Nano-particles Gene Therapy influence, in C19 vaccines, and relationship with Micro-Waves.

In physics, microwaves are electromagnetic radiations with a wavelength between the ranges of Radio Waves (RW) and Infrared Radiation (IR).

The boundary between the microwaves and the neighboring radiation ranges is in fact not clear, the microwaves are included in the wavelengths between 33 cm, which corresponds to the frequency of about 1 GHz, and 1 mm, which corresponds to about 300 GHz.

The microwave spectrum is usually defined in the frequency range between 300 MHz and 300 GHz, most applications operate between 1 and 40 GHz:

-For example, the microwave oven uses a Magnetron generator to produce microwaves at a frequency of about 2.45 GHz.

-Gsm cell phones operate on the 1.8 GHz frequency.

- The wireless communication protocols, such as bluetooth and VLAN / Wi-Fi Internet networks, in the G and B variants, use microwaves in the 2.4 GHz band (the A variant instead works in the 5 GHz band).

Numerous countries plan to use spectrum in the 4.5-5 GHz range for 5G, including China and Japan.

At the other end of the spectrum (low frequencies), Europe has favored the 700 MHz band for the capillary coverage of the 5G territory.

This preface will be useful later to understand the evolution of the state of the art of gene therapy applied in experimental vaccines against SARS CoV 2 (SC2).

Having said this, it should be noted that our DNA has evolved in harmony with the terrestrial natural resonance waves which correspond to 8 HZ.

In fact, these harmonics are identified in the ultra-low frequency range (ELF) and range from 7 Hz to 500 Hz, while otherwise the harmonics of pathogenic bacteria, or viruses, such as E. Coli, range from much higher frequencies, i.e. 'from 1000 Hz to 3000 Hz (from 10 ^ 8, to 10 ^ 13).

In the Paper by Montagnier, and Perez, the frequency / virus ratio is displayed very accurately.

https://www.researchgate.net/publication/45272907_Electromagnetic_Signals_Are_Produced_by_Aqueous_Nanostructures_Derived_from_Bacterial_DNA_Sequences

Ultrasound has the potential to damage coronaviruses, according to an MIT study.

A new study by researchers from MIT's Department of Mechanical Engineering suggests that coronaviruses may be vulnerable to ultrasound vibrations, within the frequencies used in medical diagnostic imaging.

Using computer simulations, the team modeled the virus's mechanical response to vibrations across a range of ultrasonic frequencies. They found that vibrations between 25 and 100 MHz triggered the virus's shell and spikes to collapse and began to break apart in a fraction of a millisecond.

This effect was observed in simulations of the virus in air and water.

The researchers say their findings are a first clue to a possible ultrasound-based treatment for coronaviruses, including the new SARS-CoV-2 virus.

How exactly ultrasound could be administered, and how effective it would be in harming the virus within the complexity of the human body, are among the main questions scientists will face in the future.

"We have shown that under the excitation of ultrasound, the shell and spikes of the coronavirus will vibrate and the amplitude of that vibration will be very large, producing visible damage to the outer shell and possibly invisible damage to the RNA inside," says Tomasz. Wierzbicki, professor of applied mechanics at MIT.

(The team's findings appear online in the Journal of the Mechanics and Physics of Solids.)

The researchers introduced acoustic vibrations into the simulations and observed how the vibrations propagate through the virus structure across a range of ultrasonic frequencies.

The team started with vibrations of 100 megahertz, in a fraction of a millisecond external vibrations, resonating with the frequency of the virus's natural oscillations, caused the shell and tips to bend inward.

At frequencies lower than 25 MHz and 50 MHz, the virus deformed and fractured even faster, both in simulated environments of air and water that is similar in density to fluids in the body.

The team predicts that ultrasound, which is already used to break up kidney stones and to deliver drugs through liposomes, could be used to treat and possibly prevent coronavirus infection.

The researchers also predict that miniature ultrasound transducers, inserted into phones and other portable devices, may be able to protect people from the virus.

https://news.mit.edu/2021/ultrasound-coronaviruses-damage-0316

The above is now relevant in order to introduce the latest updates regarding research on Gene Therapy (GT).

Nano-conjugates are nano-assemblies composed of nano-materials (metalloids), anti-microbial agents (graphene) and nano-polymers / nano-gels (PEI, PEG) that are linked together by chemical reactions.

Compared to nano-liposomes or nano-particles, nano-conjugates can increase drug load, improve physical stability and avoid leakage from nano-vectors in vitro or in vivo, increase drug accumulation.

The intervention therapies in which the nano-conjugates are provided are divided into 4 methodologies:

NPTT: Nano-Photo-Thermal Therapy

NMH: Nanomagnetic hyperthermia

NaRFA: Ablation with nano-radiofrequency

NUH: Nano-ultrasound hyperthermia.

Specifically, magnetic nano-particles (MNPs) act by induced magnetic hyperthermia, through a controlled release of drugs and other biomedical applications, such as vaccines.

Magnetic nano-particles are a class of nanoparticles that can be manipulated using a magnetic field.

These particles usually consist of magnetic elements such as: iron, nickel and cobalt and their chemical compounds.

Among these, the SPIONs nanoparticles (Super Paramagnetic Iron Oxide Nanoparticles), consisting of iron oxides such as Fe3O4 (magnetite) or γ-Fe2O3 (maghemite), have assumed particular importance.

https://www.nature.com/articles/ncomms15594

It should be noted that the vascular endothelium presents a natural barrier for the transport and administration of the drug, allowing only the transport of small molecules.

Thus, the enhancement of drug transport across the endothelial barrier can be enhanced by using an external magnetic field to temporarily disrupt adherent endothelial junctions through internalized iron oxide nanoparticles.

In recent years, magnetic nanoparticles (MNPs) have emerged as promising drug carriers, in magnetic drug delivery, synergistic chemotherapy, and hyperthermia.

Magnetite nanocrystals (16 and 33 nm in diameter) were synthesized through the thermo-decomposition of iron acetylacetonate

The water-dispersible MNPs were synthesized by coating the nanocrystals with phospholipid-poly (ethylene glycol) PEG.

The endothelial canals, when subjected to a magnetic field, undergo the direction flux of the magnetic force and adapt to this direction, which is not congenital, bordering on a resistance to the limit, equal to a shear stress.

This suggests how the endothelial canals can undergo unnatural tensions or deformations, which can involve the correct functioning of the circulatory system.

https://pubs.acs.org/doi/10.1021/la104479c

In the administration of experimental vaccines, the use of superparamagnetic nanoparticles (SPION), to deliver genes via magnetofection (through a ratio between PEI nitrogen and DNA phosphate), could improve the efficiency of transfection and direct the vector to the desired location.

Magnetofection, a direct electrostatic attraction between the negatively charged SPIONs (due to the presence of carboxyl groups) and the positively charged, external polyethyleneimine (PEI) polymer, was used to improve delivery of a malaria DNA vaccine that encodes for the merozoite surface protein Plasmodium yoelii MSP1.

The transfection efficiency of the magnetic nanoparticle as a vector for a malaria DNA vaccine (in vitro) in eukaryotic cells was significantly improved under the application of an external magnetic field.

The SPION system is also useful for the purification of compounds / vaccines, as it allows an ability to immobilize biological materials on their surfaces and potential for direct targeting, using external magnets.

It is very important to remember that this system allows you to work safely when, for example, biological materials are used such as: VLP particles and animal or insect cells together with adenoviral vectors; the magnetic field immobilizes these biological materials and does not allow the adenoviral vector, bound together, to escape due to its extreme volatility (as instead happened in the BSL4 of Whuan's WIV, during a failed Gain Of Function experiment).

Magnetofection arises from the concept of the release of magnetized drugs, demonstrating the applicability to gene delivery with viruses and viral vectors.

Magnetic nano-particles are generally used with any gene delivery vector; Numerous synthesis methods have been used to produce magnetic nano-particles (Ferro-Fluid Colloids) for bio-applications, including microemulsions, poly-oils, sol-gel synthesis, hydro-thermal, hydro-lysis, thermo-lysis, injection and electrospray.

However, the common method for synthesizing magnetite particles in solution is given by the chemical co-precipitation of iron / salts.

Various materials have been used as protective coatings for magnetic nano-particles; for example polyethyleneimine (PEI) is one of the most ef fi cient ionic compounds for plasmid DNA delivery in mammalian cells

It is known that the PEI polymer forms ionic complexes, with SPION, which then interact non-specifically with negatively charged DNA and enter the cell via endocytosis.

- One of the main problems related to the use of cationic polymers for DNA transfer is related to their toxicity, due to the charge of the polymer

For this reason, several laboratories are trying to improve the architecture of polymers and their biophysical properties.

In conclusion, for gene transfer mediated by chemicals such as: Polyethylene Glycol (PEG) and Dextran Sulphate (polymer loaded with anticoagulant and hypocholesterolemic properties), they induce the absorption of DNA.

Dextran Sulfate inhibits, in vitro, the binding of the human immunodeficiency virus to T lymphocytes and viral replication.

https://pubs.acs.org/doi/10.1021/acscentsci.0c01405

Another MNP example is that made by biochemists at Stanford University, California, who created a COVID-19 vaccine prototype based on nano-particles with iron-containing proteins, called ferritin.

First, they have formulated a shortened version of the Spike virus spike that is easier to synthesize and use.

They bonded these shortened peaks to ferritin nanoparticles, then used electron microscopy to confirm they had the correct structure.

They designed subunit vaccine candidates using self-assembling ferritin nanoparticles that exhibit one of two multimerized SARS-CoV-2 peaks: full-length ectodomain (S-Fer) or a C-terminal 70 amino acid deletion (SΔC-Fer).

We confirmed the correct folding and antigenicity of the spike on the ferritin surface by cryo-EM and binding with conformation-specific monoclonal antibodies.

Nano-Bots

https://onlinelibrary.wiley.com/doi/10.1002/adma.202003013

To remotely activate and control the microbots, the Max Plank labs incorporated biocompatible superparamagnetic iron oxide nanoparticles (SPION) into our photoresist formulation for 3D microprinting of zwitter-ionic magnetic nanocomposites in a single step.

A 3D micro-printed of the zwitter-ionic micro-robots was made with a helical-shaped design and micro-scale activation by magnetic torque (more efficient than the traction of the magnetic gradient at the microscale) was used, which is one of the most common strategies. to swim at the low Reynolds number regime in synthetic microbots.

External magnetic fields (10 mT) were used to induce rotation torque on the microrobots and push them through an aqueous solution.

At the optimum frequency range 10 Hz / 13 Hz, the magnetic torque overcomes the friction of the substrate and a "corkscrew" locomotion is obtained through the fluid with zero drift.

In order to overcome the innate defense mechanism of the immune system, the aim was to prevent the absorption of non-specific proteins, on the surface of the microbot, and to delay their detection by macrophages.

Poly (ethylene glycol) (PEG) and its derivatives have been widely used in drug delivery platforms, for low encrustation materials, against protein absorption; however, they are eventually recognized and cleared by immune cells due to insufficient anti-fouling properties and anti-PEG antibodies.

This expedient allows nano-robot boats to act, for at least 90 hours, with pre-established medical action (penetration of barriers, assimilation of drugs, destruction of foreign bodies, etc.) using low voltage electromagnetic fields.

We are now getting to the heart of the topic of MNP technologies implemented with LNP surfaces…

MIT is a breakthrough in the design of NP polymer nano-particles.

MIT makes it possible to produce intelligent materials in nano and larger dimensions with active sites that match the size and functionality of the target compound, the so-called model, within a polymer matrix.

The combination of sensitivity to external stimuli modulates the affinity of the polymer coating by providing the ability to release the sensitive load, such as drugs or other elements, to stimuli after the application of specific release triggers; in particular, changes in the environment such as: heat, changes in pH, light, electric or magnetic fields, enzymes, reduction waves and ultrasounds.

the application on the thin polymer shell, printed on the surface of the MNPs, of drug delivery vectors (carried out by surface modification with a medicinal ligand)

make them integrable with other similar types of nano-particles, co-polymerized, containing MRA (for example Biontech vaccine).

https://www.mdpi.com/1999-4923/12/9/831/htm

Lipid nanoparticles (LNPs) are used as carriers of messenger RNA (mRNA).

The simple mixing of PEGylated mRNA (PEG-OligoRNA) with lipofectamine LTX, a commercial lipid-based carrier, pipetted in aqueous solution, enabled the successful preparation of mRNA-loaded LNPs with a diameter of less than 100 nm.

The types of nano-platforms, which have been exploited for in vivo mRNA delivery, include: lipid nanoparticles (LNPs; including cationic liposomes / lipoplexes with lipid bilayers and more often with a lipid monolayer and lipophilic nuclei), of polymers, lipid-polymer hybrids.

Since mRNAs are negatively charged due to the presence of phosphate groups in their backbone, mRNA transport systems are typically composed of a positive cationic lipid or polymer, to allow ionization at a lower pH to facilitate encapsulation and / or complexation with an mRNA.

These lipids or polymers usually carry tertiary amino groups which allows them to be largely neutral at physiological pH, the polymer used by Biontech is: MW PEG 2000.

-Propane 1,2-dioleoyl-3-trimethylammonium (DOTAP, pKa 6.3) is one of the most used cationic lipids in mRNA and gene release with proven transfection efficiency in vitro and in vivo.

-The zwitterionic phospholipid, 1,2-dioleoylphosphatidylethanolamine (DOPE) was one of the most widely used helper lipids, due to its further fusogenicity which favors both cellular uptake and intracellular trafficking.

Other techniques such as the emulsion method (ethanol is replaced with an oily phase) usually with the help of homogenization can also be used to prepare LNP-mRNA.

The modification of the PEG lipid surface, with a specific ligand (metalloids, graphite, graphene, or even glycans, cholesterol, or targeted drugs) would involve a faster absorption but, at the same time, a sensitivity to magnetic fields.

PEGylation leads to the "PEG dilemma"; if on the one hand spherical stabilization is guaranteed, in vitro, and the protection of rapid elimination, in vivo, against the obstacle of both cellular absorption and endosomal escape, on the other hand PEG polymers can trigger the production of anti-PEG antibodies, causing the phenomenon of accelerated blood clearance (ABC), especially in subsequent inoculation.

Therefore, the LNP formulations reported for mRNA delivery rarely used MW PEG 2000 lipids, as did Biontech.

In conclusion, PEGylated vaccines can cause life-threatening anaphylactic reactions in people who previously had high levels of anti-PEG antibodies.

Severe anaphylactic reactions have been reported following administration of mRNA vaccines.

LNP-mRNA TYPES

- naked mRNA (part a);

- naked mRNA with in vivo electroporation (part b);

-mRNA complex of protamine (cationic peptide) (part c);

-mRNA associated with a positively charged oil-in-water cationic nanoemulsion (part d);

-mRNA associated with a chemically modified dendrimer and complexed with polyethylene glycol (PEG) -lipid (part e);

-mRNA protamine complex mRNA in a PEG lipid nanoparticle (part f);

-mRNA associated with a cationic polymer such as polyethyleneimine (PEI) (part g);

-mRNA associated with a cationic polymer such as PEI and a lipid component (part h);

-mRNA associated with a polysaccharide particle or gel (e.g. chitosan) (part i);

-mRNA in a cationic lipid nanoparticle (e.g., lipid 1,2-dioleoyloxy-3-trimethylammoniumopropane (DOTAP) or dioleoylphosphatidylethanolamine (DOPE)) (part j);

-mRNA complexed with cationic lipids and cholesterol (part k);

-mRNA complexed with cationic lipids, cholesterol and PEG lipids (part l).