Polyelectrolyte-coated liposomes microfluidically assembled in one-step for enhancing cell endocytosis and in-vivo immune responses.

To enhance the cellular uptake of liposomes, we prepared conventional liposomes with targeting molecules and surface-charged liposomes and evaluated their potential as nano-carriers and vaccine adjuvants by comparing their endocytosis efficiencies using immune cells. Surface-charged liposomes were synthesized via a one-step microfluidic method, which provided a novel, simple, fast, and highly reproducible method for preparing liposomes. Flow cytometry revealed that cationic polyelectrolyte-coated liposomes exhibited higher endocytosis efficiencies (of up to a factor of 100) in A774A.1 cells and JAWs II cells compared with uncoated liposomes or those coated with anionic polyelectrolytes. Positively charged liposomes exhibited some cytotoxicity at quaternary-chitosan coating concentrations higher than 6 mg/mL; however, significantly lower cytotoxicities (by a factor of almost ten) were obtained by protein mixing. Furthermore, BALB/c mice vaccinated with a mixture of Anthrax vaccine adsorbed (AVA) and quaternary chitosan-coated liposomes showed faster and stronger anti-PA IgG inductions compared to those vaccinated with AVA alone, with titers positively correlating with the amount of cationic liposome used. This finding clearly reveals that quaternary chitosan-coated liposomes act as both nano-carriers and vaccine adjuvants that significantly enhance in-vivo immune responses to vaccines with low immunogenicities.

On-chip fabrication of calcium carbonate nanoparticles loaded with various compounds using microfluidic approach.

Engineered calcium carbonate (CaCO3) particles are extensively used as drug delivery systems due to their availability, biological compatibility, biodegradability, and cost-effective production. The synthesis procedure of CaCO3 particles, however, suffers from poor reproducibility. Furthermore, reducing the size of CaCO3 particles to <100 nm requires the use of additives in the reaction, which increases the total reaction time. Here we propose on-chip synthesis and loading of nanoscaled CaCO3 particles using microfluidics. After the development and fabrication of a microfluidic device, we optimized the synthesis of CaCO3 NPs by varying different parameters such as flow rates in the microfluidic channels, concentration of reagents, and the reaction time. To prove the versatility of the used synthesis route, we performed single and double loading of CaCO3 NPs with various compounds (Doxorubicin, Cy5 or FITC conjugated with BSA, and DNA) using the same microfluidic device. Further, the on-chip loaded CaCO3 NPs were used as carriers to transfer compounds to model cells. We have developed a microfluidic synthesis method that opens up a new pathway for easy on-chip fabrication of functional nanoparticles for clinical use.

Synthesis of Submicron CaCO3 Particles in 3D-Printed Microfluidic Chips Supporting Advection and Diffusion Mixing.

Microfluidic technology provides a solution to the challenge of continuous CaCO3 particle synthesis. In this study, we utilized a 3D-printed microfluidic chip to synthesize CaCO3 micro- and nanoparticles in vaterite form. Our primary focus was on investigating a continuous one-phase synthesis method tailored for the crystallization of these particles. By employing a combination of confocal and scanning electron microscopy, along with Raman spectroscopy, we were able to thoroughly evaluate the synthesis efficiency. This evaluation included aspects such as particle size distribution, morphology, and polymorph composition. The results unveiled the existence of two distinct synthesis regimes within the 3D-printed microfluidic chips, which featured a channel cross-section of 2 mm2. In the first regime, which was characterized by chaotic advection, particles with an average diameter of around 2 µm were produced, thereby displaying a broad size distribution. Conversely, the second regime, marked by diffusion mixing, led to the synthesis of submicron particles (approximately 800-900 nm in diameter) and even nanosized particles (70-80 nm). This research significantly contributes valuable insights to both the understanding and optimization of microfluidic synthesis processes, particularly in achieving the controlled production of submicron and nanoscale particles.

Sucrose-Powered Liposome Nanosensors for Urinary Glucometer-Based Monitoring of Cancer.

Timely detection of early-stage cancer holds immense potential in enhancing prognostic outcomes. There is an increasing desire for versatile tools to enable simple, sensitive, and cost-effective cancer detection. By exploiting the extraintestinal metabolic inertness and efficiency renal clearance of sucrose, we designed a liposome nanosensor using sucrose as a messenger to convert tumor-specific esterase activity into glucose meter readout, enabling economical and sensitive urinalysis for cancer detection in point-of-care testing (POCT). Our results demonstrate that the nanosensors exhibited significant signal differences between tumor-bearing and healthy mice in both orthotopic and metastatic tumor models. Additionally, efficient elimination of the nanosensors through the hepatobiliary pathway was observed with no significant toxicity. Such a non-invasive diagnostic modality significantly assists in personalized pharmacological treatment and follow-up efficacy assessment. We envision that this modular liposome nanosensor platform might be applied for economically detecting diverse diseases via a simple urinary test.

Receptor mediated targeting of EGF-conjugated alginate-PAMAM nanoparticles to lung adenocarcinoma: 2D/3D in vitro and in vivo evaluation.

Carboplatin (cis-diamine (1,1-cyclobutandicarboxylaso)­platinum (II)) is a second-generation antineoplastic drug, which is widely used for chemotherapy of lung, colon, breast, cervix, testicular and digestive system cancers. Although preferred over cisplatin due to the lower incidence of nephrotoxicity and ototoxicity, efficient carboplatin delivery remains as a major challenge. In this study, carboplatin loaded alginate- poly(amidoamine) (PAMAM) hybrid nanoparticles (CAPs) with mean sizes of 192.13 ± 4.15 nm were synthesized using a microfluidic platform, then EGF was conjugated to the surface of CAPs (EGF-CAPs) for the receptor-targeted delivery. Hence, increased FITC+ cell counts were observed in A549 spheroids after EGF-CAP treatment compared to CAP in the 3D cellular uptake study. As such, the cytotoxicity of EGF-CAP was approximately 2-fold higher with an IC50 value of 35.89 ± 10.37 µg/mL compared to the CAPs in A549 spheroids. Based on in vivo experimental animal model, anti-tumor activities of the group treated with CAP decreased by 61 %, whereas the group treated with EGF-CAP completely recovered. Additionally, EGF-CAP application was shown to induce apoptotic cell death. Our study provided a new strategy for designing a hybrid nanoparticle for EGFR targeted carboplatin delivery with improved efficacy both in vitro and in vivo applications.

Kinetically Controlled Nucleation Enabled by Tunable Microfluidic Mixing for the Synthesis of Dendritic Au@Pt Core/Shell Nanomaterials.

The nucleation stage plays a decisive role in determining nanocrystal morphology and properties; hence, the ability to regulate nucleation is critical for achieving high-level control. Herein, glass microfluidic chips with S-shaped mixing units are designed for the synthesis of Au@Pt core/shell materials. The use of hydrodynamics to tune the nucleation kinetics is explored by varying the number of mixing units. Dendritic Au@Pt core/shell nanomaterials are controllably synthesized and a formation mechanism is proposed. As-synthesized Au@Pt exhibited excellent ethanol oxidation activity under alkaline conditions (8.4 times that of commercial Pt/C). This approach is also successfully applied to the synthesize of Au@Pd core/shell nanomaterials, thus demonstrating its generality.

Microfluidic Vaterite Synthesis: Approaching the Nanoscale Particles.

The challenge of continuous CaCO3 particle synthesis is addressed using microfluidic technology. A custom microfluidic chip was used to synthesize CaCO3 nanoparticles in vaterite form. Our focus revolved around exploring one-phase and two-phase synthesis methods tailored for the crystallization of these nanoparticles. The combination of scanning electron microscopy, X-ray diffraction, dynamic light scattering, and small-angle scattering allowed for an evaluation of the synthesis efficiency, including the particle size distribution, morphology, and polymorph composition. The results demonstrated the superior performance of the two-phase system when precipitation occurred inside emulsion microreactors, providing improved size control compared with the one-phase approach. We also discussed insights into particle size changes during the transition from one-phase to two-phase synthesis. The ability to obtain CaCO3 nanoparticles in the desired polymorph form (∼50 nm in size, 86-99% vaterite phase) with the possibility of scaling up the synthesis will open up opportunities for various industrial applications of the developed two-phase microfluidic method.

Microwave-Assisted Silanization of Magnetite Nanoparticles Pre-Synthesized by a 3D Microfluidic Platform.

Magnetite nanoparticles (Fe3O4 NPs) are among the most investigated nanomaterials, being recognized for their biocompatibility, versatility, and strong magnetic properties. Given that their applicability depends on their dimensions, crystal morphology, and surface chemistry, Fe3O4 NPs must be synthesized in a controlled, simple, and reproducible manner. Since conventional methods often lack tight control over reaction parameters and produce materials with unreliable characteristics, increased scientific interest has been directed to microfluidic techniques. In this context, the present paper describes the development of an innovative 3D microfluidic platform suitable for synthesizing uniform Fe3O4 NPs with fine-tuned properties. On-chip co-precipitation was performed, followed by microwave-assisted silanization. The obtained nanoparticles were characterized from the compositional and microstructural perspectives by X-ray diffraction (XRD) and transmission electron microscopy (TEM). Moreover, supplementary physicochemical investigations, such as Fourier Transform Infrared Spectroscopy (FT-IR), Kaiser Test, Ultraviolet-Visible (UV-Vis) Spectrophotometry, Dynamic Light Scattering (DLS), and Thermogravimetry and Differential Scanning Calorimetry (TG-DSC) analyses, demonstrated the successful surface modification. Considering the positive results, the presented synthesis and functionalization method represents a fast, reliable, and effective alternative for producing tailored magnetic nanoparticles.

Microfluidic one-step synthesis of a metal-organic framework for osteoarthritis therapeutic microRNAs delivery.

As a class of short non-coding ribonucleic acids (RNAs), microRNAs (miRNA) regulate gene expression in human cells and are expected to be nucleic acid drugs to regulate and treat a variety of biological processes and diseases. However, the issues with potential materials toxicity, quantity production, poor cellular uptake, and endosomal entrapment limit their further applications in clinical practice. Herein, ZIF-8, a metal-organic framework with noncytotoxic zinc (II) as the metal coordination center, was selected as miRNA delivery vector was used to prepare miR-200c-3p@ZIF-8 in one step by Y-shape microfluidic chip to achieve intracellular release with low toxicity, batch size, and efficient cellular uptake. The obtained miR-200c-3p@ZIF-8 was identified by TEM, particle size analysis, XRD, XPS, and zeta potential. Compared with the traditional hydrothermal method, the encapsulation efficiency of miR-200c-3p@ZIF-8 prepared by the microfluidic method is higher, and the particle size is more uniform and controllable. The experimental results in cellular level verified that the ZIF-8 vectors with low cytotoxicity and high miRNAs loading efficiency could significantly improve cellular uptake and endosomal escape of miRNAs, providing a robust and general strategy for nucleic acid drug delivery. As a model, the prepared miR-200c-3p@ZIF-8 is confirmed to be effective in osteoarthritis treatment.

Microfluidic synthesis of fibronectin-coated polydopamine nanocomplexes for self-supplementing tumor microenvironment regulation and MR imaging-guided chemo-chemodynamic-immune therapy.

Development of nanomedicines to overcome the hindrances of tumor microenvironment (TME) for tumor theranostics with alleviated side effects remains challenging. We report here a microfluidic synthesis of artesunate (ART)-loaded polydopamine (PDA)/iron (Fe) nanocomplexes (NCs) coated with fibronectin (FN). The created multifunctional Fe-PDA@ART/FN NCs (FDRF NCs) with a mean size of 161.0 â€‹nm exhibit desired colloidal stability, monodispersity, r1 relaxivity (4.96 â€‹mM-1s-1), and biocompatibility. The co-delivery of the Fe2+ and ART enables enhanced chemodynamic therapy (CDT) through improved intracellular reactive oxygen species generation via a cycling reaction between Fe3+ and Fe2+ caused by the Fe3+-mediated glutathione oxidation and Fe2+-mediated ART reduction/Fenton reaction for self-supplementing TME regulation. Likewise, the combination of ART-mediated chemotherapy and the Fe2+/ART-regulated enhanced CDT enables noticeable immunogenic cell death, which can be collaborated with antibody-mediated immune checkpoint blockade to exert immunotherapy having significant antitumor immunity. The combined therapy improves the efficacy of primary tumor therapy and tumor metastasis inhibition by virtue of FN-mediated specific targeting of FDRF NCs to tumors with highly expressed αvß3 integrin and can be guided through the Fe(III)-rendered magnetic resonance (MR) imaging. The developed FDRF NCs may be regarded as an advanced nanomedicine formulation for chemo-chemodynamic-immune therapy of different tumor types under MR imaging guidance.

Microfluidic-Generated Seeds for Gold Nanotriangle Synthesis in Three or Two Steps.

Nanoparticle synthesis has drawn great attention in the last decades. The study of crystal growth mechanisms and optimization of the existing methods lead to the increasing accessibility of nanomaterials, such as gold nanotriangles which have great potential in the fields of plasmonics and catalysis. To form such structures, a careful balance of reaction parameters has to be maintained. Herein, a novel synthesis of gold nanotriangles from seeds derived with a micromixer, which provides a highly efficient mixing and simple parameter control is reported. The impact of the implemented reactor on the primary seed characteristics is investigated. The following growth steps are studied to reveal the phenomena affecting the shape yield. The use of microfluidic seeds led to the formation of well-defined triangles with a narrower size distribution compared to the entirely conventional batch synthesis. A shortened two-step procedure for the formation of triangles directly from primary seeds, granting an express but robust synthesis is further described. Moreover, the need for a thorough study of seed crystallinity depending on the synthesis conditions, which - together with additional parameter optimization - will bring a new perspective to the use of micromixers which are promising for scaling up nanomaterial production is highlighted.

Spontaneous Phase Segregation Enabling Clogging Aversion in Continuous Flow Microfluidic Synthesis of Nanocrystals Supported on Reduced Graphene Oxide.

Eliminating clogging in capillary tube reactors is critical but challenging for enabling continuous-flow microfluidic synthesis of nanoparticles. Creating immiscible segments in a microfluidic flow is a promising approach to maintaining a continuous flow in the microfluidic channel because the segments with low surface energy do not adsorb onto the internal wall of the microchannel. Herein we report the spontaneous self-agglomeration of reduced graphene oxide (rGO) nanosheets in polyol flow, which arises because the reduction of graphene oxide (GO) nanosheets by hot polyol changes the nanosheets from hydrophilic to hydrophobic. The agglomerated rGO nanosheets form immiscible solid segments in the polyol flow, realizing the liquid-solid segmented flow to enable clogging aversion in continuous-flow microfluidic synthesis. Simultaneous reduction of precursor species in hot polyol deposits nanocrystals uniformly dispersed on the rGO nanosheets even without surfactant. Cuprous oxide (Cu2O) nanocubes of varying edge lengths and ultrafine metal nanoparticles of platinum (Pt) and palladium (Pd) dispersed on rGO nanosheets have been continuously synthesized using the liquid-solid segmented flow microfluidic method, shedding light on the promise of microfluidic reactors in synthesizing functional nanomaterials.

Microfluidic synthesis of Janus-structured QD-encoded magnetic microbeads for multiplex immunoassay.

Uniform and monodisperse quantum dot (QD)-encoded magnetic microbeads with Janus structure were produced in a microfluidic device via photopolymerization. UV light through a microscope objective was used to solidify the microbeads which showed sharp interfaces and excellent magnetic responses. QDs with different emission peaks (450 nm for blue and 640 nm for red) were mixed at different ratios to provide three spectral codes. The QD-encoded microbeads can be distinguished by analyzing their fluorescent images in HSV color space. After hydrolysis of the anhydride group in alkaline solution, protein was immobilized on microbeads via activation of carboxyl groups using (1-ethyl-3(3-dimethylaminoprophyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS). A microhole array in polydimethylsiloxane (PDMS) substrates with a specific size was fabricated to trap individual microbeads in a single microhole. The combination of Janus-structured QD-encoded magnetic microbeads and microhole arrays facilitates both flexibility, binding kinetics, sensitivity for suspension assay, and fluorescence mapping analysis for conventional biochips, thus providing a novel platform for multiplex bioanalysis. The capability of this integration for multiplex immunoassays was verified using three kinds of IgG and their corresponding anti-IgG. A detection limit of 0.07 ng/mL was achieved for human IgG, indicating practical applications in various fields.

Thiolated Chitosan Conjugated Liposomes for Oral Delivery of Selenium Nanoparticles.

This study aimed to design a hybrid oral liposomal delivery system for selenium nanoparticles (Lip-SeNPs) to improve the bioavailability of selenium. Thiolated chitosan, a multifunctional polymer with mucoadhesive properties, was used for surface functionalization of Lip-SeNPs. Selenium nanoparticle (SeNP)-loaded liposomes were manufactured by a single step microfluidics-assisted chemical reduction and assembling process. Subsequently, chitosan-N-acetylcysteine was covalently conjugated to the preformed Lip-SeNPs. The Lip-SeNPs were characterized in terms of composition, morphology, size, zeta potential, lipid organization, loading efficiency and radical scavenging activity. A co-culture system (Caco-2:HT29-MTX) that integrates mucus secreting and enterocyte-like cell types was used as a model of the human intestinal epithelium to determine adsorption, mucus penetration, release and transport properties of Lip-SeNPs in vitro. Thiolated Lip-SeNPs were positively charged with an average size of about 250 nm. Thiolated Lip-SeNPs tightly adhered to the mucus layer without penetrating the enterocytes. This finding was consistent with ex vivo adsorption studies using freshly excised porcine small intestinal tissues. Due to the improved mucoadhesion and retention in a simulated microenvironment of the small intestine, thiolated Lip-SeNPs might be a promising tool for oral selenium delivery.

A microfluidic approach for rapid and continuous synthesis of glycoprotein-imprinted nanospheres.

Many strategies have been reported for the preparation of glycoproteins imprinted polymers, but they take a long time and cannot produce imprinted polymers continuously. Herein, a microfluidic synthesis approach was developed to make glycoproteins imprinted nanospheres rapidly and continuously. By using ovalbumin as a model template and a synthesized phenylboronic acid-tagged silane reagent as the functional monomer, the synthetic conditions including the polymerization contents, the flow rate and the microfluidic reactor size were comprehensively studied. Under the optimized conditions, the glycoprotein imprinted nanospheres could be synthesized rapidly (<2 h), and exhibited high specificity with cross-reactivity factors of 1.3 (ovotransferrin), +∞ (horse-radish peroxidase), 5.1 (ß-lactoglobulin) and 101 (bovine serum albumin). The kinetic and equilibrium binding behaviors, reusability and potential applications of the glycoprotein imprinted nanosphere were investigated. Such microfluidic synthesis strategy can be easily extended to produce other target glycoproteins imprinted nanospheres, as well as non-glycoproteins by using suitable functional monomers.

Real-Time Monitoring of the In Situ Microfluidic Synthesis of Ag Nanoparticles on Solid Substrate for Reliable SERS Detection.

A sharpened control over the parameters affecting the synthesis of plasmonic nanostructures is often crucial for their application in biosensing, which, if based on surface-enhanced Raman spectroscopy (SERS), requires well-defined optical properties of the substrate. In this work, a method for the microfluidic synthesis of Ag nanoparticles (NPs) on porous silicon (pSi) was developed, focusing on achieving a fine control over the morphological characteristics and spatial distribution of the produced nanostructures to be used as SERS substrates. To this end, a pSi membrane was integrated in a microfluidic chamber in which the silver precursor solution was injected, allowing for the real-time monitoring of the reaction by UV-Vis spectroscopy. The synthesis parameters, such as the concentration of the silver precursor, the temperature, and the flow rate, were varied in order to study their effects on the final silver NPs' morphology. Variations in the flow rate affected the size distribution of the NPs, whereas both the temperature and the concentration of the silver precursor strongly influenced the rate of the reaction and the particle size. Consistently with the described trends, SERS tests using 4-MBA as a probe showed how the flow rate variation affected the SERS enhancement uniformity, and how the production of larger NPs, as a result of an increase in temperature or of the concentration of the Ag precursor, led to an increased SERS efficiency.

Microfluidic platform for synthesis and optimization of chitosan-coated magnetic nanoparticles in cisplatin delivery.

In this study, magnetic core/chitosan shell Nanoparticles (NPs) containing cisplatin were synthesized via cisplatin complexation with tripolyphosphate as the chitosan crosslinker using two different procedures: a conventional batch flow method and a microfluidic approach. An integrated microfluidic device composed of three stages was developed to provide precise and highly controllable mixing. The comparison of the results revealed that NPs synthesized in microchannels were monodisperse 104 ±â€¯14.59 nm (n = 3) in size with optimal morphological characteristics, whereas polydisperse 423 ±â€¯53.33 nm (n = 3) nanoparticles were obtained by the conventional method. Furthermore, cisplatin was loaded in NPs without becoming inactivated, and the microfluidic technique demonstrated higher encapsulation efficiency, controlled release, and consequently lower IC50 values during exposure to the A2780 cell line proving that microfluidic synthesized NPs were able to enter the cells and release the drug more efficiently. The developed microfluidic platform presents valuable features that could potentially provide the clinical translation of NPs in drug delivery.

Facile synthesis and functionalization of color-tunable Ln3+-doped KGdF4 nanoparticles on a microfluidic platform.

Hyaluronic acid (HA)-functionalized lanthanide-doped KGdF4 nanoparticles were synthesized through two steps on a microfluidic platform. This microfluidic synthesis method allows better control of experimental conditions with lower labor and energy input than traditional beaker synthesis methods for large-scale production of nanoparticles with higher uniformity. First, Ln3+-doped KGdF4 nanoparticles were ultrafast (in minutes) and continuously synthesized using a four-inlets microfluidic chip at room temperature. Then, HA is continuously functionalized on the surface of Ln3+-doped KGdF4 nanoparticles using a T-shape chip through electrostatic adsorption. The synthesized nanoparticles show good uniformity, high biocompatibility, targeted cellular uptake, photoluminescence (PL) and magnetic resonance (MR) properties. This work highlights the potential of microfluidic platform for the development of multifunctional nanoparticles in biomedicine.

Facile Actuation of Organic and Aqueous Droplets on Slippery Liquid-Infused Porous Surfaces for the Application of On-Chip Polymer Synthesis and Liquid-Liquid Extraction.

Digital microfluidics employs water-repellant surfaces to exquisitely manipulate droplets of water for chemical analysis. However, the actuation and manipulation of organic droplets is still relatively unexplored as it is significantly more difficult to synthesize organic-repellent surfaces compared to water-repellent surfaces. Here, we present the fabrication of slippery liquid-infused porous surfaces (SLIPS) based on a porous polymer monolithic approach. The synthesized SLIPS were able to repel organic liquids such as hexane and methanol with a contact angle of 42.1 ± 0.4° and 69.0 ± 1.8°, respectively, as well as water with a contact angle of 115.8 ± 0.8°. More importantly for digital microfluidic applications, the sliding angle of liquids tested was between 4° and 6°. As a result, droplets containing magnetically susceptible material could be facilely manipulated on the SLIPS surface. A systematic actuation study was carried out to explore how actuation parameters including speed, paramagnetic particle (PMP) concentrations, and droplet volume impacted the outcomes (droplet actuation, disengagement, and PMP extraction). Two different applications were used to demonstrate the utility of actuating organic droplets on SLIPS surfaces including on-chip liquid-liquid extractions of natural products (NPs) from marine bacteria and droplet-based polymer synthesis with different polymerization conditions. Both applications employ an aqueous droplet and organic droplet interface at which either phase transfer or a chemical reaction is carried out. Two NPs (prodigiosin from Pseudoalteromonas rubra and violacein from Pseudoalteromonas luteoviolacea) were extracted, from aqueous droplets containing the bacteria, into butanol droplets and characterized with matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). Nylon 6,6 was synthesized on-chip via magnetic actuation of organic droplets containing adipoyl chloride and hexamethylamine. Relative intensities of the characteristic polymer masses suggest that droplet-based microfluidic synthesis on slips can be used to probe reaction conditions. The compatibility of SLIPS with both aqueous and organic solutions opens up a wider number of droplet-based sample preparation protocols and chemical transformations.

Two-Photon Vertical-Flow Lithography for Microtube Synthesis.

Two-photon vertical-flow lithography is demonstrated for synthesis of complex-shaped polymeric microtubes with a high aspect ratio (>100:1). This unique microfluidic approach provides rigorous control over the morphology and surface topology to generate thin-walled (<1 µm) microtubes with a tunable diameter (1-400 µm) and pore size (1-20 µm). The interplay between fluid-flow control and two-photon lithography presents a generic high-resolution method that will substantially contribute toward the future development of biocompatible scaffolds, stents, needles, nerve guides, membranes, and beyond.