David J. Scurr

High loading efficiency and sustained release of siRNA encapsulated in PLGA nanoparticles: quality by design optimization and characterization.

Poly(DL-lactide-co-glycolide acid) (PLGA) is an attractive polymer for delivery of biopharmaceuticals owing to its biocompatibility, biodegradability and outstanding controlled release characteristics. The purpose of this study was to understand and define optimal parameters for preparation of small interfering RNA (siRNA)-loaded PLGA nanoparticles by the double emulsion solvent evaporation method and characterize their properties. The experiments were performed according to a 2(5-1) fractional factorial design based on five independent variables: The volume ratio between the inner water phase and the oil phase, the PLGA concentration, the sonication time, the siRNA load and the amount of acetylated bovine serum albumin (Ac-BSA) in the inner water phase added to stabilize the primary emulsion. The effects on the siRNA encapsulation efficiency and the particle size were investigated. The most important factors for obtaining an encapsulation efficiency as high as 70% were the PLGA concentration and the volume ratio whereas the size was mainly affected by the PLGA concentration. The viscosity of the oil phase was increased at high PLGA concentration, which explains the improved encapsulation by stabilization of the primary emulsion and reduction of siRNA leakage to the outer water phase. Addition of Ac-BSA increased the encapsulation efficiency at low PLGA concentrations. The PLGA matrix protected siRNA against nuclease degradation, provided a burst release of surface-localized siRNA followed by a triphasic sustained release for two months. These results enable careful understanding and definition of optimal process parameters for preparation of PLGA nanoparticles encapsulating high amounts of siRNA with immediate and long-term sustained release properties.

All-natural composite wound dressing films of essential oils encapsulated in sodium alginate with antimicrobial properties.

We present natural polymeric composite films made of essential oils (EOs) dispersed in sodium alginate (NaAlg) matrix, with remarkable anti-microbial and anti-fungal properties. Namely, elicriso italic, chamomile blue, cinnamon, lavender, tea tree, peppermint, eucalyptus, lemongrass and lemon oils were encapsulated in the films as potential active substances. Glycerol was used to induce plasticity and surfactants were added to improve the dispersion of EOs in the NaAlg matrix. The topography, chemical composition, mechanical properties, and humidity resistance of the films are presented analytically. Antimicrobial tests were conducted on films containing different percentages of EOs against Escherichia coli bacteria and Candida albicans fungi, and the films were characterized as effective or not. Such diverse types of essential oil-fortified alginate films can find many applications mainly as disposable wound dressings but also in food packaging, medical device protection and disinfection, and indoor air quality improvement materials, to name a few.

Enzyme-activated RGD ligands on functionalized poly(ethylene glycol) monolayers: surface analysis and cellular response.

We report on the design, stepwise synthesis, and surface analysis of enzyme-responsive surfaces that present cell adhesive RGD sequences on-demand, that is, by enzymatic hydrolysis of inactive RGD containing precursors that carry cleavable steric blocking groups. These surfaces, incorporating poly(ethylene glycol) (PEG) monolayers coupled via epoxy silanes to glass, are functionalized via stepwise solid phase synthesis, presenting a versatile and straightforward approach to preparation of peptide surfaces. Successive amino acid coupling and deprotection steps using fluorenylmethoxycarbonyl (Fmoc) chemistry are verified using surface analysis with time-of-flight secondary-ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS). Exposure of surfaces to elastase results in activation of cell binding ligands as demonstrated using osteoblast cells. These surfaces may have applications in spatiotemporally controlled attachment of cells as relevant for three-dimensional tissue engineering scaffolds and cell-based biosensors.

Biomaterial modification of urinary catheters with antimicrobials to give long-term broadspectrum antibiofilm activity

Catheter-associated urinary tract infection (CAUTI) is the commonest hospital-acquired infection, accounting for over 100,000 hospital admissions within the USA annually. Biomaterials and processes intended to reduce the risk of bacterial colonization of the catheters for long-term users have not been successful, mainly because of the need for long duration of activity in flow conditions. Here we report the results of impregnation of urinary catheters with a combination of rifampicin, sparfloxacin and triclosan. In flow experiments, the antimicrobial catheters were able to prevent colonization by common uropathogens Proteus mirabilis, Staphylococcus aureus and Escherichia coli for 7 to 12weeks in vitro compared with 1-3days for other, commercially available antimicrobial catheters currently used clinically. Resistance development was minimized by careful choice of antimicrobial combinations. Drug release profiles and distribution in the polymer, and surface analysis were also carried out and the process had no deleterious effect on the mechanical performance of the catheter or its balloon. The antimicrobial catheter therefore offers for the first time a means of reducing infection and its complications in long-term urinary catheter users.

Injectable and porous PLGA microspheres that form highly porous scaffolds at body temperature

Graphical abstract

Chemical and spatial analysis of protein loaded PLGA microspheres for drug delivery applications

Polymer microspheres for controlled release of therapeutic protein from within an implantable scaffold were produced and analysed using complimentary techniques to probe the surface and bulk chemistry of the microspheres. Time of Flight - Secondary Ion Mass Spectrometry (ToF-SIMS) surface analysis revealed a thin discontinuous film of polyvinyl alcohol (PVA) surfactant (circa 4.5 nm thick) at the surface which was readily removed under sputtering with C(60). Atomic Force Microscopy (AFM) imaging of microspheres before and after sputtering confirmed that the PVA layer was removed after sputtering revealing poly(lactic-co-glycolic) acid(PLGA). Scanning electron microscopy showed the spheres to be smooth with some shallow and generally circular depressions, often with pores in their central region. The occurrence of the protein at the surface was limited to areas surrounding these surface pores. This surface protein distribution is believed to be related to a burst release of the protein on dissolution. Analysis of the bulk properties of the microspheres by confocal Raman mapping revealed the 3D distribution of the protein showing large voids within the pores. Protein was found to be adsorbed at the interface with the PLGA oil phase following deposition on evaporation of the solvent. Protein was also observed concentrated within pores measuring approximately 2 μm across. The presence of protein in large voids and concentrated pores was further scrutinised by ToF-SIMS of sectioned microspheres. This paper demonstrates that important information for optimisation of such complex bioformulations, including an understanding of the release profile can be revealed by complementary surface and bulk analysis allowing optimisation of the therapeutic effect of such formulations.

ToF-SIMS analysis of chemical heterogenities in inkjet micro-array printed drug/polymer formulations.

Three different formulations comprising two drugs, felodipine and hydrochlorothiazide (HCT) and two polymers, poly(vinyl pyrolidone) (PVP) and poly(lactic-co-glycolic acid) (PLGA) were inkjet printed as micro-dot arrays and analysed on an individual micro-spot basis by time-of-flight secondary ion mass spectrometry (ToF-SIMS). For the HCT/PLGA formulation, the spots showed heterogeneity of the drug and other chemical constituents. To further investigate these heterogeneities, multivariate curve resolution was applied to the ToF-SIMS hyperspectral image datasets. This approach successfully identified distinct chemical components elucidating the HCT, PLGA, substrate material, and contaminants based on sulphur, phosphorous and sodium chloride. Spots printed using either of the drugs with PVP exhibited full substrate coverage and a uniform distribution of the active ingredient along with all other constituents within the printed spot area. This represents the preferred situation in terms of stability and controlling the release of a drug from a polymer matrix.

Surface Characterization of Carbohydrate Microarrays

Carbohydrate microarrays are essential tools to determine the biological function of glycans. Here, we analyze a glycan array by time-of-flight secondary ion mass spectrometry (ToF-SIMS) to gain a better understanding of the physicochemical properties of the individual spots and to improve carbohydrate microarray quality. The carbohydrate microarray is prepared by piezo printing of thiol-terminated sugars onto a maleimide functionalized glass slide. The hyperspectral ToF-SIMS imaging data are analyzed by multivariate curve resolution (MCR) to discern secondary ions from regions of the array containing saccharide, linker, salts from the printing buffer, and the background linker chemistry. Analysis of secondary ions from the linker common to all of the sugar molecules employed reveals a relatively uniform distribution of the sugars within the spots formed from solutions with saccharide concentration of 0.4 mM and less, whereas a doughnut shape is often formed at higher-concentration solutions. A detailed analysis of individual spots reveals that in the larger spots the phosphate buffered saline (PBS) salts are heterogeneously distributed, apparently resulting in saccharide concentrated at the rim of the spots. A model of spot formation from the evaporating sessile drop is proposed to explain these observations. Saccharide spot diameters increase with saccharide concentration due to a reduction in surface tension of the saccharide solution compared to PBS. The multivariate analytical partial least squares (PLS) technique identifies ions from the sugars that in the complex ToF-SIMS spectra correlate with the binding of galectin proteins.

Influence of the plasma sheath on plasma polymer deposition in advance of a mask and down pores

Plasma species that form plasma polymer deposits readily penetrate through small openings and are therefore well suited to coat the interior of porous objects. Here, we show how the size of the cross section of square channels influences the penetration of active species from a hexane plasma and how it affects the formation of surface chemical gradients in the interior of these model pores. WCA mapping and ToF-SIMS imaging are used to visualize the plasma polymer deposit in the interior of the model pores and demonstrate that a strong dependence of the wettability gradient profile only exists up to a channel cross section of about 1 mm. XPS data allow us to calculate a deposition rate of plasma polymerized hexane (ppHex) at discrete positions on the surface and show that the deposition rate of ppHex is reduced by the presence of the mask up to a distance of 16 mm in advance of the channel opening. A strong dependence of the ppHex deposition rate on the cross-section of the channels is found within the first 2 mm in front of the pore opening. An estimation of the sheath thickness suggests that this effect can be attributed to the plasma sheath that perturbs the plasma in front of the pores. Plasma mass spectrometry allows us to identify the nature of the plasma species penetrating from the plasma through the pores and shows that no negatively charged ions are able to penetrate through the small channels. Neutral and positively charged species penetrate several millimeters down the channels and both species are therefore likely to contribute to the formation of the deposit on the sample. In addition, the formation of positively charged higher molecular mass hexane fragments is observed in the gas phase, demonstrating the likelihood of neutral-positive reactions in the plasma.

Ambient DESI and LESA-MS Analysis of Proteins Adsorbed to a Biomaterial Surface Using In-Situ Surface Tryptic Digestion

AbstractThe detection and identification of proteins adsorbed onto biomaterial surfaces under ambient conditions has significant experimental advantages but has proven to be difficult to achieve with conventional measuring technologies. In this study, we present an adaptation of desorption electrospray ionization (DESI) and liquid extraction surface analysis (LESA) mass spectrometry (MS) coupled with in-situ surface tryptic digestion to identify protein species from a biomaterial surface. Cytochrome c, myoglobin, and BSA in a combination of single and mixture spots were printed in an array format onto Permanox slides, followed by in-situ surface digestion and detection via MS. Automated tandem MS performed on surface peptides was able to identify the proteins via MASCOT. Limits of detection were determined for DESI-MS and a comparison of DESI and LESA-MS peptide spectra characteristics and sensitivity was made. DESI-MS images of the arrays were produced and analyzed with imaging multivariate analysis to automatically separate peptide peaks for each of the proteins within a mixture into distinct components. This is the first time that DESI and LESA-MS have been used for the in-situ detection of surface digested proteins on biomaterial surfaces and presents a promising proof of concept for the use of ambient MS in the rapid and automated analysis of surface proteins. Graphical abstractᅟ