Simultaneous Encapsulation Research Articles

High-Throughput Manipulation of Circulating Tumor Cells Using a Multiple Single-Cell Encapsulation System with a Digital Micromirror Device.

Circulating tumor cells (CTCs) are potential precursors of metastatic cancer, and genomic information obtained from CTCs have the potential to provide new insights into the biology of cancer metastasis. We previously developed a technique for single-cell manipulation based on the encapsulation of a single cell in a photopolymerized hydrogel that can be used for subsequent genetic analysis. However, this technique has limitations in terms of throughput because light irradiation must be performed on each individual cell from the confocal laser-scanning microscopy. Here, we present a high-throughput cell manipulation technique using a multiple single-cell encapsulation system with a digital micromirror device. This system enables rapid cell imaging within a microcavity array, a microfilter for the recovery of CTCs from blood samples, as well as the simultaneous encapsulation of several CTCs with hydrogels photopolymerized using a multiple light-irradiation system. Furthermore, single-cell labeling using two differently shaped hydrogels was examined to distinguish between NCI-H1975 cells and A549 cells, demonstrating the utility of the system for single-cell gene mutation analysis. In addition to CTCs, our system can be widely applied for analyses of mammalian cells and microorganisms.

A nanomedicine approach enables co-delivery of cyclosporin A and gefitinib to potentiate the therapeutic efficacy in drug-resistant lung cancer

Drug resistance, accounting for therapeutic failure in the clinic, remains a major challenge to effectively manage cancer. Cyclosporin A (CsA) can reverse multidrug resistance (MDR), especially resistance to epidermal growth factor receptor tyrosine kinase inhibitors. However, the application of both drugs in cancer therapies is hampered by their poor aqueous solubility and low bioavailability due to oral administration. CsA augments the potency of gefitinib (Gef) in both Gef-sensitive and Gef-resistant cell lines. Here, we show that the simultaneous encapsulation of CsA and Gef within polyethylene glycol-block-poly(D, L-lactic acid) (PEG-PLA) produced a stable and systemically injectable nanomedicine, which exhibited a sub-50-nm diameter and spherical structures. Impressively, the co-delivery of therapeutics via single nanoparticles (NPs) outperformed the oral administration of the free drug combination at suppressing tumor growth. Furthermore, in vivo results indicated that CsA formulated in NPs sensitized Gef-resistant cells and Gef-resistant tumors to Gef treatment by inactivating the STAT3/Bcl-2 signaling pathway. Collectively, our nanomedicine approach not only provides an alternative administration route for the drugs of choice but also effectively reverses MDR, facilitating the development of effective therapeutic modalities for cancer.

Soft Nanohydrogels Based on Laponite Nanodiscs: A Versatile Drug Delivery Platform for Theranostics and Drug Cocktails

A new nanohydrogel drug delivery platform based on Laponite nanodiscs, polyacrylate, and sodium phosphate salts is described. The hybrid nanohydrogel is tailored to obtain soft and flexible nanohydrogels with G' around 3 kPa, which has been proposed as the ideal stiffness for drug delivery applications. In vitro studies demonstrate that the new nanohydrogels are biocompatible, biodegradable, nonswellable, pH-responsive, and noncytotoxic and are able to deliver antineoplastic drugs into cancer cells. The IC50 of nanohydrogels containing cisplatin, 4-fluorouracil, and cyclophosphamide is significantly lower than the IC50 of the free drugs. In vivo experiments suggest that the new nanomaterials are biocompatible and do not accumulate in crucial organs. The simple formulation procedure enables encapsulation of virtually any water-soluble molecule, without the need for chemical modification of the guests. These nanohydrogels are a versatile platform that enables the simultaneous encapsulation of several cancer drugs, yielding an efficient drug cocktail delivery system, which for instance presents a positive synergistic effect against MCF-7 cells.

A study on β-lactoglobulin-triligand-pectin complex particle: Formation, characterization and protection

There appear to be interesting market opportunities for functional foods offering multiple health benefits. This provides motivation to develop the carriers that can simultaneously encapsulate a plurality of bioactive components. Beta-lactoglobulin (β-LG) contains an internal cavity and multiple external portions as potential sites for the binding of a wide range of bioactive components. Formation of protein-polysaccharide complex may improve functional property of individual polymers. In this study, retinol, folic acid and resveratrol were used as the models of the ligands with different solubility, to investigate the formation of β-LG-triligand-pectin complex particle. The complex particle provided a better protective effect on retinol than did β-LG-retinol complex and eliminated negative effect of the protein on resveratrol. These results obtained here should be useful for the design of protein-based carriers for simultaneous encapsulation of several bioactive components with different solubility.

Development of Pluronic® nanocarriers comprising Pheophorbide, Zn-Pheophorbide, Lapachol and β-lapachone combined drugs: Photophysical and spectroscopic studies

Therapy efficiency is ruled by a single agent, which can be enhanced using an adjuvant, increasing cell inhibition in a synergetic aspect. Taking this into account, the photosensitizers Pheophorbide and Zinc-Pheophorbide, and their physical chemical and photodynamic properties, were evaluated with Lapachol and β-lapachone drugs. They were incorporated into triblock copolymers P123 and F127 as biocompatible nanocarriers. Spectrophotometric studies showed photosensitizer monomerization in the combined formulation, a fundamental aspect for optimal photodynamic results. The apparent pKa studies of Lapachol incorporated in copolymeric micelles indicated small variations when compared to the data in a homogeneous medium (water/ethanol). This demonstrated the composition of 76% and 56% for deprotonated forms of Lapachol at the physiological pH, after administration of the formulated systems in F127 and P123, respectively. This preferential location is in the poly (ethylene oxide) interfacial bulk of the Pluronic®. The characterization of the photophysical properties of the combined systems showed an expressed decrease of fluorescence quantum yield of the PS in the β-lapachone based systems. This was attributed to greater suppression by collisions with water molecules due to variations in the accommodation region of the photosensitizer after simultaneous encapsulation. The combined system showed a decrease of singlet oxygen quantum yield due to high local micellar microviscosity and low molecular oxygen concentration. However, a five-fold increase in singlet oxygen lifetime was noted in systems within the nanostructured microenvironment, which enhances the range of photosensitizer action. The combined system showed marked thermal stability when subjected to refrigeration, which was below the critical micellar temperature for nanocarriers. This was true after subsequent reheating as well. In addition, the formulations were stored for prolonged periods in the lyophilized state that demonstrated the maintenance of the monomeric state of the photosensitizers. This is a desirable effect for photodynamic applications. The majority of the combined systems incorporated into the nanostructured microenvironment showed temporal stability for more than 96 h in an aqueous solution.

Preparation and characterization of emulsion-filled gel beads for the encapsulation and protection of resveratrol and α-tocopherol

Proteins and polysaccharides have been widely used as wall materials to prepare novel carriers for the encapsulation of a single bioactive component or co-encapsulation of different bioactive components. Emulsion-filled gel beads generally contain three essential components: oil droplets, interface membrane of emulsifier, and gel matrix. In this study, emulsion-filled gel beads were prepared by the combination of heat-denatured whey protein isolate (WPI)'s emulsification and alginate's gelation at various protein and oil contents, in order to discuss simultaneous encapsulation, protection and release of hydrophobic α-tocopherol and amphiphilic resveratrol. High efficiency for the co-encapsulation of resveratrol and α-tocopherol was achieved for the emulsion-filled gel beads containing 1% WPI and 1%–4% oil. WPI content was more important than the content of oil for the protection of resveratrol while the content of oil was more important for the encapsulation and protection of α-tocopherol during storage. The release of α-tocopherol was <20% while resveratrol was rapidly released during digestion. Resveratrol remained 73% after released from the emulsion-filled gel beads containing 1% WPI and 1%–2% oil for 6 h. These results should be useful for the development of biopolymer-based carriers for the delivery of different bioactive components.

Simultaneous elimination of cancer stem cells and bulk cancer cells by cationic-lipid-assisted nanoparticles for cancer therapy

Convincing evidence indicates that the existence of cancer stem cells (CSCs) within malignant tumors is mostly responsible for the failure of chemotherapy. Therefore, instead of merely targeting bulk cancer cells, simultaneous elimination of both CSCs and bulk cancer cells is necessary to improve therapeutic outcomes. Herein, we designed cationic-lipid-assisted nanoparticles DTXLNPsiRNA for simultaneous encapsulation of the conventional chemotherapeutic agentdocetaxel (DTXL) and small interfering RNA (siRNA) targeting BMI-1 (siBMI-1). We confirmed that nanoparticles DTXLNPsiBMI-1 effectively deliver both therapeutic agents into CSCs and bulk cancer cells. The bulk cancer cells were effectively killed by the DTXL encapsulated in DTXLNPsiBMI-1. In breast CSCs, BMI-1 expression was significantly downregulated by DTXLNPsiBMI-1; consequently, the stemness was reduced and chemosensitivity of CSCs to DTXL was enhanced, resulting in the elimination of CSCs. Therefore, via DTXLNPsiBMI-1, the combination of siBMI-1 and DTXL completely inhibited tumor growth and prevented a relapse by synergistic killing of CSCs and bulk cancer cells in a murine model of an MDA-MB-231 orthotropic tumor.

Bioinspired Design of Alcohol Dehydrogenase@nano TiO₂ Microreactors for Sustainable Cycling of NAD⁺/NADH Coenzyme.

The bioinspired design and construction of enzyme@capsule microreactors with specific cell-like functionality has generated tremendous interest in recent years. Inspired by their fascinating complexity, scientists have endeavored to understand the essential aspects of a natural cell and create biomimicking microreactors so as to immobilize enzymes within the hierarchical structure of a microcapsule. In this study, simultaneous encapsulation of alcohol dehydrogenase (ADH) was achieved during the preparation of microcapsules by the Pickering emulsion method using amphiphilic modified TiO2 nanoparticles (NPs) as building blocks for assembling the photocatalytic microcapsule membrane. The ADH@TiO2 NP microreactors exhibited dual catalytic functions, i.e., spatially confined enzymatic catalysis and the membrane-associated photocatalytic oxidation under visible light. The sustainable cycling of nicotinamide adenine dinucleotide (NAD) coenzyme between NADH and NAD+ was realized by enzymatic regeneration of NADH from NAD+ reduction, and was provided in a form that enabled further photocatalytic oxidation to NAD+ under visible light. This bioinspired ADH@TiO2 NP microreactor allowed the linking of a semiconductor mineral-based inorganic photosystem to enzymatic reactions. This is a first step toward the realization of sustainable biological cycling of NAD+/NADH coenzyme in synthetic functional microsystems operating under visible light irradiation.

Immunoisolation of stem cells by simultaneous encapsulation and PEGylation

Today, cell therapy is known as an important tool in the treatment of chronic diseases where cells lose their normal function. Immunoisolation systems using microencapsulation or PEGylation have been developed to evade the problem of rejection by the immune system. The aim of the present study was to investigate a combination of microencapsulation and PEGylation methods in coating mouse embryonic stem cells (mESCs) to determine its effect in reducing the host’s immune response. Therefore, methoxy polyethylene glycol (mPEG) binding on alginate–trimethyl chitosan (TMC) microcapsules was investigated using FTIR. Furthermore, survival of the microencapsulated mESCs was confirmed using AO/PI staining and MTT assays. In addition, the effect of mESCs co-cultured with foreign lymphocytes was evaluated. Overall, interleukin-2 (IL-2) secretions as a response of the immune system revealed that mESCs microencapsulation in alginate–TMC–PEG, reduced the immune system response. The results suggested that IL-2 secretion was reduced to 62% at seventh day.

Sensitive Monitoring of Enterobacterial Contamination of Food Using Self-Propelled Janus Microsensors.

Food poisoning caused by bacteria is a major cause of disease and death worldwide. Herein we describe the use of Janus micromotors as mobile sensors for the detection of toxins released by enterobacteria as indicators of food contamination. The micromotors are prepared by a Pickering emulsion approach and rely on the simultaneous encapsulation of platinum nanoparticles for enhanced bubble-propulsion and receptor-functionalized quantum dots (QDs) for selective binding with the 3-deoxy-d-manno-oct-2-ulosonic acid target in the endotoxin molecule. Lipopolysaccharides (LPS) from Salmonella enterica were used as target endotoxins, which upon interaction with the QDs induce a rapid quenching of the native fluorescence of the micromotors in a concentration-dependent manner. The micromotor assay can readily detect concentrations as low as 0.07 ng mL-1 of endotoxin, which is far below the level considered toxic to humans (275 μg mL-1). Micromotors have been successfully applied for the detection of Salmonella toxin in food samples in 15 min compared with several hours required by the existing Gold Standard method. Such ultrafast and reliable approach holds considerable promise for food contamination screening while awaiting the results of bacterial cultures in a myriad of food safety and security defense applications.

PH-sensitive polymeric nanoparticles of mPEG-PLGA-PGlu with hybrid core for simultaneous encapsulation of curcumin and doxorubicin to kill the heterogeneous tumour cells in breast cancer

Most breast tumours are heterogeneous and not only contain the bulk of differentiated tumour cells but also a small population of highly tumorigenic and intrinsically drug-resistant cancer stem cells (CSCs). Herein, a pH-sensitive nanoparticle with simultaneous encapsulation of curcumin and doxorubicin (CURDOX-NPs) was prepared by using monomethoxy (polyethylene glycol)-b-P (D,L-lactic-co-glycolic acid)-b-P (L-glutamic acid) polymer to simultaneously target the differentiated tumor cells and CSCs. CURDOX-NPs had a mean diameter of 107.5 nm and zeta potential of −13.7 mV, determined by DLS. Drug-loading efficiency for curcumin and doxorubicin was reaching to 80.30% and 96.2%, respectively. Moreover, a cascade sustained-release profiles with the faster release of CUR followed by a slower release of DOX was observed in normal pH7.4 condition. Moreover, a pH-sensitive release profile for each cargo was seen in pH5.0 condition. The anti-tumour effect of CURDOX-NPs on CSCs-enriching MCF-7/ADR mammospheres was confirmed by in vitro. Moreover, a significant regression of tumour growth after treatment with CURDOX-NPs was also observed in Xenograft mice model. The percentage of CSCs in tumour significantly decreased from 39.9% in control group to 6.82% after treatment with CURDOX-NPs. The combinational delivery of CUR and DOX may a potentially useful therapeutic strategy for refractory breast cancer.

Cucurbit[7]uril-Directed Assembly of Colloidal Membrane and Stimuli-Responsive Microcapsules at the liquid-liquid Interface.

Colloidal microcapsules based on supramolecular architectures feature attractive properties and offer new opportunities in diverse areas such as delivery, sensing, and catalysis. Herein, we report a new strategy to fabricate the colloidal membrane and stimuli-responsive microcapsules by utilizing cucurbit[7]uril-mediated interfacial host-guest molecular recognition. In contrast to the traditionally used cross-linking approach, this method exploits the engineered interaction between a nanoparticle ligand and cucurbit[7]uril to tune the interfacial energy and stabilize the colloidal assembly at the interface. These capsules provide a versatile platform for simultaneous encapsulation of dual cargos. Additionally, the dynamic nature of the supramolecular interactions allows triggered release of the encapsulated cargos through the orthogonal presentation of a high affinity guest molecule.

Hydrogen bonded niosomes for encapsulation and release of hydrophilic and hydrophobic anti-diabetic drugs: An efficient system for oral anti-diabetic formulation

A niosomal formulation for simultaneous encapsulation and release of hydrophobic and hydrophilic anti-diabetic drugs is reported. The niosomes were prepared by thin film hydration method which involved self assembly of Tween 80 and cholesterol. Confocal laser scanning microscopy (CLSM) investigations revealed that nile red (the hydrophobic molecule) preferentially accumulated in the shell while FITC-dextran (the hydrophilic molecule) loaded in the aqueous core of the niosome. Fourier transform infra-red (FTIR) spectroscopy investigation of niosomes revealed hydrogen bond formation between tween 80 and cholesterol. The encapsulation efficiencies for glipizide and metformin hydrochloride (metformin HCl) were found to be 67.64% and 58.72%, respectively. The drug release experiments performed in buffers at different pH values (mimicking the blood plasma pH, cellular endosomal and gastric environments) revealed that the drug release followed a linear profile up to 8–10 h, slowed down, and lasted for 12–14 h. Thus, this formulation offers a promising drug delivery system for combinatorial sustained release of anti-diabetic drugs.

Plasmolysis-Inspired Nanoengineering of Functional Yolk-Shell Microspheres with Magnetic Core and Mesoporous Silica Shell.

Yolk-shell nanomaterials with a rattle-like structure have been considered ideal carriers and nanoreactors. Traditional methods to constructing yolk-shell nanostructures mainly rely on multistep sacrificial template strategy. In this study, a facile and effective plasmolysis-inspired nanoengineering strategy is developed to controllably fabricate yolk-shell magnetic mesoporous silica microspheres via the swelling-shrinkage of resorcinol-formaldehyde (RF) upon soaking in or removal of n-hexane. Using Fe3O4@RF microspheres as seeds, surfactant-silica mesostructured composite is deposited on the swelled seeds through the multicomponent interface coassembly, followed by solvent extraction to remove surfactant and simultaneously induce shrinkage of RF shell. The obtained yolk-shell microspheres (Fe3O4@RF@void@mSiO2) possess a high magnetization of 40.3 emu/g, high surface area (439 m2/g), radially aligned mesopores (5.4 nm) in the outer shell, tunable middle hollow space (472-638 nm in diameter), and a superparamagnetic core. This simple method allows a simultaneous encapsulation of Au nanoparticles into the hollow space during synthesis, and it leads to spherical Fe3O4@RF@void-Au@mSiO2 magnetic nanocatalysts, which show excellent catalysis efficiency for hydrogenation of 4-nitrophenol by NaBH4 with a high conversion rate (98%) and magnetic recycling stability.

Partition and stability of resveratrol in whey protein isolate oil-in-water emulsion: Impact of protein and calcium concentrations

Whey protein isolate (WPI) is often used in food emulsions and can also interact with resveratrol, a natural amphiphilic polyphenol, this interaction being improved by heat-denaturation. In this study, oil-in-water emulsions stabilised by heat-denatured WPI in the absence and presence of CaCl2 were characterised in terms of size, ζ-potential and protein partition. Partition and stability of resveratrol were also studied as a function of WPI and calcium concentrations. Size of WPI emulsions was dependent on the protein content at the oil–water interface. Partition of resveratrol and WPI was positively proportional at the oil–water interface and in the continuous phase. The stability of resveratrol increased as the concentration of WPI increased, but decreased when the concentration of calcium exceeded 0.20 mm. These data should be useful for simultaneous encapsulation of hydrophobic and amphiphilic bioactive components in a single emulsion and the protection of the inner oil by combination of antioxidant addition.

Cytocompatible polyion complex gel of poly(Pro-Hyp-Gly) for simultaneous rat bone marrow stromal cell encapsulation

Polyion complex (PIC) gel of poly(Pro-Hyp-Gly) was successfully fabricated by simply mixing polyanion and polycation derivatives of poly(Pro-Hyp-Gly), a collagen-like polypeptide. The polyanion, succinylated poly(Pro-Hyp-Gly), and the polycation, arginylated poly(Pro-Hyp-Gly), contain carboxy (pKa = 5.2) and guanidinium (pKa = 12.4) groups, respectively. Mixing the polyanion and the polycation at physiological pH (pH = 7.4) resulted in PIC gel. The hydrogel formation was optimum at an equimolar ratio of carboxy to guanidinium groups, suggesting that ionic interaction is the main determinant for the hydrogel formation. The hydrogel was successfully used for simultaneous rat bone marrow stromal cell encapsulation. The encapsulated cells survived and proliferated within the hydrogel. In addition, the cells exhibited different morphology in the hydrogel compared with cells cultured on a tissue culture dish as a two-dimensional (2D) control. At day one, a round morphology and homogeneous single cell distribution were observed in the hydrogel. In contrast, the cells spread and formed a fibroblast-like morphology on the 2D control. After three days, the cells in the hydrogel maintained their morphology and some of them formed multicellular aggregates, which is similar to cell morphology in an in vivo microenvironment. These results suggest that the PIC gel of poly(Pro-Hyp-Gly) can serve as a cytocompatible three-dimensional scaffold for stem cell encapsulation, supporting their viability, proliferation, and in vivo-like behavior.

Physicochemical stability of β-carotene and α-tocopherol enriched nanoemulsions: Influence of carrier oil, emulsifier and antioxidant

β-Carotene (precursor of vitamin A) and α-tocopherol (VE) are essential fat soluble vitamins (FSV) required for better body functioning and deficiencies of these vitamins are linked to many chronic diseases. Encapsulation of FSV into nanoemulsion-based delivery systems improves their aqueous solubility, oxidative stability and bioavailability. In this study, β-carotene (BC) and VE were co-encapsulated using flax seed oil (FSO) or medium chain triglycerides (MCT) as carrier oils and octenyl succinic anhydride modified starch (Purity Gum Ultra, PGU) or Tween-80 (TW) as emulsifiers. Nanoemulsions and bulk carrier oils containing bioactives were stored for 4 weeks to assess the effect of storage temperature on physicochemical properties. Oxidation behavior of bioactives was different in emulsions compared to bulk oil systems. The degradation of FSV was higher at elevated temperature, which was more profound for BC. VE worked as antioxidant for the protection of BC in MCT-based emulsions but it did not protect BC degradation in FSO-based emulsions. However, incorporation of eugenol (EU) improved the oxidative stability as well as retention of FSV in all emulsions. Nanoemulsions containing FSV and EU stabilized by PGU showed overall higher retention of BC (≈42%) and VE (≈90%) after 4 weeks of storage at 40°C as compared to TW. The higher stability of PGU was due to its ability to form a thicker protective layer around oil droplets. This research provides useful information regarding the simultaneous encapsulation of lipophilic bioactives for the development of functional foods and beverages.

Computational prediction of the endohedral metalloborofullerenes Ti n @B40 (n = 1, 2)

The recent discovery of the B40−/0 borospherenes (Zhai et al. in Nat Chem 6:727–731, 2014) has stimulated a large number of experimental/theoretical studies on the small all-boron fullerenes. At the same time, their capabilities to accommodate various metal species are predicted by calculations. However, the simultaneous encapsulation of two metal atoms has never been touched probably due to the limited cavities of these boron cages. In this work, systematic density functional theory calculations reveal that B40 tends to entrap one and even two titanium atoms. The formed Tin@B40 (n = 1, 2) complexes possess large binding energies and sizable HOMO–LUMO gap energies, and are highly robust at different temperatures (300–1000 K), suggesting their excellent stabilities. Interestingly, Ti2@B40 features two filled pentagonal (B@B5) motifs with protruding central atom, which are reminiscent of the B12 icosahedral unit in bulk boron. The analysis on the electronic properties and bonding patterns of Tin@B40 reveal that both of them have σ plus π double electron delocalization. Furthermore, various spectroscopies were simulated for the future experimental characterization.

Two-Step Assembling of Near-Infrared "OFF-ON" Fluorescent Nanohybrids for Synchronous Tumor Imaging and MicroRNA Modulation-Based Therapy.

Theranostic nanoparticles with combined imaging and therapy functions show great promise in cancer precision medicine. In this study, we constructed near-infrared (NIR) "OFF-ON" fluorescent nanohybrids (F-PNDs) for synchronous tumor imaging and microRNA (miRNA) modulation therapy against esophageal cancer. Nanodiamond clusters (NDs) were first functionalized for protamine sulfate immobilization (PNDs) on their surfaces via a noncovalent self-assembling approach and simultaneous encapsulation of NIR emitting fluorescence dye cyanine 5 (Cy-5) (F-PNDs). Tumor suppressor miRNA-203 (miR-203) was then adsorbed onto the surface of F-PNDs to form miR-203/F-PNDs via electrostatic interactions. The size, morphology, photophysical and stability properties of miR-203/F-PNDs were analyzed. We found that the NIR fluorescence of miR-203/F-PNDs could be activated to the "ON" state in intracellular environment while remaining in the "OFF" state in extracellular or blood environment. Furthermore, in vivo live imaging experiments showed that miR-203/F-PNDs could be predominantly accumulated in tumor tissues and image the tumor sites 24 h postintravenous injection. In addition, intravenous and intratumoral injection of miR-203/F-PNDs could efficiently inhibit tumor growth through down-regulation of the expressions of oncogenes Ran and Δp63. Our study indicated that miRNA/F-PNDs could serve as a promising theranostic platform for synchronous tumor imaging and miRNA-based modulation therapy against cancer.

Strengthening Gastric Cancer Therapy by Trastuzumab-Conjugated Nanoparticles with Simultaneous Encapsulation of Anti-MiR-21 and 5-Fluorouridine

Background/Aims: MicroRNA-21 is an oncogenic miR (oncomiR) frequently elevated in gastric cancer (GC). Overexpression of miR-21 decreases the sensitivity of GC cells to 5-fluorouridine (5-Fu) and trastuzumab, a humanized monoclonal antibody targeting human epidermal growth factor receptor 2 (HER2). Receptor-mediated endocytosis plays a crucial role in the delivery of biotherapeutics including anti-miRNA oligonucleotides (AMOs). This study is a continuation of earlier findings involving poly(ε-caprolactone) (PCL)-poly (ethylene glycol) (PEG) nanoparticles (PEG-PCL NPs), which were coated with trastuzumab to target GC with HER2 receptor over-expression using anti-miRNA-21 (AMO-21) and 5-Fu. Methods: HER-PEG-PCL NPs were prepared by one-step carbodiimide coupling using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAc) and Sulfo-NHS in aqueous phase. Covalent coupling of amino groups at the surface of PEG-PCL with the carboxyl groups of trastuzumab was analyzed by X-ray photoelectron spectroscopy (XPS). AMO-21/5-Fu NPs were formulated by a double-emulsion solvent evaporation technique. The cell line specificity, cellular uptake and AMO-21 delivery were investigated through the rhodamine-B-labeled 6-carboxyfluorescein (FAM)-AMO-21-PEG-PCL NPs coated with or without the antibody in both Her2-positive (NUGC4) and negative GC cells (SGC7901) visualized by fluorescence microscopy. The cytotoxicity of the HER-PEG-PCL NPs encapsulating AMO-21 was evaluated by MTT and apoptosis. Real-time reverse-transcription polymerase chain reaction (RT-PCR) was used to examine miR-21 and phosphatase and tensin homolog (PTEN) and Sprouty2 expression in GC cell lines. The antitumor effects of AMO-21/5-Fu NPs were compared with other groups in xenograft gastric cancer mice. Results: The antibody conjugates significantly enhanced the cellular uptake of NPs. The AMO-21/5-Fu NPs effectively suppressed the target miRNA expression in GC cells, which further up-regulated PTEN and Sprouty2. As a result, the sensitivity of HER2-expressing gastric cancer to trastuzumab and 5-Fu were enhanced both in vitro and in vivo. The approach enhanced the targeting by trastuzumab as well as antibody-dependent cellular cytotoxicity (ADCC) of immune effector cells Conclusions: Taken together, the results provide insight into the biological and clinical potential of targeted AMO-21 and 5-Fu co-delivery using modified trastuzumab for GC treatment.