Cargo Loading Research Articles

The biosynthesis of the odorant 2-methylisoborneol is compartmentalized inside a protein shell

Terpenoids are the largest class of natural products, found across all domains of life. One of the most abundant bacterial terpenoids is the volatile odorant 2-methylisoborneol (2-MIB), partially responsible for the earthy smell of soil and musty taste of contaminated water. Many bacterial 2-MIB biosynthetic gene clusters were thought to encode a conserved transcription factor, named EshA in the model soil bacterium Streptomyces griseus. Here, we revise the function of EshA, now referred to as Sg Enc, and show that it is a Family 2B encapsulin shell protein. Using cryo-electron microscopy, we find that Sg Enc forms an icosahedral protein shell and encapsulates 2-methylisoborneol synthase (2-MIBS) as a cargo protein. Sg Enc contains a cyclic adenosine monophosphate (cAMP) binding domain (CBD)-fold insertion and a unique metal-binding domain, both displayed on the shell exterior. We show that Sg Enc CBDs do not bind cAMP. We find that 2-MIBS cargo loading is mediated by an N-terminal disordered cargo-loading domain and that 2-MIBS activity and Sg Enc shell structure are not modulated by cAMP. Our work redefines the function of EshA and establishes Family 2B encapsulins as cargo-loaded protein nanocompartments involved in secondary metabolite biosynthesis.

Controlled enzyme cargo loading in engineered bacterial microcompartment shells.

Bacterial microcompartments (BMCs) are nanometer-scale organelles with a protein-based shell that serve to co-localize and encapsulate metabolic enzymes. They may provide a range of benefits to improve pathway catalysis, including substrate channeling and selective permeability. Several groups are working toward using BMC shells as a platform for enhancing engineered metabolic pathways. The microcompartment shell of Haliangium ochraceum (HO) has emerged as a versatile and modular shell system that can be expressed and assembled outside its native host and with non-native cargo. Further, the HO shell has been modified to use the engineered protein conjugation system SpyCatcher-SpyTag for non-native cargo loading. Here, we used a model enzyme, triose phosphate isomerase (Tpi), to study non-native cargo loading into four HO shell variants and begin to understand maximal shell loading levels. We also measured activity of Tpi encapsulated in the HO shell variants and found that activity was determined by the amount of cargo loaded and was not strongly impacted by the predicted permeability of the shell variant to large molecules. All shell variants tested could be used to generate active, Tpi-loaded versions, but the simplest variants assembled most robustly. We propose that the simple variant is the most promising for continued development as a metabolic engineering platform.

Optimization of Truck–Cargo Matching for the LTL Logistics Hub Based on Three-Dimensional Pallet Loading

This study investigates the truck–cargo matching problem in less-than-truckload (LTL) logistics hubs, focusing on optimizing the three-dimensional loading of goods onto standardized pallets and assigning these loaded pallets to a fleet of heterogeneous vehicles. A two-stage hybrid heuristic algorithm is proposed to solve this complex logistics challenge. In the first stage, a tree search algorithm based on residual space is developed to determine the optimal layout for the 3D loading of cargo onto pallets. In the second stage, a heuristic online truck–cargo matching algorithm is introduced to allocate loaded pallets to trucks while optimizing the number of trucks used and minimizing transportation costs. The algorithm operates within a rolling time horizon, allowing it to dynamically handle real-time order arrivals and time window constraints. Numerical experiments demonstrate that the proposed method achieves high pallet loading efficiency (close to 90%), near-optimal truck utilization (nearly 95%), and significant cost reductions, making it a practical solution for real-world LTL logistics operations.

An extracellular vesicle delivery platform based on the PTTG1IP protein

Extracellular vesicles (EVs) are promising therapeutic delivery vehicles, although their potential is limited by a lack of efficient engineering strategies to enhance loading and functional cargo delivery. Using an in-house bioinformatics analysis, we identified N-glycosylation as a putative EV-sorting feature. PTTG1IP (a small, N-glycosylated, single-spanning transmembrane protein) was found to be a suitable scaffold for EV loading of therapeutic cargoes, with loading dependent on its N-glycosylation at two arginine residues. Chimeric proteins consisting of PTTG1IP fused with various cargo proteins, and separated by self-cleaving sequences (to promote cargo release), were shown to enable highly efficient functional delivery of Cre protein to recipient cell cultures and mouse xenograft tumors, and delivery of Cas9-sgRNA complexes to recipient reporter cells. The favorable membrane topology of PTTG1IP enabled facile engineering of further variants with improved properties, highlighting its versatility and potential as a platform for EV-based therapeutics.

Non-lytic spread of poliovirus requires the nonstructural protein 3CD.

Non-enveloped viruses like poliovirus (PV) have evolved the capacity to spread by non-lytic mechanisms. For PV, this mechanism exploits the host secretory autophagy pathway. Virions are selectively incorporated into autophagosomes, double-membrane vesicles that travel to the plasma membrane, fuse, and release single-membrane vesicles containing virions. Loading of cellular cargo into autophagosomes relies on direct or indirect interactions with microtubule-associated protein 1B-light chain 3 (LC3) that are mediated by motifs referred to as LC3-interaction regions (LIRs). We have identified a PV mutant with a severe defect in non-lytic spread. An F-to-Y substitution in a putative LIR of the nonstructural protein 3CD prevented virion incorporation into LC3-positive autophagosomes and virion trafficking to the plasma membrane for release. Using high-angle annular dark-field scanning transmission electron microscopy to monitor PV-induced autophagosome biogenesis, for the first time, we show that virus-induced autophagic signals yield normal autophagosomes, even in the absence of virions. The F-to-Y derivative of PV 3CD was unable to support normal autophagosome biogenesis. Together, these studies make a compelling case for a direct role of a viral nonstructural protein in the formation and loading of the vesicular carriers used for non-lytic spread that may depend on the proper structure, accessibility, and/or dynamics of its LIR. The studies of PV 3CD protein reported here will hopefully provoke a more deliberate look at the presence and function of LIR motifs in viral proteins of viruses known to use autophagy as the basis for non-lytic spread.

A Versatile Magnetic Nanoplatform for Plug-and-Play Functionalization: Genetically Programmable Cargo Loading to Bacterial Magnetosomes by SpyCatcher "Click Biology".

Bacterial magnetosomes ("MAGs") represent a promising class of magnetic iron oxide nanoparticles with exceptional material characteristics and high application potential in the biomedical and biotechnological field. For the surface functionalization of MAGs with different protein cargos, their enveloping membrane can be addressed by genetic means. However, the expression of foreign polypeptides as translational fusion to magnetosome membrane proteins is still laborious and lacks versatility as the generated particles are monospecific and thus restricted to predetermined functions. Utilizing the SpyTag-SpyCatcher (ST-SC) bioconjugate system, we here establish a flexible platform for the targeted nanoassembly of multifunctional MAGs that combines the rapidity of chemical coupling (e.g., by cross-linking reactions) and the unmatched selectivity and controllability of in vivo functionalization. MAGs genetically engineered to display either SC- or ST-connectors are shown to efficiently bind a variety of complementary tagged (protein) cargo. Specifically, we cover a broad spectrum of representative functional moieties and foreign cargo (such as enzymes, antibodies, fluorophores, and silica beads) with relevance in biotechnology and biomedicine and demonstrate the interchangeability of the MAGs-adapted ST-SC system. For the controlled generation of artificial shells surrounding the particles, SC-MAGs are effectively coated by protein-corona proteins. The potential of the here-provided toolkit is even more enhanced by using SC-MAGs as an affinity tool for selective protein pulldown in vitro and in vivo. Overall, this innovative technology turns bacterial MAGs into a flexible magnetic nanoscaffold for the targeted plug-and-play display of virtually unlimited additional functionalities, thereby generating a multitude of magnetic hybrid materials that can be used in many applications.

Biomimetic Nanoparticles for Basic Drug Delivery.

Biomimetic nanoparticles (BMNPs) are innovative nanovehicles that replicate the properties of naturally occurring extracellular vesicles, facilitating highly efficient drug delivery across biological barriers to target organs and tissues while ensuring maximal biocompatibility and minimal-to-no toxicity. BMNPs can be utilized for the delivery of therapeutic payloads and for imparting novel properties to other nanotechnologies based on organic and inorganic materials. The application of specifically modified biological membranes for coating organic and inorganic nanoparticles has the potential to enhance their therapeutic efficacy and biocompatibility, presenting a promising pathway for the advancement of drug delivery technologies. This manuscript is grounded in the fundamentals of biomimetic technologies, offering a comprehensive overview and analytical perspective on the preparation and functionalization of BMNPs, which include cell membrane-coated nanoparticles (CMCNPs), artificial cell-derived vesicles (ACDVs), and fully synthetic vesicles (fSVs). This review examines both "top-down" and "bottom-up" approaches for nanoparticle preparation, with a particular focus on techniques such as cell membrane coating, cargo loading, and microfluidic fabrication. Additionally, it addresses the technological challenges and potential solutions associated with the large-scale production and clinical application of BMNPs and related technologies.

Analisis Perhitungan Weight And Balance pada Loadsheet dalam Menunjang Keselamatan Penerbangan pada Maskapai Air Asia di Bandar Udara Husein Sastranegara Bandung

The importance of calculating weight and balance on an aircraft to support flight safety is something that must be done by service providers. Mistakes in calculating weight distribution can cause overloads that risk endangering the safety of passengers, crew, and the aircraft itself. This study was conducted at AirAsia Airlines with a focus on the Airbus A320 aircraft at Husein Sastranegara Airport, Bandung. The study used a quantitative method with primary and secondary data collection. Primary data was collected through direct observation in the field, while secondary data was obtained from documents, journals, and related literature. This study analyzed data related to aircraft weight, fuel, passengers, baggage, and cargo to calculate weight and balance on the loadsheet. The results of this study indicate that calculating weight and balance on loadsheets is an important process in ensuring flight safety. Steps involving calculating the weight of passengers, baggage, cargo, fuel, and the distribution of the aircraft's center of gravity ensure that the aircraft operates within the safe limits set by the manufacturer. Based on the analysis of passenger, baggage, cargo, and fuel data, the Zero Fuel Weight (ZFW) was 60,516 kg, Takeoff Weight (TOW) was 67,516 kg, and Landing Weight (LW) was 62,516 kg. All of these parameters are within the specified safe limits. Load management on an Air Asia A320 aircraft when overloading requires special attention to parameters such as Maximum Takeoff Weight (MTOW), Maximum Landing Weight (MLW), and Maximum Landing Weight (MZFW). If an overload occurs, as recorded on the fourth day in Hold 5, the solution that can be taken is to reduce the cargo load on that Hold. For the situation on the fifth day, where the Landing Weight (LW) exceeds the MTOW, the right step is to reduce the amount of fuel.

A combined method for the evaluation of contributing factors to maritime dangerous goods transport accidents

This paper evaluates the contributing factors to maritime dangerous goods (DG) transport accidents by integrating the Entropy Weight (EW) and Grey Relational Analysis (GRA) methods. For this purpose, investigation reports of maritime DG transport accidents that occurred worldwide between 2000 and 2023 are derived from the International Maritime Organization’s Integrated Shipping Information System (IMO GISIS) database’s Marine Casualties and Incidents (MCI) module. Eleven main ship operations and thirteen primary causes were selected by analysing accident investigation reports. The weights of main ship operations are calculated utilizing the EW method. The correlational degrees of the primary causes are then calculated using the GRA method. Most maritime DG transport accidents occur during unberthing, bunkering, and pilotage operations. The most common contributing factors of maritime DG transport accidents are collisions and occupational accidents. Specifically, maritime DG transport accidents are most likely to be caused by collisions during sailing, passage, maneuvering, and bunkering operations, as well as occupational accidents during cargo loading, anchoring, berthing, and mooring operations. The results of this paper can support stakeholders in developing the needed policies to guarantee the safety of maritime DG transport.

Loading and unloading plasmid cargoes

Plasmids are vehicles for horizontal gene transfer between cells, but they also exchange genes with associated chromosomes in a process termed 'intracellular mobility'. I discuss a recent article by Kadibalban et al. mapping such plasmid-chromosomal sequence similarities across diverse bacteria.

Dual Centrifugation-based Screening for pH-responsive Liposomes.

In liposomal drug delivery development, the delicate balance of membrane stability is a major challenge to prevent leakage (during shelf-life and blood circulation), and to ensure efficient payload release at the therapeutic destination. Our composite screening approach uses the processing by dual centrifugation technique to speed up the identification ofde novoformulations of intermediate membrane stability. By screening binary lipid combinations at systemically varied ratios we highlight liposomal formulations of intermediate stability, what we termed "the edge of stability", requiring moderate stimuli for destabilization. Supplementation with a pH-sensitive cholesterol derivative (to obtain acid labile liposomes) and renewed assessment with cargo load led to the discovery of three formulations with sufficient shelf-life stability, acceptable cargo retention and efficient pH-responsive cargo release in vitro. The "lead candidates" exhibited promising in cellulo uptake with increased intracellular cargo release and revealed in vivo performance advantages compared to a control liposome. Our approach filters lipid compositions on "the edge of stability" that were introduced with a pH-sensitive cholesterol derivate leading pH-responsive liposomes, out of a multidimensional parameter space. Their discovery by rational approaches would have been highly unlikely, thus highlighting the potential of our screening approach.

Construction of Yolk@shell Nanocomposite Particles with Controlled Multisized Pore Structures by Monomicelle Confined Assembly.

Hollow nanoparticles with tunable structures and spatial and chemical specificity are considered as promising carriers. However, it remains a formidable challenge to endow hollow nanomaterials with precisely controlled multisized macro/mesoporous structures up to now. This paper demonstrates a "polydopamine (PDA) expansion-shrinkage" strategy combined with a monomicelle interfacial confined assembly method to achieve the highly controllable preparation of a series of yolk@shell PDA@SiO2 composite nanoparticles with structural asymmetry and a tunable multisized pore in the shell. The strategy allows systematic manipulation of the average pore size of large slit pores in the range of 15.4-86.5 nm by adjusting the reaction temperature. Benefiting from advantages such as an asymmetric structure and multilevel porosity, they exhibit excellent performance in the applications of on-demand loading of dual-sized cargoes, dual-propelled nanomotors, and particle size-selected encapsulation and separation. These findings provide inspiration for the construction of asymmetric yolk@shell structures with tunable multisized pores for a wide range of biological and chemical applications.

A Novel Impedance-Based Parallel Cooperative Control Method for Front and Rear Landing Gear Hydraulic Systems of UAVs

Cargo handling issues affect the ability of large heavy-duty Unmanned Aerial Vehicles (UAVs) to transport cargo and limit the development of large UAVs. Compared to conventional landing gear, hydraulically controlled landing gear can tilt the drone within a specified angle, facilitating smoother loading and unloading of goods. Therefore, it is important to study the hydraulic landing gear control system for a UAV to make the UAV’s tilt possible. In this paper, an impedance-based parallel cooperative control method for front and rear landing gear hydraulic systems of large heavy-duty UAVs is presented, which can achieve UAV tilting within a reasonable angle during the loading and unloading of cargoes by large, heavy-duty UAVs. This paper establishes the physical model of the UAV’s landing gear, the mathematical model of the hydraulic system, and the kinematic model of the airframe. Through kinematic analysis, the correlation between each hydraulic dive unit’s (HDU’s) extension length in the landing gear and the UAV’s tilt angle is established. This paper introduces a two-fold based-loop parallel control technique, featuring angle based-loop control for the UAV’s front and position based-loop control for its rear landing gear. It aims to enable the UAV to freely tilt for loading and unloading cargo at a predetermined angle, by measuring the UAV’s tilting angle, the HDU’s force exerted on the landing gear, and its positional parameters. Ultimately, the practicality of this technique is confirmed through simulations and experiments.

3D-Printed Latticed Microneedle Array Patches for Tunable and Versatile Intradermal Delivery.

Using high-resolution 3D printing, a novel class of microneedle array patches (MAPs) is introduced, called latticed MAPs (L-MAPs). Unlike most MAPs which are composed of either solid structures or hollow needles, L-MAPs incorporate tapered struts that form hollow cells capable of trapping liquid droplets. The latticestructures can also be coated with traditional viscous coating formulations, enabling both liquid- and solid-state cargo delivery, on a single patch. Here, a library of 43 L-MAP designs is generated and in-silico modeling is used to down-select optimal geometries for further characterization. Compared to traditionally molded and solid-coated MAPs, L-MAPs can load more cargo with fewer needles per patch, enhancing cargo loading and drug delivery capabilities. Further, L-MAP cargo release kinetics into the skin can be tuned based on formulation and needle geometry. In this work, the utility of L-MAPs as a platform is demonstrated for the delivery of small molecules, mRNA lipid nanoparticles, and solid-state ovalbumin protein. In addition, the production of programmable L-MAPs is demonstrated with tunablecargo release profiles, enabled by combining needle geometries on a single patch.

A bioswitchable delivery system for microRNA therapeutics based on a tetrahedral DNA nanostructure.

As microRNAs (miRNA) regulate almost all physiopathological activities in the human body, miRNA therapeutics that deliver miRNA regulators have attracted considerable attention in the field of nucleic acid drug development. The use of tetrahedral DNA nanostructures to deliver miRNA regulators is promising because of their simple fabrication, enhanced cell entry, effective tissue penetration, biocompatibility and functional editability. This protocol extension builds on our previous protocol for the use of tetrahedral DNA nanostructures and was designed to establish an updated bioswitchable delivery system (BDS) for achieving controlled cargo loading and release. A ribonuclease H-sensitive sequence is designed as a bioswitchable apparatus for the targeted release of the miRNA regulator. The functional sequence of the miRNA regulator and minimal secondary structure formation tendency during annealing are two key points in cargo design. We provide two BDS design strategies; BDS-A comprises an intact DNA tetrahedron with the RNA cargo hanging outside, offering the merits of lower cost, simplicity, and more direct structural design. In the BDS-B design, the RNA regulators are embedded into the DNA tetrahedron, which is beneficial for dermal tissue permeation applications. Following sequence design in Oligo 7 and Tiamat, the BDS assembly is completed and then ribonuclease H achieves controlled release of the miRNA regulator by triggering the bioswitchable apparatus. This is verified via polyacrylamide and agarose gel electrophoresis or fluorophore modifications. Both BDSs show promising cellular membrane permeability, tissue permeability and target inhibition in vitro and in vivo. The assembly and characterization of the BDS can be completed in 4 d, and the validation time for biostability and biological applications will depend on the specific use.

The Six-Transmembrane Enzyme GDE2 Is Required for the Release of Molecularly Distinct Small Extracellular Vesicles from Neurons.

Extracellular vesicles (EVs) are implicated in a multitude of physiological and pathophysiological processes in the nervous system; however, their biogenesis and cargoes are not well defined. Glycerophosphodiester Phosphodiesterase 2 (GDE2 or GDPD5) is a six-transmembrane protein that cleaves the Glycosylphosphatidylinositol (GPI)-anchor that tethers some proteins to the membrane and has important roles in neurodevelopment and disease-relevant pathways of neuronal survival. We show here that GDE2 regulates the number of small EVs (sEVs) released from the cell surface of neurons via its GPI-anchor cleavage activity and contributes to the loading of protein cargo through enzymatic and non-enzymatic mechanisms. Proteomic profiling reveals that GDE2 releases at least two distinct EV populations, one containing GDE2 itself and the other harboring the putative ectosomal markers CD9 and BSG. sEVs released by GDE2 are enriched in cytoskeletal and actin-remodeling proteins, suggesting a potential mechanism for GDE2-dependent EV release. Further, sEV populations released by GDE2 are enriched in proteins responsible for modulating synaptic activity and proteins that are critical for cellular redox homeostasis. These studies identify GDE2 as a novel regulator of molecularly distinct sEV populations from neurons with potential roles in the synaptic and redox pathways required for neuronal function and survival.

Towards greener city logistics: an application of agile routing algorithms to optimize the distribution of micro-hubs in Barcelona

The COVID-19 pandemic accelerated the shift towards online shopping, reshaping consumer habits and intensifying the impact on urban freight distribution. This disruption exacerbated traffic congestion and parking shortages in cities, underscoring the need for sustainable distribution models. The European Union's common transport policy advocates for innovative UFD approaches that promote intermodal transportation, reduce traffic, and optimize cargo loads. Our study addresses these challenges by proposing an agile routing algorithm for an alternative UFD model in Barcelona. This model suggests strategically located micro-hubs selected from a set of railway facilities, markets, shopping centers, district buildings, pickup points, post offices, and parking lots (1057 points in total). It also promotes intermodality through cargo bikes and electric vans. The study has two main objectives: (i) to identify a network of intermodal micro-hubs for the efficient delivery of parcels in Barcelona and (ii) to develop an agile routing algorithm to optimize their location. The algorithm generates adaptive distribution plans considering micro-hub operating costs and vehicle routing costs, and using heuristic and machine learning methods enhanced by parallelization techniques. It swiftly produces high-quality routing plans based on transportation infrastructure, transportation modes, and delivery locations. The algorithm adapts dynamically and employs multi-objective techniques to establish the Pareto frontier for each plan. Real-world testing in Barcelona, using actual data has shown promising results, providing potential scenarios to reduce CO2 emissions and improve delivery times. As such, this research offers an innovative and sustainable approach to UFD, that will contribute significantly to a greener future for cities.

Surface Engineered Osteoblast-Extracellular Vesicles Serve as an Efficient Carrier for Drug and Small RNA to Actively Target Osteosarcoma.

Osteosarcoma (OS) is a rare malignant tumor that affects soft tissue and has high rates of lung metastasis and mortality. The primary treatments for OS include preoperative chemotherapy, surgical resection of the lesion, and postoperative chemotherapy. However, OS chemotherapy presents critical challenges related to treatment toxicity and multiple drug resistance. To address these challenges, nanotechnology has developed nanosystems that release drugs directly to OS cells, reducing the drug's toxicity. Extracellular vesicles (EVs) are nanosized lipid-bilayer bound vesicles that act as cell-derived vehicles and drug delivery systems for several cancers. This study aims to utilize EVs for OS management by co-delivering Hdac1 siRNA and zoledronic acid (zol). The EVs' surface is modified with folic acid (FA) and their targeting ability is compared to that of native EVs. The results showed that the EVs' targeting ability depends on the parent cell source, and FA conjugation further enhanced it. Furthermore, EVs were used as the carrier for co-loading drug (zol) and small RNA (Hdac-1). This approach of using surface engineered EVs as carriers for cargo loading and delivery can be a promising strategy for osteosarcoma management.

Outer membrane vesicles from commensal microbes contribute to the sponge Tedania sp. development by regulating the expression level of apoptosis-inducing factor (AIF)

The transition from the swimming larval stage to the settlement stage represents a significant node in the marine sponge developmental process. Previous research has shown that the outer membrane vesicles (OMVs) from the bacterial species Tenacibaculum mesophilum associated with the sponge Tedania sp. influence larval settlement: low concentrations of OMVs increase the attachment rate, whereas high concentrations decrease the attachment rate. Here, by comparing the transcriptomes of sponge larvae in filtered seawater (FSW group) and in FSW supplemented with OMVs (FSW-OMV group), the results indicated that bacterial OMVs affected larval settlement by modulating the expression levels of apoptosis-inducing factor (AIF) in the host. Subsequently, quantitative real-time PCR revealed a decrease in aif expression near the time of settlement (SE) compared to that in the control group. RNA interference (RNAi) was used to target the aif gene, and the rate of larval settlement was significantly reduced, confirming the inhibitory effect of high concentrations of OMVs. Moreover, small RNA (sRNA) sequencing of OMVs revealed the existence of abundant AIF-sRNAs of 30 nt, further suggesting that one pathway for the involvement of sponge-associated bacteria in host development is the transport of OMVs and the direct function of cargo loading.

Orthogonal analysis reveals inconsistencies in cargo loading of extracellular vesicles.

Since extracellular vesicles (EVs) have emerged as a promising drug delivery system, diverse methods have been used to load them with active pharmaceutical ingredients (API) in preclinical and clinical studies. However, there is yet to be an engineered EV formulation approved for human use, a barrier driven in part by the intrinsic heterogeneity of EVs. API loading is rarely assessed in the context of single vesicle measurements of physicochemical properties but is likely administered in a heterogeneous fashion to the detriment of a consistent product. Here, we applied a suite of single-particle resolution methods to determine the loading of rhodamine 6G (R6G) surrogate cargo mimicking hydrophilic small molecule drugs across four common API loading methods: sonication, electroporation, freeze-thaw cycling and passive incubation. Loading efficiencies and alterations in the physical properties of EVs were assessed, as well as co-localization with common EV-associated tetraspanins (i.e., CD63, CD81 and CD9) for insight into EV subpopulations. Sonication had the highest loading efficiency, yet significantly decreased particle yield, while electroporation led to the greatest number of loaded API particles, albeit at a lower efficiency. Moreover, results were often inconsistent between repeated runs within a given method, demonstrating the difficulty in developing a rigorous loading method that consistently loaded EVs across their heterogeneous subpopulations. This work highlights the significance of how chosen quantification metrics can impact apparent conclusions and the importance of single-particle characterization of EV loading.