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Transfection efficiency of the immortalized human breast epithelial cell line MCF-10A remains an issue that needs to be resolved. In this study, it was aimed to deliver a recombinant DNA (pCMV-Azu-GFP) to the MCF-10A cells by the magnetofection method using magnetic nanoparticles (MNPs) and a simple magnet to accelerate the DNA delivery. Surface positively modified silica-coated iron oxide MNPs (MSNP-NH2) were produced and characterized via TEM, FTIR, and DLS analyses. The recombinant DNA (rDNA) was obtained by the integration of codon-optimized azurin to produce a fusion protein. Then, rDNA cloned in Escherichia coli cells was validated by sequence analysis. The electrostatically conjugated rDNA on MSNP-NH2 with an enhancer polyethyleneimine (PEI) was studied by agarose gel electrophoresis and the optimum conditions were determined to apply to the cell. A dose-dependent statistical difference was observed on treated cells based on the MTS test. The expression of the fusion protein after magnetofection was determined using laser scanning confocal microscope imaging and western blot analysis. It was observed that the azurin gene could be transferred to MCF-10A cells by magnetofection. Thus, when the azurin gene is used as a breast cancer treatment agent, it can be expressed in healthy cells without toxic effects. [ABSTRACT FROM AUTHOR]

Cancer is a disease that causes millions of deaths per year worldwide because conventional treatments have disadvantages such as unspecific tumor selectivity and unwanted toxicity. Most human solid tumors present hypoxic microenvironments and this promotes multidrug resistance. In this study, we present "Magnetogene nanoparticle vector" which takes advantage of the hypoxic microenvironment of solid tumors to increase selective gene expression in tumor cells and reduce unwanted toxicity in healthy cells; this vector was guided by a magnet to the tumor tissue. Magnetic nanoparticles (MNPs), chitosan (CS), and the pHRE-Luc plasmid with a hypoxia-inducible promoter were used to synthesize the vector called "Magnetogene nanoparticles" by ionic gelation. The hypoxic functionality of Magnetogene vector nanoparticles was confirmed in the B16F10 cell line by measuring the expression of the luciferase reporter gene under hypoxic and normoxic conditions. Also, the efficiency of the Magnetogene vector was confirmed in vivo. Magnetogene was administered by intravenous injection (IV) in the tail vein and directed through an external magnetic field at the site of tumor growth in C57Bl/6 mice. A Magnetogene vector with a size of 50 to 70 nm was directed and retained at the tumor area and gene expression was higher at the tumor site than in the others tissues, confirming the selectivity of this vector towards hypoxic tumor areas. This nanosystem, that we called the "Magnetogene vector" for systemic delivery and specific gene expression in hypoxic tumors controlled by an external magnetic designed to target hypoxic regions of tumors, can be used for cancer-specific gene therapies. [ABSTRACT FROM AUTHOR]

Abstract Background Magnetofection-mediated gene delivery shows great therapeutic potential through the regulation of the direction and degree of differentiation. Lumbar degenerative disc disease (DDD) is a serious global orthopaedic problem. However, even though intervertebral fusion is the gold standard for the treatment of DDD, its therapeutic effect is unsatisfactory. Here, we described a novel magnetofection system for delivering therapeutic miRNAs to promote osteogenesis and angiogenesis in patients with lumbar DDD. Results Co-stimulation with electromagnetic field (EMF) and iron oxide nanoparticles (IONPs) enhanced magnetofection efficiency significantly. Moreover, in vitro, magnetofection of miR-21 into bone marrow mesenchymal stem cells (BMSCs) and human umbilical endothelial cells (HUVECs) influenced their cellular behaviour and promoted osteogenesis and angiogenesis. Then, gene-edited seed cells were planted onto polycaprolactone (PCL) and hydroxyapatite (HA) scaffolds (PCL/HA scaffolds) and evolved into the ideal tissue-engineered bone to promote intervertebral fusion. Finally, our results showed that EMF and polyethyleneimine (PEI)@IONPs were enhancing transfection efficiency by activating the p38 MAPK pathway. Conclusion Our findings illustrate that a magnetofection system for delivering miR-21 into BMSCs and HUVECs promoted osteogenesis and angiogenesis in vitro and in vivo and that magnetofection transfection efficiency improved significantly under the co-stimulation of EMF and IONPs. Moreover, it relied on the activation of p38 MAPK pathway. This magnetofection system could be a promising therapeutic approach for various orthopaedic diseases. Graphical Abstract

Huntington's disease (HD) is an autosomal dominant disease affecting neurons predominantly in the striatum due to the production of the toxic huntingtin protein. Lowering the concentration of mutant huntingtin is a promising therapeutic approach, and a suitable delivery system is fascinating. Nanoparticles (NPs) minimize the host immune response and have no limit concerning the number of NPs administered. They are safe, targeted, and effective for RNA therapeutics providing a significant mode to cross the blood–brain barrier for a broad range of clinical applications. The present study generated and characterized magnetic NPs (MNPs) using the co-precipitation method with a mean particle size of around 10–20 nm. The dynamic light scattering and zeta potential measurements showed that NPs exhibited narrow size distribution and sufficient colloidal stability. These oleic acid-coated MNPs were further cross-linked with polyethyleneimine and designed to deliver interfering RNA into human embryonic kidney cells (HEK-293) driven by an external magnetic field. These MNPs showed low cytotoxicity with high transfection efficiency. Furthermore, a transient downregulation was observed in endogenous huntingtin protein obtained by RT-PCR and Western blot analysis. Thus, these MNPs may represent a promising and efficient platform for siRNA delivery and provide a potential treatment strategy for HD. [ABSTRACT FROM AUTHOR]

In recent years, nanotechnology has deployed a new set of theragnostic tools, including magnetic resonance contrast agents, nano-delivery systems and magnetic hyperthermia treatments in cancer therapy, exploiting not only the small size of nanoparticles, but also relevant nanoscale properties such as superparamagnetism. Specifically, magnetic nanostructures can be remotely manipulated by external magnetic fields, incrementing their possibilities not only for theragnosis, but also for biotech procedures. Genetic engineering processes involve a set of steps like extracting cells from complex environments, their selection and subsequent cultivation or modification by transfection and can benefit from the use of bioconjugated magnetic nanoparticles. Magnetofection of cells with genes or biological material uploaded on superparamagnetic nanoparticles attracted by a magnetic field greatly increases the efficiency, specificity and speed of the biotechnological procedure in gene transfer systems. This article presents a preliminary investigation into the enhanced transfection efficiency of fluorescent magnetic mesoporous silica nanostructures functionalized with mCherry plasmid, which were used to transfect HeLa cells in just 15 min via magnetic transfection. This method was compared to passive transfection (4 h) and conventional gene transfer using the commercial K2 Transfection System (16 h). The results demonstrated that the fluorescent magnetic mesoporous silica nanostructures were similarly effective to the commercial kit, without the need for reagents that increase costs in clinical therapy. Furthermore, viability assays conducted with HeLa cells showed negligible toxicity at concentrations of up to 50 μg/mL. [ABSTRACT FROM AUTHOR]

Background: Magnetofection-mediated gene delivery shows great therapeutic potential through the regulation of the direction and degree of differentiation. Lumbar degenerative disc disease (DDD) is a serious global orthopaedic problem. However, even though intervertebral fusion is the gold standard for the treatment of DDD, its therapeutic effect is unsatisfactory. Here, we described a novel magnetofection system for delivering therapeutic miRNAs to promote osteogenesis and angiogenesis in patients with lumbar DDD. Results: Co-stimulation with electromagnetic field (EMF) and iron oxide nanoparticles (IONPs) enhanced magnetofection efficiency significantly. Moreover, in vitro, magnetofection of miR-21 into bone marrow mesenchymal stem cells (BMSCs) and human umbilical endothelial cells (HUVECs) influenced their cellular behaviour and promoted osteogenesis and angiogenesis. Then, gene-edited seed cells were planted onto polycaprolactone (PCL) and hydroxyapatite (HA) scaffolds (PCL/HA scaffolds) and evolved into the ideal tissue-engineered bone to promote intervertebral fusion. Finally, our results showed that EMF and polyethyleneimine (PEI)@IONPs were enhancing transfection efficiency by activating the p38 MAPK pathway. Conclusion: Our findings illustrate that a magnetofection system for delivering miR-21 into BMSCs and HUVECs promoted osteogenesis and angiogenesis in vitro and in vivo and that magnetofection transfection efficiency improved significantly under the co-stimulation of EMF and IONPs. Moreover, it relied on the activation of p38 MAPK pathway. This magnetofection system could be a promising therapeutic approach for various orthopaedic diseases. [ABSTRACT FROM AUTHOR]

Nanoformulations for delivering nucleotides into cells as vaccinations as well as treatment of various diseases have recently gained great attention. Applying such formulations for a local treatment strategy, e.g., for cancer therapy, is still a challenge, for which improved delivery concepts are needed. Hence, this work focuses on the synthesis of superparamagnetic iron oxide nanoparticles (SPIONs) for a prospective "magnetofection" application. By functionalizing SPIONs with an active catechol ester (CafPFP), polyethyleneimine (PEI) was covalently bound to their surface while preserving the desired nanosized particle properties with a hydrodynamic size of 86 nm. When complexed with plasmid-DNA (pDNA) up to a weight ratio of 2.5% pDNA/Fe, no significant changes in particle properties were observed, while 95% of the added pDNA was strongly bound to the SPION surface. The transfection in A375-M cells for 48 h with low amounts (10 ng) of pDNA, which carried a green fluorescent protein (GFP) sequence, resulted in a transfection efficiency of 3.5%. This value was found to be almost 3× higher compared to Lipofectamine (1.2%) for such low pDNA amounts. The pDNA-SPION system did not show cytotoxic effects on cells for the tested particle concentrations and incubation times. Through the possibility of additional covalent functionalization of the SPION surface as well as the PEI layer, Caf-PEI-SPIONs might be a promising candidate as a magnetofection agent in future. [ABSTRACT FROM AUTHOR]

abstract: CRISPR-Cas9 genome editing systems have enormous promise in the areas of biomedical research and agriculture. While the effectiveness and utility of CRISPR have already been well established, there are many challenges in the successful application of CRISPR to plant systems. In this regard, nanotechnology could play a critical role in addressing some of the decisive challenges preventing CRISPR-mediated genome engineering in plants especially in recalcitrant species. Nanoparticles can be used as an efficient delivery agent to the targeted cell and enhance genome editing efficiency. The Nanoparticles-based delivery system can deliver functional gene, RNP, or siRNA intact into the plant cell resulting in the generation of genome-edited plants without transgenes. In this review, we have discussed various nanoparticle technologies that can overcome barriers associated with the CRISPR-mediated genetic transformation in plants, holding it to reach its full potential. CRISPR can be used to develop tolerance against various biotic and abiotic stresses. As well as, it may also be employed in improving food quality and productivity thereby providing food security to the ever-growing human population.

In recent years, nanotechnology has deployed a new set of theragnostic tools, including magnetic resonance contrast agents, nano-delivery systems and magnetic hyperthermia treatments in cancer therapy, exploiting not only the small size of nanoparticles, but also relevant nanoscale properties such as superparamagnetism. Specifically, magnetic nanostructures can be remotely manipulated by external magnetic fields, incrementing their possibilities not only for theragnosis, but also for biotech procedures. Genetic engineering processes involve a set of steps like extracting cells from complex environments, their selection and subsequent cultivation or modification by transfection and can benefit from the use of bioconjugated magnetic nanoparticles. Magnetofection of cells with genes or biological material uploaded on superparamagnetic nanoparticles attracted by a magnetic field greatly increases the efficiency, specificity and speed of the biotechnological procedure in gene transfer systems. This article presents a preliminary investigation into the enhanced transfection efficiency of fluorescent magnetic mesoporous silica nanostructures functionalized with mCherry plasmid, which were used to transfect HeLa cells in just 15 min via magnetic transfection. This method was compared to passive transfection (4 h) and conventional gene transfer using the commercial K2 Transfection System (16 h). The results demonstrated that the fluorescent magnetic mesoporous silica nanostructures were similarly effective to the commercial kit, without the need for reagents that increase costs in clinical therapy. Furthermore, viability assays conducted with HeLa cells showed negligible toxicity at concentrations of up to 50 μg/mL.

Abstract This study aimed to improve the magnetofection of MG-63 osteoblasts by integrating the use of a novel uniform magnetic field with low molecular weight polyethylenimine modified superparamagnetic iron oxide nanoparticles (PEI-SPIO-NPs). The excellent characteristics of PEI-SPIO-NPs such as size, zeta potential, the pDNA binding and protective ability were determined to be suitable for gene delivery. The novel uniform magnetic field enabled polyethylenimine-modified superparamagnetic iron oxide nanoparticles/pDNA complexes (PEI-SPIO-NPs/pDNA complexes) to rapidly and uniformly distribute on the surface of MG-63 cells, averting local transfection and decreasing disruption of the membrane caused by the centralization of positively charged PEI-SPIO-NPs, thereby increasing the effective coverage of magnetic gene carriers during transfection, and improving magnetofection efficiency. This innovative uniform magnetic field can be used to determine the optimal amount between PEI-SPIO-NPs and pDNA, as well as screen for the optimal formulation design of magnetic gene carrier under the homogenous conditions. Most importantly, the novel uniform magnetic field facilitates the transfection of PEI-SPIO-NPs/pDNA into osteoblasts, thereby providing a novel approach for the targeted delivery of therapeutic genes to osteosarcoma tissues as well as a reference for the treatment of other tumors.

Objective To prepare the polyethylenimine-superparamagnetic iron oxide nanoparticles-nanoparticles (PEI-SPION-NPs) and to observe the feasibility of PEI-SPION-NPs as targeted gene delivery vector to transefect into osteosarcoma cells and tissues in vivo. Methods We used the polyethylenimine (PEI) modified superparamagnetic iron oxide nanoparticles (SPION) to prepare the PEI-SPION-NPs by the dehydration condensation reaction. Their molecular structure, morphology, particle size, saturation magnetization and Zeta potential were detected. PEI-SPION-NPs were mixed with pDNA in different proportions，and agarose gel electrophoresis was performed to observe the binding ability and protective effect of PEI-SPION-NPs on pDNA. MG-63 cells were selected as target cells for transfection, which were cul‐ tured and then were divided into the group 1 (PEI-SPION-NPs/pDNA＋the static magnetic field), group 2（PolyMag200/ pDNA＋the static magnetic field), group 3（PEI-NPs/pDNA ), and the control group（naked pDNA)；the cell survival rate was determined by CCK-8 assay, the transfection effect was detected by inverted fluorescence microscope, and the transfection efficiency was measured by flow cytometry. The hypodermal osteosarcoma models pared, which were then divided into the control group（subcutaneous injection of normal saline), PEI-SPION-NPs group (subcutaneous injection of PEI-SPION-NPs/pGL3-Luc＋the static magnetic field), and PEI-NPs group (subcutaneous injection of PEI/pGL3-Luc complexes＋the static magnetic field). Fluorescence intensity of injection site was detected and body weight of nude mice in each group was measured at 5,6,7,8 and 9 weeks after osteosarcoma modeling, respectively. Results The hydrodynamic diameters of PEI-SPION-NPs were shorter than those of PEI-SPION-NP/pDNA complexes, the saturation magnetization of PEI-SPION-NPs was（21. 5±1. 6）emu/g and the zeta potential was (31. 2±1. 5) mV； PEI-SPION-NPs could bind to and protect pDNA，and can be successfully transfected into MG-63 cells. The cell survival rates of the group 1 and control group were higher than those of the group 2 and group3, nd the transfection efficiency of the group 1 was higher than those of the group 3 and control group（all P<0. 05). Both PEI-SPION-NPS/pGL3-Luc and PEI-NPS/pGL3-Luc could be successfully transfected into and expressed in the osteosarcoma tissues of nude mice, however, the fluorescence of the PEI-SPION-NPS group was more concentrated than that of the PEI-NPS group, and the body weight of nude mice in the PEI-SPION-NPS group was higher than that in the PEI-NPS group at 7 weeks after osteosarcoma modeling（both P<0. 01). Conclusion PEI-SPION-NP is an effective non-viral gene vector, which can be transfected into osteosarcoma cells and tissues in vivo under the guidance of the magnetic field. [ABSTRACT FROM AUTHOR]

Non-viral delivery systems are generally of low efficiency, which limits their use in gene therapy and editing applications. We previously developed a technology termed glycosaminoglycan (GAG)-binding enhanced transduction (GET) to efficiently deliver a variety of cargos intracellularly; our system employs GAG-binding peptides, which promote cell targeting, and cell penetrating peptides (CPPs), which enhance endocytotic cell internalization. Herein, we describe a further modification by combining gene delivery and magnetic targeting with the GET technology. We associated GET peptides, plasmid (p)-DNA, and iron oxide superparamagnetic nanoparticles (MNPs), allowing rapid and targeted GET-mediated uptake by application of static magnetic fields in NIH3T3 cells. This produced effective transfection levels (significantly higher than the control) with seconds to minutes of exposure and localized gene delivery two orders of magnitude higher in targeted over non-targeted cell monolayers using magnetic fields (in 15 min exposure delivering GFP reporter pDNA). More importantly, high cell membrane targeting by GET-DNA and MNP co-complexes and magnetic fields allowed further enhancement to endocytotic uptake, meaning that the nucleic acid cargo was rapidly internalized beyond that of GET complexes alone (GET-DNA). Magnetofection by MNPs combined with GET-mediated delivery allows magnetic field-guided local transfection in vitro and could facilitate focused gene delivery for future regenerative and disease-targeted therapies in vivo. [ABSTRACT FROM AUTHOR]

Bacterial magnetic particles (BMPs) are biosynthesized magnetic nano-scale materials with excellent dispersibility and biomembrane enclosure properties. In this study, we demonstrate that BMPs augment the ability of polyethylenimine (PEI) to deliver target DNA into difficult-to-transfect primary porcine liver cells, with transfection efficiency reaching over 30%. Compared with standard lipofection and polyfection, BMP-PEI gene vectors significantly enhanced the transfection efficiencies for the primary porcine liver cells and C2C12 mouse myoblast cell lines. To better understand the mechanism of magnetofection using BMP-PEI/DNA vectors, transmission electron microscopy (TEM) images of transfected Cos-7, HeLa, and HEP-G2 cells were observed. We found that the BMP-PEI/DNA complexes were trafficked into the cytoplasm and nucleus by way of vesicular transport and endocytosis. Our study builds support for the versatile BMP-PEI vector transfection system, which might be exploited to transfect a wide range of cell types or even to reach specific targets in the treatment of disease. Key Points: • We constructed a BMP-PEI gene delivery vector by combining BMPs and PEI. • The vector significantly enhanced transfection efficiencies in eukaryotic cell lines. • The transfection mechanism of this vector was explained in our study. [ABSTRACT FROM AUTHOR]

High transplant cell loss is a major barrier to translation of stem cell therapy for pathologies of the brain and spinal cord. Encapsulated delivery of stem cells in biomaterials for cell therapy is gaining popularity but experimental research has overwhelmingly used laboratory grade materials unsuitable for human clinical use - representing a further barrier to clinical translation. A potential solution is to use neurosurgical grade materials routinely used in clinical protocols which have an established human safety profile. Here, we tested the ability of Duragen Plus™ - a clinical biomaterial used widely in neurosurgical duraplasty procedures, to support the growth and differentiation of neural stem cells- a major transplant population being tested in clinical trials for neurological pathology. Genetic engineering of stem cells yields augmented therapeutic cells, so we further tested the ability of the Duragen Plus™ matrix to support stem cells engineered using magnetofection technology and minicircle DNA vectors- a promising cell engineering approach we previously reported (Journal of Controlled Release, 2016 a &b). The safety of the nano-engineering approach was analysed for the first time using sophisticated data-independent analysis by mass spectrometry-based proteomics. We prove that the Duragen Plus™ matrix is a promising biomaterial for delivery of stem cell transplant populations, with no adverse effects on key regenerative parameters. This advanced cellular construct based on a combinatorial nano-engineering and biomaterial encapsulation approach, could therefore offer key advantages for clinical translation. Unlabelled Image [ABSTRACT FROM AUTHOR]

Aim: We attempted to synthesize a magnetic gene carrier with poly(ethylenimine), dextran and iron oxide nanoparticles (PDIs) for miR-302b transfection in vitro and in vivo. Materials & methods: The nanoparticles were characterized for hydrodynamic properties, ζ potential and DNA-binding ability, evaluated by transmission electron microscopy. Cellular internalization, magnetofection efficiency and anti-osteosarcoma effects were investigated in osteosarcoma (OS) cells and OS-bearing nude mice. Results: PDIs were successfully prepared and showed mild cytotoxicity. A magnetic field efficiently enabled transport of PDI/pmiR302b to OS cells in OS-bearing nude mice, exerting the anti-osteosarcoma effect of miR-302b at the tumor site. The inhibitory effect of miR-302b on osteosarcoma-bearing nude mice may be attributed to regulation of the Hippo pathway through YOD1. Conclusion: Low-cytotoxic PDIs have potential applications as a magnetic transport carrier for future osteosarcoma treatment. [ABSTRACT FROM AUTHOR]

Nanotechnology offers the possibility of operating on the same scale length at which biological processes occur, allowing to interfere, manipulate or study cellular events in disease or healthy conditions. The development of hybrid nanostructured materials with a high degree of chemical control and complex engineered surface including biological targeting moieties, allows to specifically bind to a single type of molecule for specific detection, signaling or inactivation processes. Magnetite nanostructures with designed composition and properties are the ones that gather most of the designs as theragnostic agents for their versatility, biocompatibility, facile production and good magnetic performance for remote in vitro and in vivo for biomedical applications. Their superparamagnetic behavior below a critical size of 30 nm has allowed the development of magnetic resonance imaging contrast agents or magnetic hyperthermia nanoprobes approved for clinical uses, establishing an inflection point in the field of magnetite based theragnostic agents. [ABSTRACT FROM AUTHOR]

Abstract Background Polymer surface-modified inorganic nanoparticles (NPs) provide a multifunctional platform for assisting gene delivery. Rational structure design for enhancing colloidal stability and cellular uptake is an important strategy in the development of safe and highly efficient gene vectors. Results Heterogeneous Au-coated Fe3O4 (Fe3O4@Au) NPs capped by polyethylene glycol-b-poly1-(3-aminopropyl)-3-(2-methacryloyloxy propylimidazolium bromine) (PEG-b-PAMPImB-Fe3O4@Au) were prepared for DNA loading and magnetofection assays. The Au outer shell of the NPs is an effective platform for maintaining the superparamagnetism of Fe3O4 and for PEG-b-PAMPImB binding via Au–S covalent bonds. By forming an electrostatic complex with DNA at the inner PAMPImB shell, the magnetic nanoplexes offer steric protection from the outer corona PEG, thereby promoting high colloidal stability. Transfection efficiency assays in human esophageal cancer cells (EC109) show that the nanoplexes have high transfection efficiency at a short incubation time in the presence of an external magnetic field, due to increased cellular internalization via magnetic acceleration. Finally, after transfection with the magnetic nanoplexes EC109 cells acquire magnetic properties, thus allowing for selective separation of transfected cells. Conclusion Precisely engineered architectures based on neutral-cationic block copolymer-conjugated heterogeneous NPs provide a valuable strategy for improving the applicability and efficacy of synthesized vectors.

Magnetic nanoparticles have continuously gained importance for the purpose of magnetically-aided drug-delivery, magnetofection, and hyperthermia. We have summarized significant experimental approaches, as well as their advantages and disadvantages with respect to future clinical translation. This field is alive and well and promises meaningful contributions to the development of novel cancer therapies. [ABSTRACT FROM AUTHOR]

Genetically modified pigs play an important role in agriculture and biomedical research; hence, new efficient methods are needed to obtain genetically engineered cells and animals. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas (CRISPR-associated) system represents an effective genome editing tool. It consists of two key molecules: single guide RNA (sgRNA) and the Cas9 endonuclease that can be introduced into the cells as one plasmid. Typical delivery methods for CRISPR/Cas9 components are limited by low transfection efficiency or toxic effects on cells. Here, we describe the use of magnetic nanoparticles and gradient magnetic field to improve delivery of CRISPR/Cas9 constructs into porcine fetal fibroblasts. Polyethylenimine-coated nanoparticles with magnetic iron oxide core were used to form magnetic plasmid DNA lipoplexes. CRISPR/Cas9 construct was prepared to induce site-specific cutting at the porcine H11 locus. Quantitative assessment of genomic cleavage by sequence trace decomposition demonstrated that the magnetofection efficiency was more than 3.5 times higher compared to the classic lipofection method. The Tracking of Indels by Decomposition web tool precisely determined the spectrum of indels that occurred. Simultaneously, no additional cytotoxicity associated with the utilization of magnetic nanoparticles was observed. Our results indicate that magnetofection enables effective delivery of the CRISPR/Cas9 construct into porcine fetal fibroblasts with low cell toxicity. [ABSTRACT FROM AUTHOR]

Magnetic nanoparticles have been widely used in nanobiomedicine for diagnostics and the treatment of diseases, and as carriers for various drugs. The unique magnetic properties of “magnetic” drugs allow their delivery in a targeted tumor or tissue upon application of a magnetic field. The approach of combining magnetic drug targeting and gene delivery is called magnetofection, and it is very promising. This method is simple and efficient for the delivery of genetic material to cells using magnetic nanoparticles controlled by an external magnetic field. However, magnetofection in vivo has been studied insufficiently both for local and systemic routes of magnetic vector injection, and the relevant data available in the literature are often merely descriptive and contradictory. In this review, we collected and systematized the data on the efficiency of the local injections of magnetic nanoparticles that carry genetic information upon application of external magnetic fields. We also investigated the efficiency of magnetofection in vivo, depending on the structure and coverage of magnetic vectors. The perspectives of the development of the method were also considered.

The use of synthetic RNA for research purposes as well as RNA-based therapy and vaccination has gained increasing importance. Given the anatomical seclusion of the eye, small interfering RNA (siRNA)-induced gene silencing bears great potential for targeted reduction of pathological gene expression that may allow rational treatment of chronic eye diseases in the future. However, there is yet an unmet need for techniques providing safe and efficient siRNA delivery to the retina. We used magnetic nanoparticles (MNPs) and magnetic force (Reverse Magnetofection) to deliver siRNA/MNP complexes into retinal explant tissue, targeting valosin-containing protein (VCP) previously established as a potential therapeutic target for autosomal dominant retinitis pigmentosa (adRP). Safe and efficient delivery of VCP siRNA was achieved into all retinal cell layers of retinal explants from the RHO P23H rat, a rodent model for adRP. No toxicity or microglial activation was observed. VCP silencing led to a significant decrease of retinal degeneration. Reverse Magnetofection thus offers an effective method to deliver siRNA into retinal tissue. Used in combination with retinal organotypic explants, it can provide an efficient and reliable preclinical test platform of RNA-based therapy approaches for ocular diseases.

Cationic liposomes have been considered as potential vectors for gene delivery thanks to their ability to transfect cells with high efficiency. Recently, the combination of diagnostic agent and therapeutic agents in the same particle to form a theranostic system has been reported. Magnetic liposomes are one of these examples. Due to the magnetic nanoparticles encapsulated in the liposomes, they can act as a drug delivery system and, at the same time, a magnetic resonance imaging contrast enhancement agent or hyperthermia. In this work, nucleic acid delivery systems based on magnetic cationic liposomes (MCLs) were developed. Two different techniques, reverse phase evaporation and cosolvent sonication, were employed for liposome preparation. Both strategies produced MCLs of less than 200 nm with highly positive charge. Enhancement of their transverse and longitudinal relaxivities r2 and r1 was obtained with both kinds of magnetic liposomes compared to free magnetic nanoparticles. Moreover, these MCLs showed high capacity to form complexes and transfect CT-26 cells using the antibiotic-free pFAR4-luc plasmid. The transfection enhancement with magnetofection was also carried out in CT26 cells. These results suggested that our MCLs could be a promising candidate for image-guided gene therapy.

Genetically engineered neural stem cell (NSC) transplants offer a key strategy to augment neural repair by releasing therapeutic biomolecules into injury sites. Genetic modification of NSCs is heavily reliant on viral vectors but cytotoxic effects have prompted development of non-viral alternatives, such as magnetic nanoparticle (MNPs). NSCs are propagated in laboratories as either 3-D suspension “neurospheres” or 2-D adherent “monolayers”. MNPs deployed with oscillating magnetic fields (“magnetofection technology”) mediate effective gene transfer to neurospheres but the efficacy of this approach for monolayers is unknown. It is important to address this issue as oscillating magnetic fields dramatically enhance MNP-based transfection in transplant cells (e.g., astrocytes and oligodendrocyte precursors) propagated as monolayers. We report for the first time that oscillating magnetic fields enhanced MNP-based transfection with reporter and functional (basic fibroblast growth factor; FGF2) genes in monolayer cultures yielding high transfection versus neurospheres. Transfected NSCs showed high viability and could re-form neurospheres, which is important as neurospheres yield higher post-transplantation viability versus monolayer cells. Our results demonstrate that the combination of oscillating magnetic fields and a monolayer format yields the highest efficacy for MNP-mediated gene transfer to NSCs, offering a viable non-viral alternative for genetic modification of this important neural cell transplant population.

Gene therapy involves replacing a faulty gene or adding a new gene inside the body's cells to cure disease or improve the body's ability to fight disease. Its popularity is evident from emerging concepts such as CRISPR-based genome editing and epigenetic studies and has been moved to a clinical setting. The strategy for therapeutic gene design includes; suppressing the expression of pathogenic genes, enhancing necessary protein production, and stimulating the immune system, which can be incorporated into both viral and non-viral gene vectors. Although non-viral gene delivery provides a safer platform, it suffers from an inefficient rate of gene transfection, which means a few genes could be successfully transfected and expressed within the cells. Incorporating nucleic acids into the viruses and using these viral vectors to infect cells increases gene transfection efficiency. Consequently, more cells will respond, more genes will be expressed, and sustained and successful gene therapy can be achieved. Combining nanoparticles (NPs) and nucleic acids protects genetic materials from enzymatic degradation. Furthermore, the vectors can be transferred faster, facilitating cell attachment and cellular uptake. Magnetically assisted viral transduction (magnetofection) enhances gene therapy efficiency by mixing magnetic nanoparticles (MNPs) with gene vectors and exerting a magnetic field to guide a significant number of vectors directly onto the cells. This research critically reviews the MNPs and the physiochemical properties needed to assemble an appropriate magnetic viral vector, discussing cellular hurdles and attitudes toward overcoming these barriers to reach clinical gene therapy perspectives. We focus on the studies conducted on the various applications of magnetic viral vectors in cancer therapies, regenerative medicine, tissue engineering, cell sorting, and virus isolation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Graphical abstract Abstract The lack of efficient and cost-effective methods for gene delivery has significantly hindered the applications of gene therapy. In this paper, a simple one step and cost effective salting-out method has been explored to fabricate silk-PEI nanoparticles (SPPs) and magnetic-silk/PEI core-shell nanoparticles (MSPPs) for targeted delivery of c-myc antisense oligodeoxynucleotides (ODNs) into MDA-MB-231 breast cancer cells. The size and zeta potential of the particles were controlled by adjusting the amount of silk fibroin in particle synthesis. Lower surface charges and reduced cytotoxicity were achieved for MSPPs compared with PEI coated magnetic nanoparticles (MPPs). Both SPPs and MSPPs were capable of delivering the ODNs into MDA-MB-231 cells and significantly inhibited the cell growth. Through magnetofection, high ODN uptake efficiencies (over 70%) were achieved within 20 min using MSPPs as carriers, exhibiting a significantly enhanced uptake effect compared to the same carriers via non-magnetofection. Both SPPs and MSPPs exhibited a significantly higher inhibition effect against MDA-MB-231 breast cancer cells compared to human dermal fibroblast (HDF) cells. Targeted ODN delivery was achieved using MSPPs with the help of a magnet, making them promising candidates for targeted gene therapy applications. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Alternating magnetic field plate (AC magnet plate) for transfection is developed. • The AC magnet plate is portable, powered by the mains supply, has no moving parts. • Magnetofection of HEK 293 cells with GFP was achieved with the AC magnet plate. • Magnet-assisted transfection by AC fields improve transfection compared to DC. • GFP expression was highest for 20 min, 25 mT, 60 Hz exposure with 48 h incubation. Abstract Magnet-assisted transfection or magnetofection refers to the delivery of nucleic acids to target cells with the help of conjugated superparamagnetic iron oxide nanoparticles (SPIONs) and external magnetic fields generated by permanent magnet plates. The external magnetic field, among other parameters, directly affects the transfection efficiency. However, standard permanent magnet plates generate static magnetic fields which are not as effective compared to time-varying, dynamic fields. In this work, we show a new and novel "AC magnet plate" compatible with standard 96-well cell culture plates, which can be easily adapted for benchtop use in a typical lab setting. We provide full design details, modelling, fabrication, measurement and testing on human embryonic kidney cells (HEK 293) to show transfection improvement. We perform magnetofection under different field conditions and show that with increasing AC content, efficiency of transfection is improved. [ABSTRACT FROM AUTHOR]

This study was carried out to develop a simple and efficient method to isolate DNA directly from biological samples using iron oxide nanoparticles (IONPs) functionalized with polyethyleneimine (PEI). IONPs were synthesized via co-precipitation method followed with direct attachment of branched PEI. Nanoparticles were characterized using STEM, FT-IR spectroscopy and XRD analysis. The binding capacity of synthesized PEI-IONPs for plasmid and genomic DNA was assessed using purified DNA samples. In order to elute bound DNA, elution conditions were optimized, changing pH, salt concentration and temperature. Synthesized PEI-IONPs were subjected to isolation of DNA from bacterial cell culture and from human blood. PCR and magnetofection of the enhanced green fluorescence protein (EGFP) were carried out to verify the downstream applications of isolated DNA. The results indicated that the synthesized nanoparticles were of 5–10 nm. The binding capacity of PEI-IONPs for plasmid DNA and genomic DNA were 5.4 and 8.4 µg mg−1, respectively, which were even higher than the commercially available kits such as Mag-bind, MagJET and Magmax. The optimized condition for plasmid DNA elution was 0.1 M Tris HCl (pH 10.0), 1.5 M NaCl and 5% formamide, maintained at the temperature of 60°C. The optimized condition for genomic DNA elution was 0.1 M Tris HCl (pH 10.0), 1.5 M NaCl and 10% formamide, maintained at 60°C. PCR and magnetofection processes were successful. This study revealed that the magnetic separation of DNA using PEI-IONPs is a simple and efficient method for direct isolation of DNA from biological samples which can be then used in various downstream applications.

Nanotechnology offers the possibility of operating on the same scale length at which biological processes occur, allowing to interfere, manipulate or study cellular events in disease or healthy conditions. The development of hybrid nanostructured materials with a high degree of chemical control and complex engineered surface including biological targeting moieties, allows to specifically bind to a single type of molecule for specific detection, signaling or inactivation processes. Magnetite nanostructures with designed composition and properties are the ones that gather most of the designs as theragnostic agents for their versatility, biocompatibility, facile production and good magnetic performance for remote in vitro and in vivo for biomedical applications. Their superparamagnetic behavior below a critical size of 30 nm has allowed the development of magnetic resonance imaging contrast agents or magnetic hyperthermia nanoprobes approved for clinical uses, establishing an inflection point in the field of magnetite based theragnostic agents.

Magnetic nanoparticles have continuously gained importance for the purpose of magnetically-aided drug-delivery, magnetofection, and hyperthermia. We have summarized significant experimental approaches, as well as their advantages and disadvantages with respect to future clinical translation. This field is alive and well and promises meaningful contributions to the development of novel cancer therapies.

In these studies, we developed a novel approach of in vivo magnetofection for localized delivery of nucleic acids such as micro-RNA-139-5p (miR-139-5p; which is known to target Rho kinase2) to the circular smooth muscle layer of the internal anal sphincter (IAS). The IAS tone is known to play a major role in the rectoanal continence via activation of RhoA-associated kinase (RhoA/ROCK2). These studies established an optimized protocol for efficient gene delivery using an assembly of equal volumes of in vivo PolyMag and miR139-5p or anti-miR-139-5p (100 nM each) injected in the circular smooth muscle layer in the pinpointed areas of the rat perianal region and then incubated for 20 min under magnetic field. Magnetofection efficiency was confirmed and analyzed by confocal microscopy of FITC-tagged siRNA. Using physiological and biochemical approaches, we investigated the effects of miR-139-5p and anti-miR-139-5p on basal intraluminal IAS pressure (IASP), fecal pellet count, IAS tone, agonist-induced contraction, contraction-relaxation kinetics, and RhoA/ROCK2 signaling. Present studies demonstrate that magnetofection-mediated miR-139-5p delivery significantly decreased RhoA/ROCK2, p-MYPT1, and p-MLC20 signaling, leading to decreases in the basal IASP and IAS tone and in rates of contraction and relaxation associated with increase in fecal pellet output. Interestingly, anti-miR-139-5p transfection had opposite effects on these parameters. Collectively, these data demonstrate that magnetofection is a promising novel method of in vivo gene delivery and of nucleotides to the internal anal sphincter for the site-directed and targeted therapy for rectoanal motility disorders. NEW & NOTEWORTHY These studies for the first time demonstrate the success of topical in vivo magnetofection (MF) of nucleic acids using perianal injections. To demonstrate its effectiveness, we used FITC-tagged siRNA via immunofluorescence microcopy and functional and biochemical evidence using miR-139-5p (which is known to target ROCK2). In conclusion, MF allows safe, convenient, efficient, and targeted delivery of oligonucleotides such as siRNAs and microRNAs. These studies have direct therapeutic implications in rectoanal motility disorders especially associated with IAS. [ABSTRACT FROM AUTHOR]

Magnetic nanoparticles represent a new paradigm for molecular targeting therapy in cancer. However, the transformative targeting potential of magnetic nanoparticles has been stymied by a key obstacle-safe delivery to specified target cells in vivo. As cancer cells grow under nutrient deprivation and hypoxic conditions and decorate cell surface with excessive sialoglycans, sialic acid binding lectins might be suitable for targeting cancer cells in vivo. Here we explore the potential of magnetic nanoparticles functionalized with wheat germ lectin (WGA) conjugate, so-called nanomagnetolectin, as apoptotic targetable agents for prostate cancer. In the presence of magnetic field (magnetofection) for 15 min, 2.46 nM nanomagnetolectin significantly promoted apoptosis (∼12-fold, p value <0.01) of prostate cancer cells (LNCaP, PC-3, DU-145) compared to normal prostate epithelial cells (PrEC, PNT2, PZ-HPV-7), when supplemented with 10 mM sialic acid under nutrient deprived condition. Nanomagnetolectin targets cell-surface glycosylation, particularly sialic acid as nanomagnetolectin induced apoptosis of cancer cells largely diminished (only 2 to 2.5-fold) compared to normal cells. The efficacy of magnetofected nanomagnetolectin was demonstrated in orthotopically xenografted (DU-145) mice, where tumor was not only completely arrested, but also reduced significantly (p value <0.001). This was further corroborated in subcutaneous xenograft model, where nanomagnetolectin in the presence of magnetic field and photothermal heating at ∼42 °C induced apoptosis of tumor by ∼4-fold compared to tumor section heated at ∼42 °C, but without magnetic field. Taken all together, the study demonstrates, for the first time, the utility of nanomagnetolectin as a potential cancer therapeutic. [ABSTRACT FROM AUTHOR]

Background: Polymer surface-modified inorganic nanoparticles (NPs) provide a multifunctional platform for assisting gene delivery. Rational structure design for enhancing colloidal stability and cellular uptake is an important strategy in the development of safe and highly efficient gene vectors. Results: Heterogeneous Au-coated Fe3O4 (Fe3O4@Au) NPs capped by polyethylene glycol-b-poly1-(3-aminopropyl)-3-(2-methacryloyloxy propylimidazolium bromine) (PEG-b-PAMPImB-Fe3O4@Au) were prepared for DNA loading and magnetofection assays. The Au outer shell of the NPs is an effective platform for maintaining the superparamagnetism of Fe3O4 and for PEG-b-PAMPImB binding via Au-S covalent bonds. By forming an electrostatic complex with DNA at the inner PAMPImB shell, the magnetic nanoplexes offer steric protection from the outer corona PEG, thereby promoting high colloidal stability. Transfection efficiency assays in human esophageal cancer cells (EC109) show that the nanoplexes have high transfection efficiency at a short incubation time in the presence of an external magnetic field, due to increased cellular internalization via magnetic acceleration. Finally, after transfection with the magnetic nanoplexes EC109 cells acquire magnetic properties, thus allowing for selective separation of transfected cells. Conclusion: Precisely engineered architectures based on neutral-cationic block copolymer-conjugated heterogeneous NPs provide a valuable strategy for improving the applicability and efficacy of synthesized vectors. [ABSTRACT FROM AUTHOR]

Due to the peculiar magnetic properties and the ability to function in cell-level biological interaction, superparamagnetic nanoparticles (SMNP) have been being the attractive carrier for gene delivery. The superparamagnetic nanoparticles with surface-bound gene vector can be attracted to the surface of cells by the Kelvin force provided by external magnetic field. In this article, the influence of the oscillating magnetic field on the characteristics of magnetofection is studied in terms of the magnetophoretic velocity. The magnetic field of a cylindrical permanent magnet is calculated by equivalent current source (ECS) method, and the Kelvin force is derived by using the effective moment method. The results show that the static magnetic field accelerates the sedimentation of the particles, and drives the particles inward towards the axis of the magnet. Based on the investigation of the magnetophoretic velocity of the particle under horizontally oscillating magnetic field, an oscillating velocity within the amplitude of the magnet oscillation is observed. Furthermore, simulation results indicate that the oscillating amplitude plays an important role in regulating the active region, where the particles may present oscillating motion. The analysis of the magnetophoretic velocity gives us an insight into the physical mechanism of the magnetofection. It's also helpful to the optimal design of the magnetofection system. [ABSTRACT FROM AUTHOR]

Superparamagnetic iron oxide nanoparticles (MNPs) were prepared and coated with chitosan (CS). The chitosan-magnetic iron oxide nanoparticles (CS-MNPs) were characterized using Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), vibrating sample magnetometry (VSM), and the morphology of the particles was studied by transmission electron microscopy (TEM). Our findings show that the magnetic particles were monodisperse (10 nm mean diameter) and exhibited superparamagnetic behavior. The interaction between the particles and calf-thymus DNA (DNA) in physiological buffer was studied with UV–vis, fluorescence and circular dichroism spectroscopy and zeta potential. Spectroscopic studies were indicated DNA conformational changes in the presence of CS-MNPs. Binding and thermodynamic parameters at different temperatures were calculated using the Stern–Volmer, Hill, Scatchard and Van’t Hoff equations. The binding process was spontaneous and interactions were electrostatic with the appropriate binding constant (K b = 4.52 × 10 3 M −1 , 3.69 × 10 3 M −1 and 3.02 × 10 3 M −1 at 300 K, 310 K and 320 K, respectively). Zeta potential measurements of DNA continually increased with the addition of CS-MNPs, supporting our thermodynamic findings. Moreover, CS-MNPs were able to quench the fluorescence of DNA-intercalated ethidium bromide (DNA-EB) by a static quenching mechanism. Cytotoxicity studies show that the DNA-CS-MNP system is biocompatible with a human foreskin fibroblast cell line, HFFF2. Collectively, these results suggest that surface cationic magnetic chitosan-iron oxide nanoparticles can potentially enhance magnetofection efficiency. [ABSTRACT FROM AUTHOR]

Gene engineered mesenchymal stem cells (MSCs) have been proposed as promising tools for their various applications in biomedicine. Nevertheless, the lack of an effective and safe way to genetically modify these stem cells is still a major obstacle in the current studies. Herein, we designed novel magnetic complexes by assembling cationized pullulan derivatives with magnetic iron oxide nanoparticles for delivering target genes to MSCs. Results showed that this complexes achieved effective gene expression with the assistance of external magnetic field, and resisted the adverse effect induced by serum proteins on the gene delivery. Moreover, neither significant cytotoxicity nor the interference on the osteogenic differentiation to MSCs were observed after magnetofection. Further studies revealed that this effective and serum resistant gene transfection was partly due to the accelerated and enhanced intracellular uptake process driven by external magnetic field. To conclude, the current study presented a novel option for genetic modification of MSCs in an effective, relatively safe and serum compatible way. [ABSTRACT FROM AUTHOR]