Cargo Size Research Articles

Study on Improvement of Docking Mechanism for Docking–Undocking Drones in the Air and Docking Control Using an Onboard Camera

In this study, we developed a docking mechanism for drones that enables docking and undocking in the air for cargo transportation. This capability allows the drone configuration to be adapted depending on the cargo size and shape. We introduced a novel docking mechanism that incorporates magnetic adsorption and a newly engineered locking mechanism. We developed a drone detection system capable of estimating the relative position of a target drone by utilizing an onboard camera to identify the infrared LEDs on the target drone via image processing. Moreover, flight experiments, including detection, docking, and undocking, were performed using a mock drone to demonstrate the effectiveness of the developed system.

Enhancement of Temozolomide Stability and Anticancer Efficacy by Loading in Monopalmitolein-Based Cubic Phase Nanoparticles.

Temozolomide (TMZ) is a prodrug possessing a wide spectrum of anticancer activities. TMZ is pharmacologically inactive, but at a physiological pH, it is quickly converted to an active metabolite, 5-aminoimidazole-4-carboxamide, and a methyldiazonium cation. Due to its chemical nature, TMZ presents some capability of crossing the blood-brain barrier and therefore is used as a first-line agent in the treatment of gliomas. Here, we aimed to improve the anticancer effectiveness of TMZ by loading it into cubosomes, which are lipid nanoparticles recognized as efficient nano-based drug delivery systems. TMZ was incorporated into the monoolein (MO)- and monopalmitolein (MP)-derived cubic phases to improve its stability and half-life. It was considered that the drug release rate may vary between the MO and MP cubosomes, as the water channels of MP phases are larger than those of MO cubosomes. Therefore, we expected that due to the MPs' ability to entrap more drug molecules inside the mesophase, the concentration of TMZ available to cancer cells would be enhanced. This assumption was supported by biological analyses using the A-172 and drug-resistant T98G glioma-derived cell lines. The strongest reduction in viability was observed for A-172 cells treated with TMZ-loaded MP nanoparticles. Importantly, the TMZ-loaded MPs also caused a significant anticancer effect in the drug-resistant T98G glioma-derived cells. Both MO and MP empty cubic phases did not affect the survival of the tested cells. Concluding, TMZ-loaded cubosomes present strong anticancer properties. Encapsulating the drug within the lipid nanostructure helps to protect the drug from degradation and allows for greater accumulation of TMZ at the tumor site. Together with chemical-based features of mesophases related to increased cargo size and kinetic properties, we imply that MPs may be considered as a highly efficient nano-based drug delivery system to treat poorly curable tumors including gliomas.

Development of an Elastin-like Polypeptide-Based Nucleic Acid Delivery System Targeted to EGFR+ Bladder Cancer Cells Using a Layer-by-Layer Approach.

Nucleic acid (NA)-based therapies are revolutionizing biomedical research through their ability to control cellular functions at the genetic level. This work demonstrates a versatile elastin-like polypeptide (ELP) carrier system using a layer-by-layer (LbL) formulation approach that delivers NA cargos ranging in size from siRNA to plasmids. The components of the system can be reconfigured to modulate the biochemical and biophysical characteristics of the carrier for engaging the unique features of the biological target. We show the physical characterization and biological performance of LbL ELP nucleic acid nanoparticles (LENNs) in murine and human bladder tumor cell lines. Targeting bladder tumors is difficult owing to the constant influx of urine into the bladder, leading to low contact times (typically <2 h) for therapeutic agents delivered via intravesical instillation. LENN complexes bind to bladder tumor cells within 30 min and become rapidly internalized to release their NA cargo within 60 min. Our data show that a readily adaptable NA-delivery system has been created that is flexible in its targeting ability, cargo size, and disassembly kinetics. This approach provides an alternative path to either lipid nanoparticle formulations that suffer from inefficiency and physicochemical instability or viral vectors that are plagued by manufacturing and immune rejection challenges. This agile ELP-based nanocarrier provides an alternative route for nucleic acid delivery using a biomanufacturable, biodegradable, biocompatible, and highly tunable vehicle capable of targeting cells via engagement with overexpressed cell surface receptors.

Multi-objective multi-population simplified swarm optimization for container loading optimization with practical constraints

The container loading problem (CLP) holds significant importance in logistics and supply chain management due to its direct influence on transportation costs and times, thereby impacting the competitive advantages of enterprises across industries. Although there are existing studies for CLP, there remains a gap in addressing the optimization of multiple objectives while satisfying practical constraints. Moreover, container loading methods must also meet performance requirements such as high computational speed, explainability, and implementation capability. Although meta-heuristic-based algorithms have shown effective computational capabilities and performance, such algorithms often being trapped in the local optima especially when searching in the vast solution space of the CLP, particularly as the quantity and diversity of cargo sizes and shapes increase. Motivated by realistic needs, this study aims to develop a UNISON-based framework that integrates the merging spaces algorithm, a novel simple but effective hybrid simplified swarm optimization genetic algorithm (SSO-GA), and a multi-populations co-evolution strategy to determine the loading sequence of parcels to maximize space utilization and weight balance while satisfying to practical constraints. Specifically, the merging spaces algorithm merges fragmented small spaces resulting from loaded parcels into larger spaces, thereby facilitating the accommodation of additional parcels. The hybrid SSO-GA splits encoded solutions and updates segment using novel strategies resulting in the rearrangement of a group of parcels and their corresponding layouts for better space utilization. Furthermore, the multi-populations co-evolution strategy enhances the diversity of the search space and stabilizes solution quality by simultaneously exploiting the best solutions and exploring alternative solutions during the solution update process. An empirical study was conducted by using a public benchmark dataset which demonstrated the practical effectiveness of the proposed framework by significantly improving average space utilization while ensuring center balance and satisfying practical constraints. Furthermore, this study can also serve as a digital support system capable of assisting decision-makers in optimizing container loading operations, thereby improving productivity and saving valuable time.

Endoplasmic reticulum exit sites are segregated for secretion based on cargo size

TANGO1, TANGO1-Short, and cTAGE5 form stable complexes at the endoplasmic reticulum exit sites (ERES) to preferably export bulky cargoes. Their C-terminal proline-rich domain (PRD) binds Sec23A and affects COPII assembly. The PRD in TANGO1-Short was replaced with light-responsive domains to control its binding to Sec23A in U2OS cells (human osteosarcoma). TANGO1-ShortΔPRD was dispersed in the ER membrane but relocated rapidly, reversibly, to pre-existing ERES by binding to Sec23A upon light activation. Prolonged binding between the two, concentrated ERES in the juxtanuclear region, blocked cargo export and relocated ERGIC53 into the ER, minimally impacting the Golgi complex organization. Bulky collagen VII and endogenous collagen I were collected at less than 47% of the stalled ERES, whereas small cargo molecules were retained uniformly at almost all the ERES. We suggest that ERES are segregated to handle cargoes based on their size, permitting cells to traffic them simultaneously for optimal secretion.

Tailored assemblies of COPII proteins in secretion.

Export of secretory cargoes from the endoplasmic reticulum (ER) requires COPII proteins, which were first identified for their ability to coat small vesicles that bud from the ER. Recent data indicate that COPII proteins can also organize into a collar at the necks of tubules, as well as phase-separate into liquid-like condensates. Thus, COPII assemblies seem to be tailored to accommodate variations in the size and quantities of cargo secreted.

The vast majority of nucleic acid-loaded lipid nanoparticles contain cargo

Nucleic acid-based therapies are transforming medicine, but rely on an efficient delivery vehicle such as lipid nanoparticles (LNPs). Concerns exists in the nanomedicine field, that a large fraction of the LNPs in the ensemble does not contain any nucleic acid cargo and thus exert no functional effect. Nevertheless, how LNP lipid formulation, the LNP preparation method employed and nucleic acid cargo size correlates with the proportion of empty LNPs remains largely unexplored. Here we employ a well-established single particle based method to study nucleic acid loading heterogeneity in LNPs. We find that only a minor fraction of LNPs are “empty”, both for LNPs loaded with siRNA, mRNA and plasmids. For clinically relevant LNPs for mRNA delivery, we never detected more than 16% empty nanoparticles in the ensemble. Thus employing standard LNP lipid-cargo combinations and preparation schemes results in LNPs with the potential to serve their biomedical function.

Evolution of the clinical-stage hyperactive TcBuster transposase as a platform for robust non-viral production of adoptive cellular therapies

Cellular therapies for the treatment of human diseases, such as chimeric antigen receptor (CAR) T and NK cells have shown remarkable clinical efficacy in treating hematological malignancies, however current methods mainly utilize viral vectors which are limited by their cargo size capacities, high cost, and long timelines for production of clinical reagent. Delivery of genetic cargo via DNA transposon engineering is a more timely and cost-effective approach, yet has been held back by less efficient integration rates. Here, we report the development of a novel hyperactive TcBuster (TcB-M) transposase engineered through structure guided and in vitro evolution approaches that achieves high-efficiency integration of large, multicistronic CAR-expression cassettes in primary human cells. Our proof of principle TcB-M engineering of CAR-NK and CAR-T cells show low integrated vector copy number, a safe insertion site profile, robust in vitro function, and improves survival in a Burkitt lymphoma xenograft model in vivo. Overall, TcB-M is a versatile, safe, efficient and open-source option for the rapid manufacture and preclinical testing of primary human immune cell therapies through delivery of multicistronic large cargo via transposition.

A careful look at lipid nanoparticle characterization: analysis of benchmark formulations for encapsulation of RNA cargo size gradient

With the recent success of lipid nanoparticle (LNP) based SARS-CoV-2 mRNA vaccines, the potential for RNA therapeutics has gained widespread attention. LNPs are promising non-viral delivery vectors to protect and deliver delicate RNA therapeutics, which are ineffective and susceptible to degradation alone. While food and drug administration (FDA) approved formulations have shown significant promise, benchmark lipid formulations still require optimization and improvement. In addition, the translatability of these formulations for several different RNA cargo sizes has not been compared under the same conditions. Herein we analyze “gold standard” lipid formulations for encapsulation efficiency of various non-specific RNA cargo lengths representing antisense oligonucleotides (ASO), small interfering RNA (siRNA), RNA aptamers, and messenger RNA (mRNA), with lengths of 10 bases, 21 base pairs, 96 bases, 996 bases, and 1929 bases, respectively. We evaluate encapsulation efficiency as the percentage of input RNA encapsulated in the final LNP product (EEinput%), which shows discrepancy with the traditional calculation of encapsulation efficiency (EE%). EEinput% is shown to be < 50% for all formulations tested, when EE% is consistently > 85%. We also compared formulations for LNP size (Z-average) and polydispersity index (PDI). LNP size does not appear to be strongly influenced by cargo size, which is a counterintuitive finding. Thoughtful characterization of LNPs, in parallel with consideration of in vitro or in vivo behavior, will guide design and optimization for better understanding and improvement of future RNA therapeutics.

A flexible electrode Array for genetic transfection of different layers of the retina by electroporation.

Electroporation (in which the permeability of a cell membrane is increased transiently by exposure to an appropriate electric field) has exhibited great potential of becoming an alternative to adeno-associated virus (AAV)-based retina gene delivery. Electroporation eliminates the safety concerns of employing exogenous viruses and exceeds the limit of AAV cargo size. Unfortunately, several concerns (e.g., relatively high electroporation voltage, poor surgical operability and a lack of spatial selectivity of retina tissue) have prevented electroporation from being approved for clinical application (or even clinical trials). In this study, a flexible micro-electrode array for retina electroporation (FERE) was developed for retina electroporation. A suitably shaped flexible substrate and well-placed micro-electrodes were designed to adapt to the retina curvature and generate an evenly distributed electric field on the retina with a significantly reduced electroporation voltage of 5 V. The FERE provided (for the first time) a capability of controlled gene delivery to the different structural layers of retina tissue by precise control of the distribution of the electrical field. After ensuring the surgical operability of the FERE on rabbit eyeballs, the FERE was verified to be capable of transfecting different layers of retina tissue with satisfactory efficiency and minimum damage. Our method bridges the technical gap between laboratory validation and clinical use of retina electroporation.

Cargo Size Limits and Forces of Cell-Driven Microtransport.

The integration of motile cells into biohybrid microrobots offers unique properties such as sensitive responses to external stimuli, resilience, and intrinsic energy supply. Here, biohybrid cell-cargo systems that are driven by amoeboid Dictyosteliumdiscoideum cells are studied and how the cargo speed and the resulting viscous drag force scales with increasing radius of the spherical cargo particle are explored. Using a simplified geometrical model of the cell-cargo interaction, the findings toward larger cargo sizes, which are not accessible with the experimental setup, are extrapolated and a maximal cargo size is predicted beyond which active cell-driven movements will stall. The active forces exerted by the cells to move a cargo show mechanoresponsive adaptation and increase dramatically when challenged by an external pulling force, a mechanism that may become relevant when navigating cargo through complex heterogeneousenvironments.

Biomolecular Motor-Based Swarm Robot: An Innovation in Molecular Delivery

Biomolecular motor-based micro-sized robots have recently created an innovation in the field of science and technology as molecular transporters. Groups of these tiny robots can work substantially better than individual ones in terms of the transported distance and number or size of cargo. Site-specific molecular delivery, the main feature of these robots, has helped to improve the workability of robots in a more controllable manner.

Impact of non-ionizable lipids and phase mixing methods on structural properties of lipid nanoparticle formulations

Lipid nanoparticles (LNPs) have been widely investigated for nucleic acid therapeutic delivery, and demonstrated their potential in enabling new mRNA vaccines. LNPs are usually formulated with multi-lipid components and the composition variables may impact their structural properties. Here, we investigated the impact of helper lipids on physicochemical properties of LNPs using a Design of Experiments (DoE) definitive screening design. Phospholipid head group, degree of unsaturation, ratio to cholesterol as well as PEG-lipid content were varied and a series of 14 LNPs were prepared by microfluidic- and solvent-injection mixing. Solvent-injection mixing by a robotic liquid handler yielded 50–225 nm nanoparticles with highly ordered, ∼5 nm inter-lamellar spacing as measured by small angle X-ray scattering (SAXS) and confirmed by cryo-transmission electron microscopy (cryo-EM).In contrast, microfluidic mixing resulted in less ordered, notably smaller (50–75 nm) and more homogenous nanoparticles. Significant impacts of the stealth-lipid DSPE-PEG2000 on nanoparticle size, polydispersity and encapsulation efficiency of an oligonucleotide cargo were observed in LNPs produced by both methods, while varying the phospholipid type and content had only marginal effect on these physicochemical properties. These findings suggest that from a physicochemical perspective, the design space for combinations of helper lipids in LNPs may be considerably larger than anticipated based on the conservative formulation composition of the currently FDA-approved LNPs, thereby opening opportunities for screening and optimization of novel LNP formulations.

Cargo transport properties are enhanced by cylindrical microtubule geometry and elliptical contact zone on cargo surface

Molecular motors are responsible for carrying cellular transport of various membranous vesicles or organelles along cytoskeletal tracks. Transport of cellular cargos require high forces that are generated by motors working in groups. Hence, the properties of cargo transport can be modulated by varying various parameters such as cargo size and shape, microtubule geometry, motor number and their arrangement on cargo surface. Only those motors which are present in the contact zone on cargo surface have potential to bind to microtubule. Although earlier studies revealed the importance of cargo size, total motors attached to microtubule and their arrangement on cargo transport, yet how the contact zone influences binding of motors to microtubule largely remains unexplored. Here, it has been shown that contact zone is elliptical in shape for a spherical cargo and increases with cargo size for Kinesin-1 motors. To further understand the combined effect of elliptical contact zone and microtubule geometry on cargo transport, 3D mean-field model with uniform and clustered arrangement of motors for different cargo sizes and motor number has been used. Our findings indicate that cylindrical microtubule geometry maximizes the microtubule-bound motors which enhances the runlength and velocity of cargo transport. Our results show that microtubule-bound motors decrease with cargo size for uniform arrangement of motors on cargo thus decreasing its runlength and velocity, whereas in clustered arrangement, the number of microtubule-bound motors increase with cargo size which leads to increase in runlength and velocity.

Flagellum-driven cargoes: Influence of cargo size and the flagellum-cargo attachment geometry.

The beating of cilia and flagella, which relies on an efficient conversion of energy from ATP-hydrolysis into mechanical work, offers a promising way to propel synthetic cargoes. Recent experimental realizations of such micro-swimmers, in which micron-sized beads are propelled by isolated and demembranated flagella from the green algae Chlamydomonas reinhardtii (C. reinhardtii), revealed a variety of propulsion modes, depending in particular on the calcium concentration. Here, we investigate theoretically and numerically the propulsion of a bead as a function of the flagellar waveform and the attachment geometries between the bead and the flagellum. To this end, we take advantage of the low Reynolds number of the fluid flows generated by the micro-swimmer, which allows us to neglect fluid inertia. By describing the flagellar waveform as a superposition of a static component and a propagating wave, and using resistive-force theory, we show that the asymmetric sideways attachment of the flagellum to the bead makes a contribution to the rotational velocity of the micro-swimmer that is comparable to the contribution caused by the static component of the flagellar waveform. Remarkably, our analysis reveals the existence of a counter-intuitive propulsion regime in which an increase in the size of the cargo, and hence its drag, leads to an increase in some components of the velocity of the bead. Finally, we discuss the relevance of the uncovered mechanisms for the fabrication of synthetic, bio-actuated medical micro-robots for targeted drug delivery.

Contextually aware roadside radiation measurement testbed

We demonstrate a contextually aware multimodal roadside radiation measurement detection testbed for traffic monitoring applications in nuclear nonproliferation. Many variables in traffic such as vehicle or cargo size, mass, speed, shape, and distance of closest approach can have significant impacts on the radiation measured from a vehicle-transported radiation source. These factors can lead to uncertainties in the analysis of the radiation source, especially for lower-strength radiation sources of interest. Our testbed, known as the Multimodal Measurement System (MMS) uses non-radiation sensors including magnetometers, geophones, radiofrequency receivers, cameras, and LiDAR to extract contextual information about vehicles passing by the system. These contextual data can then be fused with data from radiation measurements to increase the system’s sensitivity and accuracy in nuclear threat detection applications. This work describes the instrumentation of the MMS and its data acquisition pipeline. Furthermore, we describe the pre-analysis performed on the raw multimodal data streams for data fusion, and the high-level machine learning analyses for detection and characterization. The variety of sensors within the MMS provides a valuable testbed that can be used to identify the combinations of contextual sensors that provide the greatest improvements to radiation source detection and characterization within the restrictions for various proliferation detection applications. The MMS is also modular so that additional combinations of sensors can be explored in the future.

Spatial dimension and binding kinetics between FG-repeats characterize the free/bound diffusion of molecules inside the nuclear pore complex.

The nuclear pore complex (NPC) is an exclusive gateway connecting the cytoplasm and the nucleus. The NPC works as a selective barrier allowing the free diffusion of small (less than 5-9 nanometer in diameter) molecules and the facilitated transport of small-to-large karyopherin-associated molecules. The selective transport is regulated by FG-Nups, intrinsically disordered proteins filling the transport channel of the NPC. FG-Nups contain many phenylalanine- and glycine-rich motifs, which interact with each other as well as with the hydrophobic pockets on karyopherins. The ability of FG-Nups to selectively transport molecules is the exquisite feature that the cell has developed, and understanding its mechanism would potentially boost their utilization for future engineering applications. In this study, we aimed to identify the detailed characteristics regarding the diffusion of molecules transported through the FG-Nups barrier. We developed a Brownian dynamics model, which represents the FG-Nups as coarse-grained bead-springs tethered to the inner wall of the cylindrical channel and simulates their motion by the overdamped Langevin equation. We placed spherical cargo in the simulation system and studied their diffusion characteristics while changing the cargo size and the binding kinetics rate between FG-motifs. The simulation result suggested that the cargo switches between two diffusive modes, free diffusion and bound diffusion, depending on its association with FG-Nups. The selectivity emerged due to the limited availability of free diffusion for oversized cargos. Besides, it showed that a specific range of the binding rate is allowed to produce a reasonably large amount of the diffusion constant for bound diffusion.

Systematic discovery of recombinases for efficient integration of large DNA sequences into the human genome

Large serine recombinases (LSRs) are DNA integrases that facilitate the site-specific integration of mobile genetic elements into bacterial genomes. Only a few LSRs, such as Bxb1 and PhiC31, have been characterized to date, with limited efficiency as tools for DNA integration in human cells. In this study, we developed a computational approach to identify thousands of LSRs and their DNA attachment sites, expanding known LSR diversity by >100-fold and enabling the prediction of their insertion site specificities. We tested their recombination activity in human cells, classifying them as landing pad, genome-targeting or multi-targeting LSRs. Overall, we achieved up to seven-fold higher recombination than Bxb1 and genome integration efficiencies of 40–75% with cargo sizes over 7 kb. We also demonstrate virus-free, direct integration of plasmid or amplicon libraries for improved functional genomics applications. This systematic discovery of recombinases directly from microbial sequencing data provides a resource of over 60 LSRs experimentally characterized in human cells for large-payload genome insertion without exposed DNA double-stranded breaks.

Reconfigurable Drone System for Transportation of Parcels With Variable Mass and Size

Cargo drones are designed to carry payloads with predefined shape, size, and/or mass. This lack of flexibility requires a fleet of diverse drones tailored to specific cargo dimensions. Here we propose a new reconfigurable drone based on a modular design that adapts to different cargo shapes, sizes, and mass. We also propose a method for the automatic generation of drone configurations and suitable parameters for the flight controller. The parcel becomes the drone's body to which several individual propulsion modules are attached. We demonstrate the use of the reconfigurable hardware and the accompanying software by transporting parcels of different mass and sizes requiring various numbers and propulsion modules' positioning. The experiments are conducted indoors (with a motion capture system) and outdoors (with an RTK-GNSS sensor). The proposed design represents a cheaper and more versatile alternative to the solutions involving several drones for parcel transportation.

Oligomerization state of the functional bacterial twin-arginine translocation (Tat) receptor complex

The twin-arginine translocation (Tat) system transports folded proteins across bacterial and plastid energy transducing membranes. Ion leaks are generally considered to be mitigated by the creation and destruction of the translocation conduit in a cargo-dependent manner, a mechanism that enables tight sealing around a wide range of cargo shapes and sizes. In contrast to the variable stoichiometry of the active translocon, the oligomerization state of the receptor complex is considered more consistently stable but has proved stubbornly difficult to establish. Here, using a single molecule photobleaching analysis of individual inverted membrane vesicles, we demonstrate that Tat receptor complexes are tetrameric in native membranes with respect to both TatB and TatC. This establishes a maximal diameter for a resting state closed pore. A large percentage of Tat-deficient vesicles explains the typically low transport efficiencies observed. This individual reaction chamber approach will facilitate examination of the effects of stochastically distributed molecules.