal transfer from either seston to zooplankton or zooplankton to fish.

Mechanistic investigations into the encapsulation and release of small molecules and proteins from a supramolecular nucleoside gel in vitro and in vivo.

Supramolecular gels have recently emerged as promising biomaterials for the delivery of a wide range of bioactive molecules, from small hydrophobic drugs to large biomolecules such as proteins. Although it has been demonstrated that each encapsulated molecule has a different release profile from the hydrogel, so far diffusion and steric impediment have been identified as the only mechanisms for the release of molecules from supramolecular gels. Erosion of a supramolecular gel has not yet been reported to contribute to the release profiles of encapsulated molecules. Here, we use a novel nucleoside-based supramolecular gel as a drug delivery system for proteins with different properties and a hydrophobic dye and describe for the first time how these materials interact, encapsulate and eventually release bioactive molecules through an erosion-based process. Through fluorescence microscopy and spectroscopy as well as small angle X-ray scattering, we show that the encapsulated molecules directly interact with the hydrogel fibres - rather than being physically entrapped in the gel network. The ability of these materials to protect proteins against enzymatic degradation is also demonstrated here for the first time. In addition, the released proteins were proven to be functional in vitro. Real-time fluorescence microscopy together with macroscopic release studies confirm that erosion is the key release mechanism. In vivo, the gel completely degrades after two weeks and no signs of inflammation are detected, demonstrating its in vivo safety. By establishing the contribution of erosion as a key driving force behind the release of bioactive molecules from supramolecular gels, this work provides mechanistic insight into the way molecules with different properties are encapsulated and released from a nucleoside-based supramolecular gel and sets the basis for the design of more tailored supramolecular gels for drug delivery applications.

BabA and LPS inhibitors against Helicobacter pylori: pectins and pectin-like rhamnogalacturonans as adhesion blockers.

The first step in the development of Helicobacter pylori pathogenicity is receptor-mediated adhesion to gastric epithelium. Adhesins of H. pylori not only enable colonisation of the epithelium, with BabA interacting with Lewisb, but also interaction of lipopolysaccharide (LPS) with galectin-3 contributes to attachment of H. pylori to the host cells. Anti-adhesive compounds against H. pylori have been described, but specific analytical assays for pinpointing the interaction with BabA are limited. LPS-galectin-3 inhibitors have not been described until now. A sandwich ELISA with recombinant BabA547-6K was developed to investigate the interaction of BabA with Lewisb-HSA. Isothermal titration calorimetry gave thermodynamic information on the interaction between BabA, Lewisb-HSA and anti-adhesive compounds. A highly esterified rhamnogalacturonan from Abelmoschus esculentus inhibited the adhesion of H. pylori to adherent gastric adenocarcinoma (AGS) cells (IC50 550 µg/mL) and interacted with BabA (IC50 17 µg/mL). Pectins with similar rhamnogalacturonan structure showed weak anti-adhesive activity. Highly branched rhamnogalacturonans with low uronic acid content and high degree of esterification are potent BabA inhibitors. BabA represents a promising target for the development of anti-adhesive drugs against H. pylori. The rhamnogalacturonan influenced also the binding affinity of H. pylori to recombinant galectin-3 in a concentration-dependent manner with an IC50 of 222 µg/mL. Similar effects were obtained with pectin from apple fruits, while pectins from other sources were inactive.

Introduction of a C-terminal hexa-lysine tag increases thermal stability of the LacDiNac binding adhesin (LabA) exodomain from Helicobacter pylori.

Helicobacter pylori is a pathogenic microorganism infecting approximately 50% of the global population, and establishes life-long colonization despite the hostile stomach environment. H. pylori employs a wide range of outer membrane proteins (adhesins) for epithelial attachment, which specifically bind to glycans or non-carbohydrate structures expressed on the gastric epithelium. A recently described adhesin from H. pylori is LabA, named after its ability to bind to a disaccharide present in gastric mucus (LacdiNAc-specific adhesin). Here, we describe the recombinant expression of LabA from H. pylori strains J99 and 26695 in E. coli. High yields of recombinant LabA were obtained using periplasmic expression. We found that the addition of a C-terminal hexalysine (6K) tag enhanced the thermal stability of LabA without affecting its secondary structure, using differential scanning fluorimetry and circular dichroism spectroscopy. In contrast to our previous report for another H. pylori adhesin (BabA), the 6K tag did not enhance recombinant protein yield or solubility. Both versions of LabA, with or without the 6K tag, were expressed and isolated from the periplasmic space of Escherichia coli, with a surprisingly high yield of at least 40 mg/L for each independent preparation, following a two-step purification protocol. The proteins were analyzed with mass spectrometry (MS). Unlike its reported effect on stability of BabA, the 6K tag did not appear to protect the N-term of recombinant LabA from partial periplasmic degradation.

Polyionic Tags as Enhancers of Protein Solubility in Recombinant Protein Expression.

Since the introduction of recombinant protein expression in the second half of the 1970s, the growth of the biopharmaceutical field has been rapid and protein therapeutics has come to the foreground. Biophysical and structural characterisation of recombinant proteins is the essential prerequisite for their successful development and commercialisation as therapeutics. Despite the challenges, including low protein solubility and inclusion body formation, prokaryotic host systems and particularly Escherichia coli, remain the system of choice for the initial attempt of production of previously unexpressed proteins. Several different approaches have been adopted, including optimisation of growth conditions, expression in the periplasmic space of the bacterial host or co-expression of molecular chaperones, to assist correct protein folding. A very commonly employed approach is also the use of protein fusion tags that enhance protein solubility. Here, a range of experimentally tested peptide tags, which present specific advantages compared to protein fusion tags and the concluding remarks of these experiments are reviewed. Finally, a concept to design solubility-enhancing peptide tags based on a protein's pI is suggested.

Chemical speciation in natural and brine sea waters.

A combined approach consisting of monitoring and thermodynamic modeling was used in order to calculate the concentration of trace element species in water samples of a broad salinity range and to explain their chemical behaviour. The study was performed on water samples (fresh, marine, hyper-saline) taken from the area of Burgas Bay, Bulgaria. The ion association model based on Debye-Hückel theory using the sst2008.dat database and the ion interaction model based on Pitzer theory using a new pit2010.dat database were compared and combined for the purposes of this study. The new pit2010.dat database combines the sst2008.dat database and the pitzer.dat database of the PHREEQCI computer program as well as the thermodynamic data for the elements Fe, Mn, Cu, Zn, Cd and Pb and their Pitzer ion interaction parameters. The results showed that: (1) the predominant species in fresh waters were free ions of Mn(2+) (73.6%), Zn(2+) (58.0%) and Cd(2+) ions (78.3%) as well as carbonate species CuCO°3 (81.8%), PbCO°3 (77.2%) and hydroxy species Fe(OH) °3(55.2%) and Fe(OH)+2(35.6%); (2) an increase in chloride species MeCl²(n)-(n)(n = 1-4, Me = Mn, Zn, Cu, Pb and Cd) and of the hydroxy species Fe(OH)⁺2 for Fe was calculated for sea and hyper-saline water.

Chemical speciation in waters influenced by lead-zinc metallurgical industry.

The Lead-Zinc Company region, Kardjali city, Bulgaria, is known to be highly polluted with heavy metals from its pyrometallurgical activities. The polluted levels and the chemical speciation in surface natural waters in the region as well as in the wastewaters of the factory were investigated in January 2008 by application of monitoring studies, thermodynamic modeling, and interpretation in terms of the "softness-hardness" factor. It was found that the levels of trace metals pollution of surface waters were lower than the legislation limits for the regions with Pb and Zn production. The wastewater treatment facilities of the company were found to operate properly, and the quality of the cleaned waters in station Kar4 was comparable to the other surface waters studied (e.g., station Kar5). The trace metals were divided into three groups: (1) Fe(3+) and Al(3+), being "hard" acids, existed in all the studied waters as hydroxy species Fe(OH)2(+), AlOH(2+), and Al(OH)2(+), followed by the phosphate species AlPO4(0) and Al(2)(OH)(2)PO4(0); (2) Mn(2+), Zn(2+), and Cd(2+) being "soft" acids with crystal field stabilization energy (CFSE) = 0 were present in natural waters mainly as free Me(2+) ions. Small concentrations of their MeSO4(0), MeCO3(0) species, and of MeCl2(0) (Me = Zn, Cd) species were also calculated. In the wastewaters, two more species [Me(SO4)2(2-) and Me(SO4)3(4-)] of the softer Zn and Cd metals were also calculated; (3) Cu(2+) and Pb(2+), as "soft" acids with CFSE not equal to 0 preferentially coordinated with softer CO3(2-) ions and in natural waters existed mainly as MeCO3(0) and PbHCO3(+), followed by free Me(2+)ions and MeOH(+). In the wastewaters, MeSO4(0) and Pb(SO4)2(2-) species increased at the expense of the free Me(2+) ions. The highest self-cleaning capability of natural waters was found with respect to Al and Fe, followed by Mn and Cd. The lowest corresponded to Pb, Cu, and Zn.

Chemical speciation of inorganic pollutants in river-estuary-sea water systems.

Monitoring studies and thermodynamic modeling were used to reveal the changes of inorganic chemical species of some water pollutants (nutrients and trace metals such as Fe, Mn, Zn, Cu, Cd and Pb) inthe river-estuary-sea water system. The case studies were two rivers, Kamchiya and Ropotamo, representing part of the Bulgarian Black Sea water catchment area, and having different flow characteristics. There were no major differences in inorganic chemical species of the two river systems. NO3(-) and NO2(-) chemical species showed no changes along the river-estuary-sea water system. Concerning phosphates six different species were calculated and differences between the three parts of the systems were established. The HPO4(2-) and H2PO4(-) species were found to be dominant in river waters. The H2PO4(-) species quickly decreased at the expense of HPO4(2-) and Ca, Mg and Na phosphate complexes in estuary and seawater. Trace metals showed a great variety of chemical species. Fe(OH)2(+) species prevailed in river waters, and Fe(OH)3(0) species--in sea waters. Me2+ and MeCO3(0) (Me = Cu, Pb) and PbHCO3(+) were dominant in river waters, while Cu(CO3)2(2-) and PbCl(-) species appear also in sea waters. Cd2+ species prevailed in river and estuary waters, and CdCln(2-n) (n = 1-3) species, in seawater. Free Zn2+ species predominated in all systems but downstream their percentage decreased at the expense of Zn phosphates, carbonates,sulfates and chlorides complexes. Only free Mn2+ species were dominant along the systems.

Chemical speciation in mining affected waters: the case study of Asarel-Medet mine.

The inorganic chemical species in Maresh and Luda Yana rivers affected by the Cu- Mo Asarel-Medet mine, Bulgaria were determined during a low-flow and a high-flow period. The mining activities, the weathering and the oxidation processes strongly influenced the physicochemical processes in the whole water system. The main pollution source was a small lake receiving the acid effluents of the mining activities. High levels of SO4(2-), Cu, Mg, Al, Mn and Fe were determined at the mining polluted and affected stations. Cu(2+) and CuCO3(0) species (1:1) were present in the reference waters and Cu(2+) and CuSO4(0) species (1:1) in the polluted and affected waters; Cu(2+) species was dominating downstream. Me(2+) followed by MeSO4(0) (Me = Mn, Zn, Cd and Pb), PbCO3(0) and PbHCO3(+) species as well as Fe(OH)2(+), Al(OH)4(-), Al(OH)2(+), Al(OH)3(0) were prevailing in the system. MeSO4(+) and Me(SO4)2(-) (Me = Fe, Al), Me(SO4)2(2-) (Me = Zn, Cd and Pb), Me(SO4)3(4-) (Me = Zn, Cd) and Cd(SO4)4(6-) species polluted and affected waters. The major elements K and Na were mainly Me(+) species, whereas Ca and Mg were Me(2+) and MeSO4(0) species in different ratios. The concentration of concentration of NO2(-), NO3(-) and NO4(+) species as well as complex phosphorous species such as H2PO4(-), FeHPO4(+), HPO4(2-), CaPO4(-), CaHPO4(0) and MgHPO4(0) were also calculated. The trace element concentrations decreased downstream due to dilution, sorption processes and precipitation, but the percentage of free metal species, which are more toxic, increased. An exception was iron and aluminum of which the dominant hydroxy colloidal and sulphate species were easily incorporated into the suspended phase.

Characterization of atmospheric particulates, particle-bound transition metals and polycyclic aromatic hydrocarbons of urban air in the centre of Athens (Greece).

The concentrations of trace metals and polycyclic aromatic hydrocarbons (PAHs) adsorbed to total suspended particulate (TSP) and finer fractions of airborne particulate matter (PM) were determined from a site in the centre of Athens (Greece), which is characterized by heavy local traffic and is densely populated, during the winter and summer periods in 2003-2004. Also, we collected and analyzed samples of diesel and gasoline exhaust particles from local vehicles (buses, taxis and private cars) and from chimney exhaust of residential central heating appliances. A seasonal effect was observed for the size distribution of aerosol mass, with a shift to larger fine fractions in winter. The most commonly detected trace metals in the TSP and PM fractions were Fe, Pb, Zn, Cu, Cr, V, Ni and Cd and their concentrations were similar to levels observed in heavily polluted urban areas from local traffic and other anthropogenic emissions. Analysis of 16 PAHs bound to PM showed that they are mostly traffic related. In general, the fine particulate PAHs concentrations were higher than coarse particles. The most common PAHs in PM(10.2) and PM(2.1) were pyrene, phenanthrene, acenapthylene and fluoranthene, which are associated with diesel and gasoline exhaust particles. The results of this study underlined the importance of local emission sources, especially vehicular traffic, central heating and other local anthropogenic emissions. Compared with other big cities, Athens has much higher levels of airborne particles, especially of the finer fractions PM(10) and PM(2.5), correlated with traffic-related air pollution.