Patent Application: US-201314087727-A

Abstract:
the present invention considers derivatized nanomolecules with proven effectiveness to bind to actinides , more specifically uranium , during in vivo , ex vivo , in vitro or in situ assays . when assayed in vivo , the invention showed a reduction in at least kidney damage due to exposition to uranium .

Description:
as previously stated , the present invention corresponds to different derivatized nanomolecules with proven effectiveness for in vivo removal of heavy metals or actinides , more specifically uranium or depleted uranium . more particularly , the derivatized nanomolecules of the present invention correspond to derivatized polyamidoamine ( pamam ) dendrimers of different generations . in a more particular embodiment of the invention , the derivatized pamam dendrimers are of generations 2nd , 3rd , 4th , 5th , or 6th . in an alternative embodiment , the derivatized nanomolecules are half - generation polyamidoamine ( pamam ) dendrimers , i . e . 0 . 5 , 1 . 5 , 2 . 5 , 3 . 5 , 4 . 5 generation dendrimers . in a more preferred embodiment , the derivatized nanomolecules of the invention are polyamidoamine ( pamam ) dendrimers of generations 4 and 5 . in a particular embodiment of the invention , the derivatization of the nanomolecules comprises a derivatization group . in a more specific embodiment of the invention , the derivatization group corresponds to an amino acid conjugated with a terminal functional group . in a more particular embodiment of the invention , the aminoacid is a basic aminoacid , such as histidine , lysine , or arginine . in a particular embodiment of the invention , the terminal functional group conjugated to the amino acid is a carbamate or a tosyl group . in a more specific embodiment , the carbamate group is a carboxybenzyl group . the present invention comprises pharmaceutical compositions comprising at least one of the dendrimers considered in the present invention , a suitable solvate , suitable salts , and / or excipients . the molecules of the invention ( i . e . active ingredient ) can be included in different dosage forms , such as for example , and with no intention of limiting the scope of the invention , oral dosage forms , such as pills , films , tablets , capsules , dragees , pastes , powders , liquid solutions or suspensions ; parenteral dosage forms , suitable for intradermal , intramuscular , intraosseous , intraperitoneal , intravenous , subcutaneous , or intrathecal administration ; topical dosage forms such as creams , gels , liniments , balms , lotions , ointments , skin patches . in a particular embodiment of the invention , the dosage for reaching an effective removal of heavy metals or actinides , more particularly uranium or depleted uranium , from a patient , correspond to a dosage of at least 5 mg / kg , at least 7 mg / kg , at least 10 mg / kg , at least 15 mg / kg , at least 20 mg / kg , at least 25 mg / kg , at least 30 mg / kg . the present invention further encompasses the use of the molecules described herein in the treatment or alleviation of damages that may be caused by heavy metal toxicity , actinide derived toxicity , and more particularly , toxicity caused by exposure to uranium or depleted uranium . in one aspect of the invention , a pharmaceutical composition comprising the molecules of the invention is injected in a patient in need thereof . in one embodiment of the invention , the injection or application of the molecules of the invention for providing in vivo chelating of heavy metals or actinides , more specifically uranium or depleted uranium , from a patient in need thereof is achieved when reaching a concentration of at least 1 μm , more preferentially at least 3 μm , and even more preferentially at least 5 μm . other higher concentrations are also considered to be in the scope of the present invention , such as up to 10 μm , or up to 15 μm . in a further embodiment , the molecules of the invention can be immobilized in a suitable matrix for extracorporeal removal of heavy metals , such as for example through dialysis , or for ex vivo removal of the heavy metals . a further embodiment of the invention considers the use of the disclosed compounds immobilized in a suitable matrix for removal of actinides or heavy metals from liquid solutions . for example , the compounds of the invention can be immobilized in a cartridge for the treatment of a body of water , wherein the water is passed through the matrix and the heavy metals , actinides , or more specifically uranium or depleted uranium , are bound to the compounds of the invention and retained in the matrix . another embodiment of the invention considers the application of the molecules described herein to contaminated environment , i . e . in situ treatment or removal of heavy metals , more particularly actinides , and more specifically uranium or depleted uranium . pamam g4 - arginine - tos - oh ( g4 - arg - tos ). conjugation of pamam - g4 dendrimer ( dendritech , midland , mich .) with boc - arginine ( tos )- oh ( sigma aldrich co . saint - louis , mo .) was carried out by a condensation between the carboxyl group of the arginine and the primary amino group of pamam . thus , 64 mg ( 0 . 208 mmoles ) of boc - arginine ( tos )- oh reacted with 150 mg ( 2 . 9 mmoles ) of edc and 150 mg of hobt in a mixture of 2 . 7 ml of dry dmf and 1 . 0 ml of dry dmso under a nitrogen atmosphere for 1 hr . the reaction mixture was added dropwise to a solution of 40 mg ( 1 . 38 × 10 − 3 mmoles ) of pamam g4 in 3 ml of water . the reaction mixture was vigorously stirred for 72 hrs . the functionalized dendrimer was purified through dialysis membranes with a cut - off of 500 da to take off the excess of the amino acids . then , the product was lyophilized and the amount of the pamam g4 - boc - arginine ( tos )- oh obtained was 55 mg . finally , a solution of hcl / dioxane ( 4 ml , 4 m ) in a 25 ml round - bottom flask equipped with a magnetic stirrer was cooled by an ice - water bath under nitrogen and pamam g4 - boc - arginine ( tos )- oh ( 0 . 2 mmol ) was added in one portion with stirring . the ice - bath was removed and the mixture was kept stirred for 1 hr ., thing layer chromatography ( tlc ) indicated that the reaction was completed . the reaction mixture was condensed by rotary evaporation under high vacuum at room temperature . the residue was then washed with dry ethyl ether and collected by filtration ( for oil products , a simple decantation was used instead ). yield : & gt ; 95 % of pamam g4 - arg - tos pamam g4 - lysine - fmoc - cbz ( g4 - lys - fmoc - cbz ). conjugation of pamam - g4 dendrimer with fmoc - cbz - lysine was carried out according to the previously reported method ( geraldo et al ., pisal et al ., 2008 ). briefly , 123 mg ( 0 . 24 mmoles ) of fmoc - cbz - lysine reacted with 38 mg ( 0 . 24 mmoles ) of edc and 33 mg of hobt ( 0 . 24 mmoles ) in a mixture of 2 . 9 ml of dry dmf and 1 . 0 ml of dry dmso under a nitrogen atmosphere for 1 hour . the reaction mixture was added dropwise to a solution of 50 mg ( 3 . 5 × 10 − 3 mmoles ) of pamam g4 in 5 ml of water . the reaction mixture was vigorously stirred for 72 hrs . the functionalized dendrimer was purified through dialysis membranes with a cut - off of 500 da to take off the excess of amino acids . after lyophilization , the amount of the pamam - lys - fmoc was 73 mg . pamam g4 - lysine - cbz - oh ( g4 - lys - cbz ). conjugation of pamam - g4 dendrimer with boc - lys - cbz - oh was carried out according to the previously reported method ( geraldo et al ., pisal et al ., 2008 ). briefly , 56 mg ( 0 . 208 mmoles ) of boc - lys - cbz - oh reacted with 150 mg ( 2 . 9 mmoles ) of edc and 150 mg of hobt in a mixture of 2 . 7 ml of dry dmf and 1 . 0 ml of dry dmso under a nitrogen atmosphere for 1 hr . the reaction mixture was added dropwise to a solution of 30 mg ( 2 . 1 × 10 − 3 mmoles ) of pamam in 3 ml of water . the reaction mixture was vigorously stirred for 72 hrs . the functionalized dendrimer was purified through dialysis membranes with a cut - off of 500 da to take off the excess of the amino acids . after the lyophilization , the amount of the pamam g4 - boc - lysine - cbz - oh obtained was 42 mg . finally , a solution of hcl / dioxane ( 4 ml , 4 m ) in a 25 ml round - bottom flask equipped with a magnetic stirrer was cooled by an ice - water bath under nitrogen , and pamam g4 - boc - lysine - cbz - oh ( 0 . 2 mmol ) was added in one portion with stirring . the ice - bath was removed and the mixture was kept stirred for 1 hr ., tlc indicated that the reaction was completed . the reaction mixture was condensed by rotary evaporation under high vacuum at room temperature . the residue was then washed with dry ethyl ether and collected by filtration ( for oil products , a simple decantation was used instead ). yield : & gt ; 95 % of pamam g4 - lys - cbz . pamam - g4 - folate ( g5 - fa ). conjugation of pamam - g5 dendrimer ( dendritech , midland , mich .) with folic acid ( fa ) ( sigma ) was carried out by a condensation between the γ - carboxyl group of fa and the primary amino group of pamam . thus , 104 mg ( 0 . 23 mmoles ) of fa reacted with 150 mg ( 1 . 28 mmoles ) of 1 -[ 3 -( dimethylamino ) propyl ]- 3 - ethylcarbodi - imide hcl ( edc ) ( sigma aldrich co . saint - louis , mo .) and 150 mg of n - hydroxybenzotriazole ( hobt ) ( sigma aldrich co . saint - louis , mo . ), in a mixture of 2 . 7 ml of dry n - dimethylformamide ( dmf ) ( sigma aldrich co . saint - louis , mo . ), and 1 . 0 ml of dry dimethyl sulfoxide ( dmso ), ( sigma aldrich co . saint - louis , mo .) under a nitrogen atmosphere for 1 hour . the reaction mixture was added dropwise to a solution of 40 mg ( 1 . 38 × 10 − 3 mmoles ) of pamam g5 in 3 ml of water . the reaction mixture was vigorously stirred for 72 hrs . the functionalized dendrimer was purified through dialysis ( using water ) membranes with a cut - off of 500 da ( spectrum laboratories , inc ., rancho dominguez , calif .) to take off the excess of folate . after the lyophilization ( freezone 6 , labconco , usa ), the amount of the pamam g5 - fa obtained was 52 mg . pamam - g5 - coumarin ( g5 - cou ). conjugation of pamam - g5 dendrimer with coumarin - 3 - carboxylic acid ( cou ) ( sigma aldrich co . saint - louis , mo .) was carried out by a condensation between the carboxyl group of cou and the primary amino group of pamam . thus , 39 . 5 mg ( 0 . 208 mmoles ) of cou reacted with 32 mg ( 0 . 208 mmoles ) of edc in a mixture of 2 . 7 ml of dry dmf and 1 . 0 ml of dry dmso under a nitrogen atmosphere for 1 hr . the reaction mixture was added dropwise to a solution of 40 mg ( 1 . 38 × 10 − 3 mmoles ) of pamam g5 in 3 ml of water . the reaction mixture was vigorously stirred for 72 hrs . the functionalized dendrimer was purified through dialysis ( using water ) membranes with a cut - off of 500 da to take off the excess of cou . after the lyophilization , the amount of the pamam g5 - cou obtained was 55 mg . maldi analysis . to confirm the molecular weight of surface modified dendrimers , mass spectral analysis of the dendrimers was performed on a maldi - tof ( matrix assisted laser desorption / ionization - time of flight ) ( bruker , usa ) with a pulsed nitrogen laser ( 337 nm ), operating in positive ion reflector mode , using 19 kv acceleration voltage and a matrix of 2 , 5 dihydroxybenzoic acid ( dhb ) ( sigma aldrich co . saint - louis , mo .). affinity assay . for each dendrimer , batch experiments were carried out to determine the extent of binding ( extb ) and fractional binding ( fbin ) of u ( vi ) in aqueous solutions . the u ( vi ) concentration was kept constant at 10 ppm ( 3 . 7 × 10 − 5 m ) in all experiments . aliquots of u ( vi ), dendrimer stock solution or deionized water were added to each tube to prepare 3 ml solutions with given u ( vi )- dendrimer molar ratio . the sealed centrifuge tubes were mixed on a rotary shaker for 120 min . then , the solution was subsequently withdrawn from each equilibrated tube and transferred into a millipore centricon filter with a molecular weight cut - off of 5000 dalton . the filters were centrifuged for 20 min at 3000 rpm to separate de uranyl - laden dendrimers from the aqueous solutions . the concentrations of uranyl in each centrifuge tube ( u 0 ) were measured by fluorescence spectrophotometry . following ( gu et al ., 2005 ), each sample was diluted 2 - fold with a 10 % wt of a h 3 po 4 solution . the complexation of u ( vi ) ions with phosphoric acid causes a large enhancement of their fluorescence emission intensity . this provides the basis of a uranyl assay method with a detection limit of aprox . 40 ppb ( gu et al ., 2005 , diallo et al ., 2008 ). all fluorescence emission spectra were collected on a steady - state rf - 5301pc spectrofluorophotometer ( shimadzu ) using an excitation wavelenght of 280 nm . the emission spectra were recorded between 480 and 545 nm . the intensity of the emission peak at 508 nm was used to develop the u ( vi ) calibration curve . the concentration of uranyl bound to a dendrimer ( u b ) ( mol / l ) was expressed as follows : where u c is the uranyl concentration of the control ( uranyl without dendrimer ) after the incubation and centrifugation . the extent of binding ( extb ) ( moles of u ( vi ) bound per mole of dendrimer ), the concentration of dendrimer ( cden ) in solution ( mol / l ) and the fractional binding ( fbin ) were expressed as follows : where m d ( g ) is the mass of dendrimer in solution , v s ( l ) is the solution volume and m wd ( g / mol ) is the molar mass of the dendrimer . sample collection . blood from healthy donors was extracted and anticoagulated with heparine tubes ( bd vacutainer , becton dickinson ). erythrocytes were separated from the plasma and leucocytes by centrifugation ( 1500 rpm , 5 min ) at 4 ° c . and washed three times with saline ( nacl 0 . 9 %) ( sigma aldrich co . saint - louis , mo .). all the protocols were authorized by the ethic committee of the university of talca in accordance with the declaration of helsinki ( approved by the 18th world medical assembly in helsinki , finland , in 1964 ). assay of red blood cells lysis . the hemolysis assay was performed according to the method of duncan et al ., ( duncan r ., 1992 ). briefly , washed rbcs at 2 % were incubated at room temperature with a concentration of 4 μm of the selected dendrimer . after 2 hours of incubation , samples were centrifuged at 2000 rpm for 10 minutes and the absorbance of the supernatant was measured at 550 nm ( clima plus , ral s . a , barcelona ). hemolysis was expressed as percentage of released hemoglobin , used as control ( 100 % of hemoglobin released ) a solution of rbcs incubated with triton x - 100 ( 0 . 2 % v / v , sigma aldrich co . saint - louis , mo .). animals . a total of 48 male c57bl / 6 mice weighing 25 - 30 g purchased from the instituto de salud pública ( isp ), were used . animal housing and experiment protocols were approved by the university of talca in adherence to conicyt experimental animal administrative regulations . all groups were maintained at a constant temperature ( 22 ± 1 ° c .) on a 12 - h light / 12 - h dark cycle ( lights - on at 08 : 00 a . m .) and had free access to food and water . animal experimental design . eight groups of study were designed ; the first group ( n = 6 ) was designated as the control group ( saline , ip ), the second group ( n = 6 ) for uranyl acute intoxication ( uo 2 ( ch 3 coo ) 2 . 2h 2 o , 5 mg / kg of uranium , ip ), the third group ( n = 6 ) for g4 - lys - fmoc - cbz ( 16 mg / kg , ip ), the fourth group ( n = 6 ) for g5 - cou ( 40 mg / kg , ip ), the fifth group ( n = 6 ) for ethylenediaminetetraacetic acid ( edta , 75 mg / kg , ip ), the sixth group ( n = 6 ) for g4 - lys - fmoc - cbz ( 16 mg / kg , ip ) immediately after u ( vi ) administration ( 5 mg / kg , ip ), the seventh group ( n = 6 ) for g5 - cou ( 40 mg / kg ) immediately after u ( vi ) administration ( 5 mg / kg , ip ) and the eighth group ( n = 6 ) for edta ( 75 mg / kg , ip ) immediately after u ( vi ) administration ( 5 mg / kg , ip ). animals were euthanized 48 h later and blood samples were collected from the heart for the assessment of serum urea , creatinine , lactate deshidrogenase ( ldh ), uric acid and calcium examination of nephrotoxicity . mice were anaesthetized with sodium pentobarbitone and blood samples were collected from the heart and the kidneys were removed . serum levels of urea , creatinine , ldh , uric acid and calcium were determined using commercially available kits according to the protocols provided by valtek ( valtek diagnostic , nuñoa , chile ). left kidneys were fixed in 10 % neutral buffered formaldehyde , embedded in paraffin wax and automatically processed . sections ( 3 μm in thickness ) of the embedded tissue were stained with hematoxylin - eosin for observation under light microscope . statistical analysis . the in vitro data were obtained from at least three independent experiments with three replicates , and all data are expressed as mean ± sd . experimental groups were compared using a one - way analysis of variance ( anova ) followed by scheffé &# 39 ; s test when the data were normally distributed and by the kruskal - wallis test when they were not normally distributed . p - values & lt ; 0 . 05 were considered significant . statistical tests were performed using graphpad prism 5 software , version 5 . 0 for mac os x . lysine , arginine and folic acid were coupled to the amine terminals of the pamam g4 dendrimers by edc and hobt coupling reaction ( geraldo et al .). a higher molar ratio of 1 : 70 was used to get all the 64 - amine groups of the pamam g4 dendrimer conjugated . nevertheless , only 16 arg - tos , 39 lys - cbz , 2 lys - fmoc - cbz and 23 fa molecules were found attached to the pamam g4 dendrimer , respectively . the difference in the number of molecules coupled to pamam g4 was due perhaps to the steric hindrance caused by the size of the molecules . to avoid dimerization and to increase the degree of conjugation , boc synthesis protocol was used . the modification of the dendrimers surface were characterized by maldi - tof spectroscopic analysis ( fig1 ). coumarin - 3 - carboxylic acid was coupled to the amine terminals of the pamam g5 dendrimers by edc and hobt coupling reaction ( geraldo et al .). a higher molar ratio of 1 : 140 was used to get all the 128 - amine groups of the pamam g5 dendrimer conjugated . however , only 64 cou molecules were found attached to the pamam g5 dendrimer . the difference in the number of molecules coupled to pamam g5 was due perhaps to steric hindrance caused by the size of the coumarin . all dendrimers synthesized had a yield over 90 %, the characteristics of each of them are listed in table 1 . each of the dendrimers synthesized was incubated with a solution of u ( vi ) ( 10 ppm ) for 2 hr ., at the end of the incubation time , the solution was centrifuged and the concentration of the uranyl unbound to the dendrimer was quantified by spectrofluorimetry . in this regard , the commercial g4 and g5 dendrimers showed a fbin rate of 93 % and 86 %, respectively , at a dendrimer concentration of 20 μm . the fbin was considerably reduced by lowering the concentration of the dendrimer in the solution ( 4 μm ), showing a binding percentage of 41 % and 63 % respectively ( fig2 ). for its part , dendrimers g4 - arg - tos , g4 - fa and g4 - lys - cbz , at a concentration of 20 μm , showed a lower fbin that the commercial dendrimers , with percentages of 62 , 65 and 77 %, respectively . however , dendrimers g4 - lys - fmoc - cbz and g5 - cou showed high percentage of trapping , reaching about 90 % of fbin at a concentration of 4 μm . also , the dendrimers g4 - lys - fmoc - cbz and g5 - cou , showed the higher extent of binding ( table 2 ), with 35 mol of u ( vi ) bound per mol of each dendrimer . the fig3 shows the hemolysis of rbcs expressed as percentage of released hemoglobin compared to the positive control triton x - 100 ( 0 . 2 %, v / v ). these findings indicate that pamam dendrimers g4 - fa and g4 - lys - fmoc - cbz ( final concentration of 4 μm in saline ) have the highest percentage of hemolysis ( 10 . 2 % and 8 . 1 %, respectively ). moreover , fig4 shows the differences between negative control and the different dendrimers tested . it can be observed that the commercial dendrimers pamam g4 and g5 cause intense agglutination of rbcs , whereas pamam g4 derivatives have no greater interaction with the rbcs , however , the pamam g5 derivatives afforded moderate agglutination of the rbcs . as shown in table 3 , after u ( vi ) administration of 5 mg / kg , the concentration of urea and uric acid increased significantly ( p & lt ; 0 . 001 ) in all groups exposed to u ( vi ), compared to control ( saline ), at the same time , none of the used dendrimers or edta caused changes in these biochemical parameters . on the other hand , when analyzing concentration of creatinine , an increase in the levels of all groups exposed to u ( vi ) ( p & lt ; 0 . 001 ) is observed , nevertheless , the concentration of creatinine in g4 - lys - fmoc - cbz - uo 2 − group was significantly lower ( p & lt ; 0 . 05 ) than positive control ( uo 2 − ) ( 1 . 2 ± 0 . 17 versus 1 . 7 ± 0 . 5 , respectively ), said effect was not observed in group g5 - cou - uo 2 − ( p & gt ; 0 . 05 , fig5 a ). regarding measurement of ldh , a wide spectre toxicity indicator , it was observed that u ( vi ) induced a significant increase of the enzyme ( p & lt ; 0 . 05 ), nevertheless , the use of different dendrimers and edta decreases said generalized toxicity , even more , g4 - lys - fmoc - cbz dendrimer was able to keep the levels in normal range obtaining a significant difference to the positive control ( uo 2 − ) and the group g4 - lys - fmoc - cbz - uo 2 − ( p & lt ; 0 . 001 ), as shown in fig5 b . using hematoxylin - eosin staining of kidneys obtained from mice from different studied groups , optical microscopy allowed to see that the control group ( saline ), preserves the structure of renal tubules and glomeruli , and on the other hand , the positive control ( uo 2 − ), showed irreversible damage with necrosis , also showing karyolysis and total eosinophilia of cytoplasm together with tubule dilation with atrophy or “ thyroidization ”. pamam g4 - lys - fmoc - cbz and g5 - cou controls , together with edta - na did not show differences compared to the positive control , standing out that low interaction with red cells and hemolysis , they also did not cause damage or toxicity ( reflected as ldh activity ) in experimental animals . nevertheless , when using these dendrimers together with u ( vi ), in the case of acute intoxication ( 5 mg / kg ), only pamam g4 - lys - fmoc - cbz dendrimer was able to protect the kidney from damage produced by this metal ( fig6 ), since the case of pamam g5 - cou dendrimer + uo 2 − and edta - na + uo 2 − , irreversible damage with necrosis and in some cases karyolysis and total eosinophilia , and thyroidization was observed , although , when observing the tissue of the group treated with pamam g4 - lys - fmoc - cbz + uo 2 − , some damage was observed but it was reversible with a slight increase of eosinophilia in cytoplasm and luminal surface vesiculations . the global distribution of uranium ( u ) contamination has remained a persistent environmental and human health problem for several decades . u is a naturally occurring radioactive heavy metal derived from the earth &# 39 ; s crust and is composed of three naturally occurring isotopes , u 234 , u 235 , and u 238 . du is a waste product of the u enrichment process wherein the more highly radioactive isotopes , u 234 and u 235 , are removed from natural u , leaving a waste material that is largely but not completely “ depleted ” of these isotopes with higher specific activities . the specific activity of du is about 60 % that of natural u because of this isotopic difference ( army environmental policy institute ( aepi ). 1995 ), but it retains the chemical toxicity of natural u as a heavy metal ( the royal society ., 2001 , society ., 2002 ). the kidney has long been recognized as the “ critical ” target of u exposure , i . e ., the organ first perturbed ( leggett , 1989 ), and is considered the primary target organ following both acute and chronic exposures to soluble u compounds ( mcdiarmid et al ., 2001 , parkhurst , 2003 ). animal evidence also documents other targets of u exposure , including the bone , reproductive , and central nervous systems ( gilman et al ., 1998 , mcdiarmid et al .). extensive experience demonstrates that acute and chronic human intoxications with a wide range of metals can be treated with considerable efficiency by the administration of a relevant chelating agent ( flora et al ., 2008 ). thus , a chelating agent forming a stable complex with a toxic metal may shield the metal ion from biological targets , thereby reducing the toxicity , even at times after administration where mobilization has not yet occurred ( andersen , 1999 ). on this context we synthesized derivatives of pamam g4 and g5 and their chelating properties and hemocompatibility were studied . in case of commercial dendrimers g4 and g5 , these showed a good capacity of trapping the uranyl ion , nevertheless , when incubated with rbcs , these dendrimers caused a high agglutination of these cells . similar results were obtained by wang et al . ( wang et al . ), when the pamam g4 dendrimer was examined as a nanocarrier candidate for gene delivery . low doses of pamam g4 dendrimer ( 10 nm - 10 μm ˜ 141 . 3 ng / ml - 141 . 3 μg / ml ) caused rbc aggregation and shape changes , from echinocytic , spindle - shaped to spherocyte - like forms , and when the concentration increased to 100 μm (˜ 1 . 41 mg / ml ), pamam g4 induced membrane rupture and disintegration ( ziemba et al .). however , dendrimers g4 - arg - tos and g4 - lys - cbz , showed a percentage entrapment of uranyl ion less than 10 %. despite this , both dendrimers showed low toxicity , with a percentage of hemolisys lower than 10 %, at a concentration of 4 μm without agglutination of red blood cells , so that , despite not be good candidates as chelating agent , positions itself as good dendrimers for biological applications because it does not affect the rbcs membranes or cause agglutination thereof . from the series of dendrimers synthesized g4 - lys - fmoc - cbz and g5 - cou , had a higher percentage of trapping even that the commercial dendrimers ( g4 and g5 ), with a percentage of trapping of 81 . 7 and 95 . 4 % each one , at a concentration of 0 . 5 μm ( 7 . 55 μg / ml y 20 . 78 μg / ml , respectively ), determining that each dendrimer ( g4 - lys - fmoc - cbz and g5 - cou ) is capable of fixing 35 mol of uranyl per mol of dendrimer in solution . also , both dendrimer g4 - lys - fmoc - cbz and the g5 - cou , do not cause hemolysis or agglutination of rbcs at a concentration of 4 μm and the uranyl ion trapping continues performing well . finally , from the series of dendrimers synthesized , only g4 - lys - fmoc - cbz and g5 - cou , at a concentration of 4 μm , have a high capacity of uranyl ion trapping without causing hemolysis or rbcs agglutination , which makes them good candidates for in vivo studies aimed to obtaining a chelating agent which can be used in case of acute poisoning by uranium . andersen , o ( 1999 ) principles and recent developments in chelation treatment of metal intoxication . chem rev 99 : 2683 - 2710 army environmental policy institute ( aepi ). 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