In vitro dissolution methods for hydrophilic and hydrophobic porous silicon microparticles

Porous silicon (PSi) is an innovative inorganic material that has been recently developed for various drug delivery systems. For example, hydrophilic and hydrophobic PSi microparticles have been utilized to improve the dissolution rate of poorly soluble drugs and to sustain peptide delivery. Previously, the well-plate method has been demonstrated to be a suitable in vitro dissolution method for hydrophilic PSi particles but it was not applicable to poorly wetting hydrophobic thermally hydrocarbonized PSi (THCPSi) particles. In this work, three different in vitro dissolution techniques, namely centrifuge, USP Apparatus 1 (basket) and well-plate methods were compared by using hydrophilic thermally carbonized PSi (TCPSi) microparticles loaded with poorly soluble ibuprofen or freely soluble antipyrine. All the methods showed a fast and complete or nearly complete release of both model compounds from the TCPSi microparticles indicating that all methods described in vitro dissolution equally. Based on these results, the centrifuge method was chosen to study the release of a peptide (ghrelin antagonist) from the THCPSi microparticles since it requires small sample amounts and achieves good particle suspendability. Sustained peptide release from the THCPSi microparticles was observed, which is in agreement with an earlier in vivo study. In conclusion, the centrifuge method was demonstrated to be a suitable tool for the evaluation of drug release from hydrophobic THCPSi particles, and the sustained peptide release from THCPSi microparticles was detected

Oligogenic basis of sporadic ALS The example of SOD1 p.Ala90Val mutation

Objective To characterize the clinical and neuropathologic features of patients with amyotrophic lateral sclerosis (ALS) with the superoxide dismutase 1 (SOD1) p.Ala90Val mutation, as well as the mutation frequency and the role of oligogenic mechanisms in disease penetrance. Methods An index patient with autopsy-proven ALS was discovered to have the SOD1 p.Ala90Val mutation, which was screened in 2 Finnish ALS cohorts (n = 453). Additional contributing variants were analyzed from whole-genome or whole-exome sequencing data. Results Seven screened patients (1.5%) were found to carry the SOD1 heterozygous mutation. Allele-sharing analysis suggested a common founder haplotype. Common clinical features included limb-onset, long disease course, and sensory symptoms. No TDP43 pathology was observed. All cases were apparently sporadic, and pedigree analysis demonstrated that the mutation has reduced penetrance. Analysis of other contributing genes revealed a unique set of additional variants in each patient. These included previously described rare ANG and SPG11 mutations. One patient was compound heterozygous for SOD1 p.Ala90Val and p.Asp91Ala. Conclusions Our data suggest that the penetrance of SOD1 p.Ala90Val is modulated by other genes and indicates highly individual oligogenic basis of apparently sporadic ALS. Additional genetic variants likely contributing to disease penetrance were very heterogeneous, even among Finnish patients carrying the SOD1 founder mutation

Oligogenic basis of sporadic ALS The example of SOD1 p.Ala90Val mutation

Objective To characterize the clinical and neuropathologic features of patients with amyotrophic lateral sclerosis (ALS) with the superoxide dismutase 1 (SOD1) p.Ala90Val mutation, as well as the mutation frequency and the role of oligogenic mechanisms in disease penetrance. Methods An index patient with autopsy-proven ALS was discovered to have the SOD1 p.Ala90Val mutation, which was screened in 2 Finnish ALS cohorts (n = 453). Additional contributing variants were analyzed from whole-genome or whole-exome sequencing data. Results Seven screened patients (1.5%) were found to carry the SOD1 heterozygous mutation. Allele-sharing analysis suggested a common founder haplotype. Common clinical features included limb-onset, long disease course, and sensory symptoms. No TDP43 pathology was observed. All cases were apparently sporadic, and pedigree analysis demonstrated that the mutation has reduced penetrance. Analysis of other contributing genes revealed a unique set of additional variants in each patient. These included previously described rare ANG and SPG11 mutations. One patient was compound heterozygous for SOD1 p.Ala90Val and p.Asp91Ala. Conclusions Our data suggest that the penetrance of SOD1 p.Ala90Val is modulated by other genes and indicates highly individual oligogenic basis of apparently sporadic ALS. Additional genetic variants likely contributing to disease penetrance were very heterogeneous, even among Finnish patients carrying the SOD1 founder mutation.Peer reviewe

Stabilization of fine fraction from landfill mining in anaerobic and aerobic laboratory leach bed reactors

Fine fraction (FF, 4/kgVS), while water addition and leachate recirculation depressed methane production slightly, to 16.1 and 16.4LCH4/kgVS, respectively. Organic matter was also removed via the leachate and was measured as chemical oxygen demand (COD). Calculated removal of organic matter in gas and leachate was highest in LBR with water addition (59gCOD/kgVS), compared with LBR without water addition or with leachate recirculation (51gCOD/kgVS). Concentrations of COD, ammonium nitrogen and anions in leachate decreased during the experiment, indicating washout mechanism caused by water additions. Aeration increased sulfate and nitrate concentrations in leachate due to oxidized sulfide and ammonium. Molecular weight distributions of leachates showed that all the size categories decreased, especially low molecular weight compounds, which were reduced the most. Aerobic stabilization resulted in the lowest final VS/TS (13.1%), lowest respiration activity (0.9-1.2mgO2/gTS), and lowest methane production after treatment (0.0-0.8LCH4/kgVS), with 29% of VS being removed from FF. Anaerobic stabilization methods also reduced organic matter by 9-20% compared with the initial amount. Stabilization reduced the quantity of soluble nitrogen in FF and did not alter concentration of soluble and insoluble phosphorus, and insoluble nitrogen. All four stabilization methods decreased organic matter and thus are possible stabilization methods for FF, but aerobic treatment was the most efficient in this study.acceptedVersionPeer reviewe

Screening biological methods for laboratory scale stabilization of fine fraction from landfill mining

Abstract Increasing interest for the landfill mining and the amount of fine fraction (FF) in landfills (40–70% (w/w) of landfill content) mean that sustainable treatment and utilization methods for FF are needed. For this study FF (<20 mm) was mined from a municipal solid waste (MSW) landfill operated from 1967 to 1989. FF, which resembles soil, was stabilized in laboratory scale reactors in two phases: first, anaerobically for 101 days and second, for 72 days using four different methods: anaerobic with the addition of moisture (water) or inoculum (sewage sludge) and aerobic with continuous water washing, with, or without, bulking material. The aim was to evaluate the effect on the stability of mined FF, which has been rarely reported, and to study the quality and quantity of gas and leachate produced during the stabilization experiment. The study showed that aerobic treatment reduced respiration activity (final values 0.9–1.1 mg O2/g TS) and residual methane potential (1.1 L CH4/kg TS) better than anaerobic methods (1.8–2.3 mg O2/g TS and 1.3–2.4 L CH4/kg TS, respectively). Bulking material mixed in FF in one aerobic reactor had no effect on the stability of FF. The benefit of anaerobic treatment was the production of methane, which could be utilized as energy. Even though the inoculum addition increased methane production from FF about 30%, but the methane production was still relatively low (in total 1.5–1.7 L CH4/kg TS). Continuous water washing was essential to remove leachable organic matter and soluble nutrients from FF, while increasing the volume of leachate collected. In the aerobic treatment, nitrogen was oxidized into nitrite and nitrate and then washed out in the leachate. Both anaerobic and aerobic methods could be used for FF stabilization. The use of FF, in landscaping for example, is possible because its nutrient content (4 g N/kg TS and 1 g P/kg TS) can increase the nutrient content of soil, but this may have limitations due to the possible presence of heavy metal and other contaminants.acceptedVersionPeer reviewe

Characterization of fine fraction mined from two Finnish landfills

A fine fraction (FF) was mined from two Finnish municipal solid waste (MSW) landfills in Kuopio (1- to 10-year-old, referred as new landfill) and Lohja (24- to 40-year-old, referred as old landfill) in order to characterize FF. In Kuopio the FF (<20mm) was on average 45±7% of the content of landfill and in Lohja 58±11%. Sieving showed that 86.5±5.7% of the FF was smaller than 11.2mm and the fraction resembled soil. The total solids (TS) content was 46-82%, being lower in the bottom layers compared to the middle layers. The organic matter content (measured as volatile solids, VS) and the biochemical methane potential (BMP) of FF were lower in the old landfill (VS/TS 12.8±7.1% and BMP 5.8±3.4m3 CH4/t TS) than in the new landfill (VS/TS 21.3±4.3% and BMP 14.4±9.9m3 CH4/t TS), and both were lower compared with fresh MSW. In the Kuopio landfill materials were also mechanically sieved in the full scale plant in two size fraction <30mm (VS/TS 31.1% and 32.9m3 CH4/t TS) and 30-70mm (VS/TS 50.8% and BMP 78.5m3 CH4/t TS). The nitrogen (3.5±2.0g/kg TS), phosphorus (<1.0-1.5g/kg TS) and soluble chemical oxygen demand (COD) (2.77±1.77kg/t TS) contents were low in all samples. Since FF is major fraction of the content of landfill, the characterization of FF is important to find possible methods for using or disposing FF mined from landfills.acceptedVersionPeer reviewe

Novel Delivery Systems for Improving the Clinical Use of Peptides

Peptides have long been recognized as a promising group of therapeutic substances to treat various diseases. Delivery systems for peptides have been under development since the discovery of insulin for the treatment of diabetes. The challenge of using peptides as drugs arises from their poor bioavailability resulting from the low permeability of biological membranes and their instability. Currently, subcutaneous injection is clinically the most common administration route for peptides. This route is cost-effective and suitable for self-administration, and the development of appropriate dosing equipment has made performing the repeated injections relatively easy; however, only few clinical subcutaneous peptide delivery systems provide sustained peptide release. As a result, frequent injections are needed, which may cause discomfort and additional risks resulting from a poor administration technique. Controlled peptide delivery systems, able to provide required therapeutic plasma concentrations over an extended period, are needed to increase peptide safety and patient compliancy. In this review, we summarize the current peptidergic drugs, future developments, and parenteral peptide delivery systems. Special emphasis is given to porous silicon, a novel material in peptide delivery. Biodegradable and biocompatible porous silicon possesses some unique properties, such as the ability to carry exceptional high peptide payloads and to modify peptide release extensively. We have successfully developed porous silicon as a carrier material for improved parenteral peptide delivery. Nanotechnology, with its different delivery systems, will enable better use of peptides in several therapeutic applications in the near future.PharmacologyBiopharmaceutic

Hyaluronan-based dissolving microneedles with high antigen content for intradermal vaccination: Formulation, physicochemical characterization and immunogenicity assessment

The purpose of this study was to optimize the manufacturing of dissolving microneedles (dMNs) and to increase the antigen loading in dMNs to investigate the effect on their physicochemical properties. To achieve this, a novel single-array wells polydimethylsiloxane mold was designed, minimizing antigen wastage during fabrication and achieving homogeneous antigen distribution among the dMN arrays. Using this mold, hyaluronan (HA)-based dMNs were fabricated and tested for maximal ovalbumin (OVA) content. dMNs could be fabricated with an OVA:HA ratio as high as 1:1 (w/w), without compromising their properties such as shape and penetration into the ex vivo human skin, even after storage at high humidity and temperature. High antigen loading did not induce protein aggregation during dMN fabrication as demonstrated by complementary analytical methods. However, the dissolution rate in ex vivo human skin decreased with increasing antigen loading. About 2.7 µg OVA could be delivered in mice by using a single array with an OVA:HA ratio of 1:3 (w/w). Intradermal vaccination with dMNs induced an immune response similar as subcutaneous injection and faster than after hollow microneedle injection. In conclusion, results suggest that (i) the polydimethylsiloxane mold design has an impact on the manufacturing of dMNs, (ii) the increase in antigen loading in dMNs affects the microneedle dissolution and (iii) dMNs are a valid alternative for vaccine administration over conventional injection.Drug Delivery Technolog

Hollow microneedle-mediated intradermal delivery of model vaccine antigen-loaded PLGA nanoparticles elicits protective T cell-mediated immunity to an intracellular bacterium

The skin is an attractive organ for immunization due to the presence of a large number of epidermal and dermal antigen-presenting cells. Hollow microneedles allow for precise and non-invasive intradermal delivery of vaccines. In this study, ovalbumin (OVA)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles with and without TLR3 agonist poly(I:C) were prepared and administered intradermally by hollow microneedles. The capacity of the PLGA nanoparticles to induce a cytotoxic T cell response, contributing to protection against intracellular pathogens, was examined. We show that a single injection of OVA-loaded PLGA nanoparticles, compared to soluble OVA, primed both adoptively transferred antigen-specific naïve transgenic CD8(+) and CD4(+) T cells with markedly high efficiency. Applying a triple immunization protocol, PLGA nanoparticles primed also endogenous OVA-specific CD8(+) T cells. Immune response, following immunization with in particular anionic PLGA nanoparticles co-encapsulated with OVA and poly(I:C), provided protection against a recombinant strain of the intracellular bacterium Listeria monocytogenes, secreting OVA. Taken together, we show that PLGA nanoparticle formulation is an excellent delivery system for protein antigen into the skin and that protective cellular immune responses can be induced using hollow microneedles for intradermal immunizations