Slgirim Lee

Electrospun nanofibrous scaffolds for controlled release of adeno-associated viral vectors

The integration of viral gene delivery with key features of biomaterial scaffolds that modulate viral delivery in a controlled manner offers a promising strategy for numerous tissue engineering applications. In this study adeno-associated virus (AAV), which is widely utilized in human gene therapy as a gene carrier due to its safety and efficient gene delivery capability, was encapsulated within electrospun nanofibrous scaffolds composed of blended mixtures of elastin-like polypeptides (ELP) and poly (ε-caprolactone) (PCL) and was employed to transduce fibroblasts adherent on the scaffolds. Combinatorial interactions between ELP and PCL chains upon physical blending significantly altered the mechanical properties (i.e. wettability, elastic modulus, strain, etc.) of the ELP/PCL composites, thus providing key tools to mediate controlled release of AAV vectors and robust cellular transduction on the fibrous scaffolds. The ability of ELP/PCL composites to manipulate the controlled release of AAV-mediated gene delivery for subsequent high-efficiency cellular transduction will provide tremendous opportunities for a variety of tissue engineering applications.

Drawing Sticky Adeno‐Associated Viruses on Surfaces for Spatially Patterned Gene Expression

In developing tissues, the spatially controlled secretion of extracellular signals creates biointerfaces that modulate cellular processes of differentiation, proliferation, and migration, and provide molecular cues to organize the structure of tissues. Systems that control spatial distribution of extracellular molecules on substrates have been developed in order to induce patterned expression of intracellular inductive factors. 5,6] Alternatively, spatially patterned gene delivery has been employed to overcome limitations found in protein delivery: short protein half-life and systemic toxicity. The development of in vitro model systems that mimic the spatial control of gene expression in tissues or organs is critical to elucidate a variety of biological mechanisms. However, the majority of gene delivery systems rely on simple additions of gene carriers directly to media, which are inherently limited in their ability to spatially control gene expression. Advances in microand nanofabrication technologies have enabled researchers to control locations of gene vectors on surfaces. 11] Existing methods include microfluidics, surface coating, and self-assembly. In general, the techniques involve multiple laborious procedures, typically chemical activation of substrates, resist depositions, pattern generation using a photomask, and gene vector immobilization. Furthermore, expensive equipment is often necessary. Unlike the aforementioned complex processes, this study describes a simple, versatile approach for spatially patterned gene delivery inspired by adhesion of marine mussels. Catecholamine, the key adhesive moiety found in the specialized adhesive proteins of mytilus edulis, was used to formulate “sticky” viruses. The adhesive catecholamine polymer used in this study is poly(ethylenimine)-catechol (PEI-C), which has been used for material-independent layerby-layer assembly and mechanical reinforcement of carbon nanotube fibers. The adeno-associated virus (AAV), which is a safe and efficient parvovirus, was complexed with the PEIC. Because of the underwater adhesive property of PEI-C, the AAV vector complexed with the PEI-C became a highly sticky virus that can stably adhere onto surfaces. Most importantly, by using the sticky viral vectors, we were able to use a micropipette as a “pen” to create viral patterns on substrates. This “genevector drawing” technique bypasses laborious multiple steps and can thus be a versatile platform to control gene expression for the establishment of complex tissues. Branched PEI was conjugated with 3-(3,4-dihydroxyphenyl) propionic acid (DPA) to generate PEI-C, which provides sticky viral vectors (Figure 1A). A novel virus, AAVr3.45, which was specifically designed for neuronal cell

Heparin-coated superparamagnetic nanoparticle-mediated adeno-associated virus delivery for enhancing cellular transduction

Superparamagnetic iron oxide nanoparticles (SPIONs) have been exploited as an elegant vehicle to enhance gene delivery efficiencies in gene therapy applications. We developed a magnetically guided adeno-associated virus (AAV) delivery system for enhancing gene delivery to HEK293T and PC12 cell lines. Wild-type AAV2 and a novel AAV vector, AAVr3.45, which was directly evolved in a previous study to possess diverse cell tropisms, were used as gene carriers. Additionally, the affinity of each viral vector to heparin was employed as a moiety to immobilize virus onto heparin-coated SPIONs (HpNPs). Magnetically guided AAV delivery resulted fast and efficient cellular transduction. Importantly, a short exposure of virus to target cells under a magnetic field (<180min) yielded comparable transduction produced by the conventional gene-delivery protocol (i.e., 24h-incubation of virus with target cells prior to replacing with fresh medium). Additionally, magnetic guidance of AAV encoding nerve growth factor (NGF) produced sufficient functional NGF, leading to robust neurite elongation by PC12 as compared to direct NGF protein delivery or non-magnetic delivery. The successful establishment of a magnetically guided AAV delivery system, with the ability to efficiently and rapidly infect target cells, will provide a powerful platform for a variety of gene therapy applications.

Electrospun nanofibers as versatile interfaces for efficient gene delivery

The integration of gene delivery technologies with electrospun nanofibers is a versatile strategy to increase the potential of gene therapy as a key platform technology that can be readily utilized for numerous biomedical applications, including cancer therapy, stem cell therapy, and tissue engineering. As a spatial template for gene delivery, electrospun nanofibers possess highly advantageous characteristics, such as their ease of production, their ECM-analogue nature, the broad range of choices for materials, the feasibility of producing structures with varied physical and chemical properties, and their large surface-to-volume ratios. Thus, electrospun fiber-mediated gene delivery exhibits a great capacity to modulate the spatial and temporal release kinetics of gene vectors and enhance gene delivery efficiency. This review discusses the powerful characteristics of electrospun nanofibers, which can function as spatial interfaces capable of promoting controlled and efficient gene delivery.

Percutaneous cryoablation of small hepatocellular carcinomas using a 17-gauge ultrathin probe

AIM To evaluate the feasibility and safety of percutaneous cryoablation (PCA) of small hepatocellular carcinomas (HCCs) using a 17 G ultrathin cryoprobe. MATERIALS AND METHODS Twenty patients (male:female ratio14:6) with 20 HCCs, who were not surgical candidates, underwent ultrasound (US)-guided PCA for treatment of HCCs. Single HCCs less than 3cm in diameter were included in this study. Ablation was performed using a 17 G cryoprobe. The effectiveness was determined by the changes in alpha-foetoprotein level and degree of tumour necrosis on follow-up computed tomography (CT); complete response (100% necrosis), partial response (100%>necrosis≥30%), stable disease (any cases not qualifying for either partial response or progressive disease) and progressive disease (increase of at least 20% in diameter of viable tumour). Haemoglobin, white blood cell count (WBC), serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), and total bilirubin were compared before and after the procedure, and the technical feasibility, complications, clinical outcomes and survival of each patient were also evaluated. RESULTS All procedures were technically successful. Each patient complained of negligible pain and there was no other procedure-related complication or mortality. The mean level of alpha-foetoprotein declined significantly from 53.2 to 20.4ng/ml 1 month after the procedure (p<0.05). At 1-month follow-up CT, there were 13 complete responses, four partial responses, three patients with stable disease, and no patients had progressive disease. Six of seven lesions that did not present with a complete response underwent further treatment. On long-term follow up (6-30 months; mean 20.7), a local recurrence was seen in one of 13 lesions (8%) with complete response revealed. Laboratory findings showed no significant changes except for the transient increase of SGOT and SGPT. CONCLUSION US-guided PCA using a 17 G cryoprobe was feasible and safe for the treatment of HCC smaller than 3cm.

Diagnosis of breast cancer at dynamic MRI in patients with breast augmentation by paraffin or silicone injection

AIM To determine the diagnostic performance of dynamic magnetic resonance imaging (MRI) for breast cancer in breasts augmented with liquid paraffin or silicone injection. MATERIALS AND METHODS Among 62 patients with breast augmentation by liquid paraffin or silicone injection who had undergone dynamic breast MRI at our institution, 27 women, who had pathological diagnosis or at least 1-year MRI follow-up, were included in this retrospective study and their MRI images were reviewed. For enhancing lesions on MRI, the morphological features, enhancement kinetics, and BI-RADS assessment category were analysed. The lesion characteristics at MRI were correlated with the final diagnosis based on the histopathological result or at least 1-year MRI follow-up. RESULTS Of the 27 patients, 17 enhancing lesions in 13 patients were found on MRI. All six lesions that were confirmed as malignancy showed suspicious morphological findings and type 2 or 3 enhancement kinetics, assigned to BI-RADS category 4 or 5. Of the remaining 11 benign lesions, 10 showed benign-favouring morphological findings, and all showed type 1 enhancement kinetics, assigned to BI-RADS category 2 or 4. CONCLUSION In patients with breasts injected with foreign material, MRI was used to successfully diagnose malignant breast lesions and could be the diagnostic method of choice. Analysis of the morphological and kinetic features at MRI in conjunction with clinical findings is essential.

Sliding Fibers: Slidable, Injectable, and Gel-like Electrospun Nanofibers as Versatile Cell Carriers

Designing biomaterial systems that can mimic fibrous, natural extracellular matrix is crucial for enhancing the efficacy of various therapeutic tools. Herein, a smart technology of three-dimensional electrospun fibers that can be injected in a minimally invasive manner was developed. Open surgery is currently the only route of administration of conventional electrospun fibers into the body. Coordinating electrospun fibers with a lubricating hydrogel produced fibrous constructs referred to as slidable, injectable, and gel-like (SLIDING) fibers. These SLIDING fibers could pass smoothly through a catheter and fill any cavity while maintaining their fibrous morphology. Their injectable features were derived from their distinctive rheological characteristics, which were presumably caused by the combinatorial effects of mobile electrospun fibers and lubricating hydrogels. The resulting injectable fibers fostered a highly favorable environment for human neural stem cell (hNSC) proliferation and neurosphere formation within the fibrous structures without compromising hNSC viability. SLIDING fibers demonstrated superior performance as cell carriers in animal stroke models subjected to the middle cerebral artery occlusion (MCAO) stroke model. In this model, SLIDING fiber application extended the survival rate of administered hNSCs by blocking microglial infiltration at the early, acute inflammatory stage. The development of SLIDING fibers will increase the clinical significance of fiber-based scaffolds in many biomedical fields and will broaden their applicability.

Bicomponent electrospinning to fabricate three-dimensional hydrogel-hybrid nanofibrous scaffolds with spatial fiber tortuosity.

Electrospun fibrous mats have emerged as powerful tissue engineering scaffolds capable of providing highly effective and versatile physical guidance, mimicking the extracellular environment. However, electrospinning typically produces a sheet-like structure, which is a major limitation associated with current electrospinning technologies. To address this challenge, highly porous, volumetric hydrogel-hybrid fibrous scaffolds were fabricated by one Taylor cone-based side-by-side dual electrospinning of poly (ε-caprolactone) (PCL) and poly (vinyl pyrrolidone) (PVP), which possess distinct properties (i.e., hydrophobic and hydrogel properties, respectively). Immersion of the resulting scaffolds in water induced spatial tortuosity of the hydrogel PVP fibers while maintaining their aligned fibrous structures in parallel with the PCL fibers. The resulting conformational changes in the entire bicomponent fibers upon immersion in water led to volumetric expansion of the fibrous scaffolds. The spatial fiber tortuosity significantly increased the pore volumes of electrospun fibrous mats and dramatically promoted cellular infiltration into the scaffold interior both in vitro and in vivo. Harmonizing the flexible PCL fibers with the soft PVP-hydrogel layers produced highly ductile fibrous structures that could mechanically resist cellular contractile forces upon in vivo implantation. This facile dual electrospinning followed by the spatial fiber tortuosity for fabricating three-dimensional hydrogel-hybrid fibrous scaffolds will extend the use of electrospun fibers toward various tissue engineering applications.

Polydopamine Inter-Fiber Networks: New Strategy for Producing Rigid, Sticky, 3D Fluffy Electrospun Fibrous Polycaprolactone Sponges

Designing versatile 3D interfaces that can precisely represent a biological environment is a prerequisite for the creation of artificial tissue structures. To this end, electrospun fibrous sponges, precisely mimicking an extracellular matrix and providing highly porous interfaces, have capabilities that can function as versatile physical cues to regenerate various tissues. However, their intrinsic features, such as sheet-like, thin, and weak structures, limit the design of a number of uses in tissue engineering applications. Herein, a highly facile methodology capable of fabricating rigid, sticky, spatially expanded fluffy electrospun fibrous sponges is proposed. A bio-inspired adhesive material, poly(dopamine) (pDA), is employed as a key mediator to provide rigidity and stickiness to the 3D poly(ε-caprolactone) (PCL) fibrous sponges, which are fabricated using a coaxial electrospinning with polystyrene followed by a selective leaching process. The iron ion induced oxidation of dopamine into pDA networks interwoven with PCL fibers results in significant increases in the rigidity of 3D fibrous sponges. Furthermore, the exposure of catecholamine groups on the fiber surfaces promotes the stable attachment of the sponges on wet organ surfaces and triggers the robust immobilization of biomolecules (e.g., proteins and gene vectors), demonstrating their potential for 3D scaffolds as well as drug delivery vehicles. Because fibrous structures are ubiquitous in the human body, these rigid, sticky, 3D fibrous sponges are good candidates for powerful biomaterial systems that functionally mimic a variety of tissue structures.

The promotion of human neural stem cells adhesion using bioinspired poly(norepinephrine) nanoscale coating

The establishment of versatile biomaterial interfaces that can facilitate cellular adhesion is crucial for elucidating the cellular processes that occur on biomaterial surfaces. Furthermore, biomaterial interfaces can provide physical or chemical cues that are capable of stimulating cellular behaviors by regulating intracellular signaling cascades. Herein, a method of creating a biomimetic functional biointerface was introduced to enhance human neural stem cell (hNSC) adhesion. The hNSC-compatible biointerface was prepared by the oxidative polymerization of the neurotransmitter norepinephrine, which generates a nanoscale organic thin layer, termed poly(norepinephrine) (pNE). Due to its adhesive property, pNE resulted in an adherent layer on various substrates, and pNE-coated biointerfaces provided a highly favorable microenvironment for hNSCs, with no observed cytotoxicity. Only a 2-hour incubation of hNSCs was required to firmly attach the stem cells, regardless of the type of substrate. Importantly, the adhesive properties of pNE interfaces led to micropatterns of cellular attachment, thereby demonstrating the ability of the interface to organize the stem cells. This highly facile surface-modification method using a biomimetic pNE thin layer can be applied to a number of suitable materials that were previously not compatible with hNSC technology.