ADVANCED TECHNOLOGY 7

RESEARCHERS TEAM

Scientific Technical Report

Summary

· PARAGRAPH 1:

Choice of the technology used for

the product

· PARAGRAPH 2:

Pain description

· PARAGRAPH 3:

Pain intensity Measurement

· PARAGRAPH 4:

Pain Management

· PARAGRAPH 5:

How an electrical impulse is

generated

· PARAGRAPH 6:

Lorentz force and mass

spectrometer

· PARAGRAPH 7:

Biological effects of high gradient

magnetic fields

· PARAGRAPH 8:

Bio-photonic Technology

· PARAGRAPH 9:

Radionic Technology

“The goal o f this research f rom the beginn ing has been to help peop le w h o suffer to improve thei r qual i t y o f l i fe, thus a l lowing greater sereni ty o n a psychological level.

T o achieve this, the team worked to p roduce an aid that works on 3 fundamen ta l aspects :

1) Pain reduction 2) Support cel l regenerat ion

3) Ba lance be tween the source of the pain and the sur round ing env i ronment .

To achieve this, we have used as m an y 3 different technologies w i th speci f ic purposes.

In the fol lowing report we wil l give you an overview of the laws of Physics, Chemistry, Magnet ism, Quantum Mechanics, Electrophysiology, Neurology, Biophotonics, Radionics.

This report was del iberately created for an aud ience that wan ts to unders tand bet ter and m o r e thorough l y h o w the idea for the product came about .

I t i s no t an in-depth scient i f ic treat ise, which wou ld l ike ly have been more confus ing and probably m a d e the in fo rmat ion underlying the project less clear.

PARAGRAPH 1: Choice of the technology

used for the product

The product is the result o f mul t ip le technologies comb ined and balanced in a speci f ic w a y in order to achieve the result o f improved funct ional i t y and an accelerat ion o f the rebalancing and regenerat ion processes o f the areas of pain.

How was it possible to achieve this?

W e took nanomolecules o f magnet ized i ron (Fe3O4) and w e harnessed them in a rubbery fabr ic. Then, through a process of U V nanocoat ing, w e compacted the mater ia l in o rder to guaran tee a h igh res is tance to scratch ing and abras ion bu t , above al l , p revent ing the detachment o f the nanomolecu les.

(a) 6.6 ± 1.0 nm (1 h); (b) 13.0 ± 0.9 nm (3 h); (c) 19.5 ± 1.7 nm (6 h) Fe3O4 nanoparticles. (Scale bar: 50 nm).

W e have chosen a magne t that has the propert ies of magnet ic anisotropy and pos i t ioned it in a speci f ic w a y . Magnet ic anisotropy is the direct ional dependence o f the magnet ic propert ies o f a mater ial. A n isotropic mater ia l f rom the magnet ic point o f v i ew does not have a preferential d i rect ion o f or ientat ion o f the magnet ic m o m en t in the absence of an external f ie ld whi le an anisotropic mater ial wi l l have a m o m en t that wi ll tend to al ign itself to a precise ax is.

PARAGRAPH 2: Pain description

T o understand w h y it is necessary to do this, w e need to k n o w wha t causes the pain.

Pain is the means b y wh ich the organ ism signals t issue damage.

According to the definit ion of the IASP (International Associat ion for the Study of Pain - 1986) and the World Health Organization, pain "is an unpleasant sensory and emotional experience associated with tissue damage, either ongoing or potential, or described in terms of damage".

It cannot real l y be descr ibed as a sensory p h en o m eno n , bu t m u s t b e seen as the composi t ion of: a percept ive par t ( the noc icept ion) that const i tu tes the sensor ia l modal i t y that a l lows recept ion and t ransport to the central nervous s ys tem o f st imul i potent ial ly harmfu l to the organism an experient ial part ( therefore comple te l y pr ivate, the real exper ience o f pain) wh i ch is the psych ic state connected to the percept ion of an unp leasant

feeling.

In fact, i t is a “scout ” that comes to in form us that someth ing i s not work ing wel l in some part of our body. The bod y in forms the brain so that this, in an inst inct ive o r med iated way, can put in place appropriate responses. Work ing as an alarm, pain s ignals the risk of loss of psychophys ical integri ty, in order to conserve or restore it. M a n y d iseases are ident if iable due, in fact, to the local izat ion o f pain and its qual it ies. But if this is true for acute i l lnesses, it is not so w h en pain becomes chronic; that is, wh en it completes i ts sent inel task, losing i ts usefu lness as an a la rm and becomi n g i tsel f m o re of a d isease than a s ympt om, thus caus ing organic or psych ic imbalance.

Types of pain

Pain can be local ized, i r radiated or reported.

W e speak of local ized pain wh en a person indicates the precise point in the bod y whe re he /she feels pain. In the event that pain f rom the point o f or igin seems to fo l low a course a long a stretch of the body ( for example , a backache w i th sciat ica i rradiat ion) it is i r radiated pain. Wh e n the sufferer indicates a more or less ex tens ive area o f sur face pain wi thout a clear locat ion, w e are deal ing w i th repor ted pain.

Pain is therefore a sensat ion t rans formed into an electr ical impulse. E lect rophys io logy is in fact the branch of phys io logy that s tud ies the funct ioning of the organism f rom the electr ical point of v iew, both in normal physiological condi t ions and under the inf luence of an external electr ical

potent ial . The h u m an bod y has a nervous s ys tem of b i l l ions of neurons, which are cel lu lar funct ional units.

PARAGRAPH 3: Pain Intensity

Measurement

T o get a more precise picture of what pain is, h e r e i s an excerpt f rom the "Frontiers in Neuroscience" of the National Center for Biotechnology Information.

Pain induction protocol

A s wi th a lot of pa in research, there are var ious wa ys to induce pain, such as the st imulat ion of thermal or cold pain (Appelhans and Luecken, 2008; Kachele et al., 2016) , the mechanica l elici tat ion of pain (Matsunaga et al., 2005 ); Shankar et al., 2009) , and electr ic shock (Ol iveira et al., 2012; Zhang et al., 2016) . In our study, pain was induced b y an electrical st imulator (Mot ionSt im8, M ed e l G m b H, Hamburg , German y) , wh i ch can generate a current square wav e wi th a certain pu lse ampl i tude. T h e ampl i tude and the f requency o f the current are ad justable. T h e va lue of the pu lse w id th can be used as an object ive index of the intensity of the pain. In the exper iment , the f requency of Mot ionSt im8 was set to 2 Hz . In order to avoid interference o f the s t imulator wi th the sensors, part icular ly the EC G sensor, st imulat ion e lect rodes were p laced on the anter ior t ibial musc le of the r ight leg, as far as poss ible f rom the sensors (Figure 1A).

In our study, the selected phys io logical s ignals were the b lood vo lume pulse (BVP), the electrocardiogram (ECG), and the skin conductance level (SCL). These signals ref lect the level of act iv i ty o f the au tonomic nervous sys tem, wh ich is related to the secretory act ivi ty o f cardiac musc les and internal

organs.

Before the st imulat ion phase, the curren t intensi ty w a s cal ibrated based o n the

sel f-assessment o f the subjects. A n electr ical st imulus w i th 20 m A induced the sensat ion of pain w h en the pain ( threshold) started. A n electr ical s t imulus p roducing intense pa in was induced b y 40 m A and was a barely to lerable pain ( tolerance). Therefore, w e d iv ided the range be tween threshold and barely to lerable in 3 equidistant intervals, st imulus 20 -mA (st im20, L1), st imulus 30 -mA (st im30, L2) and st imulus 40 -mA (st im40, L3) , and wi th a pre-st imulat ion (basel ine, L0) . Dur ing the st imulat ion exper iment , each of the di f ferent intensit ies o f current was appl ied for 1 min fo l lowed b y a recovery per iod of 1.5-2 min. T o el iminate the adaptabi l i ty o f st imulat ion and temporal correlat ions, the sequence of st imul i and the durat ion o f the recovery per iod were randomly designed. The el ici tat ion of pa in was per fo rmed for 30 sess ions for each of the 3 cal ibrated intensit ies (L1-L3) o n the sam e day. W i th the addi t ion of the basic phase, a total n umb er o f 1 20 s t imulat ion stud ies were obta ined fo r each subject . Each sess ion took about 15 minutes.

T o min imize m o v em en t arti facts, part ic ipants were asked to be as re laxed as possible dur ing the s t imulat ion phase. Fur thermore, the registrat ion scenar io that led to the mul t i -day data set was per fo rmed cont inuous ly fo r 7 days for a person in the same condi t ion.

Figure 2 shows physiological traces from a participant in the four pain states.

Therefore , w e can establ ish in reference to the abovemen t ioned that , on average, the exceed ing of the pain threshold is over 20 m A .

PARAGRAPH 4: Pain Management

Hav ing ascertained the electr ical nature o f pain, we mus t n o w dist inguish i ts

usefulness.

The pain, then, is d ist inguished in:

Acute , wh en it appears suddenly and has a l imi ted durat ion because it ceases w i th the heal ing o f the cond i t ion that caused it.

Chronic , which tends to be more insistent than acute pain: the symptom, in fact, lasts longer than expected and com promises the social l i fe and the personal i t y o f the pat ient.

In both cases, wh en it is too intense or too long, instead of faci l i tat ing heal ing it h inders it because the in fo rmat ion about the p rob lem reaches the bra in but the excess ive s t imulus prevents the resolut ion o f the prob lem. In fact, a technique cal led Nerve Block is used in medicine. NER VE B LOC K is an anesthetic and/or anti-inflammatory injection directed towards a certain ne rve or g roup of nerves to t reat pain. T h e purpose o f the injection is to "turn off" a painful signal coming f rom a specif ic posit ion in the bod y or to reduce the in f lammat ion in that area.

An imaging guide, such as f luoroscopy or computer ized tomography (C T scan or "C AT " scan), can be used to help the doctor posit ion the needle in the most appropr iate pos i t ion , so the pat ient can rece ive m ax i m u m benef i t f rom the injection.

What are some common uses of the procedure?

People suffer ing f rom acute or chronic pain m a y have a nerve b lock inject ion to get temporary pa in rel ief. Of ten , this pa in com es from the spine, but o ther co m m on l y af fected areas inc lude the neck, but tocks, legs and arms. In ject ing a nerve b lock a l lows the in jured nerve to recover f r om a state of constant

irr i tat ion. Furthermore, nerve blocks can provide diagnost ic informat ion to the doctor. B y per fo rming a nerve b lock and then moni tor ing the w a y the pat ient responds to the in ject ion, the doctor can of ten use this in format ion to he lp determine the cause o r source o f the pa in an d to gu ide fur ther t reatment .

What are the advantages compared to the risks?

Benefits:

· Temporary relief f rom pain.

· Temporary reduct ion of inf lammat ion in the region of the vertebral co lumn caus ing pain.

· It can help the doctor ident i fy a more speci f ic cause of pain.

· Improved abi l i ty to funct ion in dai ly l i fe wi thout the restrictions previously

caused b y pain.

Risks:

· In fect ion at the in ject ion si te

· h em o r rh age · Accidental del ivery o f drugs into the b loodst ream

· Unex pec ted sp read o f d rugs to other nerves

· Hi t t ing the "wro ng" nerve in an at tempt to b lock the target ne rve i f the nerves are c lose

· Wh en f luoroscopy or C T is used, there wi l l be minimal low-level radiat ion.

PARAGRAPH 5: How is an electrical

impulse generated

(Neuron)

A prolongat ion (Axon) starts f rom the neuron, wh ich has the main funct ion of t ransmit t ing the electr ical s ignal .

It connects w i th other neurons, glands, musc le f ibers and Synapses .

In th is w a y the s ignals are t ransmi t ted and propagated a long the Ax on . N o w it remains to understand wha t the electr ical intensity o f pain is.

But before th is, it is important to clar i fy that pain does not have absolute values and the s igna ls ch an ge f r o m person to pe rson an d a lso w i t h a n y i n d i v i d u a l a re probably dependent on the day. So , the qual i ty o f the s ignals is in f luenced b y the in tro- indiv idual var iance in the menta l state and env i ronmental factors.

T h e impulses o f a neu ron al l have the sam e intensi ty an d s am e durat ion regardless f rom the intensi ty o f the st imulus.

Stimulat ing a neuron wi th a st ick sensory:

· W h en t h e ep idermis is compressed m o r e deep l y, the pu lses are registered m o r e

quickly. · The pulses are al l o f the same durat ion and intensity. · The st imulus, however , to produce the Potent ial o f Act ion must exceed

a threshold value.

· The N a /K p um p pushes out o f the axon 3 N a + ions and br ings in 2 K + ions. · T h e escape channe l for K + is open and a l lows these ions to enter and leave.

· Th e channels cont ro l led b y the K + and N a + are c losed. · The number o f escape channels for N a + are negl igib le.

At rest, the concent rat ion o f K + ions is greater in the cytop lasm than in the extracel lular l iquid. K + ions spread out through escape channels for K + .

The large negat ive ions cannot also escape . T h e smal l negat ive charge present internal ly at t racts a smal l n u mber o f K + and N a + ions that pass th rough the respect ive escape channe ls .

N a + are removed th rough the N a /K p u m p , mainta in ing the concent rat ions that m a k e it poss ib le to return to the rest ing potent ial . At rest, the m em b ran e is po lar ized negat ive ly towards the inside. At the point where the st imulat ion open s the channels o f N a + control , N a + ions migra te to the interior accord ing to their grad ient o f concent rat ion. T h e inter ior o f the ax on b ecom es >0 e and the polari t y o f the m em b r an e reverses. K + ions f l o w out fo l lowing their o w n concent rat ion gradient , restor ing e to the rest ing potent ial .

The Na /K pump returns to init ial levels of ionic concentrat ions.

Propagation of the nervous impulse

· A small segment of the axon membrane becomes slightly depolarized due to the + ions present inside the membrane;

· In the depolarized region, the potential-controlled Na+ channels open up and these ions migrate inward; an action potential is created that depolarizes the adjacent region of the membrane;

· The area where the PdA has just passed opens the potential-controlled channels for the K+ ions to come out, returning the membrane to its resting potential. During this process, the potential-controlled Na+ channels are closed.

· Now the membrane is ready to respond to new stimuli with new PdA.

The PdA is propagated in only one direction.

Because of the small values of the masses of ions, it is not possible to exploit their gravitational interactions, but it is necessary to exploit the fact that a charged particle is affected by the presence of electric and magnetic fields.

For this reason, we have to analyze the motion of the ions using mass spectrometry.

PARAGRAPH 6: Lorentz force and mass

spectrometer

Lorentz force

1) Electric field

A charged part icle, in the presence o f an electr ic f ie ld E , senses a di rect force a long the di rect ion of the f ield i tself ( the d i rection depends on the s ign of the charge) given by:

mass (m), charge (q), speed (v)

2) Magnetic field

A charged part icle in mot ion wi th veloci ty v, in the presence o f a magnet ic f ie ld B, senses a di rect force or thogonal l y to the velocit y and to the magnet ic f ield itself, given by:

Since FB is always orthogonal to speed, it wi l l not per form work; that is, it wi l l on l y mod i f y the di rect ion of speed, wi thout al tering the magn i tude (speed value). A constant orthogonal force is appl ied.

T h e total force undergone b y a charge in the presence o f an electr ic and magnet ic f ield is therefore the so-cal led Lorentz force.

In mathemat ics , a straight l ine is def ined as the shortest l ine be tween t wo points, so a curved l ine is longer than a straight l ine. It therefore fo l lows that dur ing the appl icat ion o f the or thogonal magnet ic force w i th respect to the ion t rajectory, the speed remains constant , bu t w i th respect to before the appl icat ion o f this force, it undergoes a reduct ion in speed. This decrease in veloci t y t ranslates into a lower numb er of pulses per second, wh ich therefore leads to their f requency b e low the pain threshold or in an y case of lower

intensity. The brain depr ived o f the cont inuous impulse is f ree to organize itself to repair the d am a ged area o f the bod y.

Another essent ial aspect o f us ing the m agn e t o f that t ype is the abi l i ty it has to act at the cel lu lar level .

PARAGRAPH 7: Biological effects of

high gradient magnetic fields

The biological effects of high-gradient magnet ic fields (HGMFs)

The degree o f the Gradient Magne t i c f ie ld is determined b y the rate o f change.

T h e greater t he change , the greater t he grad ien t , wh i c h increases a t the

opposi te poles.

Magnet ic f ie ld gradients are the forces used in quan t um phys ics that exer t a t ranslat ional force o n bo th a stat ionary an d m o v in g loads such as a Sod ium ion

(Na+).

This is in contrast to a un i form magnet ic f ield such as a bipolar magnet , wh i ch exerts zero force on charged part icles.

The biological effects of high-gradient magnet ic f ields (HGMFs) have steadi ly ga ined the increased at tent ion o f researchers f rom di f ferent d iscipl ines, such as cel l b io logy, cel l therapy, targeted s tem cel l del ivery and nanomedic ine. W e present a theoret ica l f ramework towards a fundamenta l unders tand ing o f the effects o f H G M F s on intracellular processes, highl ight ing n ew direct ions for the study of l iv ing cel l mechanisms: changing the probabi l i ty of ion-channel on/off swi tch ing events b y m em b ran e magneto-mechan ica l st ress, suppress ion o f cel l growth b y magnet ic pressure, magnet ical l y induced cel l divis ion and cell reprograming, an d forced migrat ion of memb ran e receptor proteins. B y deriv ing a general ized fo rm for the Nernst equat ion, w e f ind that a relat ively smal l magnet ic f ield (approx imately 1 T) wi th a large gradient (up to 1 GT/m)

can sign i f icant l y ch ange the m em b r an e potent ial o f the cel l and thus have a signi f icant impact on not only the propert ies and biological funct ional i ty o f cel ls but also cel l fate.

In recent decades, the interact ion o f magnet ic f ie lds w i th l iv ing cel ls and organ isms has capt ivated the interest o f a b road scient i f ic commun i t y d rawn f rom a wide spect rum of disciplines, including biology, physics, chemistry, med ic ine and nanotechnolog ies . Ex tens ive progress in exper imental techniques and the des ign o f n e w magnet i c mater ia ls has resul ted in the bu rgeon ing deve lopment o f n ew approaches to reveal the targets of m agne t i c f ie lds o n the intracel lular and molecular levels1 ,2 ,3.

The scienti f ic l i terature is fi l led wi th thousands of works on the responses of l iv ing organisms to low, moderate and s trong magnetic f ields. However , the biological ef fects re lated to the gradient o f the magnet ic f ields are poor l y discussed. Relat ive ly f ew studies have quant i f ied magnet ic gradient act ions at the intracel lular level. Nevertheless, name l y spatial ly non-un i fo rm magnet ic f ields wi th a large enough gradient are capable of s igni f icantly al tering cel l funct ions and even organisms. Fo r example, a large-gradient magnet i c f ie ld can affect FLG29.1 cell di fferentiation to form osteoclast-l ike cel ls. Under HGMFs , significant morphologic changes in osteoblast-l ike cel ls occurred, inc lud ing expansion of the endoplasmic ret icu lum and mitochondr ia, an increased num ber o f l ysosomes, distorted microvi l l i, and aggregates of act in

f i laments. The early embryonic growth of the leopard frog (Rana pipiens) was strongly inhibited b y a 1 T magnet ic f ield wi th a high gradient of 84 Tm−1.

W h en analyz ing effects of magnet ic f ields on l iving cel ls, t issue and organisms, one should keep in m ind that in mos t cases, the biological cel ls and t issue are d iamagnet ic , w i th susceptibi l i ty very c lose to that of water. Therefore, the di f ferences in the d iamagnet ic suscept ibi l i t ies of cel lu lar components are very low, wh ich leads to t iny ef fects. In contrast, the exposure of cel ls and organisms to high-gradient magnet ic f ie lds (HGMFs) reveals man y intr iguing effects that m igh t be di rect l y re lated to the magnet i c gradient fo rce exer ted on the who le cel l and its organel les. Indeed, the magnet ic force act ing on a magnet ic dipole moment is proport ional to the field gradient , i.e., F  ∝   ∇B (where B is magnet ic induction). In the case of cells suspended in a weakl y diamagnetic med ium, the volumetric force is F  ∝  ∇B2. Thus, after achieving a suff ic ient magnet ic gradient , s igni f icant changes in cel l funct ions, shape and spat ial organizat ion migh t be possible. In spite o f the m a n y interest ing effects related to the appl icat ion of spatial ly non-uni fo rm magnet ic f ields, a key p rob lem—how high-gradient magnet ic f ields change cel l mechan isms—has never been careful l y ex amined . Specia l interest ex ists in the case w h e n the appl ied magnet ic f ie ld dramat ical l y changes in value and direct ion across the cel l body. Here, the important quest ion is: h o w wi ll the cel l respond and adapt itself to a h igh magnet ic f ield gradient? F rom point of v iew of physics, the answer is the fo l lowing. Consider ing the cel l as a droplet of d iamagnet ic l iquid placed in a non-uni form magnet ic f ield, one can conclude that such a droplet wi l l d iv ide itself into several smal ler d rops to sat isfy the m i n i m u m o f the total sys tem energy. A qualitatively similar effect—ferrofluid droplet division in a non-uni form magnet ic field (B =  68 mT) wi th gradient, dB/dz =  6.6 Tm− 1— was

recently reported. It is obv ious that l iv ing cel l mechan ics are m u ch more complex than that of a l iquid droplet . Nevertheless, in spite o f the smal l contr ibut ion o f d iamagnet ic forces in the interplay between biological and phys ical factors in the cel l mechan ism, the role of the magnet ic gradient force can increase wi th increas ing magnet ic gradient . There are n o pr incipal phys ical l imi tat ions the increase of magnet ic f ield gradients. For example, m icro -magnet arrays can produce magnet i c f ie lds that are spat ially modu la ted o n the m icron scale wi th a gradient u p to 106 T m− 1 at m icro-magnet edges . In the v ic in i ty o f a magnet i c nanost ructure, magne t i c f ie ld gradients can be large en ou gh (up to

107 T m− 1) for the f ield to vary appreciab ly over the separat ion be tween electrons in a radical pair , thereby modula t ing the intracel lular magnetocatalyt ic act ivity. Moreover , theoret ical results show that an H G M F can lead to a s igni f icant enhancemen t o f the per fo rmance o f a chemical b iocompass bel ieved to exist in certain an imals and birds. A non-uni form magnet ic f ield up to 610 T wi th a gradient on the order o f 1 06 Tm− 1 on the mil l imeter scale was recent l y generated w i th a laser-dr iven capaci tor-coi l target by proton def lectometry.

To identi fy the intracel lular targets and molecular ef fectors of magnet ic f ields and to reveal the under lying mechan isms, m a n y complex mul t id iscipl inary problems mus t be solved. A s is of ten the case wh en mult ip le discipl ines address a comp lex scient i f ic p roblem, theoret ical mode ls and mathemat ical equat ions can prov ide a uni fying p lat form to synergize the efforts. W e present a theoret ical f ramework for a fundamenta l understanding o f the ef fects o f magnet ic gradient forces o n intracel lular processes, h ighl ight ing n e w direct ions of the s tudy of l iv ing cel l mechanisms affected b y magneto-mechan ical forces.

Direct influence of a high-gradient magnetic field on the resting

membrane potential of a cell

M em b r an e vo l tage is a key parameter regulat ing cel l propert ies, mechan ism and

communicat ion. In general , electr ic i ty and the interact ion of electr ic charges play major ro les in the l i fe of a cell . Indeed, a simple est imat ion (see Methods) o f the electrostat ic energy s tored in the m em b ran e o f a spherical cel l w i th radius 10 μm and membrane vol tage 70 mV is E ≈  10−14–10−13 J, which is 6–7 orders o f magn i tude larger than thermal f luctuat ion energy and m u c h larger than the energies o f chemica l bonds an d m em b ran e bend ing, wh i ch determine m a n y membrane-m ed ia ted intracel lular processes, such as shaping, r igidi ty, endocytosis, adhesion, crawl ing, d iv is ion and apoptosis. Thus , the electrostat ic cont r ibut ion o f the bend ing energy o f charged cel l membranes is large enough, and in a first approx imat ion, the cel l membrane r igid i ty is proport ional to the square of the m em b ran e vo l tage. Qual i ta t ive analys is sho ws that cel ls (able to prol i ferate rapidly, undi f ferentiated) wi th low values of membrane potential , wh ich tend to depolar ized, are h igh l y plast ic. In contrast , cel ls that are mature, terminal l y di f ferent iated, an d quiescent tend to be hyperpolar ized. It shou ld be s t ressed here that the m e m b r an e potent ia l is no t s imp l y a ref lect ion of the cel l s tate bu t a parameter a l lowing the cont rol o f the cel l fate; for example, arti f icial depolar izat ion can prevent stem-cel l di f ferentiat ion, whereas artif icial hyperpolarizat ion can induce differentiation. Below, w e

analyt ical ly analyze the possibi l i ty o f dr iv ing the membran e potent ial wi th external ly appl ied, h igh-gradient magnet ic f ields.

W h en a high-gradient magnet ic f ie ld is appl ied to a cel l in a med ium, the magnet i c gradient fo rce acts o n ions an d can ei ther assist or oppose ion

m o v em e n t t h rough the m em b ran e . T h e magnet i c gradient force is given b y , where p is the magnet ic dipole m o m en t of the ion, B is the magnet ic induct ion, an d the der ivat ive is taken

with respect to di rect ion l , wh ich is paral lel to the magnet ic d ipo le moment o f an ion, l//p . Bear ing in m ind the fo rmer expression for the magnet ic gradient force, in this case, when the ions di f fuse in the presence o f an H G M F , the Nerns t equat ion reads as (see Methods ) where e is the elect ron charge, z is the ion valence (z =  +1 for a posit ive, univalent ion), F is the Faraday constant , R is the gas constant , T is the absolute temperature , V m is the potent ia l d i f ference be t ween the t wo m e m b r an e sides, and no and n i are the ion concentrat ions outs ide and inside a cel l , L is the hal f- cel l size. O n the r ight s ide o f Eq. 1, the second term descr ibes the magnet ic cont r ibut ion to the rest ing potent ial . Thus , Eq. 1 represents a generalized fo rm of the Nernst equat ion der ived w i th regard to the in f luence o f a high-gradient magnet ic f ield. Depend ing on the di rect ion o f the magnet ic gradient ( “+” or “−” in Equat ion 1), an H G M F can cause ei ther membrane potent ial depolar izat ion or hyperpolar izat ion, wh ich regulates not on ly the entry o f sodium, potass ium, and calc ium ions an d biological ly relevant molecules to the cel l but m an y pivotal cel l characterist ics and funct ions. The key quest ion is h o w large the gradient va lue shou ld b e to ach ieve a di rect ef fect o f the magnet i c f ie lds o n the m em b ran e potent ial . T o address th is quest ion, w e est imate the cont r ibut ion o f the magnet ic te rm to the equi l ibr ium m em b ran e potent ial g iven b y Eq. 1. For this est imat ion, the values o f the magnet ic m om en t s o f ions that create the m emb ran e potent ial should be known. Typical ion-channel species

(K +, C a2+, N a+) and nearby water mo lecu les are electron sp in paired, so they have n o sp in e lect ron magne t i c m o m e n t and their magne t i c m o m e n t is due to nuclear spin . It is interest ing that 40C a2+ions have no nuclear magnet ic mom ent . T h e magne t i c m o men t s o f these ions are very smal l an d are o n the s am e order of magni tude as the nuclear magneton , μ n =  5.05 10− 27 J/T: pNa+  =  2.22μ n (sodium-23), pK +  =  0.39μ n (potassium-39), pCl−  =  0.821μ n (chloride-35), and pC a2 + =  0 (calcium-40) . A m o n g these ions, N a+ has the largest magnet ic m o m e n t and C a2+ has zero elect ron ic an d nuclear magne t i c moments . Fo r compar ison, w e l ist the magnet ic mo m en t values of relevant molecules: for H20 (para, ant iparal lel nuclear spins) p  =  0 and H20 (ortho, paral lel nuclear spins) p   =  μ n and for hemo glob in F e2 +, the average magne t i c m o m en t meas ured for who le b lood is equal to 5.46 μ B /Heme (where μ B is the Bohr magneton, μ B /μ n ≈ 

1836). D u e to the nuclear sp ins o f the hyd rogen a toms, water consists o f a mix ture o f spin zero (para) and spin one (ortho) molecules. The equi l ibr ium

ratio of ortho to para molecules is 3∶1, making 75% of water molecules magnetical ly act ive in suff iciently strong magnet ic fields. H G M F , due to the relat ively large magnet ic m o m en ts o f N a+ ions, can affect the format ion o f the act ion potential o f a nerve cell . B y est imat ion of the magnet ic addi t ion in Eq. 1 for the above values of magnet ic moments of K+ and N a+ ions and biological ly relevant mo lecu les to the cel l , w e f ind that an external ly appl ied magnet ic f ield wi th a gradient value o n the order o f 108–109 Tm− 1 can direct ly change the cel l membrane potential b y 1–10 mV. For example, in neuron cel ls, the opening of N a+ and K+ vol tage-gated ion channe ls occurs w i th m em b ran e potent ia l depolar izat ion as smal l as 7–12 mV. In this case, the di rect effect o f the appl icat ion of H G M F s to the cel l can mani fest itself through the change of the probabi l i ty o f opening/closing the vol tage-gated ion channels. However , as es t imated above, to ach ieve m e m b r a n e potent ial depolar iza t ion or hyperpolarizat ion, one has to apply an H G M F wi th a gradient on the order of

109 Tm− 1. The possibi l i ty o f achieving such h igh values of magnet ic gradient is descr ibed in the nex t sect ion.

The current ly reachable magnet ic gradient (up to 106–107 Tm−1 ) has indirect effects related to the appl icat ion o f H G M F s to cells. First, the effects of magnet ic f ie lds w i th a gradient o n the order o f 106 T m− 1 can mani fest i tself through the change of the probabi l i ty o f opening/c los ing mechanosensi t i ve i on

channels . O n the other hand, mechan ica l st ress in the cel l m e m b r a n e can direct ly dr ive ion channel gat ing. Moreover , the memb rane potent ial can be changed th rough agi tat ion o f the m em b ran e ion channels . Recen t s tud ies have demonst ra ted the impor tance o f the m e m b r a n e potent ia l va lue in the regulat ion of cel l funct ions and signal ing at the mult icel lular level , especial ly in relat ion to ion channel act ivity. For example, cancer cel ls tend to have low m em b r an e potent ia l ( in absolute value) , wh i ch has been connected to the overexpression of speci f ic ion channels. High l y differentiated tumor cel ls (human hepatocellular carcinomas: Tong, HepG2, Hep3B, PLC/PRF/5, Mahlavu, and HA2 2 T ) have paradoxical ly smal l membrane potentials. T h e membrane potent ial cont rols the adipogenic and osteogenic dif ferent iat ion of s tem cel ls, wh ich suggests the possibi l i ty to dr ive the dif ferentiat ion pathway. Th e memb ran e potent ial p lays a key role in the spat ial organizat ion of cytoskeletal and cell division-related proteins, main ly inf luencing bacterial cell division.

Static homogeneous magnet ic f ields can also affect the di f fusion o f b io logical part ic les th rough the Loren tz force and hypothet ical l y ch an ge the m em b ran e

potential . However , the results presented show that in solut ion, the Lorentz force can suppress the di f fusion of univalent ions (e.g., N a+, K +, and C l−), but the threshold magnet ic f ield is extremely high, approx imately 5.7 ·  106 T (which is 2– 4 orders of magn i tude less than the magnet ic field at a magnetar ) . O n the other hand, the theoret ical l y predicted threshold of gradient f ie lds for p roducing a change in ion di f fusion through the magnet ic gradient st ress is m o re than 1 05 T 2m − 1 for paramagnet ic mo lecu les FeC l3 an d 02 and p lasma proteins. Thus, in l ow and moderate magnet ic f ields, the biological ef fects shou ld b e rather dependent o n the magn i tude o f the magnet ic f ie ld gradient and no t o n the s t rength of the magne t i c f ie ld, as was recent l y demonst ra ted i n exper iments w i th THP-1 cel ls. The magnet ic systems capable of generat ing H G M F s and formulas al lowing rapid est imation of the magnet ic f ield gradient

are descr ibed in Methods and Table 1. . W e n o w consider possible appl icat ions of these magnet ic sys tems to cont ro l ce l l funct ions.

Effects of an HGMF through intracellular mechanical stress

A possible alternative mechan ism of cel l response to H G M F s rel ies on the fact that magne to -mech an ica l st ress can af fect mechanosens i t i ve m e m b r a n e i on channels; for example, T R E K-1 ion channels, which are stretch-act ivated po tass ium channels . It is bel ieved that a cel l m a y have 102– 104 ion channels , and the probabi l i ty o f any of t hem being open (at an y given t ime) is typical ly in the range of a few to a few tens o f percent . Magne ti c gradient forces exer ted on cel ls impose mechan ical st ress on the p lasma m emb ran e and cel l body. T h e cel l senses th is st ress an d el ic i ts a mechano-e lec tr i c t ransduct ion cascade that ini t iates a response. In the cel l memb rane , mechanosens i t i ve ion channe ls are

responsible for t ransducing mechanical s ignals into electrical signals. Addi t ional m em b ran e tension, in our case induced b y the h igh-gradien t magnet ic f ie ld,

can increase the probabi l i ty o f mechanosensi t i ve channe l opening. Thus, p las ma m e m b r a n e mechan ica l s t ress act ivates t rans ient receptor potent ia l (TRP) channels. Be low, w e calculate the mechanical forces and stress in a cel l placed in an HGMF.

The vo lume densi ty o f the magnet ic gradient force (in N m − 3) act ing on a cel l is

where χm is the suscept ib i l i ty o f the med ium, χ c is the suscept ib i l i ty o f the cel l , and μ 0 is the vacuum permeabi l i ty. In Eq. 2, the di f ference of susceptibil i t ies, Δχ  = χm  − χ c, def ines the magnet ic force di rect ion: at t ract ion o r repuls ion of a cel l to / f rom the area wi th a high-gradient magnet ic f ield. Th is force causes

mechan ical st ress in the who le cel l and cel l membrane. Analys is o f the possible biological ef fects o f the act ion of magnet ic gradient forces wi th vo lume densi ty given b y Eq. 2; one can compare these forces wi th the gravi tat ional force

density, f g = ρg  =  104 Nm−3 (where ρ is the densi ty o f water and g is the accelerat ion of gravi ty) . Assuming Δχ to be 10 –2 0% of the d iamagnet ic susceptibil ity of water (χw  = −9 ⋅ 10−6 in SI), B =  1 T and |∇B| = 106 Tm−1, f rom Eq. 2, we obtain the magnet ic force density f  =  (0.7–1.4) ·  106 Nm−3, which yields f   ≫  fg. Because the gravitat ion force (microgravi ty) or weight lessness (e.g. , b y magnet ic levitat ion) af fect cel l development , growth and funct ion, s igni f icant ef fects o f the magne t ic gradient forces wou l d be expected. Fo r example , the appl ied magnet ic f ields wi th gradient of approx imately ∇ B 2 ≈  103 T 2m −1 were shown to change the subcel lu lar morpho logy of osteoblast- l ike cel ls, and d iamagnet ic levitat ion p lays a major ro le in the observed effects. Thus , s igni f icant ef fects on cel l mechan ism caused b y the magnet ic gradient forces are expected . T h e magnet i c fo rces that are exer ted o n the cel l b o d y are t ransmit ted to the cel l cytoskeleton an d cel l membrane . E ven t iny mechan ica l forces that are s l ight ly larger than the thermal f luctuat ion forces o f less 1 p N (see Methods) can signif icantly affect cel l functionality.

The magnet ic gradient forces given b y Eq. 2 can direct ly dr ive paramagnet ic cel ls and molecules. In general , cel ls are d iamagnet ic . However , recent research shows the ex istence of nonerythro id cel l lines der ived f rom h um an cel l cancers that are suff ic ient ly paramagnet ic . The i r paramagne t ic behav io r m ak es it possible to affect cell mot ion by application of an HGMF. Moreover, intracellular and intercel lular f ree radicals, such as O3, NO , and N O2 and molecu les FeC l3 and O2, are a lso paramagnet ic and can be redistr ibuted b y

both the Lorentz force and magnet ic gradient force, as k n o wn f rom

electrochemistry.

One of the key funct ions of cells is order ing in space and t ime. High-precis ion cel l posi t ioning w i th micromagnets is a promising approach for t issue engineer ing. Indeed, the magnet ic gradient force (Equat ion 2) is capable assist ing cel l migrat ion to areas w i th the h ighest magnet ic f ie ld gradient . It was recent l y demonst rated that m ic romagnet arrays (wi th lateral s ize o f 30–50  μm and the s am e neighbor ing d is tances) coated w i th parylene p roduce h igh magnet ic f ie ld gradients (up to 106 Tm− 1) that af fect cel l behavior in two ma in ways : i ) caus ing cel l migrat ion an d adherence to a covered magnet ic sur face and i i) e longat ing the cel ls in the di rect ion parallel to the edges o f the micromagnet . T h e resul ts o f the calculat ions o f the magnet ic f ie ld and gradient distr ibut ions above four micromagnets are shown in Figs 1 and 2. The f ield and magnet ic-gradient force distr ibut ions were calculated analyt ical ly us ing expl ici t expressions for the magnet ic stray f ields. A s seen f rom Figs 1 and 2, there are several a reas w i th the h ighest magne t i c gradient . Thus , in the exper iments ,

driven b y magnet ic gradient forces (Equat ion 2), cel l migrat ion was observed towards the areas w i th the strongest magne t i c f ie ld gradient , thereby a l lowing the bui ldup o f tunable, interconnected, s t em cel l networks .

Figure 1: Spatial distribution of the scaled modulus of the magnetic field

(B/μ0Mr) calculated in the plane 5 μm above four micromagnets (Mr is

remanent magnetization).

Several cel ls are schemat ical l y d ra wn to demonst rate that the magnet ic f ie ld var ies in the s am e length scale as the cel l m e a n size. T h e m ic romagne t s izes are 100 ×  100 μm, and the spacing is 100 μm.

Figure 2.

Spat ia l d ist r ibut ion o f the scaled p lanar componen t o f the magnet ic gradient (a ) 5 μm above the micromagnets shown in Fig. 1. (a ) Vector field

{∇ x(B/μ0M r)2,∇ y(B/μ 0M r)2 } multiplied by the micro-magnet size. Arrows indicate

the di rect ions of the magnet ic gradient forces. (b ) Scaled modu lus o f the planar magnet ic gradient (∇ x,y(B/μ 0M r)2) mult ipl ied by the micro-magnet size as a funct ion of the x-coordinate. The gradient values were calcu lated a long the O X- ax is at d is tances f r om the magne t tops: 5 μm , 7 and 10 μm .

Recent studies indicate the crucial inf luence o f external mechan ical and magnet ic forces on the cel l shape, funct ion and fate th rough phys ical interact ions w i th the cytoskeleton ne twork

Local change of membrane potential and lateral migration of membrane

receptor proteins in the vicinity of magnetic nanoparticles

A chain of magnet ic nanopart ic les (MNPs) placed on a cell membrane can create spatial ly modu la ted magnet ic f lux distr ibut ions wi th a suff icient

gradient. T he magnet ic gradient forces local ized near the M N P s affect cel l

function in two main ways: i) changing the rest ing m em b ran e potential , as predicted b y Eq. 1 , and i i) generat ing local magnetic p ressure that can cause memb ran e deformat ion, resul t ing in cel l memb rane blebbing. T h e former can occur local ly as a consequence of a very h igh f ield gradient , as given b y Eq. 15

(Methods). For magneti te (Fe3O4) MNPs with M s =  510 kAm−1 and R = 5 nm, est imat ion based o n Eq. 15 gives |∇ B r| ≈  2.6 108 T m− 1 at the m em b ran e sur face. This gradient magn i tude is enough to change the rest ing potent ial b y a few m V even though the ions dr iv ing the m em b r an e potent ial have on l y nuclear va lues of magne t i c mo m ent s . T h e second is re lated to the magnet ic p ressure due to the di f ference o f the magnet ic suscept ib i l i t ies o f the l ip id m em b ran e and cytosol . In the vicin i ty o f an M N P , the magnet ic pressure at the cel l membrane is PMN P =  fV/S =  fh, where V and S are the vo lume and areas of a small part of the m em b ran e and h is the m em b ran e thickness. T h e analyt ical express ion for this pressure is given in Methods. For chains of M NP s with paral lel and perpendicu lar or ientat ion o f the magne t i c m o m en t s wi th respect to the memb ran e sur face, the magnet ic pressure (PM N P) acts in di rect ions perpendicu lar an d paral lel to the membrane , as is il lustrated in Figs 3 (a–d) for two chains consist ing of four MNPs . The magnet ic pressure causes an imbalance in the osmot ic and hydrostat ic pressures, wh ich in turn changes the f lux of ions t ransported th rough the cel l membrane . T o est imate the magnet ic pressure, one should k n o w the magnet ic susceptibi l it ies o f the cel lular contents, in part icular, the magnet ic susceptibi l i ties o f proteins, l ipids and water are χ p =  −9 .726 1 0− 6, χ l ip   =  −8 .419 1 0− 6 and χ w   =  −9 .035 1 0− 6 (al l in SI) . Thus , prote ins are more d iamagnet ic than water , i .e., χ p <  χ w . L ip ids are less d iamagnet ic than proteins and water (χ l ip  >  χ p and χ l ip  >  χw ), resul t ing in their “quasi- paramagnet ic ” behav io r w i th respect to l ip ids and the cytosol . D u e to the di f ference of the magnet ic susceptib i l i t ies o f proteins and l ipids, the m em b ran e receptor p ro te ins are at t racted to the area w i th the h ighes t magne t i c f ie ld gradient generated b y M N P s (see Fig. 3). Est imat ions o f the lateral magnet ic pressure (Equat ion18, Methods ) act ing o n the m em b ran e receptor protein at h   =  5 nm, r  ≈ R =  5 nm, M s =  510 kAm −1(magnet i te MNPs) and Δχ  = χ p − χ l ip  = 1.3

10− 6 result in P =  1.7 Pa. Th is pressure can force the lateral migrat ion of memb ran e receptor protein towards the high-gradient f ie ld area. Moreover , cel l m em b ran es accom mod ate d om a ins w i th heterogeneous s izes ranging f r om 1 0 to 200 nm, wh ich are enr iched in cholesterol and saturated l ipids. Because the magnet ic suscept ib i l i ty o f cholesterol is c lose to that o f protein, χ c h =  −9 .236

1 0−6  , these d om a i n s are subjec ted to the lateral magn e ti c p ressure a n d fo rced di f fusion occurs. Th is redistr ibut ion o f the membrane domains can p lay a pivotal ro le in al tering membrane funct ion.

Figure 3

Vector f ields of the magnet ic induct ion (a and c ) and magnet ic gradient

(b and d ) in the vicini ty o f four magnet ic nanopart ic les magnet ized paral lel and

perpendicu lar to the mem bran e surface. In (b and d ) a r rows indicate the di rect ions o f the magnet ic gradient forces.

Magnetically assisted cell division

The first hint of the possibil i ty of cell division by an H G M F was discussed above in relat ion to an exper iment on the div is ion of ferrof lu id droplets in a moderate magnet ic f ield wi th gradient dB/dz =  6.6 Tm− 1. The diamagnet ic susceptibi l i ty o f a cel l is m u ch smal ler than that o f a ferrof lu id droplet . W h e n discussing the effects o f H G M F s o n cells, w e consider at least six orders of magni tude larger f ie ld gradients. Because the magnet i c gradient force is propor t ional to the product o f the magnet ic susceptib i l i ty and the f ield gradient (Equat ion 2), in our case, one can expect a s imilar effect, i.e., s timulat ion o f cel l d iv is ion b y

magnet ic gradient forces. Magnet ic gradient forces can be s igni f icantly increased b y loading cel ls wi th magnet ic nanopart icles. In exper iments , local ized nanopart ic le-mediated magnet ic forces were appl ied to He La cel ls th rough a magnet ic f ie ld w i th a gradien t f rom 2 .5∙103 T m− 1to 7∙104 T m− 1. Und e r the largest gradient , the cel ls loaded w i th magnet ic nanopart ic les exhibi ted ‘pull- in’ instabil i ty. However, under lower magnet ic gradients and lower intracel lu lar mechan ica l stress, b ias ing o f the metaphase p late dur ing mitosis was observed, wh ich indicates that in HGM Fs, magneto-mechanical st ress is ab le to assist in the div is ion o f cel ls free of magnet ic nanopart ic les.

Therefore, w e hypothes ize that cel l d iv is ion can be ei ther induced or assisted by a specif ical ly, spat ial ly modulated, magnet ic gradient f ield. A n example of such a magnet ic f ield conf igurat ion and magnet ic gradient force distr ibut ion is shown in Fig. 4, i l lustrating the field and its gradient (normalized ∇ B 2) d ist r ibut ions genera ted in the gap be tween two un i fo rmly magnet ized magne ts

faced pole-to-pole. T h e f ield and gradient were calculated us ing the expl ici t analyt ical express ions for the magnet ic f ie ld induct ion o f rectangular, magnet ized pr isms. F igure 4b shows that be tween the magne t i c poles, o n the left and r ight par ts o f the cent ra l area, the magnet i c grad ien t fo rces have opposi te di rect ions. If the m ea n size of this area is comparab le to the cel l s ize, a cel l p laced here wi l l be subjected to two opposi te forces, wh ich can cause magnet ic pressure that assists ei ther cel l d iv is ion or cel l compress ion . It is unknown h o w large this pressure should be to tr igger cel l div ision. In the l i terature, da ta o n th is sub ject a re rather sparse. It w a s demons t ra ted that a pressure of 100 Pa can dr ive HeLa cel l mitosis. Th is pressure is an achievable magnet ic pressure, e.g., in one of the H G M F systems l isted in Table 1.

Figure 4

Vector f ields of the magnet ic induct ion (a ) and magnet ic gradient forces (b )

between the two, po le-to-pole magnet ic s labs and cel l division. (c ) Magnet ic gradient forces (Equat ion 2) normal ized to Δχa−1μ 0M r 2 as a funct ion of the x-

coordinate. A hypothetical d iv is ion of a cell in the highly non-uni form magnet ic f ield ( the central area) is i l lustrated.

Tumor arrest by magnetic pressure

Exper iments sugges ted that mechan ica l st ress can l imit the growth of a sphero id of cancer cel ls b y restr ict ing cel l d iv is ion near the sphero id surface. Here, w e sh ow h o w magnet ic pressure can arrest t umor growth. Th e idea is based o n the fact that cancerous cel ls a re enr iched b y Fe, and therefore they are m o re paramagnet ic t han hea l thy cel ls . In such a case, magnet i c radial

pressure can l imi t tumor g rowth due to the at t ract ive magne t i c gradient fo rce act ing o n the “paramagnet ic” cancerous cel ls. A n example of magnet ic f ield and gradient d ist r ibut ions above cyl indrical magne ts wi th a ho le is shown in Fig. 5 (details o f the calculat ions can be found in Methods). Magnet ic pressure o n the tumor can be calculated as P tum = fw, where f is the force densi ty given b y Eq. 2 and w is the width o f the area corresponding to the m ax i mu m of the magnet ic f ield gradient shown in Fig. 5. Est imat ions o f the magnet ic pressure on cancerous t issue w i th magnet ic suscept ib i l i ty χ  =  6.3 10− 6 ( in S I units) for the calculated max imal value of the magnet ic gradient, B |∇ B|/

(R−1 (μ 0M r /4π )2)  ≈ 160 (see Fig. 5 (b) and (c)) and magnet radius R =  5 mm, hole radius 0 .1 mm and w =  1 mm, give pressure P tum  ≈  1 Pa =  1 pN μm − 2, which value seems to be not suff icient to affect cel l funct ions. However , |∇ B| grows as the hole rad ius decreases or the distance z goes to zero (see Tab le 1 and Eq. 13 in Methods) . Thus , ad just ing the ho le rad ius and distance, the magnet ic gradient can be increased b y hundreds o f t imes to ach ieve pressures of hundreds o f pascals, wh i ch can prevent cel ls f rom div id ing. For example , an external osmot ic pressure as weak as 500 Pa s lowed the growth rate o f a tumor

spheroid.

Distr ibut ions o f the scaled modu l i o f the magnet ic induct ion (a ) and magnet ic

gradient force (b ) in the p lane above a cyl indr ical magnet w i th an axial hole.

(c ) 2D-p lot of the magnet ic gradient force as a function of the radial coordinate. The magnet ic induct ion modu lus is normal ized to (μ 0 r/4π), whereas the modu lus of magnet ic gradient force is normal ized to R− (μ 0 r/4π)2. The

calculat ions were per fo rmed for a magne t length 1 cm, magne t rad ius 0.5 cm, ho le rad ius 0 .1 cm, an d d is tance b e tween the m agn e t top a n d the p lane o f calculat ions of 0.1 cm.

Discussion

B y summar iz ing the anal yses of the above-cons idered phenomena, models and suggested mechan isms, one can identi fy the fo l lowing intracel lular ef fectors of appl ied H G M F s . W e use the term “effector” to indicate a structural component of a cel l that responds to an appl ied high-gradient, stat ic magnet ic f ield. Thus , the fol lowing are intracellular effectors of an HGMF: i) cytoskeleton remodeling, ii) changing the probabi l i ty o f ion channel on/off swi tching events, iii) causing the mechan ica l st ress in the memb rane , iv) m em b r an e bend ing, v ) m igra t ing m em b ran e receptor proteins, and vi ) changing the ion f lux ba lance and m em b ran e potent ial due to magnet ic gradient forces. A schemat ic i l lustrat ion of the possible appl icat ions of HGM Fs and intracellular effectors is shown in Fig. 6. Work ing alone, each of these effectors can significantly af fect cel l funct ions. However , they are no t independent and can wo rk in a certain pa th wa y to alter the molecular mechanisms of a cel l and synergize its response to an H G M F . For

M

M

example, depend ing on cel l type, state and edge, an external ly appl ied H G M F can st imulate cel l division, cause cel l swel l ing fol lowed b y membrane blebbing and apoptosis , an d change the di f ferent iat ion p a thwa y o f s tem cel ls and gene

expression. Fo r these and other effects o f H G M F s , the magnet ic gradient thresholds are shown in Table 2. The cel l responses l isted in Table 2 do not occur immediately upon appl icat ion of the H G M F but can be delayed in t ime. After applying an H G M F , the cel l response arises at t imescales varying f rom a f ract ion o f a second to days, wh ich depends on cel l t ype, magnet ic gradient magn i tude and t ime of exposure (see Methods) .

Figure 6: Schematic illustration of the possible applications of HGMFs

and intracellular effectors.

Table 2: Thresholds for the effects of static HGMF.

Magnet ic s ys tems generat ing magnet ic f ie lds w i th gradients o f the order o f 109T m − 1 would a l low for signif icant al terat ion of the membrane potent ial in accordance wi th predict ions based on Eq. 1. Changes in m em b ran e potent ial have proven to be pivotal not on ly in normal cel l cyc le progress ion but also in mal ignant t ransformat ion. Thus, dr iv ing the membrane potential wi th H G M F s opens n e w opportun i t ies to s tudy intercel lu lar an d intracel lu lar p rocesses and provides n e w routes to control l ing cel l fate. B y understanding the wa ys in which H G M F s can be ut i l ized to select ively generate the required cel lular responses, w e can begin to cons ider magnet ic f ie lds as t iny non- invasive tools that can remotel y al ter the cel l mechanism, p romis ing broad appl icat ion potential in cel l therapy, neurobio logy and nanomedic ine. Ul t imately, to address the most demand ing chal lenges in med ic ine ut i l iz ing magnet ic f ields, it is necessary t o ans wer the quest ion: w h a t are the paramete rs that can re l iab ly a l low us to def ine magnet ic f ield ef fectors and cause-ef fect relat ionships between magnet ic f ield appl icat ion and cel l response? T o a large extent , b y achiev ing exper imental faci l i t ies that p rov ide the highest values of magnet ic f ield gradient, one can expect the d iscovery o f new, excit ing, b io logical effects of magnet ic fields.

Methods

Generalized Nernst equation for membrane potential

Let us consider the Nernst equi l ibr ium potent ial in the presence o f a high- gradient magnet ic field. In equi l ibr ium, wi thout a magnet ic field, the free- energy change for the di f fusion of an electrolyte into the cel l is

where z is the ion va lence (z =  +1 for a posit ive, univalent ion), F is the Faraday constant , R is the gas constant , T is the abso lu te temperature , V m is the potent ia l d i f fe rence be tween the t wo mem b r an e s ides, an d no and n i are the ion concentrat ions outs ide and inside a cel l. B y sett ing ΔG to zero, wh ich is the case w h e n the m o v e m en t o f the ions is at equi l ibr ium, o n e can arr ive at the Nernst equat ion

W h en a high-gradient magnet ic f ie ld is appl ied to a cel l in a med ium, the magnet i c gradient fo rce acts o n ions and can ei ther assist o r oppose ion m o v em en t th rough the membrane . T h e magnet ic gradient force is g iven b y

where p is the magnet ic d ipo le mom ent o f the ion, B is the magnet ic induct ion, and the der ivat ive is taken wi th respect to di rect ion l , wh i ch is paral lel to the magnet ic dipole moment o f an ion, l//p . Bear ing in m ind Eq. 5, in this case, wh en the ions di f fuse in the presence of an H G M F , the free energy change is

where L is the half-cel l size and N A is the Avogadro constant . In Eq. 6, the last te rm represents the wo rk of the magnet i c gradient forces w h e n a mo le of magnet ic ions di f fuses across a membrane ; the s igns “p lus ” and “minus ” cor respond to the two l imi t ing cases: the magne t i c gradient force ei ther assists or opposes the electr ic force exer ted o n ions m o v i ng across the membrane . In equi l ibrium ΔG =  0, and f rom Eq. 6, one can arrive at

where e is the electron charge, wh ich is Eq. 1 (see Results).

Thermal fluctuation forces

A cel l works in a no isy env i ronment created b y thermal f luctuations. Therefore, the cel lular cytoskeleton exhibi ts cont inual f luctuat ions due to thermal agitation. Th e thermal f luctuation forces of act in f i laments are given b y F th =  (kkBT)1/2, where k is the spr ing constant of a single F-act in f i lament and the thermal f luctuat ion energy is kBT  =  4.1 pN· nm at room temperature. The effect ive spr ing constant for an F-act in network was keff =  10− 5 Nm− 1. Thus, the est imated value of the thermal f luctuation force is F th =  0.2 pN. This value is s l ight ly less than the measured min imal forces (0.3–0.5 pN) generated b y act in f i lament polymerizat ion.

Estimation of the electrostatic energy stored in the membrane

For a spher ica l cel l , the electrostat ic energy can be calcu lated as the energy o f a charged capaci tor

where c is the electr ic capaci tance and U is the vol tage. Fo r a spherical cel l memb ran e w i th internal and external radi i a and b , respect ively, the electr ic capaci tance is

where ε 0 is the permit t iv i t y o f f ree space and ε is the dielectr ic constant o f the l ipid bilayer, which typical ly varies in the range 1–20. B y insert ing Eq. 9 into Eq. 8, w e obta in the electrostat ic cel l energy as

Finally, b y insert ing the fo l lowing parameters into Eq. 10: ε = 5, U  =  70 mV, a  ≈  b  =  10 μm and b  − a =  5  nm (which is the membrane thickness), one can

obtain E  ≈ 2.7 10−14 J.

Finding strength in the smallest magnets: magnetic systems

generating HGMFs

Micro- and nano-magnets are extensively used for a w ide spect rum of b iomed ical appl icat ions. Here, w e descr ibe micro- and nano- magnets that can achieve ex t remely h igh f ield gradients. O n e w a y to achieve h igh values of magne t i c grad ien t is to use smal l m agn e t s and/or to opera te near the m agn e t

edges . Th is idea is based o n the fact that the magne t i c grad ient forces benef i t great l y f rom scale reduct ion; therefore, m icro- and nanomagnets exhibi t large magnet ic gradient forces. Indeed, it can be eas i l y demonst ra ted analyt ical l y that w h e n al l d imens ions o f a pe rmanen t m ag n e t are reduced b y the s a m e factor k (wi th al l of the magnet ic character ist ics preserved) , the f ield gradient is mul t ipl ied b y the reduct ion factor k.

Magnetized slabs

The magnet ic stray f ie ld around a un i formly magnet ized s lab was calculated

elsewhere. Near the edge of a long, un i formly magnet ized s lab of width 2a, the magnet ic f ield gradient obeys

where x is the d istance to the s lab edge, n is an arbi t rary un i t vector d i rected f rom the s lab edge to the point where the f ield gradient is calculated, and M r is

the remanent magnet izat ion. Eq. 11 is val id for x«a, and the modu lus o f the magnet ic f ield gradient does not depend on the di rect ion of vector n . It fo l lows f rom Eq. 11 that when approaching the s lab edge (x → 0) , the magnet ic f ield gradient g rows and has a singulari ty. F rom Eq. 11, est imat ion wi th the value of the remanent magnet izat ion of an N d F e B magnet and x  =  1 μm gives a h igh value of magnet ic f ie ld gradient o f 5.4∙ 105 Tm−1. S imi lar va lues of magnet ic gradient we re meas ured c lose to the sur face o f m icro-magnets .

The axial componen t of the f ield gradient is

Simi larly, for a single, un i formly magnet ized, parabol ic-shaped magnet ic pole used in magnet ic tweezers, the m a x i m u m magnet ic f ield is g iven b y

where z is the distance f rom the magnet pole. Thus , in al l of the cons idered cases, the va lue o f the magne t i c gradient increases dramat ica l l y w h e n approaching the magne t edge. Fo r example, for a s ingle, parabol i c -shaped magnet ic pole of s ize 1 μm, the gradient can reach 3  ·  106  T m− 1 100 nm f rom the tip.

Magnetic nanoparticles

Let us consider a magnet ic nanopart ic le wi th a magnet ic m o m en t p =   M sV (where M s and V are the saturat ion magnetizat ion and M N P volume). We can represent a nanopart ic le as a smal l , spher ica l m agn e t w i th d iameter equa l to 2 R, that is, the s ingle domain M N P acts as a dipole wi th magnet ic moment p. Magnet ic induct ion and its gradient at the ax is paral lel to the magnet ic m o m ent di rect ion are given b y

Near the surface of the M NP , at r  =  R, the modulus of the radial magnet ic

gradient is , as fo l lows f rom (15). The perpendicular component,

B⊥ , is two t imes smal ler than B//. Thus, for the cons idered magnet geomet ry, c lose to the m agn e t sur face (edge) , the magne t i c grad ien t is the s am e order o f magn i tude: whe re r is the character ist ic length scale of the task. W e have analyt ical ly ex amined magnet ic sys tems for producing h igh-gradient magnet ic f ie lds and calculated the magnet ic f lux and gradient dist r ibut ions that m igh t enable cont ro l o f the cel l shape and funct ions. T h e magnet i c s ys tems capab le of generat ing H G M F s and formulae al lowing rapid estimation of the magnet ic f ield gradient are summar ized in Table 1.

Magnetic field distribution near a cylindrical magnet with an

Axial hole

The magnetic field and force distributions were calculated with the help of the explicit analytical expressions for magnetic field induction generated by a cylindrical permanent magnet, magnetized along its symmetry axis. For homogeneousl y magnet ized cyl inder o f the radius, a and length L , the ax ial (B z) and radial (B ρ) components o f the magnet ic f ie ld induct ion can be calculated as:

as

where Φ is the az imuthal angle, z is the coord inate a long the s ymmet r y ax is o f a cyl inder, ρ is the radial coordinate, M r is the remanent magnet izat ion and μ 0 is the permeability of free space. To calculate the magnetic field of a magnet with

the axial hole of radius, r one should make the f ield superposi t ion of two “up-” and “down-” magnetized cylinders: Bz =  Bz1(a) − Bz2(r ) and Bρ =  Bρ1(a) − Bρ2(r) , where the subscripts 1 and 2 stand for up-magnetized and down-magnetized cyl inders of the radii a and r , respectively.

Magnetic pressure in the vicinity of magnetic nanoparticles

From Eq. 2, with the help of Eq. 15, one can calculate magnetic pressure as

where Δχ is the difference of the magnetic susceptibilities of the lipid membrane and the cytosol.

Timescales of cell response to HGMFs

The HGMF-induced biological effects mediated by intracellular mechanical stress do not arise immediately upon applying the field. A time delay in cell

response to swi tching on H G M F occurs. In low and moderate magnet ic fields, the t ime de lay o f the cel l response is dependent o n the magn i tude o f the magnet ic f ie ld gradient but not on the strength o f the magne t ic f ield. T h e fo l lowing i l lustrates the hierarchy o f the t imescales of the observed cel l responses to HGM F s for different magnet ic gradients. In HG M Fs wi th magnet ic gradient o f approx imately |∇ B| ≈  109 Tm− 1, a cel l response (change o f the rest ing membrane) is expected wi th in a second. Migra t ion and adhesion o f s tem cel ls to the edges o f m icromagnets (at the edge |∇ B| ≈  106 T m− 1) w i th subsequent cytoske le ton remodel i ng and changes o f cel l shape were observed dur ing the fi rst 4 hours after cel l cul ture deposi t ion o n the magnet ic sys tem. Dur ing the fo l lowing 3 days, the cel ls m igra ted and occupied the tops of the micromagnets , c reat ing pat terns that ref lect the spat ial d ist r ibut ion o f magnet ic gradient forces generated b y m ic romagne t arrays . Ex posure o f the monocyt ic leukemia cel ls to a high-gradient magnet ic f ield (up to |∇B| ≈  103  T m −1) for 24 h induced cell swel l ing and tr iggered apoptosis. Changes in D N A organizat ion, gene express ion and the dif ferentiat ion pa th way o f s tem cel ls were detected after exposure to low-frequency (4 Hz) H G M F with |∇B| ≈  102  T m − 1 for 5 days.

Scientific Reports volume6 , Article number: 37407 (2016)

PARAGRAPH 8: Biophotonic technology

T h e advanced techno logy 7 researchers integrated th is nano techno logy w i th two other types of technologies .... Biophotonic Technology. T o understand it we must necessari ly g ive a nod to h o w l ight interacts w i th b iology through biophotons and the discoveries of prof. Fri tz-Albert Popp. Studying the effect o f radiat ion on l iv ing systems, Professor Popp cam e across some very interest ing propert ies o f carc inogenic chemica l compounds : these co mpoun ds act as " remixers o f f requencies" i n a very precise range, that o f the 380

nanometers . T h e l ight w e see around us is general l y compo sed o f a set o f inf ini te components , each w i th a precise f requency an d wave length ( the wavelength is equal to the speed o f l ight d iv ided b y f requency, then a s ingle component o f l ight can be character ized uniquely b y an y of the two

parameters) . W h a t Popp has d iscovered is that carc inogenic com po unds such as benzo [a] pyrene absorb l ight o n the wave length o f 3 80 nanomete rs , bu t re-emi t i t a t another f requency.

This does not happen wi th chemica l compounds , however s imilar, that are not carcinogens, such as benzo [e] pyrene, wh ich has a min imal di f ference f rom benzo [a] pyrene in on l y one of the r ings that compose it. Invest igat ing the part icular l ight radiat ion w i th a wavelength o f 380 nanomete rs , you’ l l not ice that it is associated w i th the photo-repai r phenomenon .

If indeed a cel l becomes damaged (and even a lmost total ly dest royed) b y ultra v iolet l ight, i t can be repaired in just one day if i t is exposed to rad iat ion of the s am e frequency but much lower intensity. Th is phenomenon occurs wi th

m ax i m u m intensity at the wave length of 380 nanometers . F u r t h e r , he d iscovered that the molecu les wi th in the cel ls respond to certain f requencies, w i t h b io-photonic radiat ions related to the d isease or heal th status o f an organ ism.

Biophotonic radiat ion is used by the cel ls of a l iving organism for one sort of very eff icient inter-cellular elect romagnetic communicat ion, wh ich is also ex changed be t ween o rgan i sms o f the s am e species ( f rom bacter ia to wate r f leas), and the l iv ing molecu le that m o re than an y other is depu ted to the reception and t ransmission of b io-photons is D NA .

Image of a body that emits Biophotons, displayed in the infrared

spectrum.

Al l of these discoveries comb ined dest royed the enti re ax iomat ic const ruct o f or thodox biology founded on the pr imacy o f D N A and opened the wa y to the new biology based on undulatory genetics and epigenetics (branch of molecular biology that s tudies genet ic muta t ions and t ransmiss ion o f inheri ted t rai ts no t d i rect l y at tr ibutable to the D N A sequence).

Speci f ical l y using the benef i t o f this techno logy in the inner part o f the product that touches the skin , t issue h as been pos it ioned w i th exact l y the wave length of 3 8 0 nanomete rs . Th is enab les the speed ing u p of t issue repai r an d the recovery an d heal ing process , as th is in format ion is constant l y t ransmit ted to the cel ls.

PARAGRAPH 9: Radionic Technology

The third technology used is radionics.

There are s o m e very interest ing and unjust l y neglected studies ( for obv ious reasons) that are no t i nves t i ga ted b y off ic ial science, bu t wh ich f ind a pract ical conf i rmat ion in the solut ion o f some problems. Let us brief ly summar i ze .

A very important contribut ion was made by Dr. Albert Abrams (1883-1924), who hypothes ized that the a tomic an d e lectron ic s t ructures o f hea l thy t issue h ad to be d i f ferent f r o m d i seased t i ssue . He no ted that the d isease created a " rad iat ion" that cou ld be neut ra l ized b y drugs or the Earth’s magnet ic f ie ld.

After a few years, researchers l ike A. de Belizal and P.A. Morel , in 1939, d iscovered interest ing phenomena descr ibed in the book of microv ibratory physics and invisible forces.

The book highl ights several important concepts, two of wh ich are fundamental ... that o f shape wav es and that o f connect ion w i th micro-rad iat ion and h o w they interact w i t h the h u m an body.

Propagat ion o f e lect romagnet ic wa ve s inside the pyramid o f Cheop s at d i f ferent lengths o f radio waves ( f rom 200 to 400 meters) . T he b lack rectangular posi t ion o f the so-cal led King 's

Chamber. Credit: ITMO University, Laser Zentrum Hannover

The knowledge f rom th is work wh ich interests us is that wh ich in radionics is cal led the " law of transfer": Some specif ic geometric or symbol ic f igures are ab le to conduct , t ransport , and prope l wav es ent rusted to t h em an d can serve as di rect ional an tennas in order to reach the chosen target . H o w can the concept b e reversed, as for ex amp le in k inemat ics (wh ich descr ibes the m o v em en t o f a body, regard less o f the causes that p rod uced it), whe re a source o f energy th rough a wav e creates a geomet r i c f igure?

Radionics has found more and more conf i rmat ion, includ ing f rom off icial science w i th the advent o f quan tum phys ics, which has s h own that the a toms of wh ich the mat ter is com pos ed are compressed energy. O u r h u m an bodies, an imals and plants (organic mat ter) are energy, as wel l as inorganic sol id matter. S o , f rom the point of v iew of energy, w e can comp are i t to the wav es that are f o rm ed ins ide water , wh i ch is energy that m o v es th rough the water .

S o , this is the t rue fo rm of the energy wav es ( f requencies) that m o v e th rough

space. Every da y w e are immers ed in these f requencies, in these wav es of

energy. But speaking o f matter, i .e. hav ing a tradit ional approach, I can div ide it and s tudy each par t separately, wh i le i f w e talk about energy waves w e cannot d o this, so w e need to have a hol is t ic approach. In fact, the n e w science says that w e mus t s tud y the wh o l e an d no t the par ts because they are fo rm ed o f energy,

and this cannot be div ided. N o w , the classic approach in address ing the p rob lems that aff l ict us is to ana lyze the part wh ere there is a p rob lem, not car ing abou t the rest , and is therefore incomplete .

The temporal evo lut ion o f a l ight b e am or an electron beam , or even a s ingle elect ron, presents the wav e character ist ic w i th inter ference p h en o m en a an d di f f ract ion. Bu t at the s am e t ime, wh e n measur ing ex tens ive quant i t ies, a con t inuous f l ow is no t obta ined bu t rather a sequence of quanta (quan tum = quant i t y) , bo th for the elect rons, wh i ch are therefore no t widespread in the who le space as their probabi l i ty d ist r ibut ion is undulatory, and both for photons, o r the quanta of the l ight b e am (Quan tum theory). T h e two wave-par t i c le aspects s eem to be in contradict ion; bu t w i th the exhaust ive formulat ion o f quantum mechan ics , w e can see the dua l nature of wave- part ic le. Mat ter is energy, as w e said:

The power of "Creat ion" is h idden in this s imple formula. Al l that we perceive wi th our senses is noth ing bu t energy, wh ich is born o f the sam e fundamenta l

part icles. T h e indiv idual "packets " o f energy, that is the "quanta" are the subatomic part icles. Energy ex ists in the fo rm of waves that propagate in space and t ime. These waves beco me local ized part ic les, in a defin i te t ime and in a def ined posi t ion, on l y wh en you becom e an observer an d you can perceive them th rough your senses. T he part icles becom e waves again if you w i thdraw you r at tent ion an d the ob ject they represent is n o longer located in space and t ime, but unex p ress ed at that mom ent . W e speak, therefore, o f "probability".

A n ew concept o f mat ter then, o f space, o f t ime, n ew equat ions l ike those of

the wav e funct ions used to measure magne t i c f ie lds are beginn ing to shed l ight on these m ech an i s ms and h o w they a re determined.

How did we apply this technology to the product?

B y insert ing a specif ic non-vis ib le geometr i c shape that has the funct ion o f channel ing m ic ro heal th radiat ions for the bod y which speed u p the recovery o f the unhealthy part or and also using E.L.F (Extremely Low Frequency) Magnetic Fields.

Alternating Magnetic Field therapy

There is substant ial in format ion o n h o w the fo rm converts the universal ether into e lect romagnet ic an d magneto -grav i tat ional forces. Fo r ex ample , the studies of quantum mechanics b y Richard Liboff descr ib ing h o w the Earth 's magnet ic f ield interacts w i th the absorpt ion of ca lc ium ions in the cel ls , of wh ich w e have a lready prov ided a substant ial art icle in the prev ious pages. T h e knowledge that ve r y w e a k e lec t romagnet ic waves , under cond i t ions where they h av e the r ight f requency and intens i ty an d , in our case, genera ted b y a speci f ic geomet r i c sym b o l o r st ructure which interacts w i th the Earth 's magnet ic f ie ld, can produce an increase in the f low of e lect romagnet ic energy towards the cel l membranes , has appl icat ion in biology, medic ine and even physics.