Patent Application: US-201314389067-A

Abstract:
there is provided a method and model insects for screening the effects of nanoparticles on brain barrier function and integrity . the method involves exposing the insect brain - barrier to the nanoparticle of interest and exposing the nanoparticle treated insect brain barrier to one or more suitable marker of the function and integrity of the brain barrier .

Description:
the present invention provides a new methodology for screening the effects of nanoparticles on brain barrier function and integrity . since the use of nps has increased tremendously during the recent years and the nps are widely spread also in the environment and there are great concerns for the potential effects on the function of living organisms as well as human beings . certain nps are taken up by the various barriers ( e . g . lungs , intestinal mucosa or skin ) and / or permeate the barriers including the brain barrier . therefore , it is of utmost importance to identify nps that are taken up by the brain barrier and affect the function and integrity of the barrier and provide experimental models for assessing the safety of the nps . the present invention provide an insect model that is generally useful for investigating the safety profile of nps including nps developed in drug discovery programs targeting a variety of diseases and disorders . in preferred embodiments the nps of the present invention is less than 100 nm , such as less than 50 nm diameter . there are several methods for creating nps , including both attrition and pyrolysis . in attrition , macro or micro scale particles are ground in a ball mill or other size reducing mechanism . thermal plasma can also deliver the energy necessary to cause evaporation of small micrometer size particles . inert - gas condensation is frequently used to make nps from metals with low melting points . the metal is vaporized in a vacuum chamber and then super cooled with an inert gas stream . the super cooled 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 a b , maömlö , 1964 ): in particular the invention relates to insect species selected from blattodea , acridoidea , cheleutoptera , brachycera , bombine , apine and lepidoptera and most particular to the acridoidea ( locusta migratoria and schistocerca 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 , schistocerca gregaria or cockroach where it is feasible to feed and inject drugs and subsequently take hemolymph samples and dissect brain tissues , for analyses . the locust has been used to develop screening models to determine nps effect on blood - brain barrier function and integrity . in accordance with a preferred embodiment of the present invention the migratoty locust , locusta migratoria and / or the desert locust , schistocerca 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 exposure of nanoparticles to the insect brain barriers 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 the present invention the insects are selected from the order acridoidea and specifically locusta migratoria and schistocerca gregaria are used . the insects may be obtained from professional breeders . the insects were reared under crowded conditions at 28 °- 38 ° and a 12 : 12 dark : light photo cycle . animals used are adult males or females between one to six weeks after adult emergence or a maximal period of 9 weeks or during the whole life span of the grasshopper . after various times of exposure to the coated or non - coated nps the effects of the nps on brain barrier function and integrity is determined by using functional marker molecules . preferably the brains are homogenized or disintegrated by ultra sound or other methods in order to obtain a homogenate reflecting the composition of the brains . the homogenate is centrifuged and the supernatant stored until analysis . the selective molecular functional markers are analyzed by liquid chromatography with mass spectrometric detection of the eluted compounds , by icp - ms . alternatively , the nps effect on the barrier function and integrity is obtained by using fluorescent functional markers . the presence of these markers in the brain are identified and / or quantified in brain slices by using fluorescence microscopy or by fluorometry . in a preferred embodiment of the present invention the insects are selected from the order acridoidea and specifically locusta migratoria and schistocerca gregaria are used . the insects may be obtained from professional breeders . animals used are adult males or females between one to six weeks after adult emergence or a maximal period of 9 weeks or during the whole life span of the grasshopper . after various times after in vivo administration ( oral , tracheal or intrahemolymphic ) the effects of the coated or non - coated nps on brain barrier function and integrity is determined by using functional marker molecules injected into the hemolymph at end of the exposure period . the selective molecular functional markers are analyzed by liquid chromatography with mass spectrometric detection of the eluted compounds , by icp - ms or are determined by using fluorescent molecule markers which are analyzed in brain slices by fluorescence microscopy or by fluorometry . in a preferred embodiment of the present invention the insects are selected from the order acridoidea and specifically locusta migratoria and schistocerca gregaria are used . the insects may be obtained from professional breeders . animals used are adult males or females between one to six weeks after adult emergence or a maximal period of 9 weeks or during the whole life span of the grasshopper . after various times after in vivo administration ( oral , tracheal or intrahemolymphic ) the insect brains are dissected out . the effects of the coated or non - coated nps on brain barrier function and integrity is determined by using functional marker molecules , which are exposed ex vivo to the dissected insect brains . the selective molecular functional markers are analyzed by liquid chromatography with mass spectrometric detection of the eluted compounds , by icp - ms or are determined by using fluorescent molecule markers which are analyzed in brain slices by fluorescence microscopy or by fluorometry . in a preferred embodiment of the present invention the insects are selected from the order acridoidea and specifically locusta migratoria and schistocerca gregaria are used . the insects may be obtained from professional breeders . animals used are adult males or females between one to six weeks after adult emergence or a maximal period of 9 weeks . a cut is 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 . the brain is dissected out and placed in a well of a microtitre plate containing the coated or non - coated np . after various times of exposure the brain is washed in cold insect buffer and the neural lamella surrounding the brain is removed . the effects of the nps on brain barrier function and integrity is determined by using functional marker molecules added to the incubation well at end of the exposure period . the selective molecular functional markers are analyzed by liquid chromatography with mass spectrometric detection of the eluted compounds , by icp - ms or are determined by using fluorescent molecule markers which are analyzed in brain slices by fluorescence microscopy or by fluorometry . in a preferred embodiment of the present invention the insects are selected from the order acridoidea and specifically locusta migratoria and schistocerca gregaria are used . the insects may be obtained from professional breeders . animals used are adult males or females between one to six weeks after adult emergence or a maximal period of 9 weeks . a cut is 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 . the brain is dissected out , the neural lamella removed and this preparation is placed in a well of a microtitre plate containing the coated or non - coated np . after various times of exposure the brain is washed in cold insect buffer . the effects of the nps on brain barrier function and integrity is determined by using functional marker molecules added to the incubation well at end of the exposure period . the selective molecular functional markers are analyzed by liquid chromatography with mass spectrometric detection of the eluted compounds , by icp - ms or are determined by using fluorescent molecule markers which are analyzed in brain slices by fluorescence microscopy or by fluorometry . locust brains were dissected in insect buffer , placed in solutions containing fluorescent polystyrene amino modified 100 nm nps and exposed for 3 hours . the brains were washed in cold insect buffer , the neural lamella removed and the brains were then exposed to evans blue for one minute and then analyzed for dye uptake . there was no significant uptake of evans blue in the locust brain barrier . locust brains were dissected in insect buffer and the neural lamella removed . the brains were placed in solutions containing fluorescent polystyrene amino modified 100 nm nps and exposed for 3 hours . the brains were washed in cold insect buffer and then exposed to evans blue for one minute and then analyzed for dye uptake . there was a marked uptake of evans blue in the locust brain barrier . locust brains were dissected in insect buffer , placed in solutions containing fluorescent polystyrene amino modified 50 nm nps and exposed for 3 hours . the brains were washed in cold insect buffer , the neural lamella removed and the brains were then exposed to evans blue for one minute and then analyzed for dye uptake . there was no uptake of evans blue in the locust brain barrier . locust brains were dissected in insect buffer and the neural lamella removed . the brains were placed in solutions containing fluorescent polystyrene amino modified 50 nm nps and exposed for 3 hours . the brains were washed in cold insect buffer and then exposed ex vivo to evans blue for one minute and then analyzed for dye uptake . there was no uptake of evans blue in the locust brain barrier . locust brains were dissected in insect buffer , placed in solutions containing bsa coated silver nps ( 80 nm ) and exposed for 3 hours . the brains were washed in cold insect buffer , the neural lamella removed and the brains were then exposed to evans blue for one minute and then analyzed for dye uptake . there was a highly significant uptake of evans blue in the locust brain barrier . locust brains were dissected in insect buffer and the neural lamella removed . the brains were placed in solutions containing bsa coated silver nps ( 80 nm ) and exposed for 3 hours . the brains were washed in cold insect buffer and then exposed ex vivo to evans blue for one minute and then analyzed for dye uptake . there was a highly significant uptake of evans blue in the locust brain barrier . polystyrene nps ( 100 nm ) were injected into the hemolymph of locusts . after 24 hours the locust brains were dissected in insect buffer , the neural lamella removed and the brains washed in cold insect buffer . the brains were treated with evans blue for 1 minute and then analyzed for dye uptake . there was no uptake of evans blue in the locust brain barrier . polystyrene nps ( 50 nm ) were injected into the hemolymph of locusts . after 24 hours the locust brains were dissected in insect buffer , the neural lamella removed and the brains washed in cold insect buffer . the brains were treated with evans blue for 1 minute and then analyzed for dye uptake . there was no uptake of evans blue in the locust brain barrier . silver nps ( 57 nm ) were injected into the hemolymph of locusts . after 24 hours the locust brains were dissected in insect buffer , the neural lamella removed and the brains washed in cold insect buffer . the brains were treated with evans blue for 1 minute and then analyzed for dye uptake . there was a marked uptake of evans blue in the locust brain barrier . conclusion : the ex vivo locust model clearly shows that different types of nps differently affect the function of the locust brain barrier . the ex vivo model , without the neural lamella , differentiate the polystyrene nps depending on size which may be related to a size dependent induction of particle incorporation of the barrier cells . silver nps affected the barrier function irrespective of the absence or presence of the neural lamella . the in vivo model also showed discrimination between the nps . silver nps affected the barrier function whereas there were no effects of polystyrene nps . abbott , n . j . 2005 dynamics of cns barriers : evolution , differentiation , and modulation . cell mol neurobiol 25 : 5 - 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