On-Demand One-Step Synthesis of Monodisperse Functional Polymeric Microspheres with Droplet Microfluidics

A simple and robust method for one-step synthesis of monodisperse functional polymeric microspheres was established by generation of reversed microemulsion droplets in aqueous phase inside microfluidic chips and controlled evaporation of the organic solvent. Using this method, water-soluble nanomaterials can be easily encapsulated into biodegradable Poly­(d,l-lactic-<i>co</i>-glycolic acid) (PLGA) to form functional microspheres. By controlling the flow rate of microemulsion phase, PLGA polymeric microspheres with narrow size distribution and diameters in the range of ∼50–100 μm were obtained. As a demonstration of the versatility of the approach, high-quality fluorescent CdTe:Zn²⁺ quantum dots (QDs) of various emission spectra, superparamagnetic Fe₃O₄ nanoparticles, and water-soluble carbon nanotubes (CNTs) were used to synthesize fluorescent PLGA@QDs, magnetic PLGA@Fe₃O₄, and PLGA@CNTs polymeric microspheres, respectively. In order to show specific applications, the PLGA@Fe₃O₄ were modified with polydopamine (PDA), and then the silver nanoparticles grew on the surfaces of the PLGA@Fe₃O₄@PDA polymeric microspheres by reducting the Ag⁺ to Ag⁰. The as-prepared PLGA@Fe₃O₄@PDA-Ag microspheres showed a highly efficient catalytic reduction of the 4-nitrophenol, a highly toxic substance. The monodisperse uniform functional PLGA polymeric microspheres can potentially be critically important for multiple biomedical applications

Graphene-Templated Synthesis of Magnetic Metal Organic Framework Nanocomposites for Selective Enrichment of Biomolecules

Successful control of homogeneous and complete coating of graphene or graphene-based composites with well-defined metal organic framework (MOF) layers is a great challenge. Herein, novel magnetic graphene MOF composites were constructed via a simple strategy for self-assembly of well-distributed, dense, and highly porous MOFs on both sides of graphene nanosheets. Graphene functionalized with magnetic nanoparticles and carboxylic groups on both sides was explored as the backbone and template to direct the controllable self-assembly of MOFs. The prepared composite materials have a relatively high specific surface area (345.4 m² g^–1), and their average pore size is measured to be 3.2 nm. Their relatively high saturation magnetization (23.8 emu g^–1) indicates their strong magnetism at room temperature. Moreover, the multifunctional composite was demonstrated to be a highly effective affinity material in selective extraction and separation of low-concentration biomolecules from biological samples, in virtue of the size-selection property of the unique porous structure and the excellent affinity of the composite materials. Besides providing a solution for the construction of well-defined functional graphene-based MOFs, this work could also contribute to selective extraction of biomolecules, in virtue of the universal affinity between immobilized metal ions and biomolecules

In Situ Caging of Biomolecules in Graphene Hybrids for Light Modulated Bioactivity

Remote and noninvasive modulation of protein activity is essential for applications in biotechnology and medicine. Optical control has emerged as the most attractive approach owing to its high spatial and temporal resolutions; however, it is challenging to engineer light responsive proteins. In this work, a near-infrared (NIR) light-responsive graphene-silica-trypsin (GST) nanoreactor is developed for modulating the bioactivity of trypsin molecules. Biomolecules are spatially confined and protected in the rationally designed compartment architecture, which not only reduces the possible interference but also boosts the bioreaction efficiency. Upon NIR irradiation, the photothermal effect of the GST nanoreactor enables the ultrafast <i>in situ</i> heating for remote activation and tuning of the bioactivity. We apply the GST nanoreactor for remote and ultrafast proteolysis of proteins, which remarkably enhances the proteolysis efficiency and reduces the bioreaction time from the overnight of using free trypsin to seconds. We envision that this work not only provides a promising tool of ultrafast and remotely controllable proteolysis for <i>in vivo</i> proteomics in study of tissue microenvironment and other biomedical applications but also paves the way for exploring smart artificial nanoreactors in biomolecular modulation to gain insight in dynamic biological transformation

Self-Assembly of Smart Multifunctional Hybrid Compartments with Programmable Bioactivity

Artificial microcompartments are highly desirable for understanding the mechanism of formation of primitive cells for the origin of life and have technological effects in broad fields such as materials science, catalysis, environmental remediation, biomedicine, and biotechnology. However, it remains a critical challenge for the construction of a structurally stable, semipermeable, and multifunctional compartment that can maintain a protective and confined internal space while allowing internalization of ingredients. Here, we present a strategy for construction of novel smart multifunctional hybrid compartments (SMHCs) with semipermeability, stimulus-response, and enzymatic bioactivity. The smart compartments were assembled by packing magnetic nanoparticles on oil/water interface, and the interstitial pores were gated by designed thermosensitive copolymer brushes. The materials characterization, multifunctionality, and on-demand permeability of prepared hybrid compartments were investigated. Notably, biological macromolecules can be easily encapsulated without sacrifice of the original bioactivity. We exploited the reversible permeability of these responsive inorganic–organic smart compartments, demonstrated temperature-triggered release of small molecules, and displayed SMHCs as a light programmable artificial microreactor

Profiling DNA Cargos in Single Extracellular Vesicles via Hydrogel-Based Droplet Digital Multiple Displacement Amplification

Due to the substantial heterogeneity among extracellular vesicle (EV) subpopulations, single-EV analysis has the potential to elucidate the mechanisms behind EV biogenesis and shed light on the myriad functions, leading to the development of novel diagnostics and therapeutics. While many studies have been devoted to reveal between-EV variations in surface proteins and RNAs, DNA cargos (EV-DNA) have received little attention. Here, we report a hydrogel-based droplet digital multiple displacement amplification approach for the comprehensive analysis of EV-DNA at the single-EV level. Single EVs are dispersed in thousands of hydrogel droplets and lysed for DNA amplification and identification. The droplet microfluidics strategy empowers the assay with single-molecule sensitivity and capability for absolute quantification of DNA-containing EVs. In particular, our findings indicate that 5–40% EVs are associated with DNA, depending on the cell of origin. Large EVs exhibit a higher proportion of DNA-containing EVs and a more substantial presence of intraluminal DNA, compared to small EVs. These DNA-containing EVs carry multiple DNA fragments on average. Furthermore, both double-stranded DNA and single-stranded DNA were able to be detected at the single-EV level. Utilizing this method, the abundance, distribution, and biophysical properties of EV-DNA in various EV populations are evaluated. The DNA level within EVs provides insight into the status of the originating cells and offers valuable information on the outcomes of anticancer treatments. The utilization of single-EV analysis for EV-DNA holds significant promise for early cancer detection and treatment response monitoring

Preoccupation of Empty Carriers Decreases Endo-/Lysosome Escape and Reduces the Protein Delivery Efficiency of Mesoporous Silica Nanoparticles

Endo-/lysosome escape is a major challenge in nanoparticle-based protein delivery for cancer therapy. To enhance the endo-/lysosomal escape and increase the efficacy of protein delivery, current strategies mainly focus on destroying endo-/lysosomes by employing modified nanoparticles, such as pH-sensitive polyplexes, cell-penetrating peptides, and photosensitive molecules. Herein, we hypothesize that pretreatment with empty nanocarriers might make endo-/lysosomes occupied and affect the endo/lysosomal escape of protein subsequently delivery by nanocarriers. We first treated breast carcinoma MDA-MB-231 cells with a high concentration of empty nanocarriers, mesoporous silica nanoparticles (MSN), to occupy the endo-/lysosome. After 2 h, we treated the cells with a lower concentration of fluorescein isothiocyanate-labeled MSN (MSN–FITC) and investigated the intracellular spatial and temporal distribution of MSN–FITC and their colocalization with endo-/lysosomes. We discovered the preoccupation of endo-/lysosomes by the empty nanocarriers did exist, mainly through changing the spatial distribution of the subsequently introduced nanocarriers. Furthermore, for the protein delivery, we observed reduced MSN–saporin delivery after endo-/lysosome preoccupation by MSN empty carriers. A similar result is observed for the delivery of cytochrome C by MSN but not for the small-molecule anticancer drug doxorubicin. The results show that the empty nanocarriers inhibit the endo-/lysosome intracellular trafficking process and decrease the endo-/lysosome escape of proteins subsequently delivered by the nanocarriers. This new discovered phenomenon of declined endo-/lysosome escape after endo-/lysosome preoccupation indicates that repeated treatment by nanomaterials with low protein-loading capacity may not yield a good cancer therapeutic effect. Therefore, it provides a new insightful perspective on the role of nanomaterial carriers in intracellular protein delivery

Self-Assembly of Extracellular Vesicle-like Metal–Organic Framework Nanoparticles for Protection and Intracellular Delivery of Biofunctional Proteins

The intracellular delivery of biofunctional enzymes or therapeutic proteins through systemic administration is of great importance in therapeutic intervention of various diseases. However, current strategies face substantial challenges owing to various biological barriers, including susceptibility to protein degradation and denaturation, poor cellular uptake, and low transduction efficiency into the cytosol. Here, we developed a biomimetic nanoparticle platform for systemic and intracellular delivery of proteins. Through a biocompatible strategy, guest proteins are caged in the matrix of metal–organic frameworks (MOFs) with high efficiency (up to ∼94%) and high loading content up to ∼50 times those achieved by surface conjunction, and the nanoparticles were further decorated with the extracellular vesicle (EV) membrane with an efficiency as high as ∼97%. <i>In vitro</i> and <i>in vivo</i> study manifests that the EV-like nanoparticles can not only protect proteins against protease digestion and evade the immune system clearance but also selectively target homotypic tumor sites and promote tumor cell uptake and autonomous release of the guest protein after internalization. Assisted by biomimetic nanoparticles, intracellular delivery of the bioactive therapeutic protein gelonin significantly inhibits the tumor growth <i>in vivo</i> and increased 14-fold the therapeutic efficacy. Together, our work not only proposes a new concept to construct a biomimetic nanoplatform but also provides a new solution for systemic and intracellular delivery of protein

Self-Assembly of Extracellular Vesicle-like Metal–Organic Framework Nanoparticles for Protection and Intracellular Delivery of Biofunctional Proteins

The intracellular delivery of biofunctional enzymes or therapeutic proteins through systemic administration is of great importance in therapeutic intervention of various diseases. However, current strategies face substantial challenges owing to various biological barriers, including susceptibility to protein degradation and denaturation, poor cellular uptake, and low transduction efficiency into the cytosol. Here, we developed a biomimetic nanoparticle platform for systemic and intracellular delivery of proteins. Through a biocompatible strategy, guest proteins are caged in the matrix of metal–organic frameworks (MOFs) with high efficiency (up to ∼94%) and high loading content up to ∼50 times those achieved by surface conjunction, and the nanoparticles were further decorated with the extracellular vesicle (EV) membrane with an efficiency as high as ∼97%. <i>In vitro</i> and <i>in vivo</i> study manifests that the EV-like nanoparticles can not only protect proteins against protease digestion and evade the immune system clearance but also selectively target homotypic tumor sites and promote tumor cell uptake and autonomous release of the guest protein after internalization. Assisted by biomimetic nanoparticles, intracellular delivery of the bioactive therapeutic protein gelonin significantly inhibits the tumor growth <i>in vivo</i> and increased 14-fold the therapeutic efficacy. Together, our work not only proposes a new concept to construct a biomimetic nanoplatform but also provides a new solution for systemic and intracellular delivery of protein