the claimed nanodiamond - glycine conjugate with no fluorine atom on the surface thereof is an ultradisperse powder , dark grey or dark grey with either a greenish or a dark blue tint and particle sizes of 2 to 10 nm ( fig2 ), wherein the size of the aggregates thereof in an aqueous suspension is 25 to 50 nm ( fig4 ). fig2 clearly demonstrates that the claimed conjugate has an ultradisperse structure created by particles with a diameter smaller than resolution ability of the used instrument ( from 20 nm ). photomicrographs of the nanodiamond - glycine conjugate particles are obtained on a super - high resolution auto emission scanning electron microscope zeiss ultra plus ( carl zeiss , germany ). the conditions of the film taking are cited on the photomicrograph . fig3 demonstrates that the nanodiamond - glycine conjugate particle size distribution is 2 - 10 nm , and the surface membrane thickness is up to 1 nm . photomicrographs of the nanodiamond - glycine conjugate particles were obtained on a transmission electron microscope jeol 1011 ( jeol , japan ). fig4 shows a distribution curve of particle sizes in the suspension of the claimed nanodiamond - glycine conjugate that shows particle sizes in the suspension being 25 - 50 nm . distribution of particle sizes in the conjugate suspension is determined by laser dynamic light scattering on a zetasizer instrument ( malvem instruments , usa ). the x - axis is the logarithmic scale of the particles in nm . the y - axis is the percent composition . fig5 shows an ir - spectrum of the claimed conjugate . the spectrum shows a broad intense band with a maximum at 3 , 400 cm − 1 ; a strong signal at 1 , 621 cm − 1 ; six bands of medium intensity at 2 , 924 , 2 , 881 , 1 , 383 , 1 , 306 , 1 , 212 , and 1 , 154 cm − 1 ; and a weak characteristic signal at 504 cm − 1 . the spectrum has maximums at 1 , 383 ; 1 , 306 ; 1 , 212 ; and 1 , 154 cm − 1 ; corresponding to the maximums of the original glycine amino acid , which shifted into the 1 , 400 - 1 , 100 cm − 1 region because of the covalent bond formation with the nanodiamond surface . ir - spectra were registered on a ftirs ir200 thermonicolet instrument ( thermo scientific , usa ). resolution − 2 cm − 1 · number of scans − 64 . for the analysis , carefully weighed samples were mixed with kbr powder and pressed into tablets . fig6 shows xpe - spectra of the claimed nanodiamond - glycine conjugate . said x - ray photoelectron spectra define the nature , energy condition , and number of surface atoms of nanodiamond particles and identify practically all the elements , except for hydrogen and helium [ 5 ]. the surface of the claimed nanodiamond - glycine conjugate is examined on a las - 3000 instrument ( riber , france ) equipped with a hemispherical analyzer opx - 150 . the non - monochromatized x - ray radiation from an aluminum anode ( a1a = 1486 . 6 ev ) ( 12 kv voltage on the tube and 20 ma emission current ) is used for photoelectron excitation . calibration of the photoelectron peaks is conducted along the c 1s carbon line with binding energy of 285 ev ( e b ). vacuum in the work chamber is 6 . 7 × 10 − 8 pa . high vacuum is achieved with an ion pump . the elemental composition on the surface of the claimed nanodiamond - glycine conjugate according to the xpe data is shown in table 1 . table 1 demonstrates that the claimed nanodiamond - glycine conjugate does not contain fluorine or any other halogen atoms in the amounts exceeding the instrument error ( 0 . 1 at %) since in the process of the nanodiamond - glycine conjugate &# 39 ; s formation ( fig1 ), all chlorine atoms are substituted with glycine molecules and leave the nanodiamond surface as hcl molecules . chlorine - modified nanodiamond particles used in the preparation method are obtained by annealing the nanodiamond at 2 - 3 l / hr . and 500 - 1 , 200 ° c . in a h 2 gas stream for 1 - 8 hours . the annealed nanodiamond is then chlorinated in liquid phase with molecular chlorine . for that , chlorine obtained in the reaction between k 2 cr 2 o 7 ( or kmno 4 ) and hydrochloric acid is dissolved in ccl 4 to 3 - 5 wt %. chlorination is conducted under photochemical exposure to visible light for 36 to 60 hours at 50 - 70 ° c . the sample is then washed with ccl 4 , the suspension is centrifuged at 6 , 000 rpm , and dried in a vacuum to constant weight . more precisely , the preparation of the nanodiamond - glycine conjugate comprises preparing a chlorinated nanodiamond suspension in a polar organic solvent , water - organic solvent , or in water , wherein glycine , as amino acetic acid nh 2 ch 2 cooh , and tertiary amine are then added to said suspension . organic solvents that dissolve glycine , such as pyridine or lower aliphatic alcohols , are preferred . the obtained mixture is treated with ultrasound ( 50 w ) for 5 - 60 min ., and kept at 50 - 80 ° c . with constant stirring for 12 - 48 hrs . the resulting product is washed with ethanol , centrifuged , and the residue is dried in a vacuum overnight at 70 ° c . thus , in the claimed method for the preparation of the nanodiamond - glycine conjugate , the hazardous , complicated , and expensive process of fluorination with fluorine gas is replaced by the readily available , safe , and substantially less expensive process of chlorination in liquid phase , while the expensive glycine derivative , glycine ethyl ester hydrochloride , is replaced by a significantly less expensive amino acid , glycine . the claimed nanodiamond - glycine conjugate dos not contain fluorine atoms , and the dispersion ability of the particles thereof in a suspension is increased 6 - 12 times the obtained nanodiamond - glycine conjugate may be useful in medicine as a system for the delivery of glycine into an organism . for that purpose , electron microscopy is used to study the interaction between the obtained conjugate and the cellular culture by cellular biology methods . a 300 mg sample of nanodiamond is annealed in a gaseous h 2 stream at 3 . 0 l / hr . and 1 , 000 ° c . for 6 hrs . the annealed nanodiamond is then subjected to liquid - phase chlorination with molecular chlorine dissolved in 40 ml of ccl 4 to 6 at % of cl 2 . chlorination is conducted under photochemical exposure to visible light for 60 hours at 60 ° c . the sample is then washed with ccl 4 , the suspension is centrifuged at 6 , 000 rpm and dried under 0 . 1 mm hg to constant weight . the chlorinated nanodiamond is then suspended in 40 ml of water - alcohol mixture ( water : alcohol = 1 : 1 ), combined with 300 mg of glycine as free amino acid nh 2 ch 2 cooh and 1 ml of triethylamine . the resulting mixture is treated with ultrasound ( 50 w ) for 60 minutes and kept at 65 ° c . with constant stirring for 30 hours . the resulting product is washed with large amounts of ethanol , centrifuged , and dried in a vacuum at 70 ° c . overnight . the residual moisture content of the product is 2 . 2 %. the yield of the final product is 279 mg ( 93 %). the obtained product is an ultradisperse powder , dark grey with a bluish tint , with 2 - 10 nm primary particle sizes and a surface layer membrane measuring up to 1 nm ; said product is characterized by ir - spectroscopy : a broad intense band at 3 , 400 cm − 1 , a strong signal at 1 , 621 cm − 1 , six moderately intense bands at 2 , 924 , 2 , 881 , 1 , 383 , 1 , 306 , 1 , 212 , and 1 , 154 cm − 1 , and a weak signal at 504 cm − 1 . the particle size in the suspension of the obtained product is 25 nm . the elemental composition of the surface is shown in table 3 . to determine the mass fraction of glycine in the obtained conjugate , three nanodiamond - glycine mixtures are prepared in a 1 : 1 . 75 : 3 . 5 ratio , respectively . a 0 . 0035 g sample of each mixture is triturated in a mortar with 0 . 09 g kbr . next , 0 . 70 g of the obtained mixture is pressed into a tablet and subjected to ir spectroscopy ( fig7 ). characteristic bands are selected at 1 , 407 , 1 , 332 , and 504 cm − 1 , respectively , and calibration graphs are then plotted therefore ( fig8 ). characteristic band intensities on the ir - spectrum of the obtained nanodiamond - glycine conjugate sample weighing 0 . 0035 g are 0 . 23 , 0 . 22 , and 0 . 10 p . u ., respectively . calibration curves a , b , and c ( fig8 ) are used to determine the glycine content in the sample , which constitutes 0 . 00057 ± 8 · 10 − 5 g of glycine in the sample . the mass fraction of glycine in the sample is therefore 21 ± 3 wt %. ultrasound treatment of the suspension for 30 - 60 minutes , followed by keeping said suspension at 70 - 80 ° c . for 30 - 48 hours , and using pyridine or lower aliphatic alcohols as polar solvents , produce a conjugate with the analogous characteristics and glycine content in the 13 - 21 wt range . ultrasound treatment of the suspension for 5 - 30 minutes , followed by keeping said suspension at 50 - 70 ° c . for 12 - 30 hours , and using water - alcohol mixtures or water as polar solvents produce a conjugate with the analogous characteristics and glycine content in the 2 - 14 wt % range . the obtained nanodiamond - glycine conjugate is used for the delivery of glycine into an organism . the presence of the nanodiamond and glycine conjugate in an organism is confirmed by electron microscopy in the reaction thereof with the lymphoblast molt - 4 cell culture ( fig9 ) after an eight - hour incubation . fig9 demonstrates that the conjugate causes invagination of the lymphoblast &# 39 ; s cellular membrane , which , while gradually penetrating deeper , leads to the absorption of the nanodiamond - glycine conjugate by the cell . cellular sections incubated with the obtained conjugate are prepared on a leica ultracut uct ultramicrotome ( leica , germany ). photomicrographs of cellular sections are prepared on a transmission electron microscope jeol 1011 ( jeol , japan ). 2 . 2 . y . liu , zh . gu , j . l . margrave , v . n . khabashesku . functionalization of nanoscale diamond powder : fluoro -, alkyl -, amino -, and amino acid - nanodiamond derivatives // chem . mater . 2004 . v . 16 . p . p . 3924 - 3930 . 3 . russian encyclopedia of job safety . 3 volumes . 2 nd edition . revised and enlarged edition , v . 3 . m : pub . nts enas . 2007 , p . 181 . 4 . t . i . shalina , l . s . vasilyeva . general issues of the toxic effect of fluorine .// siberian medical journal . 2009 . # 5 , p . p . 5 - 9 . 5 . electron and ion solid - state spectroscopy ./ edited by l . i . firmens et al .— m . 1981 , p . p . 195 - 232 .
the invention relates to the field of pharmaceutics and medicine and concerns a nano - diamond conjugate with glycine for delivering glycine into an organism , the conjugate comprising nano - diamond particles modified by glycine , with a particle size of 2 - 10 nm , and containing up to 21 % by mass of glycine which is included in the composition of the superficial shell of said particles with a thickness of up to 1 nm , and to a method for producing said conjugate .