Polyhydroxyalkanoate Decelerates the Release of Paclitaxel from Poly(lactic-co-glycolic acid) Nanoparticles.

Biodegradable nanoparticles (NPs) are preferred as drug carriers because of their effectiveness in encapsulating drugs, ability to control drug release, and low cytotoxicity. Although poly(lactide co-glycolide) (PLGA)-based NPs have been used for controlled release strategies, they have some disadvantages. This study describes an approach using biodegradable polyhydroxyalkanoate (PHA) to overcome these challenges. By varying the amount of PHA, NPs were successfully fabricated by a solvent evaporation method. The size range of the NPS ranged from 137.60 to 186.93 nm, and showed zero-order release kinetics of paclitaxel (PTX) for 7 h, and more sustained release profiles compared with NPs composed of PLGA alone. Increasing the amount of PHA improved the PTX loading efficiency of NPs. Overall, these findings suggest that PHA can be used for designing polymeric nanocarriers, which offer a potential strategy for the development of improved drug delivery systems for sustained and controlled release.

Rolling circle transcription-based polymeric siRNA nanoparticles for tumor-targeted delivery.

RNA, one of the major biological macromolecules, has been considered as an attractive building material for bottom-up fabrication of nanostructures in the past few decades due to advancements in RNA biology, RNA chemistry and RNA nanotechnology. Most recently, an isothermal enzymatic nucleic acid amplification method termed rolling circle transcription (RCT), which achieves a large-scale synthesis of RNA nanostructures, has emerged as one of fascinating techniques for RNAi-based therapies. Herein, we proposed a newly designed RCT method for synthesis of polymeric siRNA nanoflower, referred to 'RCT and annealing-generated polymeric siRNA (RAPSI)': (1) Amplification of the antisense strand of siRNA via RCT process and (2) annealing of chimeric sense strand containing 3'-terminal DNA nucleotides that provide enzyme cleavage sites. To verify its potentials in RNAi-based cancer therapy, the newly designed RAPSI nanoflower was further complexed with glycol chitosan (GC) derivatives, and systemically delivered to PC-3 xenograft tumors. The resultant RAPSI nanoparticles exhibited the improved particle stability against polyanion competition or nuclease attack. When the RAPSI nanoparticles reached to the cytoplasmic region, active mono siRNA was liberated and significantly down-regulated the expression of target VEGF gene in PC-3 cells. Excellent tumor-homing efficacy and anti-tumor effects of the RAPSI nanoparticles were further demonstrated. Overall, the proposed RCT-based polymeric siRNA nanoflower formulation can provide a new platform technology that allows further functional modifications via an advanced annealing method for systemic cancer RNAi therapy.

MicroRNA-mediated non-viral direct conversion of embryonic fibroblasts to cardiomyocytes: comparison of commercial and synthetic non-viral vectors.

Technological advances opened up new ways of directing cell fate conversion from one cell lineage to another. The direct cell conversion technique has recently attracted much attention in regenerative medicine to treat devastated organs and tissues, particularly having limited regenerative capacity such as the heart and brain. Unfortunately, its clinical application is severely limited due to a safety concern and immunogenicity of viral vectors, as human gene therapy did in the beginning stages. In this study, we examined the possibility of adopting non-viral vectors to direct cell conversion from mouse embryonic fibroblasts to induced cardiomyocytes (iCM) by transient transfection of four types of chemically synthesized micro-RNA mimics (miRNA-1, 133, 208, and 499). Herein, we tested several commercial and synthetic non-viral gene delivery carriers, which could be divided into three different categories: polymers [branched PEI (bPEI), bioreducible PEI (PEI-SS), deoxycholic acid-conjugated PEI (DA-PEI), jetPEI™, SuperFect™], lipids (Lipofectamine 2000™), and peptides (PepMute™). According to the analyses of physicochemical properties, cellular uptake, and cytotoxicity of the carrier/miRNA complexes, DA-PEI exhibited excellent miRNA delivery efficiency to mouse embryonic fibroblasts. One week after a single treatment of DA-PEI/miRNA without other adjuvants, the cells started to express cardiomyocyte-specific markers, such as α-actinin and α-MHC, indicating the formation of cardiomyocyte-like cells. Although the overall frequency of non-viral vector induced cardiomyogenic transdifferentiation was quite low (ca. 0.2%), this study can provide compelling support to develop clinically applicable transdifferentiation techniques.

Design and Characterization of a PVLA-PEG-PVLA Thermosensitive and Biodegradable Hydrogel.

A set of poly(δ-valerolactone-co-d,l-lactide)-b-poly(ethylene glycol)-b-poly(δ-valerolactone-co-d,l-lactide) (PVLA-PEG-PVLA) triblock copolymers was synthesized and the solution properties were characterized using rheology, cryo-TEM, cryo-SEM, SANS, and degradation studies. This polymer self-assembles into a low viscosity fluid with flowerlike spherical micelles in water at room temperature and transforms into a wormlike morphology upon heating, accompanied by gelation. At even higher temperatures syneresis is observed. At physiological temperature (37 °C) the hydrogel's average pore size is around 600 nm. The PVLA-PEG-PVLA gel degrades in about 45 days in cell media, making this unique hydrogel a promising candidate for biomedical applications.

The design of peptide-amphiphiles as functional ligands for liposomal anticancer drug and gene delivery.

Liposomal nanomedicine has led to clinically useful cancer therapeutics like Doxil and DaunoXome. In addition, peptide-functionalized liposomes represent an effective drug and gene delivery vehicle with increased cancer cell specificity, enhanced tumor-penetrating ability and high tumor growth inhibition. The goal of this article is to review the recently published literature of the peptide-amphiphiles that were used to functionalize liposomes, to highlight successful designs that improved drug and gene delivery to cancer cells in vitro, and cancer tumors in vivo, and to discuss the current challenges of designing these peptide-decorated liposomes for effective cancer treatment.

Simultaneous regulation of apoptotic gene silencing and angiogenic gene expression for myocardial infarction therapy: Single-carrier delivery of SHP-1 siRNA and VEGF-expressing pDNA.

Gene therapy is aimed at selectively knocking up or knocking down the target genes involved in the development of diseases. In many human diseases, dysregulation of disease-associated genes is occurred concurrently: some genes are abnormally turned up and some are turned down. In the field of non-viral gene therapy, plasmid DNA (pDNA) and small interfering RNA (siRNA) are suggested as representative regulation tools for activating and silencing the expression of genes of interest, representatively. Herein, we simultaneously loaded both siRNA (Src homology region 2 domain-containing tyrosine phosphatase-1 siRNA, siSHP-1) for anti-apoptosis and pDNA (hypoxia-inducible vascular endothelial growth factor expression vector, pHI-VEGF) for angiogenesis in a single polymeric nanocarrier and used to synergistically attenuate ischemia-reperfusion (IR)-induced myocardial infarction, which is mainly caused by dysregulating of cardiac apoptosis and angiogenesis. For dual-modality cardiac gene delivery, siSHP-1 and pHI-VEGF were sequentially incorporated into a stable nanocomplex by using deoxycholic acid-modified polyethylenimine (DA-PEI). The resulting DA-PEI/siSHP-1/pHI-VEGF complexes exhibited the high structural stability against polyanion competition and the improved resistance to digestion by nucleases. The cardiac administration of DA-PEI/siSHP-1/pHI-VEGF reduced cardiomyocyte apoptosis and enhanced cardiac microvessel formation, thereby reducing infarct size in rat ischemia-reperfusion model. The simultaneous anti-apoptotic and angiogenic gene therapies synergized the cardioprotective effects of each strategy; thus our dual-modal single-carrier gene delivery system can be considered as a promising candidate for treating ischemic heart diseases.

Targeted Nanotheranostics for Future Personalized Medicine: Recent Progress in Cancer Therapy.

Recently, many theranostic nanomaterials have been developed by integrating therapeutic and diagnostic agents in a single regimen. Real-time visualization of nano drug carrier biodistributions, drug release processes and therapeutic responses can provide critical information needed for dynamically optimizing treatment operations in a personalized manner in real time. This review highlights recent progresses in the development of multifunctional nanoparticles possessing both therapeutic and imaging functionalities for cancer therapy. The advantages of using nanoparticle platforms are discussed. Examples demonstrating various combinations of imaging and therapeutic modalities are highlighted.

Chemical and structural modifications of RNAi therapeutics.

Small interfering RNA (siRNA), a 21-23nt double-stranded RNA responsible for post-transcriptional gene silencing, has attracted great interests as promising genomic drugs, due to its strong ability to silence target genes in a sequence-specific manner. Despite high silencing efficiency and on-target specificity, the clinical translation of siRNA has been hindered by its inherent features: poor intracellular delivery, limited blood stability, unpredictable immune responses and unwanted off-targeting effects. To overcome these hindrances, researchers have made various advances to modify siRNA itself and to improve its delivery. In this review paper, first we briefly discuss the innate properties and delivery barriers of siRNA. Then, we describe recent progress in (1) chemically and structurally modified siRNAs to solve their intrinsic problems and (2) siRNA delivery formulations including siRNA conjugates, polymerized siRNA, and nucleic acid-based nanoparticles to improve in vivo delivery.

RAGE siRNA-mediated gene silencing provides cardioprotection against ventricular arrhythmias in acute ischemia and reperfusion.

Expression of receptor for advanced glycation end-products (RAGE) is suggested to play a crucial role in mediating cardiac ischemia/reperfusion (IR) injury, and the blockade of RAGE signaling has been considered as a potential therapeutic strategy for the treatment of IR-induced cardiac damage. In this study, we primarily investigated the effects of RAGE suppression particularly on IR-induced ventricular arrhythmia. To inhibit the IR-induced upregulation of RAGE, siRNA targeting RAGE (siRAGE) was delivered to myocardium by using deoxycholic acid-modified polyethylenimine (PEI-DA) as a non-viral gene carrier. The resultant PEI-DA/siRAGE nanocomplexes successfully silenced the expression of RAGE and attenuated the inflammation and apoptosis in the ischemic-reperfused myocardium. According to our results, the electrophysiological properties (e.g., action potential propagation, action potential duration, and conduction velocity), disrupted by IR injury, were restored to normal level and the induction of ventricular tachycardia was abolished by RAGE silencing. We further found that RAGE suppression led to the activation of Wnt signaling, followed by the expression of gap junction protein, connexin43. Thus it could be concluded that successful siRAGE delivery is protective against IR-induced ventricular arrhythmia.

Co-delivery of VEGF and Bcl-2 dual-targeted siRNA polymer using a single nanoparticle for synergistic anti-cancer effects in vivo.

Cancer is a multifactorial disease which involves complex genetic mutation and dysregulation. Combinatorial RNAi technology and concurrent multiple gene silencing are expected to provide advanced strategies for effective cancer therapy, but a safe and effective carrier system is a prerequisite to successful siRNA delivery in vivo. We previously developed an effective tumor-targeting siRNA delivery system for in vivo application. In response to the success of this development, herein we present a dual-gene targeted siRNA and its delivery system, to achieve synergistic effects in cancer therapy. Two different sequences of siRNA were chemically modified to be randomly copolymerized in a single backbone of siRNA polymer (Dual-poly-siRNA), and the resulting Dual-poly-siRNA was incorporated into tumor-homing glycol chitosan nanoparticles. Based on the stability in serum and delivery in a tumor-targeted manner, intravenously administered Dual-poly-siRNA carrying glycol chitosan nanoparticles (Dual-NP) demonstrated successful dual-gene silencing in tumors. Notably, co-delivery of VEGF and Bcl-2 targeting siRNA led to more effective cancer therapy for convenient application.

Enhanced Cytoplasmic Delivery of RAGE siRNA Using Bioreducible Polyethylenimine-based Nanocarriers for Myocardial Gene Therapy.

This study aims to develop bioreducible polyethylenimine (rPEI)/siRNA polyplexes with high stability, high transfection efficiency, and low cytotoxicity for efficient cytoplasmic siRNA delivery. rPEI successfully incorporated siRNA into stable and compact nanocomplexes, and the disulfide linkages in rPEI/siRNA were cleaved under reductive environments, resulting in efficient intracellular translocation and siRNA release. In this study, receptor for advanced glycation end-products (RAGE) was selected as a therapeutic target gene because it is associated with inflammatory responses in ischemia/reperfusion injury. rPEI/siRAGE exhibited high target gene silencing and low cytotoxicity in cardiomyocytes, and the treatment of rPEI/siRAGE reduced the myocardial infarction size.

Deoxycholic acid-modified polyethylenimine based nanocarriers for RAGE siRNA therapy in acute myocardial infarction.

The activation of receptor for advanced glycation end products (RAGE) signaling is mainly associated with myocardial ischemia/reperfusion injury. Thus the blockade of RAGE-ligands axis can be considered as a potential therapeutic strategy to protect myocardial infarction after ischemia/reperfusion injury. Herein, we strengthened the cardioprotective effect with combinatorial treatment of soluble RAGE (sRAGE) and RAGE siRNA (siRAGE) causing more effective suppression of RAGE-mediated signaling transduction. For pharmacological blockade of RAGE, sRAGE, the extracellular ligand binding domain of RAGE, acts as a pharmacological ligand decoy and inhibits the interaction between RAGE and its ligands. For genetic deletion of RAGE, siRAGE suppresses the expression of RAGE by participating in RNA interference mechanism. Therefore, we combined these two RAGE blockade/deletion strategies and investigated the therapeutic effects on rat ischemic and reperfused myocardium. According to our results, based on RAGE expression level analysis and infarct size/fibrosis measurement, co-treatment of sRAGE and siRAGE exhibited synergic cardioprotective effects; thus the newly designed regimen can be considered as a promising candidate for the treatment of myocardial infarction.

Tumor-targeting glycol chitosan nanoparticles as a platform delivery carrier in cancer diagnosis and therapy.

A natural based polymer, chitosan has received widespread attention in drug delivery systems due to its valuable physicochemical and biological characteristics. In particular, hydrophobic moiety-conjugated glycol chitosan can form amphiphilic self-assembled glycol chitosan nanoparticles (GCNPs) and simultaneously encapsulate hydrophobic drug molecules inside their hydrophobic core. This GCNP-based drug delivery systems exhibit excellent tumor-homing efficacy, attributed to the long blood circulation and the enhanced permeability and retention effect; this tumor-targeting drug delivery results in improved therapeutic efficiency. In this review, we describe the requisite properties of GCNPs for cancer therapy as well as imaging for diagnosis, such as their basic characteristics, in vitro delivery efficiency and in vivo tumor-targeting ability.

Cardiac RNAi therapy using RAGE siRNA/deoxycholic acid-modified polyethylenimine complexes for myocardial infarction.

Inflammatory response in myocardial ischemia-reperfusion injury plays a critical role in ventricular remodeling. To avoid deleterious effects of overwhelming inflammation, we blocked the expression of receptor for advanced glycation end-products (RAGE), a key mediator of the local and systemic inflammatory responses, via RNAi mechanism. Herein, a facial amphipathic deoxycholic acid-modified low molecular weight polyethylenimine (DA-PEI) was used as a siRNA delivery carrier to myocardium. The DA-PEI conjugate formed a stable complex with siRNA via electrostatic and hydrophobic interactions. The siRAGE/DA-PEI formulation having negligible toxicity could enhance intracellular delivery efficiency and successfully suppress RAGE expression both in vitro and in vivo. Furthermore, the cardiac administration of siRAGE/DA-PEI reduced apoptosis and inflammatory cytokine release, subsequently led to attenuation of left ventricular remodeling in rat myocardial infarction model. The potential therapeutic effects of RAGE gene silencing on myocardial ischemia-reperfusion injury may suggest that the siRAGE/DA-PEI delivery system can be considered as a promising strategy for treating myocardial infarction.

Tumor-targeting multifunctional nanoparticles for siRNA delivery: recent advances in cancer therapy.

RNA interference (RNAi) is a naturally occurring regulatory process that controls posttranscriptional gene expression. Small interfering RNA (siRNA), a common form of RNAi-based therapeutics, offers new opportunities for cancer therapy via silencing specific genes, which are associated to cancer progress. However, clinical applications of RNAi-based therapy are still limited due to the easy degradation of siRNA during body circulation and the difficulty in the delivery of siRNA to desired tissues and cells. Thus, there have been many efforts to develop efficient siRNA delivery systems, which protect siRNA from serum nucleases and deliver siRNA to the intracellular region of target cells. Here, the recent advances in siRNA nanocarriers, which possess tumor-targeting ability are reviewed; various nanoparticle systems and their antitumor effects are summarized. The development of multifunctional nanocarriers for theranostics or combinatorial therapy is also discussed.

Cross-linked iron oxide nanoparticles for therapeutic engineering and in vivo monitoring of mesenchymal stem cells in cerebral ischemia model.

Poly(ethylene glycol)-coated cross-linked iron oxide nanoparticles (PCIONs) are developed for therapeutic engineering of mesenchymal stem cells (MSCs) and their monitoring via magnetic resonance (MR) imaging at a time. PCIONs successfully combine with plasmid DNA (pDNA) via ionic interaction. Accordingly, PCION/pDNA complexes mediate superior translocations of vascular endothelial growth factor (VEGF) pDNA into intracellular regions of MSCs under external magnetic field, which significantly elevate production of VEGF from MSCs. Genetically engineered MSCs are also clearly visualized via MR imaging after administration to rat cerebrovascular ischemia models, which enable tracking of MSCs migration from injected sites to injured ischemic area.

Hyaluronic acid-siRNA conjugate/reducible polyethylenimine complexes for targeted siRNA delivery.

The clinical applications of therapeutic siRNA remain as a challenge due to the lack of efficient delivery system. In the present study, hyaluronic acid-siRNA conjugate (HA-SS-siRNA)/reducible polyethylenimine (BPEI1.2k-SS) complexes were developed to efficiently deliver the siRNA to HA receptor abundant region with the improved siRNA stability. HA and siRNA were conjugated with disulfide bonds, which are cleavable in cytoplasm. The synthesized HA-SS-siRNA was further complexed with BPEI1.2k-SS, resulting in the formation of spherical nanostructures with approximately 190 nm of size and neutral surface charge. HA-SS-siRNA/BPEI1.2k-SS complexes exhibited the improved stability against serum proteins or polyanions. These complexes were successfully translocated into intracellular region via HA receptor-mediated endocytosis, and silenced target gene expression.

Combined effect of mussel-inspired surface modification and topographical cues on the behavior of skeletal myoblasts.

The combined effect of mussel-inspired polydopamine (PDA) surface functionalization and topographical cues on the behavior of skeletal myoblasts is described. On PDA-modified nanofibers, myogenic protein expression and the fusion of myoblasts are increased significantly compared with those on unmodified nanofibers. The multinucleate myotubes on the aligned nanofibers are oriented in a direction parallel to the nanofibers.

Myoblast differentiation on graphene oxide.

Graphene-based nanomaterials have received much attention in biomedical applications for drug/gene delivery, cancer therapy, imaging, and tissue engineering. Despite the capacity of 2D carbon materials as a nontoxic and implantable platform, their effect on myogenic differentiation has been rarely studied. We investigated the myotube formation on graphene-based nanomaterials, particularly graphene oxide (GO) and reduced graphene oxide (rGO). GO sheets were immobilized on amine-modified glass to prepare GO-modified glass, which was further reduced by hydrazine treatment for the synthesis of rGO-modified substrate. We studied the behavior, including adhesion, proliferation, and differentiation, of mouse myoblast C2C12 on unmodified, GO-, and rGO-modified glass substrates. According to our analyses of myogenic protein expression, multinucleate myotube formation, and expression of differentiation-specific genes (MyoD, myogenin, Troponin T, and MHC), myogenic differentiation was remarkably enhanced on GO, which resulted from serum protein adsorption and nanotopographical cues. Our results demonstrate the ability of GO to stimulate myogenic differentiation, showing a potential for skeletal tissue engineering applications.

Carbon-based nanomaterials for tissue engineering.

Carbon-based nanomaterials such as graphene sheets and carbon nanotubes possess unique mechanical, electrical, and optical properties that present new opportunities for tissue engineering, a key field for the development of biological alternatives that repair or replace whole or a portion of tissue. Carbon nanomaterials can also provide a similar microenvironment as like a biological extracellular matrix in terms of chemical composition and physical structure, making them a potential candidate for the development of artificial scaffolds. In this review, we summarize recent research advances in the effects of carbon nanomaterial-based substrates on cellular behaviors, including cell adhesion, proliferation, and differentiation into osteo- or neural- lineages. The development of 3D scaffolds based on carbon nanomaterials (or their composites with polymers and inorganic components) is introduced, and the potential of these constructs in tissue engineering, including toxicity issues, is discussed. Future perspectives and emerging challenges are also highlighted.