Evaluation of PEG-transferrin-PEI nanocomplex as a gene delivery agent

Polyethylenimine (PEI) has been shown to be an efficient nonviral delivery vector. To improve its specificity and reduce its cytotoxicity, PEI should be modified. Transferrin (Tf) is a cell-binding ligand and Tf-receptors are expressed in malignant cells. Modification of cationic polymer by polyethylene glycol (PEG) can reduce the protein interaction and cell cytotoxicity of delivery vectors. We have synthesized PEG-Tf-PEI conjugate as an efficient and safe carrier of plasmid DNA (pDNA). Nanocomplexes of conjugates with pDNA were characterized by measuring the particle size and the surface charge. Transfection efficiency of nanocomplexes in Jurkat cells was improved and cytotoxicity was decreased compared with those of PEI complex. This was due to a reduction in the membrane damaging effect via shielding of the positive charge on the nanocomplex surface by PEG.     

Novel redox nanomedicine improves gene expression of polyion complex vector

Abstract

Gene therapy has generated worldwide attention as a new medical technology. While non-viral gene vectors are promising candidates as gene carriers, they have several issues such as toxicity and low transfection efficiency. We have hypothesized that the generation of reactive oxygen species (ROS) affects gene expression in polyplex supported gene delivery systems. The effect of ROS on the gene expression of polyplex was evaluated using a nitroxide radical-containing nanoparticle (RNP) as an ROS scavenger. When polyethyleneimine (PEI)/pGL3 or PEI alone was added to the HeLa cells, ROS levels increased significantly. In contrast, when (PEI)/pGL3 or PEI was added with RNP, the ROS levels were suppressed. The luciferase expression was increased by the treatment with RNP in a dose-dependent manner and the cellular uptake of pDNA was also increased. Inflammatory cytokines play an important role in ROS generation in vivo. In particular, tumor necrosis factor (TNF)-α caused intracellular ROS generation in HeLa cells and decreased gene expression. RNP treatment suppressed ROS production even in the presence of TNF-α and increased gene expression. This anti-inflammatory property of RNP suggests that it may be used as an effective adjuvant for non-viral gene delivery systems.     

A Highly Emissive Conjugated Polyelectrolyte Vector for Gene Delivery and Transfection

An intrinsically fluorescent cationic polyfluorene ( CCP ) has been designed, synthesized, characterized, and examined as a plasmid DNA (pDNA) delivery vector. This material facilitates nucleic acid binding, encapsulation and efficient cellular uptake. CCP can effectively protect pDNA against nuclease degradation, which is necessary for gene carriers. Green fluorescent protein (GFP) expression experiments reveal that CCP can achieve efficient delivery and transfection of pDNA encoding GFP gene with 92% efficiency, which surpasses that of commercial transfection agents, lipofectamine 2000 (Lipo) and polyethylenimine (PEI). CCP is also highly fluorescent, with 43% quantum yield in water, and exhibits excellent photostability, which allows for real‐time tracking the location of gene delivery and transfection. These features and capabilities represent a major step toward designing and applying conjugated polymers that function in both imaging and therapeutic applications.     

Encapsulation,Visualization and Expression of Genes with Biomimetically Mineralized Zeolitic Imidazolate Framework‐8 (ZIF‐8)

Recent work in biomolecule‐metal–organic framework (MOF) composites has proven to be an effective strategy for the protection of proteins. However, for other biomacromolecules such as nucleic acids, the encapsulation into nano MOFs and the related characterizations are in their infancy. Herein, encapsulation of a complete gene‐set in zeolitic imidazolate framework‐8 (ZIF‐8) MOFs and cellular expression of the gene delivered by the nano MOF composites are reported. Using a green fluorescent protein (GFP) plasmid (plGFP) as a proof‐of‐concept genetic macromolecule, successful transfection of mammalian cancer cells with plGFP for up to 4 days is shown. Cell transfection assays and soft X‐ray cryo‐tomography (cryo‐SXT) demonstrate the feasibility of DNA@MOF biocomposites as intracellular gene delivery vehicles. Expression occurs over relatively prolonged time points where the cargo nucleic acid is released gradually in order to maintain sustained expression.     

Encapsulation of Single Plasmonic Nanoparticles within ZIF‐8 and SERS Analysis of the MOF Flexibility

Hybrid nanostructures composed of metal nanoparticles and metal‐organic frameworks (MOFs) have recently received increasing attention toward various applications due to the combination of optical and catalytic properties of nanometals with the large internal surface area, tunable crystal porosity and unique chemical properties of MOFs. Encapsulation of metal nanoparticles of well‐defined shapes into porous MOFs in a core–shell type configuration can thus lead to enhanced stability and selectivity in applications such as sensing or catalysis. In this study, the encapsulation of single noble metal nanoparticles with arbitrary shapes within zeolitic imidazolate‐based metal organic frameworks (ZIF‐8) is demonstrated. The synthetic strategy is based on the enhanced interaction between ZIF‐8 nanocrystals and metal nanoparticle surfaces covered by quaternary ammonium surfactants. High resolution electron microscopy and tomography confirm a complete core–shell morphology. Such a well‐defined morphology allowed us to study the transport of guest molecules through the ZIF‐8 porous shell by means of surface‐enhanced Raman scattering by the metal cores. The results demonstrate that even molecules larger than the ZIF‐8 aperture and pore size may be able to diffuse through the framework and reach the metal core.     

Hybrid polymer-grafted multiwalled carbon nanotubes for in vitro gene delivery

Carbon nanotubes (CNTs) consist of carbon atoms arranged in sheets of graphene rolled up into cylindrical shapes. This class of nanomaterials has attracted attention because of their extraordinary properties, such as high electrical and thermal conductivity. In addition, development in CNT functionalization chemistry has led to an enhanced dispersibility in aqueous physiological media which indeed broadens the spectrum for their potential biological applications including gene delivery. The aim of this study is to determine the capability of different cationic polymer-grafted multiwalled carbon nanotubes (MWNTs) (polymer-g-MWNTs) to efficiently complex and transfer plasmid DNA (pCMV-βGal) in vitro without promoting cytotoxicity. Carboxylated MWNT is chemically conjugated to the cationic polymers polyethylenimine (PEI), polyallylamine (PAA), or a mixture of the two polymers. In order to explore the potential of these polymer-g-MWNTs as gene delivery systems, we first study their capacity to complex plasmid DNA (pDNA) using agarose gel electrophoresis. Gel migration studies confirm pDNA binding to polymer-g-MWNT with different affinities, highest for PEI-g-MWNT and PEI/PAA-g-CNT constructs. β-galactosidase expression is assessed in human lung epithelial (A549) cells, and the cytotoxicity is determined by modified LDH assay after 24 h incubation period. Additionally, PEI-g-MWNT and/or PEI/PAA-g-MWNT reveal an improvement in gene expression when compared to the naked pDNA or to the equivalent amounts of PEI polymer alone. Mechanistically, pDNA was delivered by the polymer-g-MWNT constructs via a different pathway compared to those used by polyplexes. In conclusion, polymer-g-MWNTs may be considered in the future as a versatile tool for efficient gene transfer in cancer cells in vitro, provided their toxicological profile is established.     

Gold Nanoparticles Capped with Polyethyleneimine for Enhanced siRNA Delivery

An efficient and safe delivery system for small interfering RNA (siRNA) is required for clinical application of RNA interfering therapeutics. Polyethyleneimine (PEI)‐capped gold nanoparticles (AuNPs) are successfully manufactured using PEI as the reductant and stabilizer, which bind siRNA at an appropriate weight ratio by electrostatic interaction and result in well‐dispersed nanoparticles with uniform structure and narrow size distribution. With siRNA binding, PEI‐capped AuNPs induce more significant and enhanced reduction in targeted green fluorescent protein expression in MDA‐MB‐435s cells, though more internalized PEI/siRNA complexes in cells are evidenced by confocal laser scanning microscopy observation and fluorescence‐activated cell sorting analyses. PEI‐capped AuNPs/siRNA targeting endogenous cell‐cycle kinase, an oncogene polo‐like kinase 1 (PLK1), display significant gene expression knockdown and induce enhanced cell apoptosis, whereas it is not obvious when the cells are treated with PLK1 siRNA using PEI as the carrier. Without exhibiting cellular toxicity, PEI‐capped AuNPs appear to be suitable as a potential carrier for intracellular siRNA delivery.     

An E3‐14.7K Peptide that Promotes Microtubules‐Mediated Transport of Plasmid DNA Increases Polyplexes Transfection Efficiency

Chemical vectors as cationic polymers and cationic lipids are promising alternatives to viral vectors for gene therapy. Beside endosome escape and nuclear import, plasmid DNA (pDNA) migration in the cytosol toward the nuclear envelope is also regarded as a limiting step for efficient DNA transfection with non‐viral vectors. Here, the interaction between E3‐14.7K and FIP‐1 to favor migration of pDNA along microtubules is exploited. E3‐14.7K is an early protein of human adenoviruses that interacts via FIP‐1 (Fourteen.7K Interacting Protein 1) protein with the light‐chain components of the human microtubule motor protein dynein (TCTEL1). This peptide is conjugated with pDNA and mediates interaction of pDNA in vitro with isolated microtubules as well as with microtubules in cellulo. Videomicroscopy and tracking treatment of images clearly demonstrate that P79‐98/pDNA conjugate exhibits a linear transport with large amplitude along microtubules upon 2 h transfection with polyplexes whereas control pDNA conjugate exhibits small non‐directional movements in the cytoplasm. Remarkably, P79‐98/peGFP polyplexes enhance by a factor 2.5 (up to 76%) the number of transfected cells. The results demonstrate, for the first time, that the transfection efficiency of polyplexes can be drastically increased when the microtubules migration of pDNA is facilitated by a peptide allowing pDNA docking to TCTEL1. This is a real breakthrough in the non viral gene delivery field that opens hope to build artificial viruses.     

Semiconducting Polymer Nanocavities: Porogenic Synthesis,Tunable Host–Guest Interactions,and Enhanced Drug/siRNA Delivery

Nanocavities composed of lipids and block polymers have demonstrated great potential in biomedical applications such as sensors, nanoreactors, and delivery vectors. However, it remains a great challenge to produce nanocavities from fluorescent semiconducting polymers owing to their hydrophobic rigid polymer backbones. Here, we describe a facile, yet general strategy that combines photocrosslinking with nanophase separation to fabricate multicolor, water‐dispersible semiconducting polymer nanocavities (PNCs). A photocrosslinkable semiconducting polymer is blended with a porogen such as degradable macromolecule to form compact polymer dots (Pdots). After crosslinking the polymer and removing the porogen, this approach yields semiconducting polymer nanospheres with open cavities that are tunable in diameter. Both small molecules and macromolecules can be loaded in the nanocavities, where molecular size can be differentiated by the efficiency of the energy transfer from host polymer to guest molecules. An anticancer drug doxorubicin (Dox) is loaded into the nanocavities and the intracellular release is monitored in real time by the fluorescence signal. Finally, the efficient delivery of small interfering RNA (siRNA) to silence gene expression without affecting cell viability is demonstrated. The combined features of bright fluorescence, tunable cavity, and efficient drug/siRNA delivery makes these nanostructures promising for biomedical imaging and drug delivery.     

Cholesterol tethered bioresponsive polycation as a candidate for gene delivery

The efficient unpacking of viral protein shell gave the inspiration for the synthesized vectors. In this research, novel cholesterol tethered bioresponsive polyethylenimine (PEI) was specially designed via disulfide-containing cross-linker. The cholesterol lipid had proved to increase the permeability of gene vector through cell membrane. The acid–base titration indicated that the synthesized polycation possessed efficient proton sponge effect, which was suggested to increase endosomal release of pDNA complexes into the cytoplasm. The cholesterol tethered polycation could effectively induce DNA condensation and form spherical particles with diameter about 200 nm at N/P ratio of 10. At glutathione concentration of 3 mM, the polyplexes were unpacked due to the bioresponsive cleavage of the disulfide bonds. The in-vitro experiment indicated that the polyplexes showed efficient transfection efficiency to HEK293T cells. All the results indicated that the bioresponsive polycation could be served as an effective trigger to control the release of DNA at the intracellular environment. The novel bioresponsive polycation might have great potential in non-viral gene delivery research and application.     

Smart Carbon Nanotubes with Laser‐Controlled Behavior in Gene Delivery and Therapy through a Non‐Digestive Trafficking Pathway

Near‐infrared (NIR) laser‐controlled gene delivery presents some benefits in gene therapy, inducing enhanced gene transfection efficiency. In this study, a “photothermal transfection” agent is obtained by wrapping poly(ethylenimine)‐cholesterol derivatives (PEI‐Chol) around single‐walled carbon nanotubes (SWNTs). The PEI‐Chol modified SWNTs (PCS) are effective in compressing DNA molecules and protecting them from DNaseI degradation. Compared to the complexes formed by PEI with DNA (PEI/DNA), complexes of PCS and DNA that are formed (PCS/DNA) exhibit a little lower toxicity to HEK293 and HeLa cells under the same PEI molecule weight and weight ratios. Notably, caveolae‐mediated cellular uptake of PCS/DNA occurs, which results in a safer intracellular transport of the gene due to the decreased lysosomal degradation in comparison with that of PEI/DNA whose internalization mainly depends on clathrin rather than caveolae. Furthermore, unlike PEI/DNA, PCS/DNA exhibits a photothermal conversion ability, which promotes DNA release from PCS under NIR laser irradiation. The NIR laser‐mediated photothermal transfection of PCS_10K/plasmid TP53 (pTP53) results in more apoptosis and necrosis of HeLa cells in vitro than other groups, and achieves a higher tumor‐growth inhibition in vivo than naked pTP53, PEI_25K/pTP53, and PCS_10K/pTP53 alone. The enhanced transfection efficiency of PCS/DNA can be attributed to more efficient DNA internalization into the tumor cells, promotes detachment of DNA from PCS under the mediation of NIR laser and higher DNA stability in the cells due to caveolae‐mediated cellular uptake of the complexes.     

Direct Reprogramming of Human Suspension Cells into Mesodermal Cell Lineages via Combined Magnetic Targeting and Photothermal Stimulation by Magnetic Graphene Oxide Complexes

Suspension cells can provide a source of cells for cellular reprogramming, but they are difficult to transfect by nonviral vectors. An efficient and safe nonviral vector (GO‐Fe₃O₄‐PEI complexes) based on iron oxide nanoparticle (Fe₃O₄)‐decorated graphene oxide (GO) complexed with polyethylenimine (PEI) for the first time is developed for delivering three individual episomal plasmids (pCXLE‐hOCT3/4‐shp53, pCXLE‐hSK, and pCXLE‐hUL) encoding pluripotent‐related factors of Oct3/4, shRNA against p53, Sox2, Klf4, L‐Myc, and Lin28 into human peripheral blood mononuclear cells (PBMCs) simultaneously. The combined treatment of magnetic stirring and near‐infrared (NIR)‐laser irradiation, which can promote contact between the complexes and floating cells and increase the cell membrane permeability, respectively, is used to conduct multiple physical stimulations for suspension PBMCs transfection. The PCR analysis shows that the combinatorial effect of magnetic targeting and photothermal stimulation obviously promoted the transfection efficiency of suspension cells. The transfected cells show positive expression of the pluripotency markers, including Nanog, Oct4, and Sox2, and have potential to differentiate into mesoderm and ectoderm cells. The results demonstrate that the GO‐Fe₃O₄‐PEI complex provides a safe, convenient, and efficient tool for reprogramming PBMCs into partially induced pluripotent stem cells, which are able to rapidly transdifferentiate into mesodermal lineages without full reprogramming.     

Transfection ability and intracellular DNA pathway of nanostructured gene-delivery systems

Considerable efforts have been devoted to the design of structured materials with functional properties. Polyelectrolyte multilayer films are now a well-established nanostructured concept with numerous potential applications, in particular as biomaterial coatings. This technique allows the preparation of nanostructured architectures exhibiting specific properties for cell-activation control and local drug delivery. In this study, we used a multilayered system made of poly-(l-lysine)/hyaluronic acid (PLL/HA) as a reservoir for active DNA complexes with nonviral gene-delivery vectors, PLL, beta-cyclodextrin (CD), and PLL-CD. When embedded into the multilayered films, the transfection efficiencies of the DNA complexes and the cell viability were improved. The highest transfection efficiency was obtained with the PLL-CD/plasmid DNA (pDNA) complexes. We found that this high transfection efficiency was related to an efficient internalization of the complexes in the cell cytoplasm and selected nuclei domains through a nonendocytotic pathway. For the first time, we report the intracellular pathway of the pDNA in complexes incorporated into the multilayered system.     

Preparation of dexamethasone-based cationic liposome and its application to gene delivery in vitro

In this study, dexamethasone was conjugated to PAMAM dendrimer (generation 0) and its gene transfection efficiency was investigated. To make a liposomal solution for gene delivery, DOPE was used as a fusogenic helper lipid. In gel retardation assay, PAMAM-dexamethasone conjugate (PAM-Dex)/DOPE liposome/DNA complex was completely retarded at 8:1 N/P (nitrogen/phosphate) ratio. The physicochemical characteristics are studied by measuring the average size distribution and zeta-potential values of the complexes. In vitro transfection assay showed that the PAM-Dex/DOPE liposome/DNA complex displayed higher gene delivery efficiency compared to PAMAM/DNA complex. In addition, PAM-Dex/DOPE liposome showed the lowest toxicity compared to PAMAM, PEI 25 kD and Lipofectamine. These results indicate that PAM-Dex/DOPE liposome has a potential to be used as an efficient gene carrier for gene therapy.     

Synthesis of stable silica-dye hybrid nanomaterial as DNA carrier

A new method is proposed for the fabrication of fluorescence-labeled and amine-modified silica nanoparticles for application as nonviral vectors in gene delivery. Highly monodisperse, stable fluorescent silica nanoparticles were prepared using 2,5-bis(5-tert-butyl-2-benzoxazolyl)thiophene and the water-in-oil microemulsion method. The green-fluorescent-protein gene can be easily combined onto the positively charged surfaces of nanoparticles to form a nanoparticle-DNA complex. The nanoparticle-DNA complex successfully passed through various barriers into the HeLa and HEK 293 K cells. The cytotoxicity of the PEI-coated and BBOT-encapsulated silica nanoparticles on both the HeLa and HEK 293T cell lines was found to be at an acceptable level for use as gene carriers when the particle concentration was below 125 microg/ml. The fluorescence intracellular images confirm the successful delivery of the nanoparticle-DNA complex and gene expression. The present work suggests the potential use of dye-incorporated silica nanoparticles in nonviral gene delivery.     

Spray‐Dried Nanoparticle‐in‐Microparticle Delivery Systems (NiMDS) for Gene Delivery,Comprising Polyethylenimine (PEI)‐Based Nanoparticles in a Poly(Vinyl Alcohol) Matrix

Nucleic acid‐based therapies rely on efficient formulations for nucleic acid protection and delivery. As nonviral strategies, polymeric and lipid‐based nanoparticles have been introduced; however, biological efficacy and biocompatibility as well as poor storage properties due to colloidal instability and their unavailability as ready‐to‐use systems are still major issues. Polyethylenimine is the most widely explored and promising candidate for gene delivery. Polyethylenimine‐based polyplexes and their combination with liposomes, lipopolyplexes, are efficient for DNA or siRNA delivery in vitro and in vivo. In this study, a highly potent spray‐dried nanoparticle‐in‐microparticle delivery system is presented for the encapsulation of polyethylenimine‐based polyplexes and lipopolyplexes into poly(vinyl alcohol) microparticles, without requiring additional stabilizing agents. This easy‐to‐handle gene delivery device allows prolonged nanoparticle storage and protection at ambient temperature. Biological analyses reveal further advantages regarding profoundly reduced cytotoxicity and enhanced transfection efficacies of polyethylenimine‐based nanoparticles from the nanoparticle‐in‐microparticle delivery system over their freshly prepared counterparts, as determined in various cell lines. Importantly, this nanoparticle‐in‐microparticle delivery system is demonstrated as ready‐to‐use dry powder to be an efficient device for the inhalative delivery of polyethylenimine‐based lipopolyplexes in vivo, as shown by transgene expression in mice after only one administration.     

Synthetically engineered viruses: Can synthetic chemistry tame the nature?

Gene therapy is a promising tool to tackle challenging diseases at a molecular level. However, delivery of therapeutic nucleic acids to desired tissues and cells with high efficiency, versatility, and safety has been a fundamental technological gap in gene therapy. Viral and nonviral vectors offer advantages and disadvantages that can complement each other. Viral vectors exhibit high transduction efficiency with immunogenicity, mutagenesis, and limited versatility for structural and functional tenability. On the other hand, low transfection efficiency of nonviral vectors undermines their high flexibility for modification, low immunogenicity, and easy preparation. A number of attempts have been made to hybridize viral and nonviral vectors using genetic, physical, and chemical approaches. Synthetic engineering of viral vectors is reviewed here with (1) challenges in viral nucleic acid delivery pathways, in contrast to those of nonviral vectors, (2) design goals of incorporating synthetic molecules of broad types into viral vectors, and (3) methodology to modify and re-formulate viral vectors. Recent advances in synthetically engineered viral vectors for various biomedical applications are also discussed. This review clearly emphasizes the crucial roles of interdisciplinary approaches to developing ideal vectors in order to obtain desired properties for clinical success.     

Highly Ordered N‐Doped Carbon Dots Photosensitizer on Metal–Organic Framework‐Decorated ZnO Nanotubes for Improved Photoelectrochemical Water Splitting

In spite of having several advantages such as low cost, high chemical stability, and environmentally safe and benign synthetic as well as operational procedures, the full potential of carbon dots (CDs) is yet to be explored as photosensitizers due to the challenges associated with the fabrication of well‐arrayed CDs with many other photocatalytic heterostructures. In the present study, a unique combination of metal–organic framework (MOF)‐decorated zinc oxide (ZnO) 1D nanostructures as host and CDs as guest species are explored on account of their potential application in photoelectrochemical (PEC) water splitting performance. The synthetic strategy to incorporate well‐defined nitrogen‐doped carbon dots (N‐CDs) arrays onto a zeolitic imidazolate framework‐8 (ZIF‐8) anchored on ZnO 1D nanostructures allows a facile unification of different components which subsequently plays a decisive role in improving the material's PEC water splitting performance. Simple extension of such strategies is expected to offer significant advantages for the preparation of CD‐based heterostructures for photo(electro)catalytics and other related applications.     

A DNA‐Threaded ZIF‐8 Membrane with High Proton Conductivity and Low Methanol Permeability

Natural biomolecules have potential as proton‐conducting materials, in which the hydrogen‐bond networks can facilitate proton transportation. Herein, a biomolecule/metal–organic framework (MOF) approach to develop hybrid proton‐conductive membranes is reported. Single‐strand DNA molecules are introduced into DNA@ZIF‐8 membranes through a solid‐confined conversion process. The DNA‐threaded ZIF‐8 membrane exhibits high proton conductivity of 3.40 × 10^?4 S cm^?1 at 25 °C and the highest one ever reported of 0.17 S cm^?1 at 75 °C, under 97% relatively humidity, attributed to the formed hydrogen‐bond networks between the DNA molecules and the water molecules inside the cavities of the ZIF‐8, but very low methanol permeability of 1.25 × 10^?8 cm² s^?1 due to the small pore entrance of the DNA@ZIF‐8 membranes. The selectivity of the DNA@ZIF‐8 membrane is thus significantly higher than that of developed proton‐exchange membranes for fuel cells. After assembling the DNA@ZIF‐8 hybrid membrane into direct methanol fuel cells, it exhibits a power density of 9.87 mW cm^?2 . This is the first MOF‐based proton‐conductivity membrane used for direct methanol fuel cells, providing bright promise for such hybrid membranes in this application.