New Hypothesis: Blood Clots and Magnetic nanoparticles Very Likely linked in Covid Gene Therapies. Help Us Test This!

When I first heard of blood clots in vaccinated people, I instantly recalled of a similar problem occurring while the mRNA platform was in study for a cancer therapy, by Moderna, I think, prior to Covid.
I couldn’t find that piece of information again, but during the research I discovered something even more revealing.

Blood clots in subjects of Covid gene therapies are very likely caused by defective coatings in magnetic particles used for magnetofection, which leads to cell-clogging.

Silviu “Silview” Costinescu

It has been more than plausibly theorized that the explanation for the magnetism in vaxxers is magnetofection, a method of transfection using magnetic fields.

Magnetofection is a very effective way of transfecting plasmid DNA into a variety of primary cells including primary neurons which are known to be notoriously difficult to transfect and very sensitive to toxicity.

From: Advanced Drug Delivery Reviews, 2011

For coincidence theorists, let me just add that the inventor of transfection is one of mRNA jabs inventors, Dr. Robert Malone, who has warned FDA on the dangers of these technologies, according to himself.

Scientifically trained at UC Davis, UC San Diego, and at the Salk Institute Molecular Biology and Virology laboratories, Dr. Robert Malone is an internationally recognized scientist (virology, immunology, molecular biology) and is known as one of the original inventors of mRNA vaccination and DNA Vaccination. His discoveries in mRNA non viral delivery systems are considered the key to the current COVID-19 vaccine strategies. Dr. Malone holds numerous fundamental domestic and foreign patents in the fields of gene delivery, delivery formulations, and vaccines.
Dr. Malone has close to 100 peer-reviewed publications and published abstracts and has over 11,477 citations of his peer reviewed publications, as verified by Google Scholar.  His google scholar ranking is “outstanding” for impact factors. He has been an invited speaker at over 50 conferences, has chaired numerous conferences and he has sat on or served as chairperson on numerous NIAID and DoD study sections.

Magnetofection basically involves attaching DNA onto a magnetic nanoparticle coated with a cationic polymer like polyethylenimine (PEI) [254,255]. The magnetic nanoparticles are generally made up of a biodegradable substance like iron oxide, and its coating onto the polymeric particle is done by salt-induced colloidal aggregation.
These prepared nanoparticles are then localized in the target organ by the application of an external magnetic field, which allows the delivery of attached DNA to the target organ, as shown in Figure 3.5. This method also increases the uptake of DNA into target cells as the contact time between the target organ and magnetic nanoparticles increases.
In addition, the magnetic field pulls the magnetic nanoparticles into the target cells, which also helps to increase the uptake of DNA [256,257]. In addition, the standard transfection using viral or nonviral vectors is also increased by the magnetofection.


This is a more powerful method of controlled and targeted delivery for gene therapies, in layman terms.

The problem with it is that it’s been proven to be very dangerous for lab animals and it’s not authorized for human use.

From Dr. Jane Ruby m as well as from Pfizer and Moderna we find out how these particles are packaged into the injectable concocts:

“Stew Peters interviews Dr Jane Ruby who confirms the magnetic effects that Covid vaxxed people have experienced. She says it is a deliberately made substance added to the vaccines. This shows criminal intent. It was added because it is an aggressive delivery system to get it into EVERY cell of your body. The process is called ‘Magnetofection’ and is available in scientific literature such as Pubmed. It concentrates the mRNA into people’s cells and forces your body to make these synthetic mRNA instructions even in places where they shouldn’t be located within the body.

It is a ‘forced delivery system’ and is called by the acronym of SPIONS – Supramagnetic Iron Oxide Nanoparticles. These particles use a lipid nanoparticle envelope to gain entry into the cells. It is done this way to protect mRNA because mRNA is easily degraded and this is also why the Pfizer vaccines are refrigerated at -70 degrees Fahrenheit as another form of protection.

There is a German company on the internet called ‘Chemicell’ which sells different chemicals which can make these magnetic fields around your molecules. You can buy 200 microgram vials of their product called, ‘Polymag’. These are developed and sold for research purposes only and are not to be used for human diagnostic or as a component of any drug intended for humans.

However at least Pfizer and Moderna are using this substance in their vaccines. Therefore it is vital that anyone thinking of taking a shot, obtain a full ingredient list to have full informed consent and to postpone getting the Covid Jab, as each day brings further information into the public domain. Dr Ruby is asked if this was deliberate by the manufacturers and answers that this substance doesn’t occur naturally. It had to be added into the vaccine.

Many have spoken about the Polyethelene Glycol or PEG which enables the vaccines to get through water based cell membranes as this is lipophilic – attracted to fats – but there are other places in the body where ‘God and Nature’ hadn’t intended these substances to be, but by using this delivery system of supra nanoparticles, you are creating a super delivery system which forces these substances into areas where they are not meant to be.”

. 2019 Nov;13(9):1197-1209. doi: 10.1080/17435390.2019.1650969. Epub 2019 Aug 22.

Superparamagnetic iron oxide nanoparticles (SPIONs) modulate hERG ion channel activity

Roberta Gualdani 1 2Andrea Guerrini 1Elvira Fantechi 1Francesco Tadini-Buoninsegni 1Maria Rosa Moncelli 1Claudio Sangregorio 1 3Affiliations expand

Abstract

Superparamagnetic iron oxide nanoparticles (SPIONs) are widely used in various biomedical applications, such as diagnostic agents in magnetic resonance imaging (MRI), for drug delivery vehicles and in hyperthermia treatment of tumors.

Although the potential benefits of SPIONs are considerable, there is a distinct need to identify any potential cellular damage associated with their use.

Since human ether à go-go-related gene (hERG) channel, a protein involved in the repolarization phase of cardiac action potential, is considered one of the main targets in the drug discovery process, we decided to evaluate the effects of SPIONs on hERG channel activity and to determine whether the oxidation state, the dimensions and the coating of nanoparticles (NPs) can influence the interaction with hERG channel.

Using patch clamp recordings, we found that SPIONs inhibit hERG current and this effect depends on the coating of NPs. In particular, SPIONs with covalent coating aminopropylphosphonic acid (APPA) have a milder effect on hERG activity. We observed that the time-course of hERG channel modulation by SPIONs is biphasic, with a transient increase (∼20% of the amplitude) occurring within the first 1-3 min of perfusion of NPs, followed by a slower inhibition. Moreover, in the presence of SPIONs, deactivation kinetics accelerated and the activation and inactivation I-V curves were right-shifted, similarly to the effect described for the binding of other divalent metal ions (e.g. Cd2+ and Zn2+).

Finally, our data show that a bigger size and the complete oxidation of SPIONs can significantly decrease hERG channel inhibition.

Taken together, these results support the view that Fe2+ ions released from magnetite NPs may represent a cardiac risk factor, since they alter hERG gating and these alterations could compromise the cardiac action potential.

MIT SAYS IT’S NOT JUST SPIONS, BUT ALSO LIONS:

HDT Bio, the biotechnology company in Seattle, has an alternative solution. Working with Deborah Fuller, a microbiologist at the University of Washington, it’s pioneering a different kind of protective bubble for the mRNAs. If it works, it would mean that an mRNA vaccine for covid-19 could be stable in a regular fridge for at least a month, or at room temperature for up to three weeks. 

Their method: instead of encasing the mRNA in a lipid nanoparticle, they’ve engineered molecules called lipid inorganic nanoparticles, or LIONs. The inorganic portion of the LION is a positively charged metal particle—so far they’ve been using iron oxide. The positively charged metal would bind to the negatively charged mRNA, which wraps around the LION. The resulting particle is solid, which creates more stability and reduces the reliance on refrigeration. 

A real-world study by the CDC backs up the clinical trial data from both mRNA vaccines—although the rise of the UK variant in the US is a cloud on the horizon.

“The cold chain has always been an issue for [the] distribution of vaccines, and it’s only magnified in a pandemic.”

Deborah Fuller

HDT Bio initially developed LIONs to treat liver cancer and tumors in the head and neck, but when the pandemic hit, they pivoted to trying the particles with mRNA vaccines. Early preclinical trials in nonhuman primates showed that the LION, combined with an mRNA vaccine for covid-19, worked as they’d hoped.

Carter of HDT Bio says that in an ideal situation, LIONs could be sent to clinics worldwide in advance, to be stored at room temperature or in a regular refrigerator, before being mixed into vaccine vials at clinics. Alternatively, the two could be premixed at a manufacturing facility. Either way, this method would make doses stable for at least a month in a regular refrigerator. 

Fuller says that some scientists have criticized the need for two vials—one for the LION and another for mRNA before they’re mixed together. “But I think the advantages of having an effective product more amenable to worldwide distribution outweighs those negatives,” she says.

HDT Bio is applying for permission to start human clinical trials in the US and is looking to start clinical trials in India this spring. In the US, it faces some unique challenges in FDA regulation, since the LION particles would be considered a drug separate from the vaccine. Regulators in Brazil, China, South Africa, and India—where HDT Bio is hoping to launch its product—don’t consider the LION a drug because it isn’t the active component, says Carter, meaning that there would be one less layer of regulation than in the US.

For now, it’s still very much an early-stage technology, says Michael Mitchell, a bioengineer at the University of Pennsylvania who works on drug delivery systems. He stresses that more research should reveal whether the iron oxide causes any side effects. – MIT Technology Review

Now here’s the bombshell:


This is no secret to experts, but it’s been revealed to me in the video presentation below, made in 2017 by reputed Prof Diana Borca, from Rensselaer Polytechnic Institute, who uses magnetic nanoparticles to treat diseases.
In order to get the magnetic nanoparticles into the right places, scientists like Diana have to figure out what kind of coating the nanoparticles need. Coatings help the nanoparticles get to the cells they want to treat without hurting the healthy cells.
And if the coating of the magnetic particles breaks, the result is “CLOGGING”, as Borca explains below. Which can translate as clotting, if in blood.
Who knows what they lead to when in other organs, strokes maybe?

So I think the only thing we’re missing from the puzzle is official hard evidence that they used magnetofection or magnetogentic methods.

But if it walks like a duck and quacks like a duck, only the government needs government papers to confirm it’s a duck


What each and every one of you can do until we find that evidence?

On screens we’re sound. Please help with the statistical and empirical tests!


Please help finding out if there’s a strong data and empirical correlation between blood clots and magnetism. Anyone you know that has been jabbed and experienced blood clots, heart or circulatory problems needs to take the magnet challenge right now! A strong enough correlation indicates causation.
If you make such a test, please reach us on our socials and communicate the result, whether positive or negative!
Also VAERS is exploding with reports of magnetism, please help analyzing the data to see if it pairs with clotting.
Thank you!

Also food for thought: isn’t this also related to the problems these GMO dupes experience during air-travel?
I’ll investigate this in a soon coming report.

References:

Nanoparticles in Translational Science and Medicine

Akira Ito, Masamichi Kamihira, in Progress in Molecular Biology and Translational Science, 2011

V Conclusion

This chapter highlighted magnetofection, magnetic patterning of cells, and construction of 3D tissue-like structures. Among them, Mag-TE for constructing 3D structures has been extensively studied, and various kinds of other tissues such as retinal pigment epithelial cell sheets,102 MSC sheets,44 and cardiomyocyte sheets,46 have been already generated. Tubular structures consisting of heterotypic layers of endothelial cells, smooth muscle cells, and fibroblasts have also been created.43 In this approach, magnetically labeled cells formed a cell sheet onto which a cylindrical magnet was rolled, which was removed after a tubular structure was formed. If these processes can be scaled up, there is great potential for these techniques in the treatment of a variety of diseases and defects.

In the translational research, toxicology of functional magnetite nanoparticles is an important issue. The main requisite for a cell-labeling technique is to preserve the normal cell behavior. As for biocompatibility of MCLs, no toxic effects against proliferation of several cell types were observed within the range of magnetite concentrations tested (e.g., human keratinocytes,63 < 50 pg-magnetite/cell; HUVECs,41 HAECs,42 human dermal fibroblasts,41 human smooth muscle cells,43 mouse fibroblast cells,43 canine urothelial cells,43 human MSCs,44 and rat MSCs45 < 100 pg/cell). Moreover, MCLs did not compromise MSC differentiation44,45 or electrical connections of cardiomyocytes.46 In addition, an in vivo toxicity of magnetite nanoparticles has been extensively studied. As an MRI contrast agent, ResovistR was first applied clinically for detecting liver cancer, since ResovistR is taken up rapidly by the reticuloendothelial system such as Kupffer cells of the liver compared with the uptake by cancer cells of the liver. In a preliminary study,103 the authors investigated the toxicity of systemically administered MCLs (90 mg, i.p.) in mice; none of the 10 mice injected with MCLs died during the study. Transient accumulation of magnetite was observed in the liver and spleen of the mice, but the magnetite nanoparticles had been cleared from circulation by hepatic Kupffer cells in the spleen by the 10th day after administration.103

In conclusion, magnetic nanoparticles have been developed into “functional” magnetite nanoparticles which are highly promising tools for a wide spectrum of applications in tissue engineering. The proven lack of toxicity of the functional magnetite nanoparticles is expected to provide exciting tools in the near future for clinical tissue engineering and regenerative medicine.View chapter

Viral and Nonviral Vectors for In Vivo and Ex Vivo Gene Therapies

A. Crespo-Barreda, … P. Martin-Duque, in Translating Regenerative Medicine to the Clinic, 2016

2.2.1 Magnetic Nanoparticles

One of the pioneers using magnetofection for in vitro applications was Lin et al.91 There are various cationic magnetic nanoparticles types that have the capacity to bind nucleotidic material on their surface. With this method, the magnetic nanoparticles are concentrated in the target cells by the influence of an external magnetic field (EMF). Normally, the internalization is accomplished by endocytosis or pinocytosis, so the membrane architecture stays intact. This is an advantage over other physical transfection methods. Other advantages are the low vector dose needed to reach saturation yield and the short incubation time needed to achieve high transfection efficiency. Moreover, with the application of an EMF, cells transfected with magnetic nanoparticles can be used to target the region of interest in vivo.

2.2.1.1 Iron Oxide Nanoparticles

The magnetic nanoparticles most used in magnetofection include the iron oxide nanoparticles (IONPs). IONPs are biodegradable and not cytotoxic and can be easily functionalized with PEI, PEG, or PLL. Poly-l-lysine-modified iron oxide nanoparticles (IONP–PLL) are good candidates as DNA and microRNA (miRNA) vectors because they bind and protect nucleic acids and showed high transfection efficiency in vitro. In addition, they are highly biocompatible in vivo.

Chen et al.92 used human vascular endothelial growth factor siRNA bound to superparamagnetic iron oxide nanoparticles (SPIONs) and it was capable of hepatocellular carcinoma growth inhibition in nude mice. Moreover, Li et al.93 demonstrated that the intravenous injection of IONP–PLL carrying NM23-H1 (a tumor suppressor gene) plasmid DNA significantly extended the survival time of an experimental pulmonary metastasis mouse model.

Another advantage of this kind of nanoparticles is that they can be used as MRI agents. Chen et al.94 bound siRNA to PEG-PEI SPIONs together to a gastric cancer-associated CD44v6 single-chain variable fragment. This bound permitted both cancer cell’s transfection and their visualization by MRI.

But those complexes might be used for cell therapies as well. Schade et al.95 used iron oxide magnetic nanoparticles (MNPs) to bind miRNA and transfect human mesenchymal stem cells. As the binding between the MNPs and PEI took place via biotin-streptavidin conjugation, these particles cannot pass the nuclear barrier, so they are good candidates to deliver miRNA, as it exerts its function in the cytosol. They functionalized the surface nanoparticles with PEI and were able to obtain a better transfection than PEI 72 h after transfection. Moreover, they demonstrated that magnetic polyplexes provided a better long-term effect, also when included inside of the stem cells.View chapter

Synthesis of Magnetic Iron Oxide Nanoparticles

Marcel Wegmann, Melanie Scharr, in Precision Medicine, 2018

4.1.4 Magnetofection

Another attempt to apply magnetic IONPs is the so-called magnetofection (MF) approach. Key factors enabling this method are IONPs that are coupled to vector DNA and guided by the influence of an external magnetic field. By this means, DNA can be transfected into cells of interest. One possibility to enable enhanced binding capabilities of the negatively charged DNA to magnetic IONP beads is the coating IONPs with a positively charged material such as polyethylenimine. The efficiency of the vectors has hence shown to increase up to several thousand times (Scherer et al., 2002). The above depicted engagement of IONPs in MF has shown to be universally applicable to viral and nonviral vectors. This is mostly because it is very rapid and simple. Furthermore, it is a very attractive approach since it yields saturation level transfection at low-dose in vitro (Krotz et al., 2003). Fernandes and Chari (2016) have demonstrated an approach delivering DNA minicircles (mcDNA) to neural stem cells (NSCs) by means of MF. DNA minicircles are small DNA vectors encoding essential gene expression components but devoid of a bacterial backbone, thereby reducing construct size versus conventional plasmids. This could be shown to be very beneficial for the use of genetically engineered NSC transplant populations in regenerative neurology. The aim was to improve the release of biomolecules in ex vivo gene therapy. It could be demonstrated that MF of DNA minicircles is very safe and provided for sustained gene expression for up to 4 weeks. It is described to have high potential as clinically translatable genetic modification strategy for cell therapy (Fernandes and Chari, 2016). The last in vitro application for magnetic nanoparticles to be presented in this chapter will be tissue repair.View chapter

Scientific Fundamentals of Biotechnology

Aline Do Minh, … Amine A. Kamen, in Comprehensive Biotechnology (Third Edition), 2019

1.26.2.1.7 Magnet-Mediated Transfection

Two methods rely on the application of a magnetic field for gene transfer. Magnetofection uses magnetic nanoparticles coated with DNA in presence of a magnetic field. The nucleic acid-nanoparticle complexes are driven toward and into the target cells by magnetic force application. Gene transfer is enhanced by magnetofection as DNA-loaded particles are guided and maintained in close contact with the target cells. Cellular uptake through endocytosis is thus increased as well. The process has been mainly applied to cultured cells and has been proven more efficient than other chemical methods in some cases.8 The second method is magnetoporation in which membrane permeability is increased, triggered by the applied magnetic field.9View chapter

Fabrication and development of magnetic particles for gene therapy

S. Uthaman, … C.-S. Cho, in Polymers and Nanomaterials for Gene Therapy, 2016

9.4.1 Magnectofection-based gene delivery

For gene therapy applications, magnetic particles are generally used for increasing the transfection efficiencies of cultured cells, a technique known as magnetofection [91–104] in which magnetic particles and nucleic acids are mixed together and then added to the cell culture media. The nucleic acid-bound magnetic particles then move from the media to the cell surface upon the application of an external magnetic force, as shown in Figure 9.1. The principle advantage of this approach is the rapid sedimentation of the gene-therapeutic agent onto the target area, thereby reducing the time and dose of vector to achieve highly efficient transfection, with lower cell cytotoxicity.

In in vivo magentofection, the magnetic field is focused over the target site. This method has the potential not only to enhance transfection efficiency but also to target the therapeutic gene to a specific organ or site, as shown in Figure 9.2.

Generally, magnetic particles carrying therapeutic genes are injected intravenously. As the particles flow through the bloodstream, they are captured at the target site using very strong, high-gradient external magnets. Once they are captured, the magnetic particles carrying the therapeutic gene are taken up by the tissue, followed by release of the gene via enzymatic cleavage of cross-linked molecules or degradation of the polymer matrix. If DNA is embedded inside or within the coating material, the magnetic field must be applied to heat the particles and release the gene from the magnetic carrier [105].View chapter

Nonviral Vectors for Gene Therapy

Tyler Goodwin, Leaf Huang, in Advances in Genetics, 2014

3.4 Magnetic-Sensitive Nanoparticles (Magnetofection)

In an attempt to address the transient damage caused by the invasive methods mentioned above (i.e., hydrodynamic injection and electroporation), magnetofection techniques have been introduced. This technique uses the physical method of a magnetic field to direct the deliver of genetic material to the desired target site. The concept involves attaching DNA to a magnetic nanoparticle usually consisting of a biodegradable substance such as iron oxide and coated with cationic polymer such as PEI (Mulens, Morales, & Barber, 2013). These magnetic nanoparticles are then targeted to the tissue through a magnetic field generated by an external magnet. The magnetic nanoparticles are pulled into the target cells increasing the uptake of DNA. This technique is noninvasive and can precisely target the genetic material to the desired site while increasing gene expression. The drawback to magnetofection is the need to formulate magnetic nanoparticles complexed with naked DNA, as well as the need for strong external magnets.View chapter

Small interfering RNAs (siRNAs) as cancer therapeutics

G. Shim, … Y-K. Oh, in Biomaterials for Cancer Therapeutics, 2013

11.3.5 Stimulus-guided delivery

Stimulus-guided delivery is a non-invasive and convenient approach for clinical applications. Several methods in this category, including electroporation, ultrasound and magnetofection, have been used to deliver siRNAs to specific tissue sites. Owing to constraints associated with application of external stimuli under in vivo conditions, most such studies have been done in vitro. However, in vivo applications of stimulus-guided delivery of anticancer siRNAs are increasingly being reported.

Electroporation has been studied as a means for facilitating in vivo delivery of anticancer siRNAs. Notably, an electroporation method employing a new type of ‘plate and fork’ type electrode has been applied in vivo in mice (Takei et al., 2008). In this application, a chemically modified form of VEGF-specific siRNA in phosphate-buffered saline was intratumorally administered at three doses of 0.08, 0.17 and 0.33 mg/kg, or intravenously administered at a single dose of 6.6 mg/kg. Then, an electronic pulse was applied to a pair of plate and fork electrodes pre-inserted into PC-3-xenografted tumour tissues. Application of electroporation inhibited tumour growth to a similar degree after 0.17 mg/kg intratumoral and 6.6 mg/kg intravenous doses, in each case producing a 40-fold greater inhibitory effect than a local dose. Notably, the duration of the antitumour effect was maintained for 20 days after a single injection via the local or systemic route.

Magnetically guided in vivo siRNA delivery has been investigated using magnetic crystal-lipid nanostructures (Namiki et al., 2009). In this study, a magnetite nanocrystal was coated with oleic acid and a cationic lipid shell, and complexed to EGFR-specific siRNA. Following intravenous administration to mice, siRNA complexed to the magnetic core-encapsulated cationic lipid shell showed a rank order of tissue distribution of spleen followed by liver and lung. For in vivo magnetofection, titanium nitride-coated magnets were internally implanted under the skin peripheral to tumour lesions or were externally placed onto the skin. Mice were intravenously given a total of eight 0.3 mg/kg doses of siRNA complexed to cationic nanoshells administered every other day. Both internal and external applications of a magnetic field reduced tumour (MKN-74 or NUGC-4) volume by 50% compared with the control group 28 days after the initiation of treatment.

Ultrasound-guided siRNA delivery has also been used to increase the in vivo delivery of siRNAs. Ultrasound can produce cavitation, thereby resulting in transient disruptions in cell membranes within tissues (Vandenbroucke et al., 2008). Few studies have addressed the in vivo antitumour effects of ultrasound-guided anticancer siRNAs. To date, most such studies have evaluated the feasibility of the method using siRNAs specific for reporter genes, such as enhanced green fluorescent protein (Negishi et al., 2008). In this latter study, PEG-modified cationic lipid nanobubbles entrapping the ultrasound imaging gas perfluoropropane were complexed with enhanced green fluorescent protein-specific siRNA and intramuscularly administered at a dose of 0.15 mg/kg to mice transfected 1 day prior with enhanced green fluorescent protein-encoding plasmid DNA. Three days after siRNA injection and ultrasound application, fluorescent protein levels at the injection sites were reduced.

Although the feasibility of in vivo applications of stimulus-guided delivery of anticancer siRNA has been demonstrated and positive results have been reported, the ultimate success of these delivery methods may depend on the development of devices capable of providing a sufficient stimulus to tumour tissues deep within the body. Moreover, for in vivo systemic administration, delivery systems that carry both external stimulus-responsive agents and siRNA must meet more general requirements, such as in vivo stability, low toxicity and enhanced tumour tissue accumulation. With the concurrent progress in medical device bioengineering and siRNA delivery technologies, it can be expected that stimulus-guided strategies will be used in more diverse in vivo applications to facilitate anticancer siRNA delivery.View chapter

Gene Delivery Using Physical Methods

Kaustubh A. Jinturkar, … Ambikanandan Misra, in Challenges in Delivery of Therapeutic Genomics and Proteomics, 2011

3.9 Magnetofection

Various physical methods of gene delivery have been developed, and each one has its own merits and demerits. EP is particularly important for introducing DNA to superficial areas, but to deliver DNA to particular organs, surgery is required. To overcome this problem and to enhance the introduction of gene vectors into cells [254], the new means of physical gene delivery is magnetofection, which delivers DNA to the target organ, using the magnetic field. Magnetofection basically involves attaching DNA onto a magnetic nanoparticle coated with a cationic polymer like polyethylenimine (PEI) [254,255]. The magnetic nanoparticles are generally made up of a biodegradable substance like iron oxide, and its coating onto the polymeric particle is done by salt-induced colloidal aggregation. These prepared nanoparticles are then localized in the target organ by the application of an external magnetic field, which allows the delivery of attached DNA to the target organ, as shown in Figure 3.5. This method also increases the uptake of DNA into target cells as the contact time between the target organ and magnetic nanoparticles increases. In addition, the magnetic field pulls the magnetic nanoparticles into the target cells, which also helps to increase the uptake of DNA [256,257]. In addition, the standard transfection using viral or nonviral vectors is also increased by the magnetofection.

The magnetofection has some drawbacks: a particle size below 50 nm renders it not suitable for magnetic targeting and too large a particle size (more than 5 μm) retards the entry of magnetic nanoparticles inside the blood capillaries. The blood flow rate also affects the transfection efficacy of this method; for example, the flow rate of around 20 cm/s in the human aorta makes the transfection tricky. The external magnetic flux density and gradient decreases at a distance from the magnetic pole, which also affects the transfection efficacy.

Primary endothelial cells are effectively transfected by magnetofection [254,258]. In addition, magnetofection is effective for in vitro and in vivo delivery of DNA to target cells like those in the GI tract and blood vessels [254], and for antisense ODNs delivery [259]. Other applications include advances in ex vivo tissue engineering, development of tumor vaccines, localized therapy for cancer, and cardiovascular therapy [260]. Significant enhancement in reporter gene expression in a short time has been observed in the ex vivo porcine airway model; this may be attributed to an increase in contact time with mucociliary cells, thereby reducing their clearance from the target site [261]. A study carried out using magnetic albumin microspheres with entrapped doxorubicin in the rat model for tumors resulted in a high level of tumor remission in animals compared to animals treated with free doxorubicin, placebo microspheres, or nonlocalized doxorubicin microspheres, which resulted in considerable enlargement in tumor size associated with metastases and subsequent death [262,263]. The magnetic nanoparticles with doxorubicin are also under clinical trial [264]. Magnetofection has been widely used for viral and nonviral vectors and also for the delivery of DNA, nucleic acids, and siRNA [260,265,266].

In conclusion, magnetofection is an efficient system for gene delivery and has the potential to bring in vitro and in vivo transgene transfection in the target organ. The limitations of this delivery system are overcome by the application of proper formulations and novel magnetic field skills.View chapter

Gene therapy approaches in central nervous system regenerative medicine

Assumpcio Bosch, Miguel Chillon, in Handbook of Innovations in Central Nervous System Regenerative Medicine, 2020

10.2.6 Nonviral vectors

Nonviral vectors group a heterogeneous variety of elements that can be classified as naked DNA or RNA, liposome-DNA complexes (lipoplexes), and polymer-DNA complexes (polyplexes). Since the beginning of the gene therapy field, nonviral vectors have received significant attention due to their reduced pathogenicity, lower immunotoxicity, and low cost and ease of production over viral approaches. To date, a myriad of delivery systems grouped as physical methods and chemical carriers have been reported. Physical methods such as direct injection, ballistic DNA, electroporation, sonoporation, photoporation, magnetofection, hydroporation, and mechanical massage, employ physical force to cross the cell membrane barrier. Chemical carriers such as (1) inorganic particles (calcium phosphate, silica, gold, but also magnetic nanoparticles, fullerenes, carbon nanotubes, quantum dots, and supramolecular systems); (2) lipid-based (cationic lipids, lipid-nano emulsions, solid lipid nanoparticles); (3) peptide-based; and (4) polymer-based (i.e., polyethylenimine, chitosan, dendrimers, and polymethacrylate) form small size complexes with nucleic acids to help them cross the cell membrane efficiently (see ref [29] for extensive review). However, despite the large number of different nonviral vectors still, there is poor transduction efficiency of the target cells as well as low and transient transgene expression. Due to it, nonviral vectors account for less than 25% of the clinical assays, mainly for cancer and cardiovascular diseases, being naked/plasmid DNA (452 clinical assays) and lipofection (119 clinical assays) the systems more frequently used, while all the rest of the nonviral vector account only for 3% of the assays.View chapter

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