Combining frass and fatty acid co-products derived from Black soldier fly larvae farming shows potential as a slow release fertiliser.

Use of black soldier fly larvae (BSFL) to process large volumes of organic waste is an emerging industry to produce protein. A co-product of this industry, the larval faeces (frass), has potential to be used as an organic fertiliser in a circular economy. However, BSFL frass has a high ammonium (N-NH4+) content which could result in nitrogen (N) loss following its application to land. One solution is to process the frass by combining it with solid fatty acids (FA) that have previously been used to manufacture slow-release inorganic fertilisers. We investigated the slow-releasing effect of N after combining BSFL frass with three FAs - lauric, myristic and stearic acid. Soil was amended with the three forms of FA processed (FA-P) frass, unprocessed frass or a control and incubated for 28 days. The impact of treatments on soil properties and soil bacterial communities were characterised during the incubation. Lower N-NH4+ concentrations occurred in soil treated with FA-P frass compared to unprocessed frass, and N-NH4+ release was slowest for lauric acid processed frass. Initially, all frass treatments caused a large shift in the soil bacterial community towards a dominance of fast-growing r-strategists that were correlated with increased organic carbon levels. FA-P frass appeared to enhance the immobilisation of N-NH4+ (from frass) by diverting it into microbial biomass. Unprocessed and stearic acid processed frass became enriched by slow-growing K-strategist bacteria at the latter stages of the incubation. Consequently, when frass was combined with FAs, FA chain length played an important role in regulating the composition of r-/K- strategists in soil and N and carbon cycling. Modifying frass with FAs could be developed into a slow release fertiliser leading to reduced soil N loss, improved fertiliser use efficiency, increased profitability and lower production costs.

Antibody conjugated lipid nanoparticles as a targeted drug delivery system for hydrophobic pharmaceuticals.

Cancer remains a significant health issue worldwide. The most common group of chemotherapeutic agents are small-molecule drugs, which often are associated with toxic side-effects and non-specific delivery, leading to limited therapeutic effect. This paper describes the development of a targeted drug delivery system based on lipid nanoparticles for cancer therapy. The lipid nanoparticles consist of a lipid core conjugated to an albumin stealth coating and targeting antibodies through thiol chemistry synthesized utilizing a one-step method. Applying the developed method, lipid nanoparticles with diameters down to 87 nm, capable of encapsulating small molecule compounds were synthesized. Cellular uptake studies of the lipid nanoparticles loaded with the model drug Nile red demonstrated that stealth-coating reduced non-specific cell uptake by up to a 1000-fold compared to free drug. Moreover, antibody-conjugation led to a significant cellular retargeting. Finally, it was shown that the lipid nanoparticles undergo cellular uptake through the endocytic pathway. The lipid nanoparticles are simple to synthesize, stabile in serum and have the potential to be versatile targeted towards receptors selectively expressed by diseased cells using antibodies. Thus, the system may reduce the toxic side-effects of cancer drugs while improving their delivery to cancer cells, increasing the therapeutic effect.

siRNA nanoparticle functionalization of nanostructured scaffolds enables controlled multilineage differentiation of stem cells.

The creation of complex tissues and organs is the ultimate goal in tissue engineering. Engineered morphogenesis necessitates spatially controlled development of multiple cell types within a scaffold implant. We present a novel method to achieve this by adhering nanoparticles containing different small-interfering RNAs (siRNAs) into nanostructured scaffolds. This allows spatial retention of the RNAs within nanopores until their cellular delivery. The released siRNAs were capable of gene silencing BCL2L2 and TRIB2, in mesenchymal stem cells (MSCs), enhancing osteogenic and adipogenic differentiation, respectively. This approach for enhancing a single type of differentiation is immediately applicable to all areas of tissue engineering. Different nanoparticles localized to spatially distinct locations within a single implant allowed two different tissue types to develop in controllable areas of an implant. As a consequence of this, we predict that complex tissues and organs can be engineered by the in situ development of multiple cell types guided by spatially restricted nanoparticles.

Surface functionalisation of PLGA nanoparticles for gene silencing.

This work presents a method for decorating the surface of poly (lactide-co-glycolide) (PLGA) nanoparticles with polyethyleneimine (PEI) utilising a cetyl derivative to improve surface functionalisation and siRNA delivery. Sub-micron particles were produced by an emulsion-diffusion method using benzyl alcohol. We demonstrate by x-ray photoelectron spectroscopy (XPS), 2.6 times higher surface presentation of amines using the cetyl derivative compared to non-cetylated-PEI formulations (6.5 and 2.5% surface nitrogen, respectively). The modified particles were shown by spectroscopy, fluorescent microscopy and flow cytometry to bind and mediate siRNA delivery into the human osteosarcoma cell line U2OS and the murine macrophage cell line J774.1. Specific reduction in the anti-apoptotic oncogene BCL-w in U2OS cells was achieved with particles containing cetylated-PEI (53%) with no cellular toxicity. In addition, particles containing cetylated-PEI achieved 64% silencing of TNFalpha in J774.1 cells. This rapid method for surface modification of PLGA nanoparticles promotes its application for alternative cetylated functional derivatives as a strategy to control specific biological properties of nanoparticles.

Delivery of siRNA from lyophilized polymeric surfaces.

Standard in vitro gene silencing protocols are performed using aqueous formulations of transfection reagents and small interfering RNAs (siRNA) reconstituted immediately prior to use. In this study, we describe a method for producing gene silencing-active lyophilized cationic polymer (chitosan) or lipid (TransIT-TKO) siRNA formulations. We demonstrate specific and efficient knockdown of enhanced green fluorescent protein (EGFP) in H1299 human lung carcinoma cells transfected in plates pre-coated with both TransIT-TKO/siRNA ( approximately 85%) and a chitosan/siRNA formulation containing sucrose as lyoprotectant ( approximately 70%). This method removes the necessity for both siRNA reconstitution immediately prior to use and addition onto cells. Furthermore, silencing activity of the chitosan/siRNA formulation was shown over the period studied ( approximately 2 months) when stored at room temperature. Higher cell viability was observed using the chitosan system compared to the lipid formulation. Silencing of the proinflammatory cytokine tumour necrosis factor (TNF-alpha) was also demonstrated in the RAW macrophage cell line using the lyophilized chitosan/siRNA system suggesting that the coating can improve the biocompatibility of medical implants. This work describes an efficient gene silencing methodology using freeze-dried formulations with potential applications as a high throughput screening tool for gene function, biocompatible medical implant components and longer shelf-life therapeutics.

The influence of polymeric properties on chitosan/siRNA nanoparticle formulation and gene silencing.

We have previously introduced the use of the biomaterial chitosan to form chitosan/siRNA nanoparticles for gene silencing protocols. This present study shows that the physicochemical properties (size, zeta potential, morphology and complex stability) and in vitro gene silencing of chitosan/siRNA nanoparticles are strongly dependent on chitosan molecular weight (Mw) and degree of deacetylation (DD). High Mw and DD chitosan resulted in the formation of discrete stable nanoparticles approximately 200 nm in size. Chitosan/siRNA formulations (N:P 50) prepared with low Mw (approximately 10 kDa) showed almost no knockdown of endogenous enhanced green fluorescent protein (EGFP) in H1299 human lung carcinoma cells, whereas those prepared from higher Mw (64.8-170 kDa) and DD (approximately 80%) showed greater gene silencing ranging between 45% and 65%. The highest gene silencing efficiency (80%) was achieved using chitosan/siRNA nanoparticles at N:P 150 using higher Mw (114 and 170 kDa) and DD (84%) that correlated with formation of stable nanoparticles of approximately 200 nm. In conclusion, this work confirms the application of chitosan as a non-viral carrier for siRNA and the importance of polymeric properties for the optimisation of gene silencing using chitosan/siRNA nanoparticles.

RNA interference in vitro and in vivo using a novel chitosan/siRNA nanoparticle system.

This work introduces a novel chitosan-based siRNA nanoparticle delivery system for RNA interference in vitro and in vivo. The formation of interpolyelectrolyte complexes between siRNA duplexes (21-mers) and chitosan polymer into nanoparticles, ranging from 40 to 600 nm, was shown using atomic force microscopy and photon correlation spectroscopy. Rapid uptake (1 h) of Cy5-labeled nanoparticles into NIH 3T3 cells, followed by accumulation over a 24 h period, was visualized using fluorescence microscopy. Nanoparticle-mediated knockdown of endogenous enhanced green fluorescent protein (EGFP) was demonstrated in both H1299 human lung carcinoma cells and murine peritoneal macrophages (77.9% and 89.3% reduction in EGFP fluorescence, respectively). In addition, Western analysis showed approximately 90% reduced expression of BCR/ABL-1 leukemia fusion protein while BCR expression was unaffected in K562 (Ph(+)) cells after transfection using nanoparticles containing siRNA specific to the BCR/ABL-1 junction sequence. Effective in vivo RNA interference was achieved in bronchiole epithelial cells of transgenic EGFP mice after nasal administration of chitosan/siRNA formulations (37% and 43% reduction compared to mismatch and untreated control, respectively). These findings highlight the potential application of this novel chitosan-based system in RNA-mediated therapy of systemic and mucosal disease.