Comparing the immunogenicity of glycosidase-directed resiquimod prodrugs mediated by cancer cell metabolism.

We have recently developed an enzyme-directed immunostimulant (EDI) prodrug motif, which is metabolized to active immunostimulant by cancer cells and, following drug efflux, activates nearby immune cells, resulting in immunogenicity. In this study, we synthesized several EDI prodrugs featuring an imidazoquinoline immunostimulant resiquimod (a Toll-like receptor 7/8 agonist) covalently modified with glycosidase enzyme-directing groups selected from substrates of ß-glucuronidase, α-mannosidase, or ß-galactosidase. We compared the glycosidase-dependent immunogenicity elicited by each EDI in RAW-Blue macrophages following conversion to active immunostimulant by complementary glycosidase. At a cellular level, we examined EDI metabolism across three cancer cell lines (B16 melanoma, TC2 prostate, and 4T1 breast cancer). Comparing the relative immunogenicity elicited by each EDI/cancer cell combination, we found that B16 cells produced the highest EDI prodrug immunogenicity, achieving >95% of that elicited by unmodified resiquimod, followed by TC2 and 4T1 cells (40% and 30%, respectively). Immunogenicity elicited was comparable for a given cell type and independent of the glycosidase substrate in the EDIs or differences in functional glycosidase activity between cell lines. Measuring drug efflux of the immunostimulant payload and efflux protein expression revealed that EDI/cancer cell-mediated immunogenicity was governed by efflux potential of the cancer cells. We determined that, following EDI conversion, immunostimulant efflux occurred through both P-glycoprotein-dependent and P-glycoprotein-independent transport mechanisms. Overall, this study highlights the broad ability of EDIs to couple immunogenicity to the metabolism of many cancers that exhibit drug efflux and suggests that designing future generations of EDIs with immunostimulant payloads that are optimized for drug efflux could be particularly beneficial.

Nature-inspired pretreatment of lignocellulose - Perspective and development.

As sustainability gains increasing importance in addition to cost-effectiveness as a criterion for evaluating engineering systems and practices, biological processes for lignocellulose pretreatment have attracted growing attention. Biological systems such as white and brown rot fungi and wood-consuming insects offer fascinating examples of processes and systems built by nature to effectively deconstruct plant cell walls under environmentally benign and energy-conservative environments. Research in the last decade has resulted in new knowledge that advanced the understanding of these systems, provided additional insights into these systems' functional mechanisms, and demonstrated various applications of these processes. The new knowledge and insights enable the adoption of a nature-inspired strategy aiming at developing technologies that are informed by the biological systems but superior to them by overcoming the inherent weakness of the natural systems. This review discusses the nature-inspired perspective and summarizes related advancements, including the evolution from biological systems to nature-inspired processes, the features of biological pretreatment mechanisms, the development of nature-inspired pretreatment processes, and future perspective. This work aims to highlight a different strategy in the research and development of novel lignocellulose pretreatment processes and offer some food for thought.

One-Step Oxidation of Orange Peel Waste to Carbon Feedstock for Bacterial Production of Polyhydroxybutyrate.

Orange peels are an abundant food waste stream that can be converted into useful products, such as polyhydroxyalkanoates (PHAs). Limonene, however, is a key barrier to building a successful biopolymer synthesis from orange peels as it inhibits microbial growth. We designed a one-pot oxidation system that releases the sugars from orange peels while eliminating limonene through superoxide (O2• -) generated from potassium superoxide (KO2). The optimum conditions were found to be treatment with 0.05 M KO2 for 1 h, where 55% of the sugars present in orange peels were released and recovered. The orange peel sugars were then used, directly, as a carbon source for polyhydroxybutyrate (PHB) production by engineered Escherichia coli. Cell growth was improved in the presence of the orange peel liquor with 3 w/v% exhibiting 90-100% cell viability. The bacterial production of PHB using orange peel liquor led to 1.7-3.0 g/L cell dry weight and 136-393 mg (8-13 w/w%) ultra-high molecular weight PHB content (Mw of ~1900 kDa) during a 24 to 96 h fermentation period. The comprehensive thermal characterization of the isolated PHBs revealed polymeric properties similar to PHBs resulting from pure glucose or fructose. Our one-pot oxidation process for liberating sugars and eliminating inhibitory compounds is an efficient and easy method to release sugars from orange peels and eliminate limonene, or residual limonene post limonene extraction, and shows great promise for extracting sugars from other complex biomass materials.

Acquired Drug Resistance Enhances Imidazoquinoline Efflux by P-Glycoprotein.

Multidrug-Resistant (MDR) cancers attenuate chemotherapeutic efficacy through drug efflux, a process that transports drugs from within a cell to the extracellular space via ABC (ATP-Binding Cassette) transporters, including P-glycoprotein 1 (P-gp or ABCB1/MDR1). Conversely, Toll-Like Receptor (TLR) agonist immunotherapies modulate activity of tumor-infiltrating immune cells in local proximity to cancer cells and could, therefore, benefit from the enhanced drug efflux in MDR cancers. However, the effect of acquired drug resistance on TLR agonist efflux is largely unknown. We begin to address this by investigating P-gp mediated efflux of TLR 7/8 agonists. First, we used functionalized liposomes to determine that imidazoquinoline TLR agonists Imiquimod, Resiquimod, and Gardiquimod are substrates for P-gp. Interestingly, the least potent imidazoquinoline (Imiquimod) was the best P-gp substrate. Next, we compared imidazoquinoline efflux in MDR cancer cell lines with enhanced P-gp expression relative to parent cancer cell lines. Using P-gp competitive substrates and inhibitors, we observed that imidazoquinoline efflux occurs through P-gp and, for Imiquimod, is enhanced as a consequence of acquired drug resistance. This suggests that enhancing efflux susceptibility could be an important consideration in the rational design of next generation immunotherapies that modulate activity of tumor-infiltrating immune cells.

Weak-base pretreatment to increase biomethane production from wheat straw.

Weak-base pretreatment of wheat straw was investigated for its ability to improve biomethane production. Anaerobic digestion (AD) was performed on wheat straw pretreated with 3%, 5%, or 7% Na2CO3 as a weak base. Scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier Transform Infrared Spectroscopy (FTIR) spectra demonstrated disruption of lignocellulosic structures by pretreatment. In the 5% Na2CO3 treatment group, cellulose and hemicellulose were retained effectively, with efficient removal of lignin. The removal rates of cellulose, hemicellulose, and lignin were 27.9%, 20.4%, and 31.0%, respectively, after 5% Na2CO3 pretreatment. The methane content (53.3-77.3%) was improved in the 5% Na2CO3 treatment group, with maximum methane production (307.9 L/kg VS) that was 41.6% higher than that of the untreated sample. Cellulose and hemicelluloses were degraded 59.3% and 56.3% after AD. It took 20 days to reach 80% of the maximum cumulative methane production for the 5% Na2CO3 pretreatment group, which was 4 days faster than the untreated group. These results indicate that 5% Na2CO3 pretreatment improve the lignocellulose structure of wheat straw, allowing better biodegradability of wheat straw in AD for increased biogas production, enhanced methane content, and decreased digestion time.

Directional Structure Modification of Poplar Biomass-Inspired High Efficacy of Enzymatic Hydrolysis by Sequential Dilute Acid-Alkali Treatment.

A major challenge in converting lignocellulose to biofuel is overcoming the resistance of the biomass structure. Herein, sequential dilute acid-alkali/aqueous ammonia treatment was evaluated to enhance enzymatic hydrolysis of poplar biomass by removing hemicellulose first and then removing lignin with acid and base, respectively. The results show that glucose release in sequential dilute acid-alkali treatments (61.4-71.4 mg/g) was 7.3-24.8% higher than sequential dilute acid-aqueous ammonia treatments (57.2-61.8 mg/g) and 283.8-346.3% higher than control (16.0 mg/g), respectively. Dilute acid treatment removed most hemicellulose (84.9%) of the biomass, followed by alkaline treatment with 27.5% removal of lignin. Roughness, surface area, and micropore volume of the biomass were crucial for the enzymatic hydrolysis. Furthermore, the ultrastructure changes observed using crystallinity, Fourier transform infrared spectroscopy, thermogravimetric analysis, and pyrolysis gas chromatography/mass spectrometry support the effects of sequential dilute acid-alkali treatment. The results provide an efficient approach to facilitate a better enzymatic hydrolysis of the poplar samples.

pH-Responsive Nanoparticles Targeted to Lungs for Improved Therapy of Acute Lung Inflammation/Injury.

Dysregulated vascular inflammation is the underlying cause of acute lung inflammation/injury (ALI). Bacterial infections and trauma cause ALI that may rapidly lead to acute respiratory distress syndrome (ARDS). There are no pharmacological therapies available to patients with ALI/ARDS, partially as drugs cannot specifically target the lungs. Herein, we developed a stimuli-responsive nanoparticle (NP) to target inflammatory lungs for ALI therapies. The NP is composed of a sharp acid-sensitive segment poly(ß-amino esters) as a core for drug loading and controlled release and a polyethylene glycol-biotin on the particle surface available for bioconjugation, enabling lung targeting and extended circulation. The studies on dissipative particle dynamics simulation and characteristics of NPs suggest that anti-ICAM-1 antibodies can be coated to the particle surface and this coating is required to enhance lung targeting of NPs. A model drug of anti-inflammatory agent TPCA-1 is encapsulated in NPs with a high drug-loading content at 24% (w/w). In the mouse ALI model, our TPCA-1-loaded NPs coated with anti-ICAM-1 can target inflamed lungs after intravenous injection, followed by drug release triggered by the acid environment, thus mitigating lung inflammation and injury. Our studies reveal the rational design of nanotherapeutics for improved therapy of ALI, which may be applied to treating a wide range of vascular inflammation.