Targeted Dual Small Interfering Ribonucleic Acid Delivery via Non-Viral Polymeric Vectors for Pulmonary Fibrosis Therapy.

Inhibiting the myofibroblast differentiation of lung-resident mesenchymal stem cells (LR-MSCs) is a promising yet challenging approach for pulmonary fibrosis (PF) therapy. Here, micelles formed by a graft copolymer of multiple PEGs modified branched polyethylenimine are used for delivering runt-related transcription factor-1 (RUNX1) small interfering RNA (siRNA) (siRUNX1) to the lung, aiming to inhibit the myofibroblast differentiation of LR-MSCs. LR-MSC targeting is achieved by functionalizing the micelle surface with an anti-stem-cell antigen-1 antibody fragment (Fab'). Consequently, therapeutic benefits are obtained by successful suppression of myofibroblast differentiation of LR-MSCs in bleomycin-induced PF model mice treated with siRUNX1-loaded micelles. Furthermore, an excellent synergistic effect of PF therapy is achieved for this micelle system loaded siRUNX1 and glioma-associated oncogene homolog-1 (Gli1) small interfering RNA (siGli1), a traditional anti-PF siRNA of glioma-associated oncogene homolog-1. Hence, this work not only provides RUNX1 as a novel PF therapeutic target, but also as a promising dual siRNA-loaded nanocarrier system for the therapy of PF.

Co-delivery of siPTPN13 and siNOX4 via (myo)fibroblast-targeting polymeric micelles for idiopathic pulmonary fibrosis therapy.

Rationale: (Myo)fibroblasts are the ultimate effector cells responsible for the production of collagen within alveolar structures, a core phenomenon in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Although (myo)fibroblast-targeted therapy holds great promise for suppressing the progression of IPF, its development is hindered by the limited drug delivery efficacy to (myo)fibroblasts and the vicious circle of (myo)fibroblast activation and evasion of apoptosis. Methods: Here, a dual small interfering RNA (siRNA)-loaded delivery system of polymeric micelles is developed to suppress the development of pulmonary fibrosis via a two-arm mechanism. The micelles are endowed with (myo)fibroblast-targeting ability by modifying the Fab' fragment of the anti-platelet-derived growth factor receptor-α (PDGFRα) antibody onto their surface. Two different sequences of siRNA targeting protein tyrosine phosphatase-N13 (PTPN13, a promoter of the resistance of (myo)fibroblasts to Fas-induced apoptosis) and NADPH oxidase-4 (NOX4, a key regulator for (myo)fibroblast differentiation and activation) are loaded into micelles to inhibit the formation of fibroblastic foci. Results: We demonstrate that Fab'-conjugated dual siRNA-micelles exhibit higher affinity to (myo)fibroblasts in fibrotic lung tissue. This Fab'-conjugated dual siRNA-micelle can achieve remarkable antifibrotic effects on the formation of fibroblastic foci by, on the one hand, suppressing (myo)fibroblast activation via siRNA-induced knockdown of NOX4 and, on the other hand, sensitizing (myo)fibroblasts to Fas-induced apoptosis by siRNA-mediated PTPN13 silencing. In addition, this (myo)fibroblast-targeting siRNA-loaded micelle did not induce significant damage to major organs, and no histopathological abnormities were observed in murine models. Conclusion: The (myo)fibroblast-targeting dual siRNA-loaded micelles offer a potential strategy with promising prospects in molecular-targeted fibrosis therapy.

Producing protein-nanoparticle co-assembly supraparticles by the interfacial instability process.

Originally discovered in fundamental research of nanomaterial-biomolecule interactions, protein-nanoparticle co-assembly supraparticles (PNCAS) have become an emerging class of nanomaterials with various biological applications. We apply the interfacial instability process, which was originally reported for forming nanoparticles-encapsulated polymeric micelles, to produce PNCAS. By doing so hydrophobic nanoparticles, which are often the product formed from the upstream nanoparticle synthesis step, can be directly used as the raw materials of the production process of PNCAS. On the other hand, we take advantage of the structural features of protein molecules, in comparison with amphiphilic block copolymers, to mitigate two common problems encountered in the original interfacial instability-mediated nanoparticle encapsulation process, namely (1) poor encapsulation number control and (2) inconvenience and high cost to vary the assembly size. Additionally, we achieve semi-continuous and scalable production of PNCAS by combining the electrospray process and the interfacial instability process. We also conduct proof-of-concept studies of biological applications of the PNCAS products.

Intracellular targeted delivery of quantum dots with extraordinary performance enabled by a novel nanomaterial design.

Quantum dots (QDs) have emerged as a major class of fluorescent probes with unique optical properties, but applying QDs for imaging specific intracellular entities in live cells has been hindered by the poor performance of targeted intracellular delivery of QDs due to various cellular transport barriers. We describe a novel QD nanoprobe design, which is termed a cosolvent-bare hydrophobic QD-biomolecule (cS-bQD-BM, or 'SDot' for short), combining a cosolvent, a bare hydrophobic nanoparticle surface, ultrasmall size and biomolecular function. SDots show extraordinary intracellular targeting performance with the nucleus as the model target, including near-perfect specificity, excellent efficiency and reproducibility, high-throughput ability, minimal toxicity, and ease of operation, as well as superb optical properties and colloidal stability. We introduce integrated single-particle tracking and pair-correlation function analysis of a spinning-disk confocal microscope platform (iSPT-pCF-SDCM) to study SDot's cellular transport. Endocytosed SDots can undergo a highly potent and noninvasive process of vesicle escape, yielding complete vesicle escape with no serious vesicle disruption. We exploit SDots' unprecedented ability to overcome cellular transport barriers to enhance drug and macromolecule delivery.

Direct and Noninvasive Penetration of Bare Hydrophobic Quantum Dots through Live Cell Membranes.

Semiconductor quantum dots (QDs) possess outstanding optical properties as fluorescent probes, but their applications in live cell intracellular imaging are hindered by various cellular transport barriers. Inspired by membrane proteins inserting their nanometer-scale hydrophobic surface into biomembranes, the present work aims to investigate the possibility that bare hydrophobic QDs could penetrate through live cell membranes without disrupting the membrane integrity. We utilize live cell spinning disk confocal microscopy to image and track the cellular transport process of bare hydrophobic QDs in the presence of a small percentage of three different organic cosolvents, namely, tetrahydrofuran (THF), chloroform, and hexane. A major finding is that, under certain cosolvent conditions, bare hydrophobic QDs can indeed penetrate through biomembranes in a noninvasive manner. Results of this work offer us guidance to design a new class of nanobioprobes based on combining hydrophobic nanoscale surface and cosolvent, and they provide key new pieces to the emerging complex and sophisticated picture of nanostructure-biosystem interactions.

A potent, minimally invasive and simple strategy of enhancing intracellular targeted delivery of Tat peptide-conjugated quantum dots: organic solvent-based permeation enhancer.

Targeted delivery of nanomaterials to specific intracellular locations is essential for the development of many nanomaterials-based biological applications. Thus far the targeting performance has been limited due to various intracellular transport barriers, especially intracellular vesicle trapping. Here we report the application of permeation enhancers based on organic solvents in small percentage to enhance the intracellular targeted delivery of nanomaterials. Previously permeation enhancers based on organic solvents and ionic liquids have been used in overcoming biological transport barriers at tissue, organ, and cellular levels, but this strategy has so far rarely been examined for its potential in facilitating transport of nanometer-scale entities across intracellular barriers, particularly intracellular vesicle trapping. Using the cell nucleus as a model intracellular target and Tat peptide-conjugated quantum dots (QDs-Tat) as a model nanomaterial-based probe, we demonstrate that a small percentage (e.g. 1%) of organic solvent greatly enhances nucleus targeting specificity as well as increasing endocytosis-based cellular uptake of QDs. We combine vesicle colocalization (DiO dye staining), vesicle integrity (calcein dye release), and single-particle studies (pair-correlation function microscopy) to investigate the process of organic solvent-enhanced vesicle escape of QDs-Tat. The organic solvent based vesicle escape-enhancing approach is found to be not only very effective but minimally invasive, resulting in high vesicle escape efficiency with no significant disruption to the membrane integrity of either intracellular vesicles or cells. This approach drastically outperforms the commonly used vesicle escape-enhancing agent (i.e., chloroquine, whose enhancement effect is based on disrupting vesicle integrity) in both potency and minimal invasiveness. Finally, we apply organic solvent-based targeting enhancement to improve the intracellular delivery of the anticancer drug doxorubicin (DOX).

Fabrication and in vitro biocompatibility of biomorphic PLGA/nHA composite scaffolds for bone tissue engineering.

In this study, biomorphic poly(dl-lactic-co-glycolic acid)/nano-hydroxyapatite (PLGA/nHA) composite scaffolds were successfully prepared using cane as a template. The porous morphology, phase, compression characteristics and in vitro biocompatibility of the PLGA/nHA composite scaffolds and biomorphic PLGA scaffolds as control were investigated. The results showed that the biomorphic scaffolds preserved the original honeycomb-like architecture of cane and exhibited a bimodal porous structure. The average channel diameter and micropore size of the PLGA/nHA composite scaffolds were 164 ± 52 µm and 13 ± 8 µm, respectively, with a porosity of 89.3 ± 1.4%. The incorporation of nHA into PLGA decreased the degree of crystallinity of PLGA, and significantly improved the compressive modulus of biomorphic scaffolds. The in vitro biocompatibility evaluation with MC3T3-E1 cells demonstrated that the biomorphic PLGA/nHA composite scaffolds could better support cell attachment, proliferation and differentiation than the biomorphic PLGA scaffolds. The localization depth of MC3T3-E1 cells within the channels of the biomorphic PLGA/nHA composite scaffolds could reach approximately 400 µm. The results suggested that the biomorphic PLGA/nHA composite scaffolds were promising candidates for bone tissue engineering.

Preparation and characterization of bimodal porous poly(γ-benzyl-L-glutamate) scaffolds for bone tissue engineering.

An ideal scaffold in bone tissue-engineering strategy should provide biomimetic extracellular matrix-like architecture and biological properties. Poly(γ-benzyl-L-glutamate) (PBLG) has been a popular model polypeptide for various potential biomedical applications due to its good biocompatibility and biodegradability. This study developed novel bimodal porous PBLG polypeptide scaffolds via a combination of biotemplating method and in situ ring-opening polymerization of γ-benzyl-L-gIutamate N-carboxyanhydride (BLG-NCA). The PBLG scaffolds were characterized by proton nuclear magnetic resonance spectroscopy, X-ray diffraction, differential scanning calorimetry, scanning electron microscope (SEM) and mechanical test. The results showed that the semi-crystalline PBLG scaffolds exhibited an anisotropic porous structure composed of honeycomb-like channels (100-200 µm in diameter) and micropores (5-20 µm), with a very high porosity of 97.4±1.6%. The compressive modulus and glass transition temperature were 402.8±20.6 kPa and 20.2°C, respectively. The in vitro biocompatibility evaluation with MC3T3-E1 cells using SEM, fluorescent staining and MTT assay revealed that the PBLG scaffolds had good biocompatibility and favored cell attachment, spread and proliferation. Therefore, the bimodal porous polypeptide scaffolds are promising for bone tissue engineering.

Reduction/pH dual-sensitive PEGylated hyaluronan nanoparticles for targeted doxorubicin delivery.

To minimize the side effect of chemotherapy, a novel reduction/pH dual-sensitive drug nanocarrier, based on PEGylated dithiodipropionate dihydrazide (TPH)-modified hyaluronic acid (PEG-SS-HA copolymer), was developed for targeted delivery of doxorubicin (DOX) to hepatocellular carcinoma. The copolymer was synthesized by reductive amination via Schiff's base formation between TPH-modified HA and galactosamine-conjugated poly(ethylene glycol) aldehyde/methoxy poly(ethylene glycol) aldehyde. Conjugation of DOX to PEG-SS-HA copolymer was accomplished through the hydrazone linkage formed between DOX and PEG-SS-HA, and confirmed by FTIR and (1)H NMR spectra. The polymer-DOX conjugate could self-assemble into spherical nanoparticles (~150 nm), as indicated by TEM and DLS. In vitro release studies showed that the DOX-loaded nanoparticles could release DOX rapidly under the intracellular levels of pH and glutathiose. Cellular uptake experiments demonstrated that the nanoparticles could be efficiently internalized by HepG2 cells. These results indicate that the PEG-SS-HA copolymer holds great potential for targeted intracellular delivery of DOX.