Patent Application: US-201615259610-A

Abstract:
a claimed invention relates to the field of medicine or biology , namely the methods using nanotechnology objects , in particular colloidal solutions of gold nanoparticles which demonstrate a strongly marked effect on cellular proteins and can be used to restore the contractile function of mammalian vascular smooth muscle cells . further , the invention relates to medicinal products which comprise gold nanoparticles and may be used for remote opening of voltage - gated ion channels in mammalian vascular smooth muscle cells . a method for remote opening of voltage - gated ion channels in mammalian vascular smooth muscle cells comprises extracellular application of a colloidal solution of gold nanoparticles in population of mammalian vascular smooth muscle cells . a medicinal product opening voltage - gated ion channels in mammalian vascular smooth muscle cells comprises the colloidal solution of gold nanoparticles at a concentration required for remote opening of voltage - gated ion channels in mammalian vascular smooth muscle cells .

Description:
the effect of remote opening of voltage - gated ion channels in mammalian vascular smooth muscle cells by extracellular application of the colloidal solution of gold nanoparticles in the population of mammalian vascular smooth muscle cells was studied in 6 - 8 week old male rats wistar ( body weight : 250 - 350 g ) which are held under controlled environmental conditions (+ 21 ° c ., day - night cycle : 12 h / 12 h ) and have free access to water and standard food for rats . for the purposes of the study , smooth muscle cells isolated from rat thoracic aorta using collagenase and pronase were taken . rats were anesthetised with ketamine ( 37 . 5 mg / kg body weight , intraperitoneally ) and xylazine ( 10 mg / kg body weight , intraperitoneally ), and thoracic aorta about 1 . 0 . . . 1 . 5 cm long was removed and denuded to make it free from connective tissue . aorta was cut into small pieces ( 1 . 5 × 1 . 5 mm ) in the cooled low ca 2 + ion content solution comprising ( in mm ): nacl 140 , kcl 6 , mgcl 2 3 , d - glucose 10 , hepes 10 ( ph 7 . 4 ) for 15 minutes . vascular tissues were placed in fresh low ca 2 + ion content solution comprising collagenase type ia 2 mg / ml ( 417 u / mg ), pronase type e xxv 0 . 5 mg / ml , and bovine albumin serum 2 mg / ml . then tissues were incubated for 30 minutes at a temperature of + 37 ° c . after incubation , tissues were washed ( for 2 - 3 minutes ) twice in fresh low ca 2 + ion content solution to remove enzymes . cells were dispersed by stirring using a glass pipette and then placed in modified krebs bicarbonate buffer . a certain number of muscle cells was maintained at a temperature of + 40 ° c . and remained functional for at least 4 hours . the studies were conducted using au np colloidal solutions with different np size and various stabilisers ; however ascorbate - stabilised au nps 4 . . . 7 nm in size , more preferably ≈ 5 nm in size , demonstrated strongly marked ability to relax vascular smooth muscle cells in rats by remote control ( see table 1 below ). fig2 shows dose - effect curves for au nps 5 nm in diameter in rat thoracic aorta sm narrowed by norepinephrine ( ne , 10 - 6 m ). au nps at a concentration range between 10 − 6 and 3 × 10 − 4 m apparently have the ability to significantly reduce the amplitude of norepinephrine - induced contraction in a dose - dependent manner ( pd 2 = 4 . 2 ± 03 , e max = 55 ± 4 %), i . e . au nps demonstrated strongly marked vasodilating activity . endothelial disorder had no significant impact on au nps induced sm relaxation . it is important to note that stabilisers alone had no effect on the tone of thoracic aorta sm narrowed with norepinephrine . to induce excitation of surface plasmon resonance in gold nps , a cell with organ tissues was irradiated with “ green ” laser 5 mw at a wavelength of 532 nm over the entire period of au nps induced relaxation . in this case , addition of au nps resulted in a significant increase in the amplitude of maximum au np induced relaxation , from 55 %± 4 to 85 ± 5 . 0 % ( n = 10 , p & lt ; 0 . 05 ), while sm sensitivity to au nps did not increase , and the average value of pd 2 was ± 0 . 013 ( n = 10 , p & gt ; 0 . 05 ). it is important to note that intensity of laser irradiation was so that it did not lead to any significant changes in temperature of krebs solution in a bath . in the presence of paxillin ( 500 nm ), a powerful bk ca channel blocker , au nps lost their vasodilating activity in isolated aortic sm segments almost completely ( fig2 ) demonstrating the critical involvement of bk ca channels in this case . the next panel of tests was conducted to study original k + channel activity in control thoracic aorta sm cells . single cells isolated enzymatically from healthy rat thoracic aorta were kept at resting potential − 60 mv , and outgoing currents were then caused by stepwise increase of depolarizing voltage . the respective original current curves and current - voltage ( i - v ) relationship are shown on fig3 . the total amplitude of outgoing current ( i k ) density in an intact sm cell was on average 32 ± 2 pa / pf ( n = 12 ) at + 70 mv . all cells clearly demonstrated marked trace currents confirming that measured currents were recorded mainly from external potassium channels ( i k ). further , spontaneous current fluctuations on a horizontal site of curve i k resembled spontaneous transient outgoing currents ( stocs ) placed on the total current of bk ca induced activation ( fig3 b ). current to voltage ratio ( curve iv ) shows that outgoing currents in intact aorta cells are absent at potentials below − 40 mv and markedly rise in response to exposure of depolarizing voltage . curve iv shows nonlinear dependence at potentials above − 40 mv which can be more clearly traced at more positive potentials than + 25 mv ( fig3 a ). it is important to note that outgoing currents are activated quite slowly , and in control conditions inactivation was poor or was not observed during 300 ms impulses . fig3 a , b shows that the original curve ik and the curve iv obtained for sm cells treated with au nps ( 10 − 4 m ) showed a significant increase in amplitude k + current density from 32 ± 2 pa / pf to 59 ± 5 pa / pf + 70 mv , respectively ( p & lt ; 0 , 05 , n = 10 ). external irradiation with “ green ” laser increased au np - induced increment i k from 59 ± pa / pf to 74 ± 1 pa / pf ( n = 10 , p & lt ; 0 . 05 ). after that , various components of total potassium current activated by action of au nps were determined pharmacologically ( fig3 c , d ). it is clear that total outgoing current in rat thoracic aorta sm cells , mainly due to current activation , is provided through paxillin - sensitive bk ca channels . this conclusion is based on the following evidence . paxillin ( 500 nm ) added to external solution in a bath reduced outgoing current in untreated rat thoracic aorta sm cells significantly ( from 32 ± 2 to ± 6 1 pa / pf , n = 10 , p & lt ; 0 . 05 ). additional administration of tetraethylammonium ( 10 mm ) resulted in a decrease of outgoing current to 3 ± 1 pa / pf ( n = 10 , p & lt ; 0 . 05 ). application of paxillin ( as well as tea ) stopped au nps - induced i k increment ( fig3 d ). it is known that the relationship between potential applied to the channel and its current ( conductivity ) is described by boltzmann equation . thus , the standard boltzmann equation can be used for static assessment of the impact of au nps on aortic i k of sm cells before and after administration of au nps ( fig4 ). the curve conductivity - voltage thus built shows the nature of channel opening , specifically movement of charges in an area of channel protein sensitive to potential changes . thus , values of fixed data points of activation correspond to boltzmann equation , allowing to obtain half - maximal activation voltage ( v 1 / 2 ) and showing a significant increase in values v 112 for au nps and combinations of au nps and “ green ” laser irradiation ( v 112 = 8 . 5 ± 0 . 5 and 14 ± 0 . 8 mv , control condition and upon au np administration , respectively , n = 10 , p & lt ; 0 . 05 ). laser irradiation enhanced this effect ( v 112 = 16 + 0 . 8 mv , n = 10 , p & lt ; 0 . 05 ). these results clearly show that k + channel activation after au np administration does not switch the general bk ca activation mechanism through a negative shift in the activation curve ( i . e . changing voltage sensitivity was achieved by growth of free intracellular calcium concentration ), and increasing conductivity of the entire cell is likely associated with changes in the channel conductivity ( e . g . increasing the amplitude of single current or probability of channel opening ( p o )). the data shows a significant increase in conductivity by activating k + channel in the whole range of potentials above − 50 mv . this effect was studied experimentally using single channel recordings , the most direct approach to establish a mechanism of channel modulation by au nps . in supracellular areas formed on rat aortic myocytes , activity of 3 - 5 bk ca channels typically occurs at potentials ranging from − 20 to + 40 mv . depending on the extent of channel activity in each separate membrane segment , the optimal confining potential was set within this range so that the initial channel activity ( typically expressed in npo ) would not be too high , for example about 0 . 05 - 0 . 1 . adding au nps potentiates bk ca activity with a delay for 1 - 2 minutes ( fig5 a ), as it was originally observed for more frequent openings of channels with subsequent progressive emergence of additional open levels corresponding to multiple openings of channels with identical single - channel current amplitudes ( fig5 a , the middle panel ). in the presence of au nps for 10 - 15 minutes and especially in combination with “ green ” laser irradiation , a significant increase in channel activity is observed with activation of more than 10 channels ( e . g ., fig5 v , the upper curve , recording for about 35 minutes ). accurate measurement of npo in such high channel activity is problematic due to emergence of noises on the curve ; however 2 - 11 - fold increase of npo values was observed in all 3 sites obtained depending on the au np concentration irrespective of whether laser irradiation was applied or not . therefore , au np impact on channel activity likely depends on their concentration ( fig5 b ); however it is difficult to quantify the maximum effect , especially at a concentration of 10 − 4 m , more precisely due to the above - mentioned problems of extremely high channel activity and a large number of channels in membrane areas . in terms of evaluation , mean fold increase in channel npo was on average 3 . 4 ± 0 . 7 at an au np concentration of 10 − 5 m and 8 . 5 ± 2 . 5 at a concentration of 10 − 4 m , which indicates a dependence of au np opening effect on au np concentration . similar results were obtained for bk ca channels in mouse iliac myocytes ( n = 3 , data not shown ), indicating the effect of au nps as “ openers ” of bkca channels both in vascular and visceral smooth muscle . the mechanism of interaction au nps with potassium channels and strengthening of the achieved effect by exposing cells to laser irradiation may be associated with plasmon resonance effect , i . e . excitation of surface plasmons on the surface of au nps , which may be increased by irradiation with light at a resonance frequency . au nps , as nanosized structures of a noble metal , are known to have a distinctive feature , such as collective oscillation of electronic “ gas ” on np surface ( surface plasmon resonance ) leading to photo - induced local electric fields near the surface of nanoparticles . in case of laser irradiation , this local electric field can be significantly enhanced by local plasmon resonance , which in turn affects ion channel voltage sensor , increases outgoing current , and leads to sm relaxation . in the absence of external irradiation , such plasmon may be excited by chemiluminescence of natural tissue . the strength of an electric field in a cell membrane depends on au np concentration , and k + channels may be activated when threshold au np concentration is reached . therefore , channel recordings shown on fig5 provide direct confirmation of single - channel bk ca activation using au nps . “ green ” laser irradiation is an additional intensifying factor in the presence of au nps and is ineffective in the absence of gold nps . the medicinal product opening voltage - gated ion channels in mammalian vascular smooth muscle cells containing the colloidal solution of gold nanoparticles may prepared as follows . the au np colloid solution with maximum plasmon resonance absorption band within 532 nm is synthesised by reducing sodium tetrachloroaurate with sodium ascorbate in aqueous solutions at a room temperature . colloidal solutions thus prepared are then neutralised with acetic acid to ph 6 - 7 . dynamic light scattering spectroscopy of the au np colloidal solution thus obtained ( 200 mg / l , i . e . ˜ 10 − 3 m or 6 . 02 × 10 20 particles / l ) shows that the average hydrodynamic size of au nps is approximately 5 nm ( fig1 ). this result is confirmed by sem method ( see a fragment of sem microphotography on the insert to fig1 ). au np surface stabilised with ascorbate anions are negatively charged and provide electrokinetic ( zeta ) potential − 35 mv to np surface . the colloidal solutions thus prepared remained stable for many months and did not show noticeable changes in au np size and distribution by size over time . following extracellular application of the colloidal solution of gold nanoparticles , population of mammalian vascular smooth muscle cells may be exposed to pulsed nanosecond irradiation allowing to increase irradiation capacity without side effects . according to another embodiment , fibre optic devices for endoscopic or interstitial irradiation may be used for irradiation ( dykman , klebtsov , 2012 ). the claimed invention helps to achieve the effect of relaxation of mammalian vascular smooth muscle cells when au nps are used as colloidal solution with nps 4 . . . 7 nm in size by remote opening of voltage - gated ion channels ( in particular bk ca channels ). the claimed method helps to renew normal vascular contractile activity in a number of diseases associated with the development of vascular hypercontractility in vascular sm . 1 . cox r , folande k ., swanson r . differential expression of voltage - gated k + channel genes in arteries from spontaneously hypertensive and wistar - kyoto rats . hypertension 2001 ; 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