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Superparamagnetic Nanoparticle Delivery of DNA Vaccine

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Abstract

The efficiency of delivery of DNA vaccines is often relatively low compared to protein vaccines. The use of superparamagnetic iron oxide nanoparticles (SPIONs) to deliver genes via magnetofection shows promise in improving the efficiency of gene delivery both in vitro and in vivo. In particular, the duration for gene transfection especially for in vitro application can be significantly reduced by magnetofection compared to the time required to achieve high gene transfection with standard protocols. SPIONs that have been rendered stable in physiological conditions can be used as both therapeutic and diagnostic agents due to their unique magnetic characteristics. Valuable features of iron oxide nanoparticles in bioapplications include a tight control over their size distribution, magnetic properties of these particles, and the ability to carry particular biomolecules to specific targets. The internalization and half-life of the particles within the body depend upon the method of synthesis. Numerous synthesis methods have been used to produce magnetic nanoparticles for bioapplications with different sizes and surface charges. The most common method for synthesizing nanometer-sized magnetite Fe3O4 particles in solution is by chemical coprecipitation of iron salts. The coprecipitation method is an effective technique for preparing a stable aqueous dispersions of iron oxide nanoparticles. We describe the production of Fe3O4-based SPIONs with high magnetization values (70 emu/g) under 15 kOe of the applied magnetic field at room temperature, with 0.01 emu/g remanence via a coprecipitation method in the presence of trisodium citrate as a stabilizer. Naked SPIONs often lack sufficient stability, hydrophilicity, and the capacity to be functionalized. In order to overcome these limitations, polycationic polymer was anchored on the surface of freshly prepared SPIONs by a direct electrostatic attraction between the negatively charged SPIONs (due to the presence of carboxylic groups) and the positively charged polymer. Polyethylenimine was chosen to modify the surface of SPIONs to assist the delivery of plasmid DNA into mammalian cells due to the polymer's extensive buffering capacity through the "proton sponge" effect.

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... VLPs and NPs have been widely used in research on virus vaccines [27][28][29]. Many studies have recently been published that focus on developing VLPs and NPs vaccines against picornaviruses (Tables 1 and 2) [29][30][31][32][33][34]. This review describes the immune responses related to picornaviruses. ...
... In 2008, Cubillos et al. designed and synthesised a dendritic peptide, B4T, which comprises four B-cell epitopes from the G-H loop (amino acid residues 136-154 in VP1) and one T-cell epitope from the 3A region (amino acid residues [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35]. This design was based on the sequence of the FMDV serotype C isolate C-S8c1 [130]. ...
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Picornaviridae are non-enveloped ssRNA viruses that cause diseases such as poliomyelitis, hand-foot-and-mouth disease (HFMD), hepatitis A, encephalitis, myocarditis, and foot-and-mouth disease (FMD). Virus-like particles (VLPs) vaccines mainly comprise particles formed through the self-assembly of viral capsid proteins (for enveloped viruses, envelope proteins are also an option). They do not contain the viral genome. On the other hand, the nanoparticles vaccine (NPs) is mainly composed of self-assembling biological proteins or nanomaterials, with viral antigens displayed on the surface. The presentation of viral antigens on these particles in a repetitive array can elicit a strong immune response in animals. VLPs and NPs can be powerful platforms for multivalent antigen presentation. This review summarises the development of virus-like particle vaccines (VLPs) and nanoparticle vaccines (NPs) against picornaviruses. By detailing the progress made in the fight against various picornaviruses such as poliovirus (PV), foot-and-mouth disease virus (FMDV), enterovirus (EV), Senecavirus A (SVA), and encephalomyocarditis virus (EMCV), we in turn highlight the significant strides made in vaccine technology. These advancements include diverse construction methods, expression systems, elicited immune responses, and the use of various adjuvants. We see promising prospects for the continued development and optimisation of VLPs and NPs vaccines. Future research should focus on enhancing these vaccines' immunogenicity, stability, and delivery methods. Moreover, expanding our understanding of the interplay between these vaccines and the immune system will be crucial. We hope these insights will inspire and guide fellow researchers in the ongoing quest to combat picornavirus infections more effectively.
... Multiple studies since 2007 describe research with highly magnetic nanoparticles thus fueling allegations that the COVID-19 vaccine was imbedded with these substances [5][6][7][8][9][10][11][12][13][14][15][16][17]. Theories ascribing magnet attachment to highly magnetic nanoparticles embedded in the COVID-19 vaccine are clearly refuted by this study and could not be rationally challenged. ...
... If such materials were at play, attraction would be expected to occur only at vaccination sites and nowhere else. There are numerous reports documenting research using highly magnetic nanoparticles, ferromaterials, and graphenes in medical research and in our environment [5][6][7][8][9][10][11][12][13][14][15][16]. It is also impossible to determine if highly magnetic nanoparticles in the environment may have contributed to the magnetic effect observed; there are no historical references to document human magnetism before highly magnetic nanoparticles were described. ...
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... Iron oxide nanoparticles (Fe 3 O 4 ) especially their superparamagnetic variation (SPION) paved the way for use of nanoparticles in the broad medical field: Feraheme ® , Feridex ® and GastroMARK ® are just a few of the registered medical products [1][2][3]. After many years of research, these nanoparticles still find innovative use in many research fields, e.g., drug delivery [4][5][6], magnetic resonance imaging (MRI) [7,8], gene magnetofection [9,10], magnetic hyperthermia [11,12], and radionuclide therapy [13][14][15]. The last two are under our group's particular attention. ...
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... Superparamagnetic iron oxide nanoparticles (SPIONs) exhibit unique physicochemical properties [1][2][3], making them an attractive material for various biomedical applications including drug delivery [4][5][6], chemo-photothermal therapy [7,8], magnetic hyperthermia [9,10], magnetic resonance imaging (MRI) [11,12], and gene magnetofection [13,14]. Due to their properties, iron oxide-based nanoparticles, also known as ferumoxytol, have already gained approval for use as MRI contrast agents and as iron deficiency therapeutics by the Food and Drug Administration (FDA). ...
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... SPIONs have received much attention in gene delivery applications because of their low toxicity and a high ability for direct targeting using the exterior magnets. Accordingly, SPIONs can be applied as therapeutic and diagnostic agents due to their ideal properties 42 . In contrast, due to the agglomeration of SPION colloidal suspensions, their utilization in the clinic have been restricted 43 . ...
Article
HIF-1α and STAT3 are two of the critical factors in the growth, proliferation, and metastasis of cancer cells and play a crucial role in inhibiting anti-cancer immune responses. Therefore, we used superparamagnetic iron oxide (SPION) nanoparticles (NPs) coated with thiolated chitosan (ChT) and trimethyl chitosan (TMC) and functionalized with hyaluronate (H) and TAT peptide for delivery of siRNA molecules against STAT3 and HIF-1α to cancer cells both in vivo and in vitro. The results indicated that tumor cell transfection with siRNA-encapsulated NPs robustly inhibited proliferation and migration and induced apoptosis in tumor cells. Furthermore, simultaneous silencing of HIF-1α and STAT3 significantly repressed cancer development in two different tumor types (4 T1 breast cancer and CT26 colon cancer) which was associated with upregulation of cytotoxic T lymphocytes and IFN-γ secretion. The findings suggest inhibiting the HIF-1α/STAT3 axis by SPION-TMC-ChT-TAT-H NPs as an effective way to treat cancer.
... Superparamagnetic iron oxide nanoparticles (SPIONs) have attracted attention in nucleic acid delivery applications due to their low cost, low toxicity, and potential for direct targeting by using external magnets. Therefore, SPIONs could be used as both diagnostic and therapeutic agents because of their unique characteristics [6]. Moreover, the mechanism of magnetic hyperthermia of SPIONs can be applied to suppress the HIV viral load, since hyperthermic temperatures increase the activity of cytotoxic T lymphocytes (CTLs), which can inhibit HIV replication [7]. ...
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Introduction: To date, while many studies have investigated antiviral agents or vaccines against HIV, success has been limited. In this field, mutagenesis of the viral genome mostly contributes to the viral escape from the antiretroviral therapies as well as the emergence of resistant strains of HIV-1 to pharmaceutical therapy. Therefore, developing alternative methods, including more effective vaccines, antiviral therapies (such as RNAi therapy) and delivery systems, seem to be necessary to compensate for these issues. The aim of this research was to establish an efficient system for siRNA delivery as a safe anti-HIV therapeutic approach. Methods: Chitosan-coated superparamagnetic iron oxide nanoparticles (SPION) was investigated as a method for RNA delivery. After generating HEK293 stable cells (expressing HIV-1 tat), a potent siRNA against HIV-1 tat was designed and the effectiveness of the modified SPION in siRNA delivery to HEK293 cells was evaluated. Results: The optimal concentration (50 µg/mL) of the modified SPION-containing anti-tat siRNA (with a range size of 50-70 nm and average zeta potential of +25 mV) was significantly internalized into the cells and decreased the expression of HIV-1 tat, more than 80%. Moreover, the nanoparticles showed no considerable toxicity on the cells. Conclusion: SPION could be optimized as a probable RNA/vaccine delivery system into target cells. Therefore, this study offers a therapeutic strategy against HIV or other infectious diseases.
... Magnetic NPs (MNPs) are another group of inorganic NPs approved by the FDA. These biocompatible NPs have been used in vaccine delivery [58]. MNPs can be easily handled with the aid of an external magnetic field, the possibility of using passive and active cargo delivery strategies, the ability of visualization (in MRI), and enhanced uptake [59]. ...
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In spite of the progress of conventional vaccines, improvements are required due to concerns about the low immunogenicity of these vaccines, toxicity, instability, and the need for multiple administrations. To overcome mentioned problems, nanotechnology have recently been incorporated into vaccine development. Nanotechnology increasingly plays an important role in vaccine development Nanocarrier-based delivery systems offer an opportunity to increase the cellular and humoral immune responses. The use of nanoparticles in vaccine formulations allows not only enhanced immunogenicity and stability of antigen, but also targeted delivery and slow release. Over the past decade nanoscale size materials such as virus-like particles, liposomes, ISCOMs, polymeric, inorganic nanoparticles and emulsions have received attention as potential delivery vehicles for vaccine antigens which can both stabilize vaccine antigens and act as adjuvants. This advantage is attributable to the nanoscale particle size, which facilitates uptake by antigen presenting cells (APCs) then leading to efficient antigen recognition and presentation. Modifying the surfaces of nanoparticles with a different of targeting moieties permits the delivery of antigens to specific receptors on the cell surface, thereby stimulating selective and specific immune responses. This review provides an overview of recent advances in nanovaccinology.
... To address these issues, modification with other components such as polycationic polymer can be used to provide the surface with sufficient capacity for functionalization and antigen binding. Superparamagnetic iron oxide nanoparticles (SPIONs) modified with cationic polyethyleneimine (PEI) polymer are promising DNA vaccine vectors due to their high buffering capacity [106]. Magnetic fields offer the capacity of enhanced cellular uptake and DC maturation. ...
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As effective tools for public health, vaccines prevent disease by priming the body's adaptive and innate immune responses against an infection. Due to advances in understanding cancers and their relationship with the immune system, there is a growing interest in priming host immune defenses for a targeted and complete antitumor response. Nanoparticle systems have shown to be promising tools for effective antigen delivery as vaccines and/or for potentiating immune response as adjuvants. Here, we highlight relevant physiological processes involved in vaccine delivery, review recent advances in the use of nanoparticle systems for vaccines and discuss pertinent challenges to viably translate nanoparticle-based vaccines and adjuvants for public use.
... Silica nanoparticles, due to properties like biodegradability, biocompatibility, possibility of surface modification, possibility of fabrication in any desired size, and, of course, due to promising results, seem to be potent nanocarriers for vaccine delivery. Magnetic nanoparticles are another group of the inorganic nanoparticles that have been used for vaccine delivery [82][83][84]. These nanoparticles are biocompatible and FDA-approved [85]. ...
... Magnetofection, is a gene delivery technique where a magnetic field is applied to concentrate Iron Oxide particles containing nucleic acid desired target. In this way, the magnetic force allows a rapid concentration of the entire applied vector dose onto cells [140][141][142][143][144][145] . ...
... Magnetofection, is a gene delivery technique where a magnetic field is applied to concentrate Iron Oxide particles containing nucleic acid desired target. In this way, the magnetic force allows a rapid concentration of the entire applied vector dose onto cells [140][141][142][143][144][145] . ...
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IntroductionPreparationOther Methods
Article
A ‘size sorting’ method performed on ionic magnetic fluids, constituted by polydisperse anionic particles dispersed in water at pH 7 allows ‘monodisperse’ samples to be obtained. Here results on phase transitions observed in these samples with decreasing temperature, application of a magnetic field or increasing ionic strength are presented.
Article
Superparamagnetic iron oxide nanoparticles (SPIONs) were coated with polyethylenimine. Here, we briefly describe the synthesis as well as DNA:PEI:SPION complexes and the characterization of the compounds according to their particle size, ζ-potential, morphology, DNA complexing ability, magnetic sedimentation, and colloidal stability. PEI coating of SPIONs led to colloidally stable beads even in high salt concentrations over a wide pH range. DNA plasmids and PCR products encoding for green fluorescent protein were associated with the described beads. The complexes were added to cells and exposed to permanent and pulsating magnetic fields. Presence of these magnetic fields significantly increased the transfection efficiency.
Article
Results of work on the preparation of aqueous magnetic liquids without using organic stabilizing agents are presented.
Article
Low efficiency is often observed in the delivery of DNA vaccines. The use of superparamagnetic nanoparticles (SPIONs) to deliver genes via magnetofection could improve transfection efficiency and target the vector to its desired locality. Here, magnetofection was used to enhance the delivery of a malaria DNA vaccine encoding Plasmodium yoelii merozoite surface protein MSP1(19) (VR1020-PyMSP1(19)) that plays a critical role in Plasmodium immunity. The plasmid DNA (pDNA) containing membrane associated 19-kDa carboxyl-terminal fragment of merozoite surface protein 1 (PyMSP1(19)) was conjugated with superparamagnetic nanoparticles coated with polyethyleneimine (PEI) polymer, with different molar ratio of PEI nitrogen to DNA phosphate. We reported the effects of SPIONs-PEI complexation pH values on the properties of the resulting particles, including their ability to condense DNA and the gene expression in vitro. By initially lowering the pH value of SPIONs-PEI complexes to 2.0, the size of the complexes decreased since PEI contained a large number of amino groups that became increasingly protonated under acidic condition, with the electrostatic repulsion inducing less aggregation. Further reaggregation was prevented when the pHs of the complexes were increased to 4.0 and 7.0, respectively, before DNA addition. SPIONs/PEI complexes at pH 4.0 showed better binding capability with PyMSP1(19) gene-containing pDNA than those at neutral pH, despite the negligible differences in the size and surface charge of the complexes. This study indicated that the ability to protect DNA molecules due to the structure of the polymer at acidic pH could help improve the transfection efficiency. The transfection efficiency of magnetic nanoparticle as carrier for malaria DNA vaccine in vitro into eukaryotic cells, as indicated via PyMSP1(19) expression, was significantly enhanced under the application of external magnetic field, while the cytotoxicity was comparable to the benchmark nonviral reagent (Lipofectamine 2000).
Article
In order to reveal the biocompatibility of Fe(3)O(4) nanoparticles and bipolar surfactant tetramethylammonium 11-aminoundecanoate cytotoxicity tests were performed as a function of concentration from low (0.1 microg ml(-1)) to higher concentration (100 microg ml(-1)) using various human glia, human breast cancer and normal cell lines. Cytotoxicity tests for human glia (D54MG, G9T, SF126, U87, U251, U373), human breast cancer (MB157, SKBR3, T47D) and normal (H184B5F5/M10, WI-38, SVGp12) cell lines exhibited almost nontoxicity and reveal biocompatibility of Fe(3)O(4) nanoparticles in the concentration range of 0.1-10 microg ml(-1), while accountable cytotoxicity can be seen at 100 microg ml(-1). The results of our studies suggest that Fe(3)O(4) nanoparticles coated with bipolar surfactant tetramethylammonium 11-aminoundecanoate are biocompatible and promising for bio-applications such as drug delivery, magnetic resonance imaging and magnetic hyperthermia.
Article
Ethylenimine polymers (PEIs) belong to one of the most efficient family of cationic compounds for delivery of plasmid DNA into mammalian cells. The high transfection efficiencies are obtained even in the absence of endosomolytic agents such as fusogenic peptides or chloroquine, which is in contrast to most of the other cationic polymers. It has been hypothesized that the efficiency of PEI is due to its capacity to buffer the endosomes. To investigate the importance of the acidification of endosomes during PEI-mediated DNA transfer we used proton pump inhibitors such as bafilomycin A1 and concanamycin A. Moreover, we tested whether PEI is able to destabilize natural membranes per se at neutral or acidic pH by performing erythrocyte lysis assays. PEI-mediated transfection in the presence of bafilomycin A1 resulted in a 7-74-fold decrease in reporter gene expression depending on the cell line used. In contrast, the efficiency of the monocationic lipid, DOTAP, was not importantly altered in the presence of the drug. Furthermore, the present data show that PEI cannot destabilize erythrocyte membranes, even at acidic pH, and that PEI, complexed or not to DNA, can increase the transfection efficiency of the cationic polymer, polylysine, when added at the same time to the cells. The transfection efficiency of PEIs partially relies on their ability to capture the protons which are transferred into the endosomes during their acidification. In addition, PEI is able to deliver significant amounts of DNA into cells and the DNA complexes involved in the expression of the transgene escape within 4 h from the endosomes.
Article
Low efficiencies of nonviral gene vectors, the receptor-dependent host tropism of adenoviral or low titers of retroviral vectors limit their utility in gene therapy. To overcome these deficiencies, we associated gene vectors with superparamagnetic nanoparticles and targeted gene delivery by application of a magnetic field. This potentiated the efficacy of any vector up to several hundred-fold, allowed reduction of the duration of gene delivery to minutes, extended the host tropism of adenoviral vectors to nonpermissive cells and compensated for low retroviral titer. More importantly, the high transduction efficiency observed in vitro was reproduced in vivo with magnetic field-guided local transfection in the gastrointestinal tract and in blood vessels. Magnetofection provides a novel tool for high throughput gene screening in vitro and can help to overcome fundamental limitations to gene therapy in vivo.
Article
Modification of cellular functions by overexpression of genes is increasingly practised for research of signalling pathways, but restricted by limitations of low efficiency. We investigated whether the novel technique of magnetofection (MF) could enhance gene transfer to cultured primary endothelial cells. MF of human umbilical vein endothelial cells (HUVEC) increased transfection efficiency of a luciferase reporter gene up to 360-fold compared to various conventional transfection systems. In contrast, there was only an up to 1.6-fold increase in toxicity caused by MF suggesting that the advantages of MF outbalanced the increase in toxicity. MF efficiently increased transfection efficiency using several commercially available cationic lipid transfection reagents and polyethyleneimine (PEI). Using PEI, even confluent HUVEC could be efficiently transfected to express luciferase activity. Using a green fluorescent protein vector maximum percentages of transfected cells amounted up to 38.7% while PEI without MF resulted in only 1.3% transfected cells. Likewise, in porcine aortic endothelial cells MF increased expression of a luciferase or a beta-galactosidase reporter, reaching an efficiency of 37.5% of cells. MF is an effective tool for pDNA transfection of endothelial cells allowing high efficiencies. It may be of great use for investigating protein function in cell culture experiments.
Article
Superparamagnetic iron oxide nanoparticles (SPION) with appropriate surface chemistry have been widely used experimentally for numerous in vivo applications such as magnetic resonance imaging contrast enhancement, tissue repair, immunoassay, detoxification of biological fluids, hyperthermia, drug delivery and in cell separation, etc. All these biomedical and bioengineering applications require that these nanoparticles have high magnetization values and size smaller than 100 nm with overall narrow particle size distribution, so that the particles have uniform physical and chemical properties. In addition, these applications need special surface coating of the magnetic particles, which has to be not only non-toxic and biocompatible but also allow a targetable delivery with particle localization in a specific area. To this end, most work in this field has been done in improving the biocompatibility of the materials, but only a few scientific investigations and developments have been carried out in improving the quality of magnetic particles, their size distribution, their shape and surface in addition to characterizing them to get a protocol for the quality control of these particles. Nature of surface coatings and their subsequent geometric arrangement on the nanoparticles determine not only the overall size of the colloid but also play a significant role in biokinetics and biodistribution of nanoparticles in the body. The types of specific coating, or derivatization, for these nanoparticles depend on the end application and should be chosen by keeping a particular application in mind, whether it be aimed at inflammation response or anti-cancer agents. Magnetic nanoparticles can bind to drugs, proteins, enzymes, antibodies, or nucleotides and can be directed to an organ, tissue, or tumour using an external magnetic field or can be heated in alternating magnetic fields for use in hyperthermia. This review discusses the synthetic chemistry, fluid stabilization and surface modification of superparamagnetic iron oxide nanoparticles, as well as their use for above biomedical applications.
Article
Gene therapy has become a promising strategy for the treatment of many inheritable or acquired diseases that are currently considered incurable. Non-viral vectors have attracted great interest, as they are simple to prepare, rather stable, easy to modify and relatively safe, compared to viral vectors. Unfortunately, they also suffer from a lower transfection efficiency, requiring additional effort for their optimization. The cationic polymer polyethylenimine (PEI) has been widely used for non-viral transfection in vitro and in vivo and has an advantage over other polycations in that it combines strong DNA compaction capacity with an intrinsic endosomolytic activity. Here, we give some insight into strategies developed for PEI-based non-viral vectors to overcome intracellular obstacles, including the improvement of methods for polyplex preparation and the incorporation of endosomolytic agents or nuclear localization signals. In recent years, PEI-based non-viral vectors have been locally or systemically delivered, mostly to target gene delivery to tumor tissue, the lung or liver. This requires strategies to efficiently shield transfection polyplexes against non-specific interaction with blood components, extracellular matrix and untargeted cells and the attachment of targeting moieties, which allow for the directed gene delivery to the desired cell or tissue. In this context, materials, facilitating the design of novel PEI-based non-viral vectors are described.
Article
Research of the molecular and cellular biology of mammalian cells would be highly restricted if not for the development of methods to deliver exogenous DNA into cultivated cells. Transient transfection of mammalian cells became a routine research tool following a landmark publication by Graham and Van der Eb, who presented the calcium phosphate method as an assay to test the infectivity of purified viral DNA in 1973 (1). For the first time, this technique allowed the delivery of genes into animal cells without the help of a virus. Novel techniques for nonviral gene transfer have been developed and improved since then. Today, numerous commercial transfection agents for a wide range of cell lines are available (2). This chapter focuses on the two leading methods for large-scale applications of transient gene expression (TGE): calcium phosphate DNA coprecipitates (3,4) and polyethylenimine (PEI)-DNA polyplexes (5,6). Although calcium phosphate DNA coprecipitation is a well-established method that has been modified for high efficiency by many authors (7–12), PEI has only recently been developed as a transfection vehicle (13–15), but it has achieved rapid acceptance for large-scale suspension transfections because of its simplicity in handling and its efficacy of gene transfer to many different cell types.
Polyethylenimine-mediated gene delivery: a mechanistic study
  • A Kichler
  • C Leborgne
  • E Coeytaux
Kichler A, Leborgne C, Coeytaux E et al (2001) Polyethylenimine-mediated gene delivery: a mechanistic study. J Gene Med 3: 135–144
Iron oxides in the laboratory: preparation and characterization
  • U Schwertmann
  • R M Cornell
Schwertmann U, Cornell RM (1991) Iron oxides in the laboratory: preparation and characterization. Wiley-VCH, Weinheim, NY