Patent Application: US-201013387094-A

Abstract:
there is provided an insect model for determining the penetration of nanoparticles through the bbb . the model is also directed to the use of insects for studying the environmental safety of nanoparticles . the particles are administered into the hemolymf of an insect having a bbb , such as the locust , and the uptake into the brain is analysed . thus , in this model it is possible to investigate the potential up - take into the brain of nanoparticles circulating in the blood .

Description:
the present invention provides new methodology for screening for bbb penetration of nanoparticles , which enter the blood stream . the invention is generally useful for medium to high throughput screening for nanoparticles developed in drug discovery programs targeting a variety of diseases and disorders , specifically degenerative disorders , including : parkinson &# 39 ; s disease , alzheimer &# 39 ; s disease , huntington &# 39 ; s disease , diseases with motor neuron inclusions , tauopathies , corticobasal degeneration neuropsychiatric disorders , including : depression bipolar disease , schizophrenia , anxiety , aggression , and brain tumours . a nanoparticle in accordance with the present invention is a particle sized between 1 and 100 nanometers . there are several methods for creating nanoparticles , including both attrition and pyrolysis . in attrition , macro or micro scale particles are ground in a ball mill or other size reducing mechanism . a thermal plasma can also deliver the energy necessary to cause evaporation of small micrometer size particles . inert - gas condensation is frequently used to make nanoparticles from metals with low melting points . the metal is vaporized in a vacuum chamber and then supercooled with an inert gas stream . the supercooled metal vapor condenses into nanometer - sized particles , which can be entrained in the inert gas stream and deposited on a substrate or studied in situ . the present invention relates to but is not restricted to the use of insects selected from the following orders : ( taxonomy according to : djurens värld , ed b . hanström ; förlagshuset norden ab , maölmö , 1964 ): in particular the invention relates to insect species selected from blattoidea , acridoidea , cheleutoptera , brachycera and lepidoptera and most particular to the acridoidea ( locusta migratoria and schistocera gregaria ). the invention will also relate to the following orders comprising insect species relevant for the method of the present invention : the present invention preferably uses large insects , such as the migratoty locust , locusta migratoria and the desert locust , schistocera gregaria or cockroach where it is feasible to feed and inject nanoparticles and subsequently take hemolymph samples and dissect brain tissues , for analyses . the locust has been used to develop screening models to compare this model with existing literature data from conventional in vivo vertebrate studies . in accordance with a preferred embodiment of the present invention the migratoty locust , locusta migratoria and / or the desert locust , schistocera gregaria , is used since it is easy to breed and it is a relatively large insect ( 40 - 60 mm long , weight : approx . 2 g , hemolymph volume : approx . 300 μl , brain weight : approx . 2 mg ). the application of nanoparticles to insects of the present invention in a screening method may be as follows , in accordance with a preferred embodiment of the present invention . in a preferred embodiment of present invention the insects are selected from the order acridoidea and specifically locusta migratoria and schistocera gregaria are used . the insects may be obtained from local suppliers or bred in house . the grasshoppers were reared under crowded conditions at 28 ° c . and a 12 : 12 dark : light photocycle and fed fresh grass and bran . before experiments the grasshoppers were fed ecologically cultivated wheat for two weeks . animals used are adult males ( in some experiments females ) between two to four weeks after adult emergence . nanoparticles are administrated into the haemolymph in a way similar to that described by goldworthy et al . ( 2003 ). for quantitative determination of brain nanoparticle concentration the brains are dissected ( a cut was made through the frontal part of the locust head comprising the most frontal parts including the antennae , the compound eyes , the brain and all neural connections between the brain and the antennae and the eyes ), washed , snap - frozen and stored until analyses . at analysis the brains are homogenised / vortexed and centrifuged . the nanoparticle containing supernatant is analysed for its drug concentration by hplc , lc / msms or other relevant methods . the effect of drug treatment may be documented by recording the pharmacological effects on behaviour or by use of recordings of central nervous system nerve signalling . amine modified fluorescent polystyrene nanoparticles ( 100 nm ) were injected ( 20 ul ) into the hemolymph of locusts ( locusta migratoria ). after 60 minutes hemolymph was harvested and analyzed for uptake of nanoparticles by hemolymph cells . there was a highly selective uptake of the nanoparticles into macrophage like cells and no accumulation in other cell types . it was also found that the particles were accumulated into the lysosomes strongly indicating that the particles were taken up by endocytosis . conclusion : the uptake of nanoparticles into endocytic cells of the locust hemolymph is identical with the in vivo uptake of nanoparticles by vertebrate macrophages ( sadauskas et al ., 2007 ). this observation is important since the clearance of nanoparticles from the circulation by macrophages is very efficient ( about 90 % at 60 minutes after administration in a mouse study ) but also dependent on the shape of the nanoparticle and this clearance mechanism is therefore a determinant of the exposure of the nanoparticle at the brain barrier . silver nanoparticles ( snp ; 80 nm ) were injected into the hemolymph of locusts . after 3 and 24 hours the locust brains were dissected in saline , the neural lamella removed and the brains washed twice in saline and then placed in tubes containing 100 ul nitric acid . the content of ag was analyzed by icp - ms . at 3 hours there was an average uptake of 0 . 58 ng ag per brain and at 24 hours the uptake had increased to 0 . 86 ng ag per brain . average background content of ag in control brains was 0 . 36 ng per brain . a cut was made through the frontal part of the head of female locusts . each brain in its cuticle was placed in a snp ( 80 nm ) solution for 3 hours . the brains were dissected in saline , the neural lamella removed , washed twice in saline and then placed in tubes containing 100 ul nitric acid . the average content of ag per per brain was 4 . 82 ng as measured by icp - ms . a cut was made through the frontal part of the head of female locusts . each brain in its cuticle shell was placed in a snp ( 80 nm ) solution for 3 hours . the brains were dissected in saline , the neural lamella removed , washed twice in saline and then placed in tubes containing 100 ul nitric acid . the average content of ag per per brain was 6 . 40 ng as measured by icp - ms . locust brains were dissected in saline and the neural lamella removed . the brains were placed in a snp ( 80 nm ) solution for 3 hours . the brains were washed twice in saline and then placed in tubes containing 100 ul nitric acid . the average content of ag per per brain was 21 . 7 ng as measured by icp - ms . silver nanoparticles ( snp ; 57 nm ) was injected into the hemolymph of locusts . after 24 hours the locust brains were dissected in saline , the neural lamella removed and the brains exposed to evans blue . there was a marked uptake of evans blue in snp treated locusts whereas there was no uptake in control brains . conclusions : similar to the findings in vertebrates , snp are taken up in vivo by the locust brain and thus pass the locust brain barrier . the observation of evans blue uptake after 24 hours exposure in vivo is similar to that found for snp in a rat model showing deterioration of the bbb integrity 24 hours after i . v administration ( sharma et al ., 2010 ). ex vivo exposure markedly increased brain uptake and the uptake was further increased in the locust brain when exposed to the snp after removal of the neural lamella . a cut was made through the frontal part of the head of female locusts . each brain in its cuticule shell was placed in a solution of amino modified fluorescent polystyrene nanoparticles ( 100 nm ) for 60 min . the brains were dissected in saline , washed twice in saline and then placed in tubes with paraformaldehyde . microscopic analysis of nanoparticle fluorescence in tissue slices showed a marked uptake in the neural lamella . conclusion . this study support the difference found in the snp studies with and without the neural lamella at exposure to the snp solution . locust brains were dissected in saline and the neural lamella was removed . the brains were placed in solutions containing amino modified fluorescent polystyrene ( 100 nm ) nanoparticles and exposed for 3 hours . the brains were then placed in tubes with paraformaldehyde . microscopic analysis of nanoparticle fluorescence in tissue slices showed a marked uptake in the locust brains of the 100 nm amino modified nanoparticles and there was a marked deterioration of the barrier . locust brains were dissected in saline and the neural lamella was removed . the brains were placed in solutions containing amino modified fluorescent polystyrene ( 50 nm ) nanoparticles and exposed for 3 hours . the brains were then placed in tubes with paraformaldehyde . microscopic analysis of nanoparticle fluorescence in tissue slices showed a marked uptake in the locust brain barrier region of the 50 nm amino modified nanoparticles but there was no deterioration of the barrier . locust brains were dissected in saline and the neural lamella was removed . the brains were placed in solutions containing carboxymodified fluorescent polystyrene ( 30 nm ) nanoparticles and exposed for 3 hours . the brains were then placed in tubes with paraformaldehyde . microscopic analysis of nanoparticle fluorescence in tissue slices showed no uptake in the locust brain of the 30 nm carboxy modified nanoparticles . in vivo exposure of various nanoparticles to the locust hemolymph results in the same elimination by endocytic cell uptake and uptake over the brain barrier as in vertebrate models . ex vivo modeling in the locust clearly indicate the utility of the model in studies of brain permeation with reference to nanoparticle size and chemical properties . the observations of effects on brain barrier integrity is of extraordinary importance since the effects of nanoparticles on brain barrier permeation due to functional deterioration is often not fully understood and also the polysorbate 80 coated pbca nanoparticle ( widely used for carrying drugs into the brain ) is suggested to deteriorate the vertebrate bbb ( olivier , 2005 ). banerjee and baht ( 2007 ), neuron - glial interactions in blood - brain barrier formation annual review of neuroscience 30 : 235 - 258 . di , l . and kerns , e . h . 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