Patent Application: US-200913059950-A

Abstract:
an application of monoolein - based cationic lipid systems can be used for the complexation , transport and transfection of genetic material into target cells . the lipid systems can be prepared through the conjugation of the neutral lipid monooleoyl - rac glycerol with the cationic tensioactives derived from dioctadecyldimethylammonium as , for example , dioctadecyldimethylammonium bromide , dioctadecyldimethylammonium chloride and dioctadecyldimethylammonium phosphate . the lipofection method can present high levels of biocompatibility and reduced acquisition costs , and is applicable for molecular biology purposes , as well as in the health area in gene therapy .

Description:
being the cationic lipid / monoolein molar ratios applicable between the range of 0 . 1 to 0 . 9 , an application example of the invention consists in the use of a lipid formulation with dodab / monoolein molar ratio 2 : 1 and charge ratios (+/−) 2 . 0 and 4 . 0 , corresponding to different concentrations of the positive ammonium headgroups ( dodab ) for the same concentration of negative phosphate units of the plasmid dna . the relative efficiency of the systems ( in terms of their transfection effectiveness and citotoxicity level ) was determined by direct comparison with the lipofectamin ® commercial system , in the conditions proposed by the manufacturer ( invitrogen ). the transfection efficiency was determined through the measuring of the β - galactosidase reporter gene activity ( absorbance at 420 nm ) ( fig4 ). the citotoxicity level was determined through the measuring of the lactate dehydrogenase activity ( absorbance at 340 nm ) ( fig5 ). in the presented example , the use of two different charge ratios (+/−) ( 2 . 0 and 4 . 0 ) for the dodab : mo ( 2 : 1 ) system was made with the purpose of determining the minimum lipid concentration that could be used to obtain the maximum transfection effectiveness without citotoxicity . for the tested formulation , although it was expected that charge ratio (+/−) 4 . 0 presented higher transfection efficiency than charge ratio (+/−) 2 . 0 because it had twice the concentration of the lipofection agent , that was not experimentally observed ( fig4 ), probably due to the citotoxicity increase at 48 h ( 5 % to 7 % cell mortality , respectively at charge ratios (+/−) 2 . 0 and 4 . 0 ) ( fig5 ). it must also be referred that the commercial system ( lipofectamin ) presents a higher toxicity level ( 7 % cell mortality ) than the dodab : mo ( 2 : 1 ) system at charge ratio (+/−) 2 . 0 ( 5 % mortality ). nevertheless , the demonstration that dodab : mo ( 2 : 1 ) formulation at distinct charge ratios (+/−) present comparable transfection efficiencies ( c . r . (+/−) 2 . 0 ) or even superior ( c . r . (+/−) 2 . 0 ) to the commercial system , indicate that this model of invention is effective at variable concentrations of lipid and dna . this versatility can reveal itself useful in the application of this lipid system in other cell lines where other charge ratios (+/−) and transfection conditions are more productive to increase the process efficiency . for preparing the mixed liposome solutions , defined volumes from the stock solutions of dodab and mo in ethanol ( 20 mm ) were injected under vigorous vortexing to an aqueous buffer solution at 70 ° c . ( 30 mm tris - hcl ), so that the final lipid concentration ([ dodab more mo ]) was 1 mm and the cationic lipid ( dodab ): helper lipid ( mo ) molar ratio of 2 : 1 was obtained . the cationic lipoplexes were prepared through the addition of defined volumes of the mixed liposome solutions ( variable concentrations of positive ammonium groups ) to a constant volume of plasmid dna solution ( concentration of negative phosphate units = number of wells × 1 μg dna / well ). different volumes of opti - mem i reduced serum medium ( gibco ) were used to complete a total constant volume of solution in the several formulations and charge ratios (+/−) tested ( 100 μl / 19 mm well ). the resulting lipoplexes were incubated at room temperature during 30 min without agitation . the genetic material used was the psv - β - gal ( invitrogen ) plasmid , which was amplified by escherichia coli dhb4 competent cell line . the extraction and purification of the plasmid dna was made using the “ wizard ® plus midipreps dna purification system ” extraction kit , commercialized by promega . the final phosphate dna concentration ( 1 . 75 μg / μl ) was measured through to absorbance at 260 nm in a shimadzu uv - 3101 - pc spectrophotometer . for transfection and citotoxicity determination assays , the 293 t cell line was used and cells were cultivated in t75 cm 3 flasks with dmem medium supplemented with l - glutamin ( 4 mm ), sodium bicarbonate ( 1 . 5 g / l ), glucose ( 4 . 5 g / l ), fetal bovine serum ( 10 % v / v ) and antibiotics ( 1 uni / ml ). the cells were regularly subcultivated between passage numbers 18 and 24 , being always kept at confluences lower than 90 % ( 37 ° c . and 5 % co 2 ). twelve to sixteen hours prior to transfection , each t75 cm 3 flask was washed once with pbs ( 1 ×) solution and the cells were released through the addition of trypsin solution ( 0 . 05 %) ( 3 minutes incubation at 37 ° c . and 5 % co 2 ). the number of viable cells was quantified in a hemocytometer through the trypan blue exclusion method . cells were resuspended in culture medium at a density of 2 × 10 5 cells / 19 mm well , and were transferred to 24 - multiwell plates at a final volume of 500 μl / 19 mm well ( approximately 50 - 80 % confluence twelve hours later ). in the day of transfection , the culture medium was replaced by fresh medium , and the recently prepared lipoplexes ( see section “ preparation of cationic lipoplexes ) were homogeneously added to each well , in a quantity that did not exceed 20 % of the final volume of the solution ( 1 μg dna / well ). the cells were then once more incubated at 37 ° c . and 5 % co 2 , for a 24 h - 48 h period . the transfection efficiency was determined through the determination of β - galactosidase activity , the reporter gene used . the citotoxicity level was determined by measuring the activity of the lactate dehydrogenase . in order to determine the transfection efficiency , recently incubated cells ( 24 h - 48 h ) were washed twice with pbs ( 1 ×) solution and disrupted with lysis buffer solution ( rlb 1 ×) for 15 minutes . the cells were then recollected and centrifuged for 2 minutes at 14000 rpm to remove cellular debris . β - galactosidase enzyme activity was determined in the several samples ( in arbitrary units of absorbance , a . u .) with the “ β - galactosidase enzyme assay system with reporter lysis buffer ” expression kit , accordingly with the manufacturer instructions ( 1 μl of cellular extract + 49 μl of rlb ( 1 ×) solution + 50 μl of assay ( 2 ×) buffer solution + 150 μl of sodium carbonate solution , to stop the colorimetric reaction ). the absorbance reading was performed at 420 nm in a 96 multi - well plate with a spectramax 340pc microplate reader . for determination of the citotoxicity level in the analyzed systems , the intra and extracellular amounts of the lactate dehydrogenase enzyme were quantified . the collection of the culture medium in each well allowed a posterior measurement of the extracellular ldh . to obtain the value of intracellular ldh , the wells were washed with 500 μl of tris - hcl buffer solution ( 15 mm ), followed by mechanic scraping and ultrasound lysis of the cell samples for 3 periods of 30 seg sonication . all samples ( intra and extracellular ) were centrifuged for 1 minute at 13000 rpm to remove cellular debris . the enzymatic activity of the intracellular ldh was determined [ 10 μl of intracellular sample + 250 μl of nadh ( 0 . 31 mm ) solution + 10 μl of pyruvate ( 8 . 96 mm ) solution to stop the colorimetric reaction ]. the absorbance reading was performed at 340 nm in a 96 multi - well plate with a spectramax 340pc microplate reader . the enzymatic activity of the extracellular ldh was determined [ 40 μl of extracellular sample + 250 μl of nadh ( 0 . 31 mm ) solution + 10 μl of pyruvate ( 8 . 96 mm ) solution to stop the colorimetric reaction ]. the absorbance reading was performed at 340 nm in a 96 multi - well plate with a spectramax 340pc microplate reader . the balance between the enzymatic activities of the intra and extracellular ldh allowed the determination of the survival and mortality percentages . m . a . maslov , e . i . syicheva , n . g . morozova , g . a . serebrennikova , ( 2002 ), “ cationic amphiphiles of both lipid and non - lipid nature in gene therapy ”, ( 2000 ), russian chemical bulletin , v . 49 , n . 3 , pp . 385 - 401 . g . gregoriadis , ( 2007 ), “ liposome technology — ii — entrapment of drugs and other materials into liposomes ”, 3rd edition , editions i . healthcare , new york ( usa ), pp . 424 . p . l . felgner , t . r . gadek , m . holm , r . roman , h . w . chan , m . wenz , j . p . northrop , r . m . ringold , m . danielson , ( 1987 ), “ lipofection : a highly efficient , lipid - 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