Patent Application: US-58777505-A

Abstract:
therapeutic drug delivery and diagnostics systems comprise biologically active compounds associated with particulate carriers of less than 20 nm . these systems can be utilised for targeted modification of growth , development and functions , such as gene expression , protein synthesis , intracellular energy production and transport mechanisms in prokaryotic and eukaryotic organisms . the systems are also applicable for controlled modification of structural and functional properties of extracellular components and tissue constituents . the characteristics of a biological site are evaluated and an entity is provided which is dependent on the site characteristics . the entity comprises nanoparticles of less than 20 nm . a probe comprising nanoparticles of less than 5 nm is also provided .

Description:
living cell ( cell )— refers to the self - replicating biological structure enclosed by an outer membrane and containing cytoplasm , organelles and nucleic acids ( i . e . viruses , prokaryotic bacterial cells , protozoa and eukaryotic cells of higher species and multicellular organisms ). drugs — any chemical substances of therapeutic and / or diagnostic application . nanoparticles are nanosized ( between 1 . 0 and 100 nm ) inorganic or organic particles with size dependent physical properties . these may include metal semiconductor , magnetic , organic or inorganic ( e . g . polyhedral silsesquioxane ) polymer nanoparticles . extracellular matrix — refers to the amorphous and fibrillar components of tissues and blood including collagen , laminin , fibronectin , vitronectin , their subtypes and combinations and other components thereof . coagulation components — refers to the entire plurality of factors participating in the process of blood clot formation , whether in soluble or fibrillar form . biologically active compounds — substances which are able to interact with the cells , biological membranes , subcellular components or nuclei and / or are capable of affecting cell or organelle function , proliferation or development as a result of such interactions . ii - vi colloidal quantum dots — are semiconductor nanoparticles of ii - vi compounds prepared as a colloidal solution with size - dependent optical and electronic properties . optical illuminators / emitters — any source of ultraviolet , visible or infrared light and combinations thereof . chemical or physical linking — bond via covalent , noncovalent , hydrophobic , hydrophilic , electrostatic , van der waals , hydrogen bonding , magnetic or electromagnetic interactions . cytoplasmic and nuclear components — refers to the plurality of proteins and protein derivatives ( glycoproteins , nucleoproteins and other complex protein derivatives ), nucleic acids ( dna , rna ), carbohydrates , lipids , glycolipids and other molecular cell constituents . cdte nanocrystals capped with thioglycolic acid used in the experiments were synthesized in aqueous medium as reported earlier ( gaponik et al , 2002 ). briefly , demineralised aqueous solutions containing cd ( clo 4 ) 2 . 6h 2 o and a stabilizer ( thioglycolic acid , tga ) at ph 11 . 8 were treated by h 2 te gas , which was generated by the reaction of al 2 te 3 lumps with 0 . 5 m h 2 so 4 under nitrogen . the mixture of was then heated under reflux under open - air conditions . this method enabled us to prepare good quality cdte nanocrystals with a narrow (& lt ; 10 %) size distribution . variation of the temperature and the duration of the heating during the preparation of cdte nanocrystals determines the final size of the nanocrystals and as a result the colour and luminescence maximum of the solution . thus green ( with photoluminescence maximum at 563 nm ) cdte nanoparticles were produced after 15 min of heating under reflux , while red ( with photoluminescence maximum at 602 nm ) cdte colloid solution were produced after 24 hours of heating . we have utilised water - soluble thioglycolic capped cdte nanoparticles of varying sizes for selective nuclear and nucleolar localisation of green cdte qds and cytoplasmic compartmentalisation of red qds , dependent on size and surface chemistry . cdte nanoparticles showed limited cytotoxicity and proved to be suitable for biological systems as demonstrated by fig1 to 10 . the entity may comprise a stabiliser such as any thiol based organic stabiliser with different functionalities such as carboxylic acids , amines , alcohols , aldehydes , esters , amides , phosphines , alkyl - phosphates , their derivatives or any other functional groups . table 1 lists examples of stabilizers , which may be used particularly with cdte nanoparticles . in the studies referred to in fig1 - 10 , human thp - 1 monocytes , the transformed epithelial cell lines ht - 29 , hct - 116 and t cell lymphoma cell line hut - 78 were obtained from the european collection of animal cell cultures ( ecacc , salisbury , uk ). cells were grown in rpmi 1640 medium supplemented with 10 % heat - inactivated fetal bovine serum ( fbs ), 2 mm l - glutamine / l , 100 μg penicillin / ml and 100 mg streptomycin / ml , and incubated at 37 ° c . in 5 % co 2 . to induce monocyte to macrophage differentiation , thp - 1 cells were cultured in the presence of 100 ng / ml pma for 72 h . live cell imaging was performed in lab - tek chambered coverglass slides ( nunc ). microinjection was carried on an inverted nikon te300 microscope with narishige hydraulic micromanipulation and microinjection equipment and utilising ex tempore fabricated glass capillary microneedles . images were acquired by fluorescence microscopy ( nikon eclipse te 300 ) and on the ultraview live cell imager confocal microscopy workstation ( perkin - elmer life sciences , warrington , uk ) ( nikon eclipse te 2000 - u ). processing and 3 - d image analysis was performed using ultra view lci and volocity - 2 software .). fig1 illustrates intracellular distribution of red - emitting 4 . 4 nm thiol - capped cdte quantum dots in human primary macrophages ( upper and middle panel ) and corresponding phase contrast image ( lower panel ). primary human macrophages were derived from the peripheral blood of healthy volunteers by initial positive selection of monocytes from the mononuclear blood fraction by adhesion to the surface of borosilicate glass chambered coverslips and subsequent maturation over 7 - 14 days in the presence of complete tissue culture medium supplemented with 10 % fetal calf serum and antibiotics . quantum dots were added to the differentiated macrophages at the stage of established cell spreading and incubated in complete culture medium over 1 - 24 hour intervals . macrophages in these conditions did not show signs of excessive cell death even after the longest incubation intervals used thereby indicating suitability of nanoparticles for this type of biological application and limited cytotoxicity . fig1 shows fluorescent images of the macrophages following an 18 hour incubation in the presence of quantum dots taken with a 25 min interval ( upper panel reflect starting point , lower panel — end point ). a significant change in localization of quantum dots over time will be noted which reflects their active intracellular transport . fig2 illustrates phase contrast ( upper ) and confocal ( lower ) images of cdte nanocrystals ( green - emitting 2 . 2 nm and red - emitting 4 . 4 nm sizes , synthesised as described above ) with two distinctive fluorescence spectra microinjected into cultured transformed epithelial cell line ht29 . human colonic epithelial carcinoma cells ht29 were split and seeded after the 4 th passage into the compartments of the 4 - well borosilicate chambered coverslips and allowed to grow to a sub - confluent state . nanoparticles were delivered into the cells via a direct intracytoplasmic injection with an ex tepmore pulled glass microinjection needle ( inner diameter of the injecting tip 0 . 1 - 0 . 15 μm ). two types of quantum dots as described above with distinctive spectral properties were utilized in this experiment : lower left panel , red 4 . 4 nm - size quantum dots ; lower right panel , green - emitting 2 . 2 nm size nanodots . particles readily distribute inside the cells giving a bright fluorescent signal . visualization , 100 × oil immersion objective lens on a nikon eclipse te 300 microscope with leica dc - 100 colour digital camera . fig3 illustrates selective intracellular distribution of green versus red quantum dots in the living phagocytic cells visualized by confocal microscopy . green cdte qds localise in the nucleus of thp - 1 cells while red cdte qds are concentrated in the cytoplasm . differentiated thp - 1 cells were washed three times with hbss prior to their incubation with green or red cdte particles . particles were added in full media ( 2 μl green cdte nanoparticles + 2 μl red cdte nanoparticles into 150 μl cell culture chamber ). cells were analysed after 30 mins . right panel , fluorescence of red qds detected in the red channel ; left panel , fluorescence of green qds detected in the green channel . areas of pronounced quantum dots accumulation in close proximity to the nucleus correspond to the typical location of rough endoplasmic reticulum in mammalian cells . fig4 illustrates red - emitting 4 . 4 nm particles do not undergo nuclear accumulation even if the primary barrier ( cell membrane barrier ) in macrophages is omitted by direct cytoplasmic microinjection of cdte nanoparticles ( fig1 ) thereby suggesting the primary importance of active intracellular transport mechanisms , not necessary directly dependent on the phagocytic activity . lower panel shows green - emitting 2 . 2 nm cdte particles are retained in the nucleus following intranuclear microinjection . nanoparticles were delivered into the cells via a direct injection with an ex tepmore pulled glass microinjection needle ( inner diameter of the injecting tip 0 . 1 - 0 . 15 μm ). fig5 illustrates thapsigargin - induced blockade of intranuclear accumulation of quantum dots in human primary macrophage cells . nuclear import inhibitor thapsigargin ( 100 mm ) was added to the cells for 30 min and subsequently washed out with pre - warmed culture medium prior to incubation with 2 . 2 nm size ( green emitting ) cdte particles . fig6 illustrates brefeldin a - induced dispersion of quantum dots and partial block of intranuclear accumulation in macrophages . prior to incubation with cdte particles thp - 1 differentiated macrophages were incubated with the golgi complex disrupter brefeldin a for 30 min at 20 μg / ml . following a wash out , green 2 . 2 nm size cdte particles were added to the cells . fig7 illustrates accumulation of 2 . 2 nm size green - emitting quantum dots in freshly formed fibrin filaments . left panel , fluorescence in the green channel . right panel , corresponding microscopic field in transmitted light showing two large polymorphonuclear cells ( neutrophils ) and six red blood cells . neutrophils were isolated from peripheral blood by adhesion onto the glass surface in chambered coverslips with subsequent washout of the unbound cells . immediately after the washout , chambers were filled with fresh warm culture medium containing quantum dots and incubated at 37 ° c . for 30 minutes . fibrin filaments start building up in these conditions after 5 - 10 min incubation period with neutrophils serving as primary sites initiating fibrin formation . fig8 illustrates accumulation of aspirin — functionalised siloxane nanoparticles in normal peripheral blood polymorphonuclear cell ( neutrophil ). cells were incubated in the presence of the nanoparticles for 30 mins and subsequently analysed by live cell confocal microscopy . a , upper optical plane ( top of the cell ), b , middle plane showing highlighted segmented nucleus of the polymorphonuclear cell ( arrow ), due to accumulation of fluorescent drug - coupled nanoparticles ; c , lower optical plane ( at the level of cell contact with glass support ). fig9 illustrates the uptake of red cdte nanoparticle in mitochondria in macrophages . red cdte nanoparticle were incubated with monocyte - derived macrophages for 15 mins in culture . live cell confocal microscopy was then performed to examine red cdte nanoparticle localisation . fig1 . illustrates the accumulation of red and green cdte nanoparticles within the elements of the blood coagulation system . fresh blood was allowed to clot for 15 mins at 37 ° c . in glass chambered slides . immediately after clot formation the glass chambers were washed out with warm culture medium containing quantum dots and incubated at 37 ° c . for 15 minutes . the upper panel shows red cdte nanoparticles highlighting groups of platelets while the 2 . 2 nm size green - emitting quantum dots decorate freshly formed fibrin filaments in the middle panel . the lower panel corresponds to the overlay of red green and blue channels , where by blue nuclei in the lower panel are dyed with hoechst nuclear stain . referring to fig1 and 3 , as an example without limitation to the present invention , a nanocarrier - based drug delivery system is used for treatment of an inflammatory condition accompanied by abnormally enhanced functional activity of phagocytic cells . anti - inflammatory drugs are known to modulate macrophage function , but possessing non - specific undesirable side effects for the different cell types , are used in complex with medium - sized nanocarriers ( 3 - 8 nm diameter ), which are subject to active engulfment and uptake by human macrophages . the system can be deployed at the site of inflammation by local application , e . g . direct injection into the inflamed joint . overactive phagocytic cells at the site of inflammation will be exposed to enhanced drug uptake with subsequent moderation and / or resolution of inflammation . phagocytic cells have differential ability to uptake nanoparticles depending on their maturation and cell cycle status thereby increasing the opportunity of selective targeting of cell sub populations . the same principles can be applied to the cells of the non phagocytic lineages . as other examples without limitation to the present invention , the nanodrug system is used for the treatment of inflammatory conditions accompanied by over - activity of polymorphonuclear phagocytes , protozoa - related infections , such as disenteria caused by amoebal parasites of the large intestine and infections caused by bacterial intestinal flora . the nanodrug system of the invention can be composed of the drug conjugated to the nanoparticles of the 5 - 10 nm size . uptake of the drug by the cells , parasites and epithelial cells can be facilitated by active non - specific phagocytosis of the particles , at the same time creating the uptake barrier for the intestinal cells due to the carrier size . for examples of the drug - particle system within microphagocytes and epithelial cells refer to fig1 - 3 . in another preferred embodiment , referring to the fig7 , the nanosize drug delivery system is implemented for treatment of blood coagulation disorders associated with excessive clot formation and thrombosis . fibrin - destabilising drugs coupled to nanoparticles can be delivered intravenously and due to the unique avidity of nanoparticle carriers to build into the biopolymers , the drug is selectively targeted to the intravascular sites of fibrin formation and exerts its fibrinolytic effects . another example of preferred embodiment of the invention without limitation to the one described here referring to fig3 is the regulation of gene expression ( gene therapy ) using the nanosystems for targeted nuclear drug delivery . drug - carrier complexes are applied to the living cells , selectively uptaken into the cytoplasm and subsequently into the nuclei on the basis of their size , charge and surface functionalisation specificity . the nanosystems are bound to the dna or rna in the nucleus and deliver the signal for the selectively targeted gene resulting in altered protein synthesis or changes in cell functional responses . in another example of preferred embodiment referring to fig4 and 5 , nanosystems are used for verification of intracellular drug transport and delivery efficiency . fluorescent quantum dots can be coupled to the drugs under study and brought in contact with the living cells as described in the previous example . the efficiency of the drug delivery and specificity of intracellular distribution is subsequently evaluated by live cell confocal imaging . in yet another preferred embodiment referring to fig8 , salicyl and aspirin - based drug systems can be constructed and delivered into cell . as other example of preferred embodiment fig7 and 10 nanoparticle based drug delivery system may be used to target fibrin filaments . the nanodrug system of the invention can be composed of the drug conjugated to nanoparticles of 2 - 10 nm size , the drug thereby being incorporated into the fibrin clot during its development can selectively modify ( enhance or reduce ) the speed of its formation or composition . yet as other example of preferred embodiment fig9 , nanodrug delivery systems are used to target mitochondria . selective targeting of such systems to mitochondria can be utilised for modifying cell functions e . g . targeting of both , small drug molecules and large macromolecules to and into mitochondria may provide the basis for a large variety of future cytoprotective and cytotoxic therapies : the delivery of therapeutic dna and rna such as antisense oligonucleotides , ribozymes , plasmid dna expressing mitochondrial encoded genes as well as wild - type mtdna may provide the basis for treatment of mitochondrial dna diseases ; the targeting of antioxidants into the mitochondrial matrix may protect mitochondria from oxidative stress caused by a variety of insults , perhaps even contribute to slowing down the natural aging process ; the mitochondria - specific targeting of naturally occurring toxins or synthetic drugs such as photosensitizers may open up avenues for new anticancer therapies . in another preferred embodiment delivering molecules known to trigger apoptosis by directly acting on mitochondria may overcome the apoptosis - resistance of many cancer cells and drugs able to target mitochondrial uncoupling proteins may become a basis for treating obesity . in another example of preferred embodiment of nanodrug systems can be used to modify the conductivity or speed of electrochemical signal transduction mediators , ( ions , synaptic vesicles , etc .) or polarisation events in nerve cells , cardiomyocytes , and other signal transducting cells . in this embodiment the semiconducting nature of the nanosystems can be exploited to alter and measure and manipulate the status of cell potential ( relevant to the ratio between the cell membrane and cytosol ). consequently , a number of cell functions dependent on this parameter can be selectively targeted , inhibited or restored in case of a pre existing damage . the nanosized drug delivery systems of the invention may include individual drugs or complex mixtures thereof . the compounds of therapeutic nature which have the potential and are anticipated to be used in such systems include but not limited to anti - inflammatory compounds such as aspirin , ibuprofen , and naproxen , mobic , celebrex , disease - modifying anti - rheumatic drugs ( dmards , methotrexate and sulphasalazine , anti - malarials ( hydroxychloroquine ), d - penicillamine , azathioprine and gold salts , transcription modulating drugs such as thiazolidinediones , tamoxifen , anti cancer , anti bacterial drugs and antibiotics . structures of salycyl - and aspirin - functionalised nanoparticles are presented in scheme 1 . to prepare these nanoparticles an appropriate salycyl - or aspirin - containing precursor was synthesised first . the salicylamidopropyltriethoxysilane precursor was made by mixing 3 - aminopropyltriethoxysilane with ethyl salicylate under argon and heating at ca . 120 ° c . for at least 24 hours to ensure complete migration of the salicylyl group from the ester to the amide ( scheme 2 ). this precursor was used for the preparation of the correspondent siloxane nanoparticles and functionalisation of cdte and magnetite nanoparticles . in a similar manner , salycyl can be linked to cysteamine or to any appropriate compound containing amine functionality to give correspondent precursors for capping of nanoparticles . the aspirin containing precursor was made by reaction of aspirin chloride with cysteamine ( scheme 3 ). in a similar manner aspirin can be linked to any appropriate compound containing amine functionality to give precursors for capping of nanoparticles . 3 - salicylamidopropyltriethoxysilane or a corresponding aspirin precursor was hydrolysed at room temperature in thf with h 2 o . the white precipitate was dispersed in water to form a colloidal suspension , which is suitable for experiments performed in aqueous phase . salycyl - and aspirin - conjugated cdte nanodrug systems have been prepared similarly to the procedures above using an appropriate thiol precursor salycyl - and aspirin - functionalised magnetite nanoparticles have been prepared by the addition of 3 - salicylamidotriethoxysilane or correspondent aspirin precursor to a suspension of fe 3 o 4 nanoparticles ( size 9 - 11 nm ) in thf . this was followed by the addition of degassed , deionised water , and the reaction mixture was left stirring vigorously at room temperature for 12 hours . the precipitate of functionalised magnetite nanocrystals was washed with thf and then dispersed in water using ultrasound . then the samples were suitable for testing in cell culture systems . uv - vis absorption spectra of the colloidal solutions of nanocrystals were measured using a shimadzu uv - 3101 pc spectrometer and the photoluminescence ( pl ) spectra were recorded using a spex fluorolog spectrometer equipped with a r943 hamamatsu photomultiplier . the optical density of all samples was kept the same and below 0 . 1 at the first absorption feature of the nanocrystals for a 1 - cm path length . other non limiting examples of nanoparticles which can be used in relation to the invention may comprise semi conductor nanoparticles ii - vi semiconductor nanoparticles : zno , zns , znse , znte , cds , cdse , cdte , hgs , hgse , hgte . iii - v semiconductor nanoparticles : aln , alp , alas , alsb , gan , gap , gaas , gasb , inn , inp , inas , insb . other possible nanoparticles include sio 2 ( silica ), any transition metal oxide ( e . g . tio 2 , zro 2 , hfo 2 , moo 2 , fe 2 o 3 , fe 3 o 4 , co 3 o 4 , ferrites ), siloxane nanoparticles , dendrimers ( dendritic polymers ) and organic polymer nanoparticles . two aqueous colloidal solutions of cdte nanocrystals of 2 - 5 nm mean size were used for studies in biological systems . the entity to be delivered to a target site may be indirectly linked to a nanoparticle using an organic linker . such a linker may be organic group , which can serve to link the stabiliser , drug or biomolecule to the nanoparticle surface such as : alkyl chain e . g . (— ch 2 —) n , polyethyleneglycole e . g . (— ch 2 — o — ch 2 —) n , peptide e . g . 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