Study of the Synergistic Immunomodulatory and Antifibrotic Effects of Dual-Loaded Budesonide and Serpine1 siRNA Lipid-Polymer Nanoparticles Targeting Macrophage Dysregulation in Tendinopathy.

Musculoskeletal diseases involving tissue injury comprise tendon, ligament, and muscle injury. Recently, macrophages have been identified as key players in the tendon repair process, but no therapeutic strategy involving dual drug delivery and gene delivery to macrophages has been developed for targeting the two main dysregulated aspects of macrophages in tendinopathy, i.e., inflammation and fibrosis. Herein, the anti-inflammatory and antifibrotic effects of dual-loaded budesonide and serpine1 siRNA lipid-polymer hybrid nanoparticles (LPNs) are evaluated in murine and human macrophage cells. The modulation of the gene and protein expression of factors associated with inflammation and fibrosis in tendinopathy is demonstrated by real time polymerase chain reaction and Western blot. Macrophage polarization to the M2 phenotype and a decrease in the production of pro-inflammatory cytokines are confirmed in macrophage cell lines and primary cells. The increase in the activity of a matrix metalloproteinase involved in tissue remodelling is proven, and studies evaluating the interactions of LPNs with T cells proved that dual-loaded LPNs act specifically on macrophages and do not induce any collateral effects on T cells. Overall, these dual-loaded LPNs are a promising combinatorial therapeutic strategy with immunomodulatory and antifibrotic effects in dysregulated macrophages in the context of tendinopathy.

Rheological insights into 3D printing of drug products: Drug nanocrystal-poloxamer gels for semisolid extrusion.

The importance of ink rheology to the outcome of 3D printing is well recognized. However, rheological properties of printing inks containing drug nanocrystals have not been widely investigated. Therefore, the objective of this study was to establish a correlation between the composition of nanocrystal printing ink, the ink rheology, and the entire printing process. Indomethacin was used as a model poorly soluble drug to produce nanosuspensions with improved solubility properties through particle size reduction. The nanosuspensions were further developed into semisolid extrusion 3D printing inks with varying nanocrystal and poloxamer 407 concentrations. Nanocrystals were found to affect the rheological properties of the printing inks both by being less self-supporting and having higher yielding resistances. During printing, nozzle blockages occurred. Nevertheless, all inks were found to be printable. Finally, the rheological properties of the inks were successfully correlated with various printing and product properties. Overall, these experiments shed new light on the rheological properties of printing inks containing nanocrystals.

Mycophenolic Acid-loaded Naïve Macrophage-derived Extracellular Vesicles Rescue Cardiac Myoblast after Inflammatory Injury.

Exosomes are natural endogenous extracellular vesicles with phospholipid-based bilayer membrane structures. Due to their unique protein-decorated membrane properties, exosomes have been regarded as promising drug carriers to deliver small molecules and genes. A number of approaches have been developed for exosome-based drug loading. However, the drug loading capability of exosomes is inconsistent, and the effects of loading methods on the therapeutic efficacy have not been investigated in detail. Herein, we developed anti-inflammatory drug-loaded exosomes as an immunomodulatory nanoplatform. Naïve macrophage-derived exosomes (Mϕ-EVs) were loaded with the anti-inflammatory drug mycophenolic acid (MPA) by three major loading methods. Loading into exosomes significantly enhanced anti-inflammatory and antioxidation effects of MPA in vitro compared to free drugs. These findings provide a scientific basis for developing naïve macrophage-secreted exosomes as drug carriers for immunotherapy.

Fabrication of hydrogel microspheres via microfluidics using inverse electron demand Diels-Alder click chemistry-based tetrazine-norbornene for drug delivery and cell encapsulation applications.

Microfluidic on-chip production of polymeric hydrogel microspheres (MPs) can be designed for the loading of different biologically active cargos and living cells. Among different gelation strategies, ionically crosslinked microspheres generally show limited mechanical properties, meanwhile covalently crosslinked microspheres often require the use of crosslinking agents or initiators with limited biocompatibility. Inverse electron demand Diels Alder (iEDDA) click chemistry is a promising covalent crosslinking method with fast kinetics, high chemoselectivity, high efficiency and no cross-reactivity. Herein, in situ gellable iEDDA-crosslinked polymeric hydrogel microspheres are developed via water-in-oil emulsification (W/O) glass microfluidics. The microspheres are composed of two polyethylene glycol precursors modified with either tetrazine or norbornene as functional moieties. Using a single co-flow glass microfluidic platform, homogenous MPs of sizes 200-600 µm are developed and crosslinked within 2 minutes. The rheological properties of iEDDA crosslinked bulk hydrogels are maintained with a low swelling degree and a slow degradation behaviour under physiological conditions. Moreover, a high-protein loading capacity can be achieved, and the encapsulation of mammalian cells is possible. Overall, this work provides the possibility of developing microfluidics-produced iEDDA-crosslinked MPs as a potential drug vehicle and cell encapsulation system in the biomedical field.

In Vitro Study of the Anti-inflammatory and Antifibrotic Activity of Tannic Acid-Coated Curcumin-Loaded Nanoparticles in Human Tenocytes.

Tendinitis is a tendon disorder related to inflammation and pain, due to an injury or overuse of the tissue, which is hypocellular and hypovascular, leading to limited repair which occurs in a disorganized deposition of extracellular matrix that leads to scar formation and fibrosis, ultimately resulting in impaired tendon integrity. Current conventional treatments are limited and often ineffective, highlighting the need for new therapeutic strategies. In this work, acetalated-dextran nanoparticles (AcDEX NPs) loaded with curcumin and coated with tannic acid (TA) are developed to exploit the anti-inflammatory and anti-fibrotic properties of the two compounds. For this purpose, a microfluidic technique was used in order to obtain particles with a precise size distribution, aiming to decrease the batch-to-batch variability for possible future clinical translation. Coating with TA increased not only the stability of the nanosystem in different media but also enhanced the interaction and the cell-uptake in primary human tenocytes and KG-1 macrophages. The nanosystem exhibited good biocompatibility toward these cell types and a good release profile in an inflammatory environment. The efficacy was demonstrated by real-time quantitative polymerase chain reaction, in which the curcumin loaded in the particles showed good anti-inflammatory properties by decreasing the expression of NF-κb and TA-coated NPs showing anti-fibrotic effect, decreasing the gene expression of TGF-ß. Overall, due to the loading of curcumin and TA in the AcDEX NPs, and their synergistic activity, this nanosystem has promising properties for future application in tendinitis.

Rational design of a polysaccharide-based viral mimicry nanocomplex for potent gene silencing in inflammatory tissues.

Rational design and fabrication of small interfering RNA (siRNA) delivery system with simple production scheme, specific targeting capability, responsiveness to endogenous stimuli and potential multi-functionalities remains technically challenging. Herein, we screen and design a virus-mimicking polysaccharide nanocomplex that shows specific gene delivery capability in a selective subset of leukocytes. A virus-inspired poly (alkyl methacrylate-co-methacrylic acid) fragment was conjugated on barley ß-glucans (EEPG) to endow the nanocomplex with pH-dependent endosomal membrane destabilization capabilities, as confirmed both biologically and computationally. siRNA loaded EEPG nanocomplex is feasibly fabricated in a single-step manner, which exhibit efficient gene silencing efficacy towards Dectin-1+ monocytes/macrophages. The inherent targeting affinity and feasible gene silencing potency of EEPG nanocomplex are investigated in three independent murine inflammation models, including myocardial infarction, lung fibrosis and acute liver damage. Significant enhanced accumulation level of EEPG nanocomplex is observed in cardiac lesion site, indicating its exclusive targeting capability for ischemic heart diseases. As a proof of concept, siTGF-ß based gene therapy is confirmed in murine model with heart fibrosis. Overall, our findings suggest the designed EEPG nanocomplex is favorable for siRNA delivery, which might have translational potential as a versatile platform in inflammation-related diseases.

Controlled Interfacial Polymer Self-Assembly Coordinates Ultrahigh Drug Loading and Zero-Order Release in Particles Prepared under Continuous Flow.

Microparticles are successfully engineered through controlled interfacial self-assembly of polymers to harmonize ultrahigh drug loading with zero-order release of protein payloads. To address their poor miscibility with carrier materials, protein molecules are transformed into nanoparticles, whose surfaces are covered with polymer molecules. This polymer layer hinders the transfer of cargo nanoparticles from oil to water, achieving superior encapsulation efficiency (up to 99.9%). To control payload release, the polymer density at the oil-water interface is enhanced, forming a compact shell for microparticles. The resultant microparticles can harvest up to 49.9% mass fraction of proteins with zero-order release kinetics in vivo, enabling an efficient glycemic control in type 1 diabetes. Moreover, the precise control of engineering process offered through continuous flow results in high batch-to-batch reproducibility and, ultimately, excellent scale-up feasibility.

Microfluidic preparation and optimization of sorafenib-loaded poly(ethylene glycol-block-caprolactone) nanoparticles for cancer therapy applications.

The use of amphiphilic block copolymers to generate colloidal delivery systems for hydrophobic drugs has been the subject of extensive research, with several formulations reaching the clinical development stages. However, to generate particles of uniform size and morphology, with high encapsulation efficiency, yield and batch-to-batch reproducibility remains a challenge, and various microfluidic technologies have been explored to tackle these issues. Herein, we report the development and optimization of poly(ethylene glycol)-block-(ε-caprolactone) (PEG-b-PCL) nanoparticles for intravenous delivery of a model drug, sorafenib. We developed and optimized a glass capillary microfluidic nanoprecipitation process and studied systematically the effects of formulation and process parameters, including different purification techniques, on product quality and batch-to-batch variation. The optimized formulation delivered particles with a spherical morphology, small particle size (dH < 80 nm), uniform size distribution (PDI < 0.2), and high drug loading degree (16 %) at 54 % encapsulation efficiency. Furthermore, the stability and in vitro drug release were evaluated, showing that sorafenib was released from the NPs in a sustained manner over several days. Overall, the study demonstrates a microfluidic approach to produce sorafenib-loaded PEG-b-PCL NPs and provides important insight into the effects of nanoprecipitation parameters and downstream processing on product quality.

A pH-Responsive Cluster Metal-Organic Framework Nanoparticle for Enhanced Tumor Accumulation and Antitumor Effect.

As a result of the deficient tumor-specific antigens, potential off-target effect, and influence of protein corona, metal-organic framework nanoparticles have inadequate accumulation in tumor tissues, limiting their therapeutic effects. In this work, a pH-responsive linker (L) is prepared by covalently modifying oleylamine (OA) with 3-(bromomethyl)-4-methyl-2,5-furandione (MMfu) and poly(ethylene glycol) (PEG). Then, the L is embedded into a solid lipid nanoshell to coat apilimod (Ap)-loaded zeolitic imidazolate framework (Ap-ZIF) to form Ap-ZIF@SLN#L. Under the tumor microenvironment, the hydrophilic PEG and MMfu are removed, exposing the hydrophobic OA on Ap-ZIF@SLN#L, increasing their uptake in cancer cells and accumulation in the tumor. The ZIF@SLN#L nanoparticle induces reactive oxygen species (ROS). Ap released from Ap-ZIF@SLN#L significantly promotes intracellular ROS and lactate dehydrogenase generation. Ap-ZIF@SLN#L inhibits tumor growth, increases the survival rate in mice, activates the tumor microenvironment, and improves the infiltration of macrophages and T cells in the tumor, as demonstrated in two different tumor-bearing mice after injections with Ap-ZIF@SLN#TL. Furthermore, mice show normal tissue structure of the main organs and the normal serum level in alanine aminotransferase and aspartate aminotransferase after treatment with the nanoparticles. Overall, this pH-responsive targeting strategy improves nanoparticle accumulation in tumors with enhanced therapeutic effects.

Microfluidics Fabrication of Micrometer-Sized Hydrogels with Precisely Controlled Geometries for Biomedical Applications.

Micrometer-sized hydrogels are cross-linked three-dimensional network matrices with high-water contents and dimensions ranging from several to hundreds of micrometers. Due to their excellent biocompatibility and capability to mimic physiological microenvironments in vivo, micrometer-sized hydrogels have attracted much attention in the biomedical engineering field. Their biological properties and applications are primarily influenced by their chemical compositions and geometries. However, inhomogeneous morphologies and uncontrollable geometries limit traditional micrometer-sized hydrogels obtained by bulk mixing. In contrast, microfluidic technology holds great potential for the fabrication of micrometer-sized hydrogels since their geometries, sizes, structures, compositions, and physicochemical properties can be precisely manipulated on demand based on the excellent control over fluids. Therefore, micrometer-sized hydrogels fabricated by microfluidic technology have been applied in the biomedical field, including drug encapsulation, cell encapsulation, and tissue engineering. This review introduces micrometer-sized hydrogels with various geometries synthesized by different microfluidic devices, highlighting their advantages in various biomedical applications over those from traditional approaches. Overall, emerging microfluidic technologies enrich the geometries and morphologies of hydrogels and accelerate translation for industrial production and clinical applications.

High drug-loaded microspheres enabled by controlled in-droplet precipitation promote functional recovery after spinal cord injury.

Drug delivery systems with high content of drug can minimize excipients administration, reduce side effects, improve therapeutic efficacy and/or promote patient compliance. However, engineering such systems is extremely challenging, as their loading capacity is inherently limited by the compatibility between drug molecules and carrier materials. To mitigate the drug-carrier compatibility limitation towards therapeutics encapsulation, we developed a sequential solidification strategy. In this strategy, the precisely controlled diffusion of solvents from droplets ensures the fast in-droplet precipitation of drug molecules prior to the solidification of polymer materials. After polymer solidification, a mass of drug nanoparticles is embedded in the polymer matrix, forming a nano-in-micro structured microsphere. All the obtained microspheres exhibit long-term storage stability, controlled release of drug molecules, and most importantly, high mass fraction of therapeutics (21.8-63.1 wt%). Benefiting from their high drug loading degree, the nano-in-micro structured acetalated dextran microspheres deliver a high dose of methylprednisolone (400 µg) within the limited administration volume (10 µL) by one single intrathecal injection. The amount of acetalated dextran used was 1/433 of that of low drug-loaded microspheres. Moreover, the controlled release of methylprednisolone from high drug-loaded microspheres contributes to improved therapeutic efficacy and reduced side effects than low drug-loaded microspheres and free drug in spinal cord injury therapy.

Surface Adsorption-Mediated Ultrahigh Efficient Peptide Encapsulation with a Precise Ratiometric Control for Type 1 and 2 Diabetic Therapy.

A surface adsorption strategy is developed to enable the engineering of microcomposites featured with ultrahigh loading capacity and precise ratiometric control of co-encapsulated peptides. In this strategy, peptide molecules (insulin, exenatide, and bivalirudin) are formulated into nanoparticles and their surface is decorated with carrier polymers. This polymer layer blocks the phase transfer of peptide nanoparticles from oil to water and, consequently, realizes ultrahigh peptide loading degree (up to 78.9%). After surface decoration, all three nanoparticles are expected to exhibit the properties of adsorbed polymer materials, which enables the co-encapsulation of insulin, exenatide, and bivalirudin with a precise ratiometric control. After solidification of this adsorbed polymer layer, the release of peptides is synchronously prolonged. With the help of encapsulation, insulin achieves 8 days of glycemic control in type 1 diabetic rats with one single injection. The co-delivery of insulin and exenatide (1:1) efficiently controls the glycemic level in type 2 diabetic rats for 8 days. Weekly administration of insulin and exenatide co-encapsulated microcomposite effectively reduces the weight gain and glycosylated hemoglobin level in type 2 diabetic rats. The surface adsorption strategy sets a new paradigm to improve the pharmacokinetic and pharmacological performance of peptides, especially for the combination of peptides.

Quantitative Analysis of Porous Silicon Nanoparticles Functionalization by 1H NMR.

Porous silicon (PSi) nanoparticles have been applied in various fields, such as catalysis, imaging, and biomedical applications, because of their large specific surface area, easily modifiable surface chemistry, biocompatibility, and biodegradability. For biomedical applications, it is important to precisely control the surface modification of PSi-based materials and quantify the functionalization density, which determines the nanoparticle's behavior in the biological system. Therefore, we propose here an optimized solution to quantify the functionalization groups on PSi, based on the nuclear magnetic resonance (NMR) method by combining the hydrolysis with standard 1H NMR experiments. We optimized the hydrolysis conditions to degrade the PSi, providing mobility to the molecules for NMR detection. The NMR parameters were also optimized by relaxation delay and the number of scans to provide reliable NMR spectra. With an internal standard, we quantitatively analyzed the surficial amine groups and their sequential modification of polyethylene glycol. Our investigation provides a reliable, fast, and straightforward method in quantitative analysis of the surficial modification characterization of PSi requiring a small amount of sample.

Neonatal Fc receptor-targeted lignin-encapsulated porous silicon nanoparticles for enhanced cellular interactions and insulin permeation across the intestinal epithelium.

Oral insulin delivery could change the life of millions of diabetic patients as an effective, safe, easy-to-use, and affordable alternative to insulin injections, known by an inherently thwarted patient compliance. Here, we designed a multistage nanoparticle (NP) system capable of circumventing the biological barriers that lead to poor drug absorption and bioavailability after oral administration. The nanosystem consists of an insulin-loaded porous silicon NP encapsulated into a pH-responsive lignin matrix, and surface-functionalized with the Fc fragment of immunoglobulin G, which acts as a targeting ligand for the neonatal Fc receptor (FcRn). The developed NPs presented small size (211 ± 1 nm) and narrow size distribution. The NPs remained intact in stomach and intestinal pH conditions, releasing the drug exclusively at pH 7.4, which mimics blood circulation. This formulation showed to be highly cytocompatible, and surface plasmon resonance studies demonstrated that FcRn-targeted NPs present higher capacity to interact and being internalized by the Caco-2 cells, which express FcRn, as demonstrated by Western blot. Ultimately, in vitro permeability studies showed that Fc-functionalized NPs induced an increase in the amount of insulin that permeated across a Caco-2/HT29-MTX co-culture model, showing apparent permeability coefficients (P app ) of 2.37 × 10-6 cm/s, over the 1.66 × 10-6 cm/s observed for their non-functionalized counterparts. Overall, these results demonstrate the potential of these NPs for oral delivery of anti-diabetic drugs.

Multifunctional Biomimetic Nanovaccines Based on Photothermal and Weak-Immunostimulatory Nanoparticulate Cores for the Immunotherapy of Solid Tumors.

An alternative strategy of choosing photothermal and weak-immunostimulatory porous silicon@Au nanocomposites as particulate cores to prepare a biomimetic nanovaccine is reported to improve its biosafety and immunotherapeutic efficacy for solid tumors. A quantitative analysis method is used to calculate the loading amount of cancer cell membranes onto porous silicon@Au nanocomposites. Assisted with foreign-body responses, these exogenous nanoparticulate cores with weak immunostimulatory effect can still efficiently deliver cancer cell membranes into dendritic cells to activate them and the downstream antitumor immunity, resulting in no occurrence of solid tumors and the survival of all immunized mice during 55 day observation. In addition, this nanovaccine, as a photothermal therapeutic agent, synergized with additional immunotherapies can significantly inhibit the growth and metastasis of established solid tumors, via the initiation of the antitumor immune responses in the body and the reversion of their immunosuppressive microenvironments. Considering the versatile surface engineering of porous silicon nanoparticles, the strategy developed here is beneficial to construct multifunctional nanovaccines with better biosafety and more diagnosis or therapeutic modalities against the occurrence, recurrence, or metastasis of solid tumors in future clinical practice.

Inhibiting Phase Transfer of Protein Nanoparticles by Surface Camouflage-A Versatile and Efficient Protein Encapsulation Strategy.

Engineering a system with a high mass fraction of active ingredients, especially water-soluble proteins, is still an ongoing challenge. In this work, we developed a versatile surface camouflage strategy that can engineer systems with an ultrahigh mass fraction of proteins. By formulating protein molecules into nanoparticles, the demand of molecular modification was transformed into a surface camouflage of protein nanoparticles. Thanks to electrostatic attractions and van der Waals interactions, we camouflaged the surface of protein nanoparticles through the adsorption of carrier materials. The adsorption of carrier materials successfully inhibited the phase transfer of insulin, albumin, ß-lactoglobulin, and ovalbumin nanoparticles. As a result, the obtained microcomposites featured with a record of protein encapsulation efficiencies near 100% and a record of protein mass fraction of 77%. After the encapsulation in microcomposites, the insulin revealed a hypoglycemic effect for at least 14 d with one single injection, while that of insulin solution was only ∼4 h.

Prospective Cancer Therapies Using Stimuli-Responsive DNA Nanostructures.

Nanostructures based on DNA self-assembly present an innovative way to address the increasing need for target-specific delivery of therapeutic molecules. Currently, most of the chemotherapeutics being used in clinical practice have undesired and exceedingly high off-target toxicity. This is a challenge in particular for small molecules, and hence, developing robust and effective methods to lower these side effects and enhance the antitumor activity is of paramount importance. Prospectively, these issues could be tackled with the help of DNA nanotechnology, which provides a route for the fabrication of custom, biocompatible, and multimodal structures, which can, to some extent, resist nuclease degradation and survive in the cellular environment. Similar to widely employed liposomal products, the DNA nanostructures (DNs) are loaded with selected drugs, and then by employing a specific stimulus, the payload can be released at its target region. This review explores several strategies and triggers to achieve targeted delivery of DNs. Notably, different modalities are explained through which DNs can interact with their respective targets as well as how structural changes triggered by external stimuli can be used to achieve the display or release of the cargo. Furthermore, the prospects and challenges of this technology are highlighted.

Multistage signal-interactive nanoparticles improve tumor targeting through efficient nanoparticle-cell communications.

Communication between biological components is critical for homeostasis maintenance among the convergence of complicated bio-signals. For therapeutic nanoparticles (NPs), the general lack of effective communication mechanisms with the external cellular environment causes loss of homeostasis, resulting in deprived autonomy, severe macrophage-mediated clearance, and limited tumor accumulation. Here, we develop a multistage signal-interactive system on porous silicon particles through integrating the Self-peptide and Tyr-Ile-Gly-Ser-Arg (YIGSR) peptide into a hierarchical chimeric signaling interface with "don't eat me" and "eat me" signals. This biochemical transceiver can act as both the signal receiver for amantadine to achieve NP transformation and signal conversion as well as the signal source to present different signals sequentially by reversible self-mimicking. Compared with the non-interactive controls, these signal-interactive NPs loaded with AS1411 and tanespimycin (17-AAG) as anticancer drugs improve tumor targeting 2.8-fold and tumor suppression 6.5-fold and showed only 51% accumulation in the liver with restricted hepatic injury.

Non-viral nanoparticles for RNA interference: Principles of design and practical guidelines.

Ribonucleic acid interference (RNAi) is an innovative treatment strategy for a myriad of indications. Non-viral synthetic nanoparticles (NPs) have drawn extensive attention as vectors for RNAi due to their potential advantages, including improved safety, high delivery efficiency and economic feasibility. However, the complex natural process of RNAi and the susceptible nature of oligonucleotides render the NPs subject to particular design principles and requirements for practical fabrication. Here, we summarize the requirements and obstacles for fabricating non-viral nano-vectors for efficient RNAi. To address the delivery challenges, we discuss practical guidelines for materials selection and NP synthesis in order to maximize RNA encapsulation efficiency and protection against degradation, and to facilitate the cytosolic release of oligonucleotides. The current status of clinical translation of RNAi-based therapies and further perspectives for reducing the potential side effects are also reviewed.

A Hydrogen-Bonded Extracellular Matrix-Mimicking Bactericidal Hydrogel with Radical Scavenging and Hemostatic Function for pH-Responsive Wound Healing Acceleration.

Generation of reactive oxygen species, delayed blood clotting, prolonged inflammation, bacterial infection, and slow cell proliferation are the main challenges of effective wound repair. Herein, a multifunctional extracellular matrix-mimicking hydrogel is fabricated through abundant hydrogen bonding among the functional groups of gelatin and tannic acid (TA) as a green chemistry approach. The hydrogel shows adjustable physicochemical properties by altering the concentration of TA and it represents high safety features both in vitro and in vivo on fibroblasts, red blood cells, and mice organs. In addition to the merit of facile encapsulation of cell proliferation-inducing hydrophilic drugs, accelerated healing of skin injury is obtained through pH-dependent release of TA and its multifaceted mechanisms as an antibacterial, antioxidant, hemostatic, and anti-inflammatory moiety. The developed gelatin-TA (GelTA) hydrogel also shows an outstanding effect on the formation of extracellular matrix and wound closure in vivo via offered cell adhesion sites in the backbone of gelatin that provide increased re-epithelialization and better collagen deposition. These results suggest that the multifunctional GelTA hydrogel is a promising candidate for the clinical treatment of full-thickness wounds and further development of wound dressing materials that releases active agents in the neutral or slightly basic environment of infected nonhealing wounds.