Cargo-selective and adaptive delivery of nucleic acid therapeutics by bola-amphiphilic dendrimers.

Nucleic acid therapeutics are becoming an important drug modality, offering the unique opportunity to address "undruggable" targets, respond rapidly to evolving pathogens, and treat diseases at the gene level for precision medicine. However, nucleic acid therapeutics have poor bioavailability and are chemolabile and enzymolabile, imposing the need for delivery vectors. Dendrimers, by virtue of their well-defined structure and cooperative multivalence, represent precision delivery systems. We synthesized and studied bola-amphiphilic dendrimers for cargo-selective and on-demand delivery of DNA and small interfering RNA (siRNA), both important nucleic acid therapeutics. Remarkably, superior performances were achieved for siRNA delivery with the second-generation dendrimer, yet for DNA delivery with the third generation. We systematically studied these dendrimers with regard to cargo binding, cellular uptake, endosomal release, and in vivo delivery. Differences in size both of the dendrimers and their nucleic acid cargos impacted the cooperative multivalent interactions for cargo binding and release, leading to cargo-adaptive and selective delivery. Moreover, both dendrimers harnessed the advantages of lipid and polymer vectors, while offering nanotechnology-based tumor targeting and redox-responsive cargo release. Notably, they allowed tumor- and cancer cell-specific delivery of siRNA and DNA therapeutics for effective treatment in different cancer models, including aggressive and metastatic malignancies, outperforming the currently available vectors. This study provides avenues to engineer tailor-made vectors for nucleic acid delivery and precision medicine.

Glycogen nanoparticles for efficient mRNA transduction to T lymphocytes.

T lymphocyte therapies demonstrate significant promise in the treatment of cancer and infectious diseases. An efficient gene delivery system is essential for the safe and reliable introduction of exogenous genes, especially mRNA, into cells to achieve therapeutic purposes. Commercial transfection reagents are suitable for the transduction of plasmids to adherent cells, whereas they are ineffective for suspension cells such as T lymphocytes and for unstable mRNA. Moreover, the cytotoxicity of transfection reagents themselves constitutes an impediment to their application. The challenge of mRNA transduction to T lymphocytes with high efficiency is notably formidable. An innovative transfection strategy is urgently needed. In this study, we synthesized aminated glycogen (AGly) nanoparticles as gene vectors, encapsulating mRNA to facilitate the efficient transfection of T lymphocytes. Compared to commercial transfection reagent polyethylenimine (PEI), the AGly demonstrated favorable biocompatibility. The positive charge provided AGly with pH buffering ability and mRNA-binding capacity. AGly formed stable nanoparticles with mRNA, which were readily internalized by suspension cells and enhanced the cellular uptake of mRNA. In the T lymphocyte model cell lines (Jurkat cells and HuT 78 cells), AGly demonstrated superior transfection efficiency than that of PEI. Consequently, AGly can emerge as a viable mRNA vector for the efficient transfection of T lymphocytes whilst circumventing the issue of cytotoxicity. The AGly designed in this study provides a novel concept for the exploitation of transfection reagents and proposes a promising methodology for the proficient transfection of T lymphocytes which may significantly contribute to the treatment of cancer and other complex diseases.

Construction of GSH-responsive polyethyleneimine-based delivery vector for effective gene transfection.

The rise of gene therapy has solved many diseases that cannot be effectively treated by conventional methods. Gene vectors is very important to protect and deliver the therapeutic genes to the target site. Polyethyleneimine (PEI) modified with mannitol could enhance the gene transfection efficiency reported by our group previously. In order to further control and improve the effective gene release to action site, disulfide bonds were introduced into mannitol-modified PEI to construct new non-viral gene vectors PeiSM. The degrees of mannitol linking with disulfide bonds were screened. Among them, moderate mannitol-modified PEI with disulfide bonds showed the best transfection efficiency, and significantly enhanced long-term systemic transgene expression for 72 hin vivoeven at a single dose administration, and could promote caveolae-mediated uptake through up-regulating the phosphorylation of caveolin-1 and increase the loaded gene release from the nanocomplexes in high glutathione intracellular environment. This functionalized gene delivery system can be used as an potential and safe non-viral nanovector for further gene therapy.

Micromotor-based localized electroporation and gene transfection of mammalian cells.

Herein, we studied localized electroporation and gene transfection of mammalian cells using a metallodielectric hybrid micromotor that is magnetically and electrically powered. Much like nanochannel-based, local electroporation of single cells, the presented micromotor was expected to increase reversible electroporation yield, relative to standard electroporation, as only a small portion of the cell's membrane (in contact with the micromotor) is affected. In contrast to methods in which the entire membrane of all cells within the sample are electroporated, the presented micromotor can perform, via magnetic steering, localized, spatially precise electroporation of the target cells that it traps and transports. In order to minimize nonselective electrical lysis of all cells within the chamber, resulting from extended exposure to an electrical field, magnetic propulsion was used to approach the immediate vicinity of the targeted cell, after which short-duration, electric-driven propulsion was activated to enable contact with the cell, followed by electroporation. In addition to local injection of fluorescent dye molecules, we demonstrated that the micromotor can enhance the introduction of plasmids into the suspension cells because of the dielectrophoretic accumulation of the plasmids in between the Janus particle and the attached cell prior to the electroporation step. Here, we chose a different strategy involving the simultaneous operation of many micromotors that are self-propelling, without external steering, and pair with cells in an autonomic manner. The locally electroporated suspension cells that are considered to be very difficult to transfect were shown to express the transfected gene, which is of significant importance for molecular biology research.

Peritoneal Gene Transfection of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand for Tumor Surveillance and Prophylaxis.

Peritoneal metastasis is very common in gastrointestinal, reproductive, and genitourinary tract cancers in late stages or postsurgery, causing poor prognosis, so effective and nontoxic prophylactic strategies against peritoneal metastasis are highly imperative. Herein, we demonstrate the first gene transfection as a nontoxic prophylaxis preventing peritoneal metastasis or operative metastatic dissemination. Lipopolyplexes of TNF-related-apoptosis-inducing-ligand (TRAIL) transfected peritonea and macrophages to express TRAIL for over 15 days. The expressed TRAIL selectively induced tumor cell apoptosis while exempting normal tissue, providing long-term tumor surveillance. Therefore, tumor cells inoculated in the pretransfected peritoneal cavity quickly underwent apoptosis and, thus, barely formed tumor nodules, significantly prolonging the mouse survival time compared with chemotherapy prophylaxis. Furthermore, lipopolyplex transfection showed no sign of toxicity. Therefore, this peritoneal TRAIL-transfection is an effective and safe prophylaxis, preventing peritoneal metastasis.

A Fluorophore-Labeled Lysine Dendrimer with an Oxo-Anion-Binding Motif for Tracking Gene Transfection.

A transfection vector based on a peptide dendrimer (1) has been developed and its abilities for DNA binding and transport have been investigated. By attaching a fluorophore to the vector system (1*), several steps in the transfection process could be monitored directly. As DLS and AFM studies showed, the labeled vector 1* condensed DNA into tightly packed aggregates able to enter eukaryotic cells. Co-localization experiments revealed that the ligand/plasmid complex is taken up by the endosomal pathway followed by an endosomal escape or lysosomal degradation. Afterwards, the plasmid DNA seems to enter the nucleus due to a breakdown of the nuclear envelope during mitosis, as only cells that have recently undergone mitosis showed H2B-GFP expression.

Lipoic Acid-Based Poly(disulfide)s as Versatile Biomolecule Delivery Vectors and the Application in Tumor Immunotherapy.

Intracellular delivery of therapeutic biomacromolecules, including nucleic acids and proteins, attracts extensive attention in biotherapeutics for various diseases. Herein, a strategy is proposed for the construction of poly(disulfide)s for the efficient delivery of both nucleic acids and proteins into cells. A convenient photo-cross-linking polymerization was adopted between disulfide bonds in two modified lipoic acid monomers (Zn coordinated with dipicolylamine analogue (ZnDPA) and guanidine (GUA)). The disulfide-containing main chain of the resulting poly(disulfide)s was responsive to reducing circumstance, facilitating the release of cargos. By screening the feeding ratio of ZnDPA and GUA, the resulting poly(disulfide)s exhibited better performance in the delivery of nucleic acids including plasmid DNA and siRNA than commercially available transfection reagents. Cellular uptake results revealed that the polymer/cargo complexes entered the cells mainly following a thiol-mediated uptake pathway. Meanwhile, the polymer could also efficiently deliver proteins into cells without an obvious loss of protein activity, showing the versatility of the poly(disulfide)s for the delivery of various biomacromolecules. Moreover, the in vivo therapeutic effect of the materials was verified in the E.G7-OVA tumor-bearing mice. Ovalbumin-based nanovaccine induced a strong cellular immune response, especially cytotoxic T lymphocyte cellular immune response, and inhibited tumor growth. These results revealed the promise of the poly(disulfide)s in the application of both gene therapy and immunotherapy.

Endothelial progenitor cells overexpressing platelet derived growth factor-D facilitate deep vein thrombosis resolution.

To assess the therapeutic efficacy of PDGF-D-overexpressing endothelial progenitor cells (EPCs) in deep vein thrombosis. Inferior vena cava thrombosis was induced in female Sprague Dawley (SD) rats. Animals were injected via the distal vena cava with EPCs overexpressing PDGF-D after transfection with a lentiviral vector containing the PDGF-D gene. The effect on thrombosis in animals who received EPCs was evaluated using MSB staining, immunohistochemistry, immunofluorescence, and venography; the steady-state mRNA and protein levels of PDGF-D and its receptor (PDGF-Rß) were determined by RT-PCR and Western blotting, respectively; and the PDGF-D-induced mobilization of circulating EPCs was estimated by flow cytology. Compared with controls, injection of EPCs overexpressing PDGF-D was associated with increased thrombosis resolution; recanalization; PDGF-D and PDGF-Rß expression; induction of monocyte homing; and mobilization of EPCs to the venous circulation. In a rat model, transplantation of PDGF-D-overexpressing EPCs facilitated the resolution of deep vein thrombosis.

Cellular nanomechanics derived from pattern-dependent focal adhesion and cytoskeleton to balance gene transfection of malignant osteosarcoma.

Gene transfection was supposed to be the most promising technology to overcome the vast majority of diseases and it has been popularly reported in clinical applications of gene therapy. In spite of the rapid development of novel transfection materials and methods, the influence of morphology-dependent nanomechanics of malignant osteosarcoma on gene transfection is still unsettled. In this study, cell spreading and adhesion area was adjusted by the prepared micropatterns to regulate focal adhesion (FA) formation and cytoskeletal organization in osteosarcoma cells. The micropattern-dependent FA and cytoskeleton could induce different cellular nanomechanics to affect cell functions. Our results indicated that transfection efficiency was improved with enlarging FA area and cell nanomechanics in micropatterned osteosarcoma. The difference of gene transfection in micropatterned cells was vigorously supported by cellular internalization capacity, Ki67 proliferation ability and YAP mechanotranduction through the regulation of focal adhesion and cytoskeletal mechanics. This study is an attempt to disclose the relationship of cell nanomechanics and gene transfection for efficient gene delivery and develop multifunctional nanomedicine biomaterials for accurate gene therapy in osteosarcoma cells.

A Novel Combination of Keratinocyte and Autologous Microskin Grafting to Repair Full-Thickness Skin Loss.

INTRODUCTION: The high mortality of patients with extensive deep burns is mainly attributed to the extensive burn wound and the scarce autologous skin left for wound repair. The purpose of this study was to explore how to effectively use the limited remaining autologous skin to repair the extensive deep wound. METHODS: Human keratinocytes harvested from the foreskin were cultured and transfected with epidermal growth factors (EGFs) by an adenovirus vector (Ad-EGF). The expression and the biological activity of EGF in both the normal human keratinocytes and the EGF gene-modified human keratinocytes were quantified by ELISA assay and CCK-8 assay, respectively. The differentiated phenotype of epidermal cells was detected by immunofluorescence staining via CK10, CK14, and CK19 expressions. Rats were subjected to a full-thickness skin loss (3.3 cm × 3.0 cm) on the dorsum, which was repaired with the EGF gene-modified human keratinocyte suspension and autologous microskin and covered with the allogeneic skin. The wound healing was quantified, and the expression of EGF mRNA was measured by RT-PCR. RESULTS: The EGF gene-modified human keratinocytes highly expressed EGF. CK10, CK14, and CK19 as keratinocyte differentiation markers were increased in the EGF gene-modified human keratinocytes. Wound healing was accelerated remarkably by the combination of autologous microskin grafting and EGF gene-modified human keratinocytes in vivo, and a very high EGF mRNA expression was observed in EGF gene-modified human keratinocytes groups on days 7 and 14 compared with other groups. DISCUSSION/CONCLUSION: The EGF gene-modified human keratinocyte suspension may serve as promising seed cells which can effectively secrete EGF to accelerate wound repair in combination with autologous microskin grafting and reduce the autologous skin requirement for wound repair.

High-Intensity Pulsed Electromagnetic Field-Mediated Gene Electrotransfection In Vitro.

A high-intensity pulsed electromagnetic field (HI-PEMF) is a non-invasive and non-contact delivery method and may, as such, have an advantage over gene electrotransfer mediated by conventional electroporation using contact electrodes. Due to the limited number of in vitro studies in the field of gene electrotransfection by HI-PEMF, we designed experiments to investigate and demonstrate the feasibility of such a technique for the non-viral delivery of genetic material into cells in vitro. We first showed that HI-PEMF causes DNA adsorption to the membrane, a generally accepted prerequisite step for successful gene electrotransfection. We also showed that HI-PEMF can induce gene electrotransfection as the application of HI-PEMF increased the percentage of GFP-positive cells for two different combinations of pDNA size and concentration. Furthermore, by measuring the uptake of larger molecules, i.e., fluorescently labelled dextrans of three different sizes, we showed endocytosis to be a possible mechanism for introducing large molecules into cells by HI-PEMF.

MicroRNA-22-3p Restrains the Proliferation, Phenotypic Transformation, and Migration of Vascular Smooth Muscle Cells by Manipulating TOMM40.

microRNA (miR) -22-3p has been confirmed to be engaged in the phenotype transformation and proliferation of vascular smooth muscle cells (VSMCs), which is intimately correlated with restenosis. The current research set out to explore the detailed mechanism and function of miR-22-3p in VSMC proliferation, phenotype transformation, and migration via the translocase of outer mitochondrial membrane (TOMM40). Peripheral blood samples were acquired from patients with in-stent restenosis (ISR) after percutaneous coronary intervention (PCI), with subsequent quantitative reverse transcription (qRT) -polymerase chain reaction (PCR) and Western blot analyses of miR-22-3p and TOMM40 expression. After miR-22-3p-inhibitor, oe-TOMM40, and sh-TOMM40 were transfected into VSMCs, Cell Counting Kit (CCK) -8 assay, scratch test, and Western blot analysis were implemented to measure the VSMC proliferation, migration, and matrix metallopeptidase 9 (MMP9), α-smooth muscle actin (SMA), smooth muscle-myosin heavy chain (SM-MHC), and osteopontin (OPN) expressions, respectively. In addition, human umbilical vein endothelial cell (HUVEC) proliferation was examined by CCK-8 assay. The binding relationship between miR-22-3p and TOMM40 was assessed by dual luciferase reporter and RNA immunoprecipitation assays. The peripheral blood of patients with ISR after PCI had low expression of miR-22-3p and high expression of TOMM40. The mechanistic analysis reported the negative targeting relationship between miR-22-3p and TOMM40. Down-regulating miR-22-3p or up-regulating TOMM40 elevated the proliferation, migration, and phenotype transformation of VSMCs. miR-22-3p inhibitor had no evident impact on HUVEC proliferation. In addition, rescue assays displayed that TOMM40 silencing annulled miR-22-3p inhibition-enhanced VSMC proliferation, migration, and phenotype transformation. Conclusively, miR-22-3p could repress VSMC proliferation, phenotypic transformation, and migration by targeting TOMM40, which might be a possible treatment candidate for restenosis after PCI in patients with cardiovascular disease.

Ultrasound-Targeted Microbubble Destruction Mediates Gene Transfection for Beta-Cell Regeneration and Glucose Regulation.

Ultrasound-targeted microbubble destruction (UTMD) mediates gene transfection with high biosafety and thus has been promising toward treatment of type 1 diabetes. However, the potential application of UTMD in type 2 diabetes (T2D) is still limited, due to the lack of systematic design and dynamic monitoring. Herein, an efficient gene delivery system is constructed by plasmid deoxyribonucleic acid (DNA) encoding glucagon-like peptide 1 (GLP-1) in ultrasound-induced microbubbles, toward treatment of T2D in macaque. The as designed UTMD afforded enhancement of cell membrane penetration and GLP-1 expression in macaque, which is characterized by ultrasound-guided biopsy to monitor the dynamic process of islet cells for 6 months. Also, improvement of pancreatic beta cell regeneration, and regulation of plasma glucose in macaque with T2D is achieved. The approach would serve as promising alternatives for the treatment of T2D.

Structure of LINC00511-siRNA-conjugated nanobubbles and improvement of cisplatin sensitivity on triple negative breast cancer.

The drug resistance of triple negative breast cancer (TNBC) is considered as a major obstacle for the curative effect of chemotherapy. Long intergenic noncoding RNA 00511 (LINC00511) has been considered as a target gene of drug resistance. A novel theranostic agent loaded with LINC00511-siRNA to deliver siRNA was structured, and the responses of drug sensitivity in TNBC were detected. Next-generation high-throughput RNA sequencing (RNA-Seq) was performed to accurately analyze the differential expression of mRNAs and lncRNA targets after LINC00511-siRNA transfection with low-frequency ultrasound (LFUS). The LINC00511-siRNA conjugated nanobubble complexes showed appropriate characterization, with a mean diameter of 516.1 ± 24.7 nm and a zeta potential of -38.05 ± 0.24 mV. The transfection efficiency of nanobubble complexes was approximately 50% with LFUS. By RNA-Seq, the differential expressions of lncRNA transcripts and mRNA transcripts were identified, and then analyzed. The GO and KEGG enrichment analyses revealed the TNBC drug resistance related target genes and pathways. The combination of LFUS irradiation and nanobubble complexes is regarded as an efficient and safe method for siRNA transfection. The TNBC drug resistance occurs as a result of synergistic reactions between a variety of genes and a variety of pathways.

Continuous monitoring of cell transfection efficiency with micropatterned substrates.

The effect of cell-cell contact on gene transfection is mainly unknown. Usually, transfection is carried out in batch cell cultures without control over cellular interactions, and efficiency analysis relies on complex and expensive protocols commonly involving flow cytometry as the final analytical step. Novel platforms and cell patterning are being studied to control cellular interactions and improve quantification methods. In this study, we report the use of surface patterning of fibronectin for the generation of two types of mesenchymal stromal cell patterns: single-cell patterns without cell-to-cell contact, and small cell-colony patterns. Both scenarios allowed the integration of the full transfection process and the continuous monitoring of thousands of individualized events by fluorescence microscopy. Our results showed that cell-to-cell contact clearly affected the transfection, as single cells presented a maximum transfection peak 6 h earlier and had a 10% higher transfection efficiency than cells with cell-to-cell contact.

Effects of combination TGF-B1 transfection and platelet rich plasma (PRP) on three-dimension chondrogenic differentiation of rabbit dental pulp-derived mesenchymal stem cells.

Aim: The aim of this study was to evaluate the effects of standard culture medium and chondrogenic differentiation medium with PRP on chondrogenic differentiation of rabbit dental pulp-derived mesenchymal stem cells (rabbit DPSCs) that are transfected with transforming growth factor-beta 1 (TGF-B1) gene, based on the hypothesis of TGF- B1 and PRP can be effective on the chondrogenesis of stem cells. Materials and Methods: Rabbit DPSCs were characterized by using flow cytometry, immunofluorescent staining, quantitative Real Time Polymerase Chain Reaction (qRT-PCR) and differentiation tests. For the characterization, CD29, CD44 and CD45 mesenchymal cell markers were used. Rabbit DPSCs were transfected with TGF-B1 gene using electroporation technique in group 1; with PRP 10% in group 2; with chondrogenic medium in group 3; with both chondrogenic medium and PRP in group 4. DPSCs were cultured in medium with 10% inactive PRP in group 5, chondrogenic medium in group 6, chondrogenic medium with PRP 10% in group 7. SOX9, MMP13 and Aggrecan gene expression levels were evaluated in 3, 6, 12. and 24. days by qRT-PCR. Results: The expression levels of SOX9, MMP13 and Aggrecan were higher in group 2, 3 and group 7 in 3th day however in 24th day group 7 and group 2 were found higher. The expression levels changed by time-dependent. The extracellular matrix of the cells in experimental groups were positively stained with safranin O and toluidine blue. Conclusion: The combination in culture medium of TGF-B1 gene transfection and 10% PRP accelerates the chondrogenic differentiation of DPSCs.

Multicomponent Polymerization toward Cationic Polymers for Efficient Gene Delivery.

A new class of cationic polymers containing tertiary amine, thioether, and hydroxyl groups are prepared via a catalyst-free, multicomponent polymerization method using dithiol, formaldehyde, and di-sec-amine with a ratio of 1:2:1, to access a library of water-soluble polymers with well-defined structures and suitable molecular weights (Mw ranging from 5000 to 8000 Da) in high yields (up to 90%). Such polycations are demonstrated to be promising nonviral gene delivery vectors with high transfection efficiency (up to 3.5-fold of PEI25k) and low toxicity with multiple functionalities: 1) efficient gene condensation by tertiary amine groups; 2) reactive oxygen species scavenging by thioether groups; and 3) positive charge shielding by hydroxyl groups. Both the thioether and hydroxyl groups are contributed to reduce the cytotoxicity of the polycations by tuning the oxidative stress and preventing the undesired serum binding. The optimized polycations can achieve high transfection efficiency under the serum conditions, indicating the great potential as a nonviral gene delivery vector candidate for clinical application.

Degradable cationic polyesters via ring-opening copolymerization of valerolactones as nanocarriers for the gene delivery.

The development of cationic polymers as non-viral gene vectors has been hurdled by their high toxicity, thus degradable and biocompatible polymers are urgently demanded. Herein, five polyesters (B3a-B3e) were synthesized based on the ring-opening copolymerization between α-allyl-δ-valerolactone and δ-valerolactone derivatives decorated with alkyl or alkoxyl chains of different lengths, followed by the modification with 1,5,9-triazacyclododecyl ([12]aneN3) through thiol-ene click reactions. The five polyesters effectively condensed DNA into nanoparticles. Of them, B3a with a shorter alkyl chain and B3d with more positive charged units showed stronger DNA condensing performance and can completely retard the migration of DNA at N/P = 1.6 in the presence of DOPE. B3b/DOPE with a longer alkyl chain exhibited the highest transfection efficiency in HeLa cells with 1.8 times of 25 kDa PEI, while B3d/DOPE with more positive charged units exhibited highest transfection efficiency in A549 cells with 2.3 times of 25 kDa PEI. B3b/DOPE and B3d/DOPE successfully delivered pEGFP into zebrafish, which was superior to 25 kDa PEI (1.5 folds and 1.1 folds, respectively). The cytotoxicity measurements proved that the biocompatibility of these polyesters was better than 25 kDa PEI, due to their degradable property in acid environment. The results indicated that these cationic polyesters can be developed as potential non-viral gene vectors for DNA delivery.

Integration of [12]aneN3 and Acenaphtho[1,2-b]quinoxaline as non-viral gene vectors with two-photon property for enhanced DNA/siRNA delivery and bioimaging.

Two-photon fluorescent Acenaphtho[1,2-b]quinoxaline (ANQ) and the hydrophilic di-(triazole-[12]aneN3) moieties were combined through an alkyl chain (ANQ-A-M) or a ß-hairpin motif with two aromatic γ-amino acid residues (ANQ-H-M) to explore their capabilities for in vitro and in vivo gene delivery and tracing. ANQ-A-M and ANQ-H-M showed the same maximum absorption at 420 nm, and their fluorescent intensities around 650 nm were varied in different solvents and became poor in the protic solvents. Gel electrophoresis assays indicated that both compounds completely retarded the migration of pDNA at 20 µM in the presence of DOPE. However, the DNA condensation with ANQ-H-M was not reversible, and the particle size of the corresponding complexes were larger indicated from the SEM and DLS measurements. In vitro transfections indicated ANQ-A-M/DOPE achieved Luciferase and GFP expressions were to be 7.9- and 5.7-fold of those by Lipo2000 in A549 cells respectively. However, ANQ-H-M showed very poor transfection efficiency in Luciferase expression. With the help of single/two-photon fluorescence imaging it clearly demonstrated that the successful transfection of ANQ-A-M was attributed to its cellular uptake, apparent lysosomal escape, and reversible release of DNA; and the poor transfection of ANQ-H-M was resulted from the aggregation of the DNA complexes which prevented them from the cellular uptake, and also the strong binding ability which is not easy to release DNA. ANQ-A-M/DOPE also exhibited robust gene silencing (83% knockdown of Luciferase) and GFP expression (2.47-fold higher) efficiency compared with Lipo2000 in A549 and zebrafish, respectively. The work demonstrated that the linkage structure between fluorescent and di(triazole-[12]aneN3) played the important role for their gene delivery performance, and that ANQ-A-M represents a vector with the strong transfection efficiency in vitro and in vivo as well as the efficient real time bioimaging properties, which is potential for the development in biomedical research.

Non-viral, direct neuronal reprogramming from human fibroblast using a polymer-functionalized nanodot.

Among various strategies to treat neurodegenerative disorders, cell replacement therapies have drawn much attention recently. Such a trend led to the increase in demand for the rare and specialized cells, followed by the outburst development of various cell reprogramming strategies. However, several limitations on these conventional methods remain to be solved, including the genetic instability of the viral vectors and the high cytotoxicity or poor performance of the non-viral carriers. Therefore, non-viral methods need to be developed to ensure safe and efficient cell reprogramming. Here, we introduce a polymer-modified nano-reagent (Polymer-functionalized Nanodot, PolyN) for the safe and efficient, non-viral direct cell reprogramming. PolyN facilitated the highly efficient contemporary overexpression of the transgene compared to the conventional reagent. With our nano-reagent, we demonstrated the SOX2-mediated cell reprogramming and successfully generated the neuron-like cell from the human fibroblast.