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Hepatic stellate cell-targeted chemo-gene therapy for liver fibrosis using fluorinated peptide-lipid hybrid nanoparticles

Exploring precise and effective treatments for liver fibrosis is urgent. The effective therapy for liver fibrosis depends on the specific delivery of antifibrotic drugs to activated hepatic stellate cells (aHSCs). However, this is a challenging task due to pathological barriers, primarily caused by collagen deposition. This study developed vitamin A-functionalized fluorinated peptide/lipid hybrid nanoparticles to co-deliver sorafenib and siRNA against HSP47 (SF-siHSP47@VFPL NPs). This nanoparticle formulation offers significant advantages due to its fluorine‑fluorine and electrostatic interactions, allowing for high SF and siHSP47 loading efficiency and sustained drug release. Importantly, in vitro cell uptake and in vivo biodistribution revealed that VA functionalization significantly improved aHSC-targeted delivery efficiency by engaging retinol-binding protein receptors on HSCs. Furthermore, it dramatically reduced extracellular matrix deposition, as evidenced by diminished levels of liver fibrosis-associated genes (HSP47, TIMP-1, and collagen I), promoting collagen breakdown and preventing collagen production, thus overcoming drug delivery barriers. Thus, SF-siHSP47@VFPL NPs demonstrated optimal antifibrotic effects by triggering apoptosis and ferroptosis in aHSCs. In liver fibrosis mouse models, SF-siHSP47@VFPL NPs remodeled the pathological environment and restored liver functionality through a marked reduction in serum liver transferases, hydroxyproline content, collagen deposition, and α-SMA and CD31 expression in liver tissue, resulting in alleviated liver fibrosis. Consequently, SF-siHSP47@VFPL NPs showed significant potential for HSC-targeted, chemo-gene therapy in the treatment of liver fibrosis.

Synthesis of dumbbell-like heteronanostructures encapsulated in ferritin protein: Towards multifunctional protein based opto-magnetic nanomaterials for biomedical theranostic

Dumbbell-like hetero nanostructures based on gold and iron oxides is a promising material for biomedical applications, useful as versatile theranostic agents due the synergistic effect of their optical and magnetic properties. However, achieving precise control on their morphology, size dispersion, colloidal stability, biocompatibility and cell targeting remains as a current challenge. In this study, we address this challenge by employing biomimetic routes, using ferritin protein nanocages as template for these nanoparticles’ synthesis. We present the development of an opto-magnetic nanostructures using the ferritin protein, wherein gold and iron oxide nanostructures were produced within its cavity. Initially, we investigated the synthesis of gold nanostructures within the protein, generating clusters and plasmonic nanoparticles. Subsequently, we optimized the conditions for the superparamagnetic nanoparticles synthesis through controlled iron oxidation, thereby enhancing the magnetic properties of the resulting system. Finally, we produce magnetic nanoparticles in the protein with gold clusters, achieving the coexistence of both nanostructures within a single protein molecule, a novel material unprecedented to date. We observed that factors such as temperature, metal/protein ratios, pH, dialysis, and purification processes all have an impact on protein recovery, loading efficiency, morphology, and nanoparticle size. Our findings highlight the development of ferritin-based nanomaterials as versatile platforms for potential biomedical use as multifunctional theranostic agents.

PH-Responsive MOF Nanoparticles Equipped with Hydrophilic "Armor" Assist Fungicides in Controlling Peanut Southern Blight.

The development of novel, safe, and efficient pest and disease control technologies for agricultural crops remains a pivotal area of research. In this study, by combining ZIF-8 and ZIF-90, a water-stable, pH-responsive bilayer MOF nanoparticle (NP) named Z8@Z90 was created, and tebuconazole (TEB) was added to form T@Z8@Z90, used for controlling peanut southern blight. The loading efficiency of TEB within the T@Z8@Z90 reached 26.15%, enabling rapid release in acidic environments triggered by oxalic acid (OA) secreted by Sclerotium rolfsii. In vitro experiments showed that T@Z8@Z90 can regulate the oxalic acid secretion of S. rolfsii and destroy its cell membrane structure. Additional experiments revealed that T@Z8@Z90 reduced sclerotial formation, decreased the total protein content of sclerotia, and influenced their sensitivity to pesticides, thereby mitigating the risk of reinfection by S. rolfsii. Notably, T@Z8@Z90 exhibited efficient translocation within peanut seedlings, being absorbed through the roots and transported to the leaves. At a concentration of 200 mg/L, T@Z8@Z90 exhibited high safety profiles for peanut seedling growth compared to the TEB suspension. Moreover, T@Z8@Z90 is safer for earthworms than TEB SC. Overall, this study offers valuable insights for the management of soil-borne diseases in agriculture and contributes to the advancement of sustainable agricultural practices.

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.

Thiacloprid-silica nano-delivery system enhances toxicity against Aphis gossypii and improves non-target biosafety

Nanotechnology aligns with the requirements of sustainable development of agriculture, and nano-pesticides offer a promising approach to controlling agricultural pests and increasing productivity. Non-target predators also play a crucial role in pest controls and enhancing the efficacy of pesticide on target organisms. Reducing the toxicity of pesticides to non-target organisms is key to of coordinating chemical control and biological control methods. Therefore, it is essential to assess the toxicity of nano-pesticides on non-target predators. In this study, a carbon dots-doped mesoporous silica nano-delivery system (Thi@CD-MSN) was successfully developed using CD-MSN as carrier material and thiacloprid (Thi) as a model pesticide. The results demonstrated that the synthesized Thi@CD-MSN exhibited a relatively high loading efficiency (33.58%). Laboratory bioassay experiments revealed that Thi@CD-MSN demonstrated effective insecticidal activity (LC50 = 21.67 mg/L) in controlling Aphis gossypii Glover. Besides, the acute toxicity of Thi@CD-MSN on Chrysoperla pallens larvae was significantly lower than that of Thi, as was its toxicity to 4T1 cells. These findings suggest that CD-MSN can serve as an ecological safety carrier for pesticide delivery, improving the effective utilization of Thi while reducing the risks to non-target predators. These results are essential for comprehending the effects of nano-pesticides on non-target predators, providing informative data for implementing biological and chemical control strategies. It strengthens the safety evaluation of nano-pesticides.

Correlation among Yield, Quality and Mechanization Amicable Traits in Sugarcane Clones

The objective of this study was to identify superior clones suitable for mechanical harvesting, which is essential to improve the production efficiency, cost effectiveness, cane loading efficiency infield losses and also to investigate the relationships among the yield and quality parameters with cane yield and sugar yield in sugarcane. This experiment was conducted at Regional sugarcane and Rice Research Station, Rudrur, Nizamabad district, Telangana state in Randomized Block Design (RBD) with three replications. The analysis of variance revealed significant differences among genotypes for fifteen yield, quality and mechanization amicable characters. Correlation coefficient results indicated that cane yield was positively correlated with commercial cane sugar, number of tillers at 120 days after planting, cane height, cane girth, single cane weight. So, selection for cane yield alone will also increase the other characters because these traits are positively correlated with cane yield. The Angle of inclination is negatively correlated with cane yield and positively correlated with ratio of crown weight to cane weight, brix%, sucrose%, purity% and CCS%. Results indicate that the genotypes should be selected on the basis of number of millable canes, cane length, cane girth and single cane weight for getting higher sugarcane yield. Furthermore, mechanization amicable genotypes can be selected which are near to erect nature and also having less crown weight.

Metal‐organic frameworks‐based nanomedicines to promote cancer immunotherapy: Recent advances and future directions

AbstractCancer immunotherapy uses the body's immune system to fight tumors by restoring natural antitumor responses. Metal‐organic frameworks (MOFs), characterized by their unique crystalline porous structures formed from metal ions linked by organic ligands, offer a promising solution. Recent studies have unveiled the potential of MOFs in cancer immunotherapy. The exceptional porosity and surface area, coupled with their extraordinary thermal and chemical stability, bring significant advantages for efficient drug loading and delivery of immunotherapeutic agents. The adaptability of MOFs further enhances the controlled release of immunotherapeutic drugs within target cells and increases tumor sensitivity to other therapies such as photodynamic, photothermal, and radiotherapy. This multifunctional carrier contributes to modulating the tumor microenvironment and reactivating antitumor immunity, providing a comprehensive strategy for cancer treatment. In this review, we summarize the applications of MOFs in immune checkpoint blockade, immunomodulator delivery, and cancer vaccine delivery, and discuss existing challenges in their use for immunotherapy. This discussion aims to offer insights for developing better treatments and enhancing the efficacy of immunotherapy.

Inhalable cyclodextrin metal-organic framework dry powder enhanced direct pulmonary delivery of paclitaxel for the treatment of lung cancer

Inhalation dry powders are in great demand for the treatment of lung diseases due to their unique advantages such as excellent direct delivery, rapid onset of action, and reduced systemic side effects. Herein, this work highlights the successful development of an inhalable paclitaxel dry powder, which is facilely constructed through the optimization of the formulation between chemotherapy components (paclitaxel) and a metal-organic framework (MOF) carrier (γ-cyclodextrin and KOH). Moderate pore and particle size distribution, as well as low hygroscopicity endow the dry powder with excellent loading efficiency and resistance to degradation. All ingredients and their interactions confer favorable emptying rates, flowability, and pulmonary deposition, as well as direct lung drug delivery and release. These attractive characteristics enable the dry powder to effectively enhance cytotoxicity, improve pharmacokinetics, promote lung accumulation, and reduce the side effects of paclitaxel. Thus, this paclitaxel dry powder may open a new avenue for the inhalation treatment of diseases, greatly advancing its clinical application.

Novel combination therapy using recombinant oncolytic adenovirus silk hydrogel and PD-L1 inhibitor for bladder cancer treatment

Recombinant oncolytic adenovirus offers a novel and promising cancer treatment approach, but its standalone efficacy remains limited. This study investigates a combination treatment strategy by co-administering recombinant oncolytic Adv-loaded silk hydrogel with a PD-L1 inhibitor for patients with bladder cancer to enhance treatment outcomes. Bladder cancer tissues from mice were collected and subjected to single-cell sequencing, identifying CRB3 as a key gene in malignant cells. Differential expression and functional enrichment analyses were performed, validating CRB3’s inhibitory role through in vitro experiments showing suppression of bladder cancer cell proliferation, migration, and invasion. Recombinant oncolytic adenoviruses encoding CRB3 and GM-CSF were constructed and encapsulated in silk hydrogel to enhance drug loading and release efficiency. In vivo experiments demonstrated that the nano-composite hydrogel significantly inhibited tumor growth and increased immune infiltration in tumor tissues. Co-administration of adenovirus silk hydrogel (Adv-CRB3@gel) with a PD-L1 inhibitor significantly enhanced T-cell infiltration and tumor killing. The combination of recombinant oncolytic Adv-loaded nano-composite hydrogel encoding CRB3 and GM-CSF with a PD-L1 inhibitor improves bladder cancer treatment outcomes by effectively recruiting T cells, providing a novel therapeutic strategy.

Assessment of merits and demerits of perpendicular and slanted photovoltaic/thermal facades

This study compares the thermal and electrical energy production potential and wind load assessment of perpendicular versus slanted photovoltaic (PV) facades. The goal is to identify the optimal facade orientation for societal adoption based on energy efficiency and wind load considerations. Using Hybrid RANS/LES models, the aerodynamic performance and energy yield of both orientations are analyzed under varying angles and environmental conditions. The findings show that while the slanted façade is better designed to capture solar irradiance, it produces 4 to 8 % less energy than the perpendicular façade and yields approximately 2 % less exergy efficiency due to wind regime. However, the slanted design reduces wind load by 9.8 %, enhancing structural stability. The maximum thermal energy output of 217 kW is recorded from the perpendicular façade at a wind speed of 1 m/s, while the slanted façade generates a peak electrical power of 68 kW at 8 m/s wind speed. Overall, the perpendicular façade underscores more efficient in equal weather conditions, making it a sustainable choice for PV installation. These results provide valuable insights for architects, engineers, and policymakers in optimizing PV facade design for urban environments.

Metal-Organic Nanomaterials for Tumor Metabolic Blockade and Image to Increase Tumor Therapy.

The abnormal energy metabolism level of a tumor reduces the efficiency of chemotherapy. Metal-organic nanomaterials (MONs) with high drug loading efficiency, easy processes of synthesis, and controlled drug release have shown great potential in metabolic blocking and enhancement of tumor therapy. These metal-organic nanomedicines have been reported to modulate glycolysis or oxidative phosphorylation to provide monotherapy or combined therapies in tumorous treatments. In addition, the encapsulation or coordination of fluorescent dyes into MONs endowed them with the imaging ability of tumor metabolism. Herein, this Perspective summarizes the progress of MONs as therapeutic agents or imaging probes for application during tumor metabolic blocking or imaging, providing solid inspiration for biomedical applications of effective biomaterials. In addition, the current drawbacks of MONs for further biological applications in the future were discussed, giving stimulation of innovation and development in biomedical applications of MONs.

Doxorubicin-loaded methoxy-intercalated kaolinite as a repackaging of doxorubicin for an enhanced breast cancer treatment: in vitro and in vivo investigation

Doxorubicin is one of the most common wide-spectrum chemotherapeutics. However, its efficacy is limited due to off-target accumulation and selectivity issues. In this study, we compared the anti-cancer effect and biocompatibility of KaoliniteMeOH-Dox, a doxorubicin repackaging, to doxorubicin monotherapy. The formulation was extensively tested using transmission electron microscopy, dynamic light scattering, zeta potential, Fourier transform infrared, x-ray diffraction, and in vitro drug release. The MTT assay measured MCF-7 cell growth inhibition in vitro. In vivo testing involved 20 naïve mice and 40 Ehrlich solid tumor-inoculated mice. The tumor size was monitored for 18 days. In all experimental groups, tumor and cardiac tissues were evaluated for cytotoxicity and genotoxicity by addressing oxidative stress, histopathology, and comet assay. We found that kaoliniteMeOH-Dox has many advantages in terms of size, charge, shape, high loading efficiency (90.16%), and pH-dependent release. The MTT assay showed that the formulation outperformed doxorubicin in growth inhibition and selectivity. In vivo, research showed that kaoliniteMeOH-Dox suppressed tumors by 86.075% compared to 60.379% for free doxorubicin. Histological analysis showed that kaoliniteMeOH-Dox reduced tumor size, metastasis, and carcinogenic oxidative stress and inflammation in mice without harming naive mice. Based on the obtained data, the kaoliniteMeOH-Dox formulation holds promise for breast cancer treatment and warrants further investigation.

Pesticide delivery microcapsule based on maleic anhydride‐functionalized cellulose nanocrystals‐stabilized pickering emulsion

AbstractThe efficient use of pesticides is conducive to the harmonious coexistence of humans and nature, and pesticide transport carriers can enhance the utilization value of pesticides. However, design flaws in many pesticide carriers have resulted in numerous environmental and toxicity problems. Therefore, this paper proposed a method for synthesizing pesticide microcapsules using Pickering emulsion templates stabilized by maleic anhydride‐functionalized cellulose nanocrystals‐g‐poly (methyl methacrylate) (MACNCs‐g‐MMA), viz., simple radical polymerization of maleic anhydride‐functionalized cellulose nanocrystals with methyl methacrylate in Pickering emulsion. The structure and morphology of microcapsules were characterized via the FT‐IR, XRD, TG‐DTG, and SEM techniques. The imidacloprid‐loaded MACNCs‐g‐MMA (IMI@MACNCs‐g‐MMA) presented a high encapsulation efficiency (~94.13%) and drug loading efficiency (~24.00%). The release behavior was affected by temperature, viz., higher temperature promoted the release of IMI. Regarding the spread and retention on the leaf surface, the IMI@MACNCs‐g‐MMA demonstrated significant advantages over commercial IMI water‐dispersible granules (CG) (36.9° vs. 104.9° for contact angle, 14.3 vs. 30.0 mg cm−2 for retention). This study provides a promising pesticide formula for sustainable agriculture.

Ultra-High Sensitivity Anisotropic Piezoelectric Sensors for Structural Health Monitoring and Robotic Perception

HighlightsA novel anisotropic sensor with oriented piezoelectric filaments was prepared, capable of detecting both the magnitude and direction of micro-deformations.Due to the efficient load transfer of continuous fibers and the formation of porous ferroelectrets, an ultra-low strain detection limit of 0.06% was achieved in the sensor.Given the sensor's ultra-low detection limit and deformation direction sensing capability, we developed the sensor for detecting micron-scale deformations in thin-film structures and for robotic tactile sensing applications.

FORMULATION OF SELF-NANO EMULSIFYING SYSTEM (SNES) CONTAINING SNAKEHEAD FISH EXTRACT PROTEIN INCORPORATED INTO CORN OIL BY HYDROPHOBIC ION PAIRING METHOD

Objective: The purpose of this study was to determine whether the protein complexation of snakehead fish extract using the hydrophobic ion pairing method with Sodium Dodecyl Sulfate (SDS) as a complexing ligand could be loaded into the corn oil component of the SNES and to explore the ratio of oil, surfactant, and cosurfactant in the Self-Nanoemulsifying System (SNES) formula that can produce good SNES characteristics. Methods: Snakehead fish proteins were extracted using pressurized hot water and then complexed with SDS at acidic pH. The SDS-protein complex was loaded into the oil component of the SNES and then combined with the surfactant component (Tween 80) and cosurfactant (propylene glycol) in a ratio of 1:9:1, which, based on the preliminary miscibility screening, has the best miscibility among other screening results. Results: The results of the study were as follows. The binding efficiency of the SDS-protein-complex was 31.74% and the loading efficiency into SNES was 83.17%. The nanoemulsion formed had visible light transmittance of 100.4%, particle size 72.7 nm, zeta potential-53.03 mV, and emulsification time 71.67 seconds. The nanoemulsion was stable at storage and 100 and 1000 times dilution challenges using aqueous media, Simulated Gastric Fluid (SGF), and Simulated Intestinal Fluid (SIF). Conclusion: The hydrophobic ion-pair complexation of snakehead fish protein extract with SDS as the complexing ligand was able to load the protein into the oil component of SNES and the ratio of oil, surfactant, and cosurfactant in the SNES formula that produce good SNES characteristics was corn oil, Tween 80, and propylene glycol at a ratio of 1:9:1

Developing a Container Ship Loading-Planning Program Using Reinforcement Learning

This study presents an optimized container-stowage plan using reinforcement learning to tackle the complex logistical challenges in maritime shipping. Traditional stowage-planning methods often rely on manual processes that account for factors like container weight, unloading order, and balance, which results in significant time and resource consumption. To address these inefficiencies, we developed a two-phase stowage plan: Phase 1 involves bay selection using a Proximal Policy Optimization (PPO) algorithm, while Phase 2 focuses on row and tier placement. The proposed model was evaluated against traditional methods, demonstrating that the PPO algorithm provides more efficient loading plans with faster convergence compared to Deep Q-Learning (DQN). Additionally, the model successfully minimized rehandling and maintained an even distribution of weight across the vessel, ensuring operational safety and stability. This approach shows great potential for enhancing stowage efficiency and can be applied to real-world shipping scenarios, improving productivity. Future work will aim to incorporate additional factors, such as container size, type, and cargo fragility, to further improve the robustness and adaptability of the stowage-planning system. By integrating these additional considerations, the system will become even more capable of handling the complexities of modern maritime logistics.

Development of Chitosan-Coated Graphene Oxide and Iron Oxide Nanocomposites for Targeted Delivery of Camptothecin to Liver Cancer Cells.

Innovative drug delivery platforms for selective, regulated, and sustained release of anticancer drugs are crucial in cancer treatment. This study presents nanoparticles developed from chitosan (CS), graphene oxide (GO), and magnetite (Fe3O4), and their nanocomposites, to enhance the loading and release efficiency of camptothecin (CPT). Nanostructures were characterized using imaging microscopy, FT-IR, and X-ray diffraction, with an average crystallite size of 5.5 nm. Camptothecin binding proportions were 70% for CS, 81% for CS@Fe3O4, 58% for CS@GO, and 74% for CS@GO/Fe3O4. At pH 5.0, CPT release ratios were 87%, 80%, 88%, and 90%, respectively, and at pH 7.4, 84%, 72%, 89%, and 87%. Cytotoxicity was assessed using the MTT assay against HepG2 and SMMC-7721 cancer cells. CPT-CS@GO/Fe3O4 exhibited the highest survival at 5 µM and 12.5 µM concentrations, indicating it as the most effective nanocarrier for camptothecin delivery. The study demonstrates CS@GO/Fe3O4's potential as a superior drug delivery system.

A natural polyphenolic nanoparticle--knotted hydrogel scavenger for osteoarthritis therapy

Exploring highly efficient and cost-effective biomaterials for osteoarthritis (OA) treatment remains challenging, as current therapeutic strategies are difficult to eradicate the excessive reactive oxygen species (ROS) and nitric oxide (NO) at damaged sites. Tea polyphenol (TP) nanoparticles (NPs), a nature-inspired antioxidant in combination with 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), a NO scavenger, could provide maximized positive therapeutic effects on OA by eradicating both ROS and NO. Notably, this combination not only improves the half-life of the TP monomer and the drug loading efficiency of carboxy-PTIO but also prevents nitrite from being harmful to tissue. Moreover, the protonation ability of carboxy-PTIO allows smart acid-responsive release in response to environmental pH, which provides conditioned treatment strategies for OA. In in vitro experiments, TP/PTIO NPs downregulated proinflammatory cytokine release via synergistic removal of ROS and NO and suppression of ROS/NF-κB and iNOS/NO/Caspase-3 signaling. For in vivo experiments, NPs were cross-linked with 4-arm-PEG-SH to form an injectable hydrogel system. The release of TP and carboxy-PTIO from the system efficiently prevents cartilage inflammation and damage via similar signaling pathways. Overall, the proposed system provides an efficient approach for OA therapy.

Curcumin-loaded pickering emulsions based on soy protein isolate aggregates enhance diabetic wound healing

Wound healing in diabetic patients is more complex and prolonged in comparison to nondiabetic persons, and dealing with this problem has attracted considerable attention from researchers. According to its anti-inflammatory and anti-infective properties, Curcumin has a valued place in therapeutics, including wound healing. Still, its low stability and poor bioavailability are significant barriers to using its desirable properties. Hence, various biocompatible and biodegradable delivery systems have been developed to address this issue. In this study, Pickering emulsion (PE) prepared from soybean oil containing curcumin and modified SPI solution at 85 °C at pH 2.0 with 130 mM NaCl for different durations were used as a delivery system for curcumin. Changes in SPI properties were analyzed using fluorescence spectroscopy, Fourier transform infrared spectroscopy, and tensiometry methods. The prepared PEs were studied using microscopic techniques. Curcumin loading efficiency by PE was measured by UV–visible spectroscopy, and the impact of curcumin-loaded PE on wound healing was tested in diabetic rats. Spectroscopic studies and microscopy images revealed SPI amyloid-like aggregates suitable for stabilizing oil-in-water PEs. Tensiometry and creaming stability studies indicated that at least 6 h of heating (HSPI6) is necessary for optimal stability. Curcumin encapsulation efficiency was 95.7 ± 2.1 %. Curcumin-loaded PE-HSPI6 increased the healing rate of diabetic wounds in male Wistar rats by 1.46-fold. This study presents an approach for using biocompatible and biodegradable PEs to deliver hydrophobic compounds like curcumin for accelerating diabetic wound healing.

A Mem-dELISA platform for dual color and ultrasensitive digital detection of colocalized proteins on extracellular vesicles

Accurate, multiplex, and ultrasensitive measurement of different colocalized protein markers on individual tumor-derived extracellular vesicles (EVs) and dimerized proteins with multiple epitopes could provide insights into cancer heterogeneity, therapy management and early diagnostics that cannot be extracted from bulk methods. However, current digital protein assays lack certain features to enable robust colocalization, including multi-color detection capability, large dynamic range, and selectivity against background proteins. Here, we report a lithography-free, inexpensive (< $0.1) and ultrasensitive dual-color Membrane Digital ELISA (Mem-dELISA) platform by using track-etched polycarbonate (PCTE) membranes to overcome these shortcomings. Their through-pores remove air bubbles through wicking before they are sealed on one side by adhesion to form microwells. Immunomagnetic bead-analyte complexes and substrate solution are then loaded into the microwells from the opposite side, with >80% loading efficiency, before sealing with oil. This enables duplex digital protein colorimetric assay with beta galactosidase and alkaline phosphatase enzymes. The platform achieves 5 logs of dynamic range with a limit of detection of 10 aM for both Biotinylated β-galactosidase (B-βG) and Biotin Alkaline Phosphatase Conjugated (B-ALP) proteins. We demonstrate its potential by showing that a higher dosage of paclitaxel suppresses EpCAM-positive EVs but not GPC-1 positive EVs from breast cancer cells, a decline in chemo-resistance that cannot be detected with Western blot analysis of cell lysate. The Mem-dELISA is poised to empower researchers to conduct ultrasensitive, high throughput protein colocalization studies for disease diagnostics, treatment monitoring and biomarker discovery.