Cargo Loading Research Articles

A mitochondria-targeted biocompatible photothermal nanoscale hydrogen-bonded organic framework for effective multimodal-imaging guided breast cancer therapy

Organic small-molecule photothermal agents (smPTAs) have been extensively studied for biomedical applications due to their good biocompatibility comparing with inorganic materials. However, most smPTAs suffer from water insolubility, even organic solvents insolubility, poor bio- and photo-stability, and short blood half-life. Herein, a new type of salinized 4,4′-(benzo[1,2-d:4,5-d']bis(thiazole)-2,6-diyl)dibenzoic acid (BBTDB-Na) smPTA is synthesized via a cyano group of 2-cyano-benzothiazole (CBT)-1,2-aminothiol group of cysteine (Cys) click condensation reaction, and photothermal nanoscale hydrogen-bonded organic frameworks (BT-nHOFs) is further constructed through a facile self-assembly strategy. The prepared BT-nHOFs exhibit a fusiform shape and bisthiazole structure, resulting in good cellular uptake and mitochondrial targeting effects. The well-organized structure of HOFs enables excellent photostability and high photothermal efficiency (51.4 %). Under 808 nm laser irradiation, the BT-nHOFs show favorable photothermal effects on breast cancer cells in vitro and in vivo, indicating that BT-nHOFs can act as a biocompatible PTA candidate for clinical applications. Besides, the fluorescence features, photoacoustic ability, cargo loading capacity of BT-nHOFs furnish multimodal imaging-guided precise photothermal therapy. In summary, this work paves a new way for the development of photothermal HOFs by salinizing insoluble smPTA and demonstrates their great potential for clinical application in phototheranostics.

Swelling, Rupture and Endosomal Escape of Biological Nanoparticles Per Se and Those Fused with Liposomes in Acidic Environment.

Biological nanoparticles (NPs), such as extracellular vesicles (EVs), exosome-mimetic nanovesicles (EMNVs) and nanoghosts (NGs), are perspective non-viral delivery vehicles for all types of therapeutic cargo. Biological NPs are renowned for their exceptional biocompatibility and safety, alongside their ease of functionalization, but a significant challenge arises when attempting to load therapeutic payloads, such as nucleic acids (NAs). One effective strategy involves fusing biological NPs with liposomes loaded with NAs, resulting in hybrid carriers that offer the benefits of both biological NPs and the capacity for high cargo loads. Despite their unique parameters, one of the major issues of virtually any nanoformulation is the ability to escape degradation in the compartment of endosomes and lysosomes which determines the overall efficiency of nanotherapeutics. In this study, we fabricated all major types of biological and hybrid NPs and studied their response to the acidic environment observed in the endolysosomal compartment. In this study, we show that EMNVs display increased protonation and swelling relative to EVs and NGs in an acidic environment. Furthermore, the hybrid NPs exhibit an even greater response compared to EMNVs. Short-term incubation of EMNVs in acidic pH corresponding to late endosomes and lysosomes again induces protonation and swelling, whereas hybrid NPs are ruptured, resulting in the decline in their quantities. Our findings demonstrate that in an acidic environment, there is enhanced rupture and release of vesicular cargo observed in hybrid EMNVs that are fused with liposomes compared to EMNVs alone. This was confirmed through PAGE electrophoresis analysis of mCherry protein loaded into nanoparticles. In vitro analysis of NPs colocalization with lysosomes in HepG2 cells demonstrated that EMNVs mostly avoid the endolysosomal compartment, whereas hybrid NPs escape it over time. To conclude, (1) hybrid biological NPs fused with liposomes appear more efficient in the endolysosomal escape via the mechanism of proton sponge-associated scavenging of protons by NPs, influx of counterions and water, and rupture of endo/lysosomes, but (2) EMNVs are much more efficient than hybrid NPs in actually avoiding the endolysosomal compartment in human cells. These results reveal biochemical differences across four major types of biological and hybrid NPs and indicate that EMNVs are more efficient in escaping or avoiding the endolysosomal compartment.

Abstract 154: Hierarchical Redundancy And Contextual Roles Of Vesicle-associated Membrane Proteins (VAMPs) In Platelet Function

Background: Platelet granule secretion, driven by the SNARE complex involving Vesicle-Associated Membrane Proteins (VAMPs) and t-SNAREs, is essential for vascular integrity. We dissected individual and combinatorial contributions of all four platelet VAMPs to granule secretion, hemostasis, and intracellular cargo trafficking. Research Questions: 1. Are specific VAMPs critical for granule fusion, or does total VAMP quantity (mass action) dictate secretion? 2. How do differential VAMP deficiencies impact hemostasis in arterial and venous microenvironments? 3. Do VAMPs influence platelet cargo loading and gene expression? Goals: Investigate individual and combinatorial roles of VAMPs in platelet biology and their context dependent contributions to hemostasis. Approach: We generated V7 -/- 8 -/- and V2 Δ 3 Δ 7 -/- 8 -/- mice, assessing granule secretion (dense, alpha, lysosomal) via ATP, PF4/P-selectin, and β-hexosaminidase/LAMP1 release assays, respectively. Hemostasis in arterial and venous models was evaluated with tail bleeding, FeCl3, and venous puncture injury assays. Cargo levels were analyzed using proteomic arrays, and platelet RNA sequencing identified transcriptional changes. Results: Global V7/V8 deletion modestly reduced secretion compared to isolated V8 deficiency. Surprisingly, while platelet-specific V2 Δ 3 Δ 7 -/- 8 -/- exhibited the most dramatic decrease, complete abolition was absent (p<0.01). Alpha and lysosomal secretion were more affected than densegranule release. Both knockout models bled profusely and failed to form stable arterial thrombi (p<0.01). Notably, only V2 Δ 3 Δ 7 -/- 8 -/- mice displayed significantly prolonged venous bleeding (p<0.01). RNA sequencing revealed >800 upregulated and >500 downregulated transcripts in V7 - /- 8 -/- and V2 Δ 3 Δ 8 -/- platelets, suggesting shared transcriptional control by V7 and secondary VAMPs (V2 and V3). Conclusions: VAMP functions exhibit hierarchical redundancy, with V8 presence minimally impacting dense granule secretion and arterial hemostasis. Alpha granule release is more sensitive to VAMP loss. The requirements for platelet granule secretion for hemostasis are context dependent. The deletion of VAMPs disrupts platelet cargo trafficking and transcription.

Novel loading protocol combines highly efficient encapsulation of exogenous therapeutic toxin with preservation of extracellular vesicles properties, uptake and cargo activity

Extracellular vesicles (EVs) have mostly been investigated as carriers of biological therapeutics such as proteins and RNA. Nevertheless, small-molecule drugs of natural or synthetic origin have also been loaded into EVs, resulting in an improvement of their therapeutic properties. A few methods have been employed for EV cargo loading, but poor yield and drastic modifications of vesicles remain unsolved challenges. We tested a different strategy based on temporary pH alteration through incubation of EVs with alkaline sodium carbonate, which resulted in conspicuous exogenous molecule incorporation. In-depth characterization showed that vesicle size, morphology, composition, and uptake were not affected. Our method was more efficient than gold-standard electroporation, particularly for a potential therapeutic toxin: the plant Ribosome Inactivating Protein saporin. The encapsulated saporin resulted protected from degradation, and was efficiently conveyed to receiving cancer cells and triggered cell death. EV-delivered saporin was more cytotoxic compared to the free toxin. This approach allows both the structural preservation of vesicle properties and the transfer of protected cargo in the context of drug delivery.Graphic

Mussel-inspired interface deposition strategy for mesoporous metal-phenolic nanospheres with superior antioxidative, photothermal and antibacterial performance

Metal-phenolic networks (MPNs) have emerged as a versatile and multifunctional platform applied in bioimaging, disease treatment, electrocatalysis, and water purification. The synthesis of MPNs with mesoporous frameworks and ultra-small diameters (<200 nm), crucial for post-modification, cargo loading, and mass transport, remains a formidable challenge. Inspired by mussel chemistry, mesoporous metal-phenolic nanospheres (MMPNs) are facilely prepared by direct deposition of the metal-polyphenol complex on the interface of oil nano-droplets composed of block copolymers/1,3,5-trimethylbenzene followed by a spontaneous template-removal process. Due to the penetrable and stable networks, the oil nano-droplets gradually leak from the networks driven by shear stress during the stirring process. As a result, MMPNs are obtained without additional template removal procedures such as solvent extraction or high-temperature calcination. The materials have a large pore size (∼12.1 nm), uniform spherical morphology with a small particle size (∼99 nm), and a large specific surface area (49.8 m2 g−1). Due to the abundant phenolic hydroxyl groups, the MMPNs show excellent antioxidative property. The MMPNs also have excellent photothermal property, whose photothermal conversion efficiency was 40.9 %. Moreover, the phenolic hydroxyl groups can reduce Ag+ in situ to prepare Ag nanoparticles loaded MMPNs composites, which have excellent inhibition performance of drug-resistant bacteria biofilm.

Design and performance analysis of hybrid electric class 8 heavy-duty regional-haul trucks with a micro-pilot natural gas engine in real-world highway driving conditions

Diesel-fueled heavy-duty (HD) trucks are a key component of global freight transport, but consume 25% of the transportation industry's energy and emitting substantial greenhouse gas (GHG) emissions. Utilizing low-carbon fuels like natural gas (NG), combined with hybridization, presents an excellent opportunity for reducing GHG emissions and fuel consumption. Micro-pilot dual fuel diesel/NG engines offer high engine brake thermal efficiency (BTE) with an efficient three-way catalyst (TWC) to limit pollutant emissions like CO2 without the need for costly exhaust aftertreatment systems. This engine is particularly well-suited for hybridization, as it features high-efficiency under stable, mid-, and high-load conditions, but with reduced performance at low loads. This paper validates two conventional baseline models (X15 Cummins diesel engine and micro-pilot NG engine) based on a tested class 8 regional-haul truck on highway. The study investigates the combined effect of low-carbon fuels and hybridization on improving the fuel economy and GHG emissions of class 8 regional-haul trucks. This involves designing pre-transmission parallel and plug-in hybrid configurations with downsized 6.7-liter micro-pilot NG engines. The cargo load and performance requirements are maintained equivalent to the conventional diesel baseline. Results show comparable fuel economy and engine BTE between conventional diesel baseline and NG models on highways with full cargo load. The parallel and series plug-in hybrid NG designs exhibit fuel economy improvements of 2.9% and 26%, respectively, compared to the micro-pilot NG over highway with full cargo load. In the same conditions, the CO2 emissions reductions for parallel and series plug-in hybrid NG designs are 15% and 51%, respectively, relative to the conventional diesel baseline. In addition, this article provides a detailed comparison and analysis of non-plug-in and plug-in hybrids based on component energy losses and internal combustion engine (ICE) operating points.

Chromosomal 3q amplicon encodes essential regulators of secretory vesicles that drive secretory addiction in cancer.

Cancer cells exhibit heightened secretory states that drive tumor progression. Here, we identify a chromosome 3q amplicon that serves as a platform for secretory regulation in cancer. The 3q amplicon encodes multiple Golgi-resident proteins, including the scaffold Golgi integral membrane protein 4 (GOLIM4) and the ion channel ATPase Secretory Pathway Ca2+ Transporting 1 (ATP2C1). We show that GOLIM4 recruits ATP2C1 and Golgi phosphoprotein 3 (GOLPH3) to coordinate calcium-dependent cargo loading and Golgi membrane bending and vesicle scission. GOLIM4 depletion disrupts the protein complex, resulting in a secretory blockade that inhibits the progression of 3q-amplified malignancies. In addition to its role as a scaffold, GOLIM4 maintains intracellular manganese (Mn) homeostasis by binding excess Mn in the Golgi lumen, which initiates the routing of Mn-bound GOLIM4 to lysosomes for degradation. We show that Mn treatment inhibits the progression of multiple types of 3q-amplified malignancies by degrading GOLIM4, resulting in a secretory blockade that interrupts pro-survival autocrine loops and attenuates pro-metastatic processes in the tumor microenvironment. Potentially underlying the selective activity of Mn against 3q-amplified malignancies, ATP2C1 co-amplification increases Mn influx into the Golgi lumen, resulting in a more rapid degradation of GOLIM4. These findings show that functional cooperativity between co-amplified genes underlies heightened secretion and a targetable secretory addiction in 3q-amplified malignancies.

IPSC-derived healthy human astrocytes selectively load miRNAs targeting neuronal genes into extracellular vesicles

Astrocytes are in constant communication with neurons during the establishment and maturation of functional networks in the developing brain. Astrocytes release extracellular vesicles (EVs) containing microRNA (miRNA) cargo that regulates transcript stability in recipient cells. Astrocyte released factors are thought to be involved in neurodevelopmental disorders. Healthy astrocytes partially rescue Rett Syndrome (RTT) neuron function. EVs isolated from stem cell progeny also correct aspects of RTT. EVs cross the blood-brain barrier (BBB) and their cargo is found in peripheral blood which may allow non-invasive detection of EV cargo as biomarkers produced by healthy astrocytes. Here we characterize miRNA cargo and sequence motifs in healthy human astrocyte derived EVs (ADEVs). First, human induced Pluripotent Stem Cells (iPSC) were differentiated into Neural Progenitor Cells (NPCs) and subsequently into astrocytes using a rapid differentiation protocol. iPSC derived astrocytes expressed specific markers, displayed intracellular calcium transients and secreted ADEVs. miRNAs were identified by RNA-Seq on astrocytes and ADEVs and target gene pathway analysis detected brain and immune related terms. The miRNA profile was consistent with astrocyte identity, and included approximately 80 miRNAs found in astrocytes that were relatively depleted in ADEVs suggestive of passive loading. About 120 miRNAs were relatively enriched in ADEVs and motif analysis discovered binding sites for RNA binding proteins FUS, SRSF7 and CELF5. miR-483-5p was the most significantly enriched in ADEVs. This miRNA regulates MECP2 expression in neurons and has been found differentially expressed in blood samples from RTT patients. Our results identify potential miRNA biomarkers selectively sorted into ADEVs and implicate RNA binding protein sequence dependent mechanisms for miRNA cargo loading.

Force-controlled release of small molecules with a rotaxane actuator

Force-controlled release of small molecules offers great promise for the delivery of drugs and the release of healing or reporting agents in a medical or materials context1–3. In polymer mechanochemistry, polymers are used as actuators to stretch mechanosensitive molecules (mechanophores)4. This technique has enabled the release of molecular cargo by rearrangement, as a direct5,6 or indirect7–10 consequence of bond scission in a mechanophore, or by dissociation of cage11, supramolecular12 or metal complexes13,14, and even by ‘flex activation’15,16. However, the systems described so far are limited in the diversity and/or quantity of the molecules released per stretching event1,2. This is due to the difficulty in iteratively activating scissile mechanophores, as the actuating polymers will dissociate after the first activation. Physical encapsulation strategies can be used to deliver a larger cargo load, but these are often subject to non-specific (that is, non-mechanical) release3. Here we show that a rotaxane (an interlocked molecule in which a macrocycle is trapped on a stoppered axle) acts as an efficient actuator to trigger the release of cargo molecules appended to its axle. The release of up to five cargo molecules per rotaxane actuator was demonstrated in solution, by ultrasonication, and in bulk, by compression, achieving a release efficiency of up to 71% and 30%, respectively, which places this rotaxane device among the most efficient release systems achieved so far1. We also demonstrate the release of three representative functional molecules (a drug, a fluorescent tag and an organocatalyst), and we anticipate that a large variety of cargo molecules could be released with this device. This rotaxane actuator provides a versatile platform for various force-controlled release applications.

Enhanced plant-derived vesicles for nucleotide delivery for cancer therapy

Small RNAs (microRNAs [miRNAs] or small interfering RNAs [siRNAs]) are effective tools for cancer therapy, but many of the existing carriers for their delivery are limited by low bioavailability, insufficient loading, impaired transport across biological barriers, and low delivery into the tumor microenvironment. Extracellular vesicle (EV)–based communication in mammalian and plant systems is important for many physiological and pathological processes, and EVs show promise as carriers for RNA interference molecules. However, some fundamental issues limit their use, such as insufficient cargo loading and low potential for scaling production. Plant-derived vesicles (PDVs) are membrane-coated vesicles released in the apoplastic fluid of plants that contain biomolecules that play a role in several biological mechanisms. Here, we developed an alternative approach to deliver miRNA for cancer therapy using PDVs. We isolated vesicles from watermelon and formulated a hybrid, exosomal, polymeric system in which PDVs were combined with a dendrimer bound to miRNA146 mimic. Third generation PAMAM was chosen due to its high branching structure and versatility for loading molecules of interest. We performed several in vivo experiments to demonstrate the therapeutic efficacy of our compound and explored in vitro biological mechanisms underlying the anti-tumor effects of miRNA146, which are mostly related to its anti-angiogenic activity.

An integrated intermodal freight transportation system to avoid container supply chain disruptions in Chattogram Port of Bangladesh

An integrated intermodal freight transportation system is a derived demand to transport shipping containers from/to the seaport to/from the hinterland, instead of undertaking cargo loading and unloading activities in port-protected areas. The concept of the intermodal system is to transfer containers to nearby hinterland nodes, such as dry ports, inland rail, road, and river terminals as a part of port development, as well as increasing port efficiency and productivity. Lloyd’s List ranked Chattogram Port as the 67th busiest container port in the world as per container port throughput in 2022. Chattogram Port has small-scale intermodal facilities for off-docks/ICDs in the port city only. In addition, a pure intermodal system has been developed by one rail ICD in the capital city, Dhaka, and two RICTs nearby Dhaka, but less than 5 % of port throughput. A large-scale intermodal for serving the major cities, SEZs, EPZs, and industrial areas is absent due to planning and policy problems, as well as the unavailability of intermodal infrastructure and network. Due to an improper hinterland intermodal chain, the port faced container supply chain disruptions at the port yard and exceeded port capacity, resulting in container vessel congestion at the outer anchorage of Chattogram Port. By developing rail and road infrastructure, and utilizing natural river connectivity, it is possible to develop road, rail, and river intermodal systems, and shape an integrated intermodal freight transportation system to avoid container supply chain disruptions in Chattogram Port. To develop an integrated intermodal system, this research chose a quantitative research methodology for port throughput data analysis and forecasting future throughputs that are advanced to the intermodal model for Chattogram Port. Overall, this paper aims to develop an integrated intermodal freight transportation system to support Chattogram Port to keep and increase the record of port productivity, efficiency, and competitiveness actively. Highlights Intermodal system is a derived demand for managing hinterland and port terminals of a seaport that needs to be implemented in Chattogram Port Container supply chain disruptions to be avoided to increase the port productivity and efficiency Inland intermodal connectivity will help to increase hinterland of a seaport It is necessary to develop an integrated intermodal freight transportation system to support Chattogram Port to keep and increase the record of port productivity, efficiency, and competitiveness globally

Dual Noncovalent Binding Modes Enabled via Single Thiourea Motif: An Emerging Arsenal for Supramolecular Polymeric Nanomedicine

AbstractThe structural rigidity of thiourea (TU) motifs has made them useful in supramolecular (bio)materials. However, the role of the TU motif in a single system endowing dual noncovalent interactions, i.e., Hydrogen‐bonding (H‐bonding) association and metal‐coordination interaction, to afford nanomedicine is still unexplored. Herein a smart supramolecular polymeric nanomedicine constructed via TU motifs privileged dual noncovalent interactions, toward synergistic chemo/chemodynamic (CT/CDT) cancer therapy is reported. The study first synthesized a six‐arm star‐shaped amphiphilic polymer vehicle containing pendant TU motifs, poly(acylthiourea‐co‐oligo(ethylene glycol) ethyl acrylate)6 (P(TU‐co‐OEGEA)6), followed by addressing both H‐bonding association and metal‐coordination to fabricate supramolecular nanomedicine (e.g., Dox/Cu@P(TU‐co‐OEGEA)6). Structural privilege and functional diversity of TU motifs constitute an outstanding scaffold, not only offering an H‐bonding site to associate doxorubicin (Dox) but also acting as a ligand to coordinate copper (Cu). Thereby, one TU motif can enable dual noncovalent binding modes, triggering multiple curative outcomes. TU/Dox and TU/Cu noncovalent interactions can induce intermolecular configuration, yielding prompted cargo loading and in vivo stability. Moreover, benefiting from pH‐responsive Dox release and Fenton‐like copper redox chemistry, accompanied by Dox‐induced intratumoral H2O2 elevation and prompted •OH generation, synergistic CT/CDT with extraordinary anti‐tumor efficacy is indeed accomplished. This work provides a new paradigm using TU motifs regulated dual supramolecular forces to meet therapeutic goals.

Fuzzy-Set QCA on Performance and Sustainability Determinants of Ports Supporting Floating Offshore Wind Farms

The present study explores the relationship between the characteristics, performance, and sustainability of ports that will, in the future, support the logistical chains of new floating offshore wind farms, considering the crucial advancement and expansion of the offshore wind energy sector for the transition to a low-carbon economy. Through a detailed analysis, which includes international case studies in America and Europe utilizing expert interviews and quantitative methods through surveys, the importance of the location, new types of infrastructure and superstructure, and new planning and governance models for the performance and sustainability of ports that will be involved in this new energy industry is highlighted. Also, the context in which ports associated with floating wind turbines are located influences the performance and sustainability. This research employs Fuzzy-Set Qualitative Comparative Analysis (fsQCA), based on a survey of 22 European sector company experts, to emphasize the critical port characteristics for the performance and sustainability. This study reveals the significant contribution of supporting ports for the future floating offshore wind sector for the gross added value and the expansion of regional employment, and the need for new planning, construction, operation, and management models for ports instead of the traditional models applied to simple cargo loading and unloading ports, offering valuable new insights for port managers, policymakers, and academics. However, a future research trajectory with a more geographically diverse sample is suggested to enhance the applicability and generalizability of the results.

Analyzing the impact of bicycle geometry and cargo loading on the rideability and safety of cargo bikes: An investigative study

IntroductionElectric cargo bikes have become popular for transporting goods and people due to their small size and strong carrying capacity. However, the way they perform, handle, and operate safely can be affected by the weight of the cargo, where it is placed on the bike, and the bike's design. MethodThis paper analyzes the rideability and safety of eight different cargo bikes representing three different design categories, Retrofitted, Long-john, and Long-tail bikes, also considering three different cargo loading locations. We quantitatively examined the rideability by computing the minimum speed for self-stability, the maximum possible acceleration and deceleration without losing wheel-ground contact, the handlebar torque for steady-state turning, and the force required to overcome obstacles. The effect of using powerful motorized wheels has also been discussed. ResultsLong-john cargo bikes are unstable for lightweight cargo loads, more sensitive to cargo loads, and therefore may not be suitable for riding in narrow, crowded spaces like footpaths. Moreover, retrofitted cargo bikes should only be used to carry lightweight cargo as a combination of heavy cargo load and a powerful rear wheel motor poses a potential risk of accidents. Long-tail cargo bikes are less affected by changes to the cargo load and are thus safer than retrofitted bikes. Their relatively compact length also makes for a smaller turning radius. ConclusionRideability and safe handling of the cargo bikes strongly depend on the bike design and load and loading position. Retrofitted bikes are not suitable for carrying heavy loads and any load at the front has an adverse effect on the overall rideability and safety. Practical applicationThe results highlight the benefits and limitations of different cargo bike designs and, therefore, could have implications for the cargo bike manufacturers, service providers, and policymakers.

Milk Extracellular Vesicles: Natural Nanoparticles for Enhancing Oral Drug Delivery against Bacterial Infections.

Oral drug delivery is typically preferred as a therapeutic intervention due to the complexities and expenses associated with intravenous administration. However, some drugs are poorly absorbed orally, requiring intravenous administration to bypass the gastrointestinal tract and deliver the drug directly into the bloodstream. Thus, there is an urgent need to develop novel drug delivery platforms to overcome the challenges of oral drug delivery with low solubility, low permeability, oral degradation, and low bioavailability. Advances in extracellular vesicles (EVs) as natural carriers have provided emerging approaches to improve potential therapeutic applications. Milk not only contains traditional nutrients but is also rich in EVs. In this Review, we focus mainly on the purification of milk EVs (mEVs), their safety, and the advantages of mEV-based drug carriers in combatting intestinal infections. Additionally, we summarize several advantages of mEVs over conventional synthetic carriers, such as low immunogenicity, high biocompatibility, and the ability to transfer bioactive molecules between cells. Considering the unmet gaps of mEVs in clinical translation, it is essential to review the cargo loading into mEVs and future perspectives for their use as natural drug carriers for oral delivery. This overview of mEV-based drug carriers for oral delivery sheds light on alternative approaches to treat clinical infections associated with intestinal pathogens and the development of novel oral delivery systems.

Inter-coat protein loading of active ingredients into Tobacco mild green mosaic virus through partial dissociation and reassembly of the virion

Chemical pesticide delivery is a fundamental aspect of agriculture. However, the extensive use of pesticides severely endangers the ecosystem because they accumulate on crops, in soil, as well as in drinking and groundwater. New frontiers in nano-engineering have opened the door for precision agriculture. We introduced Tobacco mild green mosaic virus (TMGMV) as a viable delivery platform with a high aspect ratio and favorable soil mobility. In this work, we assess the use of TMGMV as a chemical nanocarrier for agriculturally relevant cargo. While plant viruses are usually portrayed as rigid/solid structures, these are “dynamic materials,” and they “breathe” in solution in response to careful adjustment of pH or bathing media [e.g., addition of solvent such as dimethyl sulfoxide (DMSO)]. Through this process, coat proteins (CPs) partially dissociate leading to swelling of the nucleoprotein complexes—allowing for the infusion of active ingredients (AI), such as pesticides [e.g., fluopyram (FLP), clothianidin (CTD), rifampicin (RIF), and ivermectin (IVM)] into the macromolecular structure. We developed a “breathing” method that facilitates inter-coat protein cargo loading, resulting in up to ~ 1000 AIs per virion. This is of significance since in the agricultural setting, there is a need to develop nanoparticle delivery strategies where the AI is not chemically altered, consequently avoiding the need for regulatory and registration processes of new compounds. This work highlights the potential of TMGMV as a pesticide nanocarrier in precision farming applications; the developed methods likely would be applicable to other protein-based nanoparticle systems.

Double-Network structure chemically programmed DNA Cryogel: Enzyme immobilization platform with enhanced catalytic activity for portable biosensing

Enzyme-based hydrogels have conferred multiple benefits, particularly for disease diagnosis and environmental monitoring due to their pronounced biocompatibility, ability to load cargo, and optical properties. However, traditional hydrogels suffer from some significant limitations, such as enzyme leakage and catalytic activity decreased, restricting the extent to which hydrogels can be beneficially used. Herein, chemically programmed DNA-DNA interactions were designed as a promising solution for enzyme immobilization that substantially maintains biological functionality with remarkable leakage reduction. Additionally, their responsiveness and mechanical properties are dramatically enhanced after adjusting the porous structure of hydrogels via freezing. As a proof-of-concept, a DNA cryogel with a hierarchical structure was developed with adjustable distance among cascade enzymes, increasing the local concentration of intermediates and reducing their mass transfer energy barriers for direct glucose monitoring and glutathione (GSH) sensing. Moreover, after being integrated with a smartphone system, a universal sensing platform (immunosensor) was fabricated for rapid detection of environmental chemicals (i.e., bisphenol A) without expensive instruments and sophisticated procedures. This strategy provided some novel insights on hydrogel- based bioassays, implying their great potentials for conducting onsite analysis against various targets.

Structural basis for peroxidase encapsulation inside the encapsulin from the Gram-negative pathogen Klebsiella pneumoniae

Encapsulins are self-assembling protein nanocompartments capable of selectively encapsulating dedicated cargo proteins, including enzymes involved in iron storage, sulfur metabolism, and stress resistance. They represent a unique compartmentalization strategy used by many pathogens to facilitate specialized metabolic capabilities. Encapsulation is mediated by specific cargo protein motifs known as targeting peptides (TPs), though the structural basis for encapsulation of the largest encapsulin cargo class, dye-decolorizing peroxidases (DyPs), is currently unknown. Here, we characterize a DyP-containing encapsulin from the enterobacterial pathogen Klebsiella pneumoniae. By combining cryo-electron microscopy with TP and TP-binding site mutagenesis, we elucidate the molecular basis for cargo encapsulation. TP binding is mediated by cooperative hydrophobic and ionic interactions as well as shape complementarity. Our results expand the molecular understanding of enzyme encapsulation inside protein nanocompartments and lay the foundation for rationally modulating encapsulin cargo loading for biomedical and biotechnological applications.

The encapsulation and controlled release of proteins from “meltable” chemically cross‐linked hydrogels

AbstractChemically crosslinked hydrogels can be used for the controlled delivery of therapeutic proteins, however, cargo loading can be challenging. A common approach involves performing the hydrogel crosslinking step in the presence of the cargo proteins, however, this can require the utilization of selective chemistries to ensure the cargos do not chemically react with the forming hydrogel network. We demonstrate that chemically crosslinked hydrogels based upon the protein polymer Capsular antigen fragment 1 (Caf1) can be used to encapsulate proteins within a chemically crosslinked hydrogel network. This encapsulation exploits the “meltable” feature of Caf1 hydrogels, where upon heating the Caf1 protein unfolds with concomitant depolymerization of the hydrogel network. Protein cargos can be loaded into the “molten” form of the hydrogel which cools with protein refolding and concomitant reformation of the hydrogel network to entrap the cargos, ensuring a more homogenous dispersion of the cargo. Analysis of release profiles indicated lower burst release in comparison to release from identical hydrogels loaded by an absorption method. This work is significant because it suggest that the “meltable” feature of Caf1 hydrogels may present an improved method of loading protein cargos into crosslinked hydrogel networks whilst avoiding the need for crosslinking chemistries.

Air cargo load and route planning in pickup and delivery operations

In the aerial pickup and delivery of goods in a distribution network, transport aviation faces risks of load imbalance due to the urgency required for loading, immediate take-off, and mission accomplishment. Transport planners deal with trip itineraries, prioritization of items, building up pallets, and balanced loading, but there are no commercially available systems that can integrally assist in all these requirements. This enables other risks, such as improper delivery, excessive fuel burn, and possible safety issues due to cargo imbalance, as well as a longer than necessary turn-around time. This NP-hard problem, named Air Cargo Load Planning with Routing, Pickup, and Delivery Problem (ACLP+RPDP), is mathematically modelled using standardized pallets in fixed positions. We developed a strategy to solve this problem, considering historical transport data from some Brazilian hub networks, and performed several experiments with a commercial solver, five known meta-heuristics, and a new heuristic designed specifically for this problem. By using a portable computer, our strategy quickly found practical solutions to a wide range of real problems in much less than operationally acceptable time.