Patent Application: US-200913143049-A

Abstract:
the invention is applied in the therapy of brain and spinal cord tumors , degenerative , hypoxic , ischemic diseases and traumatic injuries of the central nervous system and other diseases of humans and animals . object of invention is to provide target delivery of signaling substance to the pathological area of an organism , triggering in due time and long - time support of active signaling intercellular action of specific therapeutic character of a healthy and well controlled stem cell onto controlling system of defective cells . the preparation of sc with reprogrammed cell signaling comprises the basic preparation of sc , the membrane , and / or nucleus , and / or cytoplasm of which contains implanted protein or pharmaceutical able to regulate signaling pathways of sc and cells of pathological focus in a mammal organism , provisionally encapsulated in nanocontainers of less than 100 nm size , obtained from biodegradable material , intact for organelles and compartments of sc of the basic preparation . the material has a set biodegradation period in a mammal organism to provide programmed exit of protein or pharmaceutical in intra - or intercellular space thus reprogramming signal transduction of key genes in the desired therapeutic orientation of physiologic events in the cell cycle directly in the pathological area or tissue of an organism .

Description:
the basic cell preparation was produced on the basis of the standard preparation of hematopoietic sc , their method of production being described in patent ru 2283119 ( mπκ 35 / 14 , . 09 . 2006 ) for “ preparation of autologous hematopoietic stem cells , their method of production , cryopreservation and application for traumatic disease of central nervous system ”. autologous neural stem cells ( nsc ) obtained from olfactory sheath of a patient &# 39 ; s nose can be used for basic preparation . the method and protocol of nsc production are published [ 11 - 14 ]. the tissue of olfactory sheath including olfactory epithelium and the layer of connective tissue ( lamina propria ) is obtained from the patients and processed according to standard cultural protocol described in academic literature [ 15 , 16 ]. size 10 · 5 mm fragments of mucosa dissected under local anesthesia from the upper part of superior nasal meatus were accepted for research . sampled tissue was delivered to laboratory in cool hank &# 39 ; s solution without ca 2 + and mg 2 + ( hbss ) containing antibiotic and antimycotic agents ( 1 : 100 ; gibco ). delivery time was no more than 2 hours . after repeated wash in the same solution blood vessels were removed from mucosa , then the tissue was minced and incubated for 40 minutes at 36 . 5 ° c . in 0 . 25 % trypsin / edta solution prepared on m phosphate buffer ( pbs , ph 7 . 4 ). activity of ferments was blocked by dmem medium ( gibco ) that contained 3 % of serum , tissue was washed three times in new hanks &# 39 ; balanced salt solution ( hbss , sigma ) and dissociated by repeated pipetting in nutritive medium . medium composition : 90 % minimum eagle medium ( mem , sigma ), fetal bovine serum ( fbs , gibco , invitrogen ), 0 . 8 % glucose , 2 mm glutamine ( gibco ), b27 supplement ( sigma ), hepes 20 , growth factors ( only for primary cultures ), fibroblast growth factor ( fgf2 , 1 ng / ml , sigma ), neural growth factor ( ngf 2 ng / ml , sigma ). received cell suspension was centrifuged ( 3 minutes at 1200 revolutions per minute ), the sediment was resuspended in nutritive medium of the same composition . the number and viability of cells were checked in gorjaev &# 39 ; s chamber after 0 . 1 % trypan blue staining . cell suspensions with 85 - 95 % of viable cells were used for further culturing . dissociated cells ( 5 · 10 5 ) cells per ml ) were cultured in 12 - well trays on polylysine / laminine substratum for 14 days ( 36 . 5 ° c ., 5 % co 2 ). partial change of ⅓ nutritive medium was done 2 times a week . primary culture after confluent cell monolayer was removed by trypsin / edta solution . after wash in hbss and centrifuging the cells were resuspended in nutritive medium . cell suspension ( 10 000 - 12 000 cells per cm 2 ) was placed into 12 - well trays or dishes ( square 25 cm 2 ). so the cultures were passed 4 times till confluent monolayer was formed . free floating and attached to substratum neurospheres formed in cell monolayer were selected by pastuer pipette and dissociated by the method of ferment processing described above . this permitted separation of neurospheres from accessory glial cells , fibroblasts and stromal ( foot ) cells . cell suspension of neurospheres after washing and centrifuging was resuspended in culture medium and cultured in 12 - well trays ( 10000 - 12000 cells per cm 2 ) and on cover slides ( 18 · 18 mm ) in petri dishes till confluent monolayer was formed . received cultures were used for cytological and immunocytochemical tests . part of the cells of last passages was frozen in cryopreservation medium ( 90 % serum , 10 % dmso ) and stored in liquid nitrogen . cell monolayer was fixated in 4 % solution of paraformaldehyde prepared on 0 . 01 phosphate buffer ( ph 7 . 4 ) for 30 minutes . after pbs wash ( 3 · 10 min ) cells were incubated for 24 hours at 4 ° c . with primary antibodies to β - tubulin ( 1 : 300 ; chemicon ), nestin ( 1 : 100 , chemicon ) and neural specific enolase ( 1 : 100 , antibodies received in our laboratory ). after pbs wash the cells were successively processed by biotinylated antibodies with avidin - biotin complex ( abc , vector laboratories , inc ), diaminobenzidine solution , prepared on phosphate buffer ( dba 0 . 5 mg / ml , hydrogen dioxide 0 . 03 %). preparations were dehydrated and placed into synthetic resin under cover slides ( entellan , merk ). to avoid graft - versus - host reaction it is necessary to apply only autologous biomaterial , for example nsc isolated from olfactory sheath of the patient &# 39 ; s nose or the preparation of hematopoietic stem cells ( cd34 +) isolated from highly purified mixture of mononuclears isolated from leukoconcentrate of mobilized sc of peripheral blood . the culture of mesenchymal sc isolated from biopsy sample of the patient &# 39 ; s bone marrow can also be applied . however , the opportunity to apply basic preparation of allogeneic sc cannot be completely excluded . signaling proteins or pharmaceutical able to regulate signaling pathways of sc and cells of pathologic site are selected individually in each case depending on the objective of inductive cell therapy . the table below shows the examples of the therapy using available signaling agents ( substances that influence cancer sc or its niche ) that can be used in the claimed invention . considering that apoptosis is the best examined cell process , it was chosen to study new cell preparation with reprogrammable properties on the model of apoptosis . the general scheme of signaling pathways of the formation of apoptosis is shown at fig1 . the scheme demonstrates that apoptosis processes trigger extracellular molecules of signaling proteins of necrosis of factor of tumor ( tnfα ), proteins of death ( fas - l ), insulin growth factor ( igf ). the process can be activated on a cell level affecting caspases and p53 gene . thus , to program inductive instructive apoptosis in brain tumor , the substances participating in signaling chains of apoptosis must be chosen as signaling proteins . vicin and viscumin can serve as the example of such substances . the authors of the claimed invention organized laboratory research to examine cytotoxic action of ricin and viscumin on the cells of c6 neuroglioma . these proteins were demonstrated to be functionally active in relation to the cells of this line . ld50 value made 5 × 10 − 12 mol for ricin and 5 × 10 − 11 mol for viscumin . four specific methods showed that both protein toxins initiated apoptosis of c6 neuroglioma cells . microscopy tests detected nuclei fragmentation and phosphatydil serine exposition at cell membrane . flow cytofluorimeter detected the percentage of cells with fragmented dna being processed with these toxins . electrophoresis showed specific fragmentation of dna characteristic for caspase - 3 - dependant apoptotic endonuclease cad . rapamicin can be mentioned as the example of a pharmaceutical applied in the claimed invention , as the substance to suppress growth of cancer sc and to stimulate growth of normal sc ( see the table ). the authors of the claimed invention developed technical specifications for the production of nanocontainers ( nanocapsules ): the size of nanocapsules must not exceed 100 nm . nanocapsules must hermetically contain signaling agent inside and be covered by biodegradable biopolymer or liposomal coating biologically neutral for the body and have preset period of biodegradation inside the body . nanocapsules must be neutral for all internal cell compartments , and easy in production . biodegradation time of nanocapsules must begin at rigidly set time after administration of sc preparation to a patient . depending on specific conditions and objectives of the therapy the period can vary from one or two hours to several days and months . the nanocapsule ( nanocontainer ) should permit controllable ( programmed ) biodegradation under the influence of the method of ionizing radiation , laser radiation and / or chemical agents , in order to control cell signaling following prior determined algorithm ( program ) depending on the exact conditions and objectives of the therapy . the nanocapsule must biodegrade into safe components for a cell . the nanocapsule ( nanocontainer ) must be contrast for the methods of radiological diagnostics and be easily diagnosed by computer and magnetic resonance tomography . the molecules of signaling substance inside the nanocapsules must fully preserve their potential activity to fully release it after the exit from nanocapsules due to it biodegradation . the nanocapsule must be easily implanted through cytoplasm membrane of a cell by endocytosis , by direct intracellular injection or by physiologic approaches or physical - chemical methods . in certain cases biodegradable material of nanocapsule coating must be able to conjugate with lipids to provide for the nanocapsules the opportunity to attach to plasmatic membrane of sc . the claimed cell preparation with nanocapsules must meet safety criteria and be non - toxic . to produce the nanocapsules biodegradable material must be properly chosen . depending on the tasks of the claimed invention nanocapsule of three types have been chosen to deliver signaling protein to target cells : 1 ) biodegradable nanocapsules produced from biopolymer materials ( co - polymers of polylactid glycolid and co - polymers polyhydroxybutirate - valerate ); in all cases the size of the obtained nanoparticles varied from 10 to 100 nm thus enabling passive diffusion into stem cell or active capture by the cells through pinocytosis , while nanoparticles over 100 nm were distributed only in intercellular space . obtained nanocapsules with molecules of signaling protein inside were placed in plastic 1 . 5 ml tubes and stored at − 12 ° c . till further analysis and use . signaling protein was encapsulated into biopolymer ( polylactide - glycolide or hydroxybutyrate - valerate ) carrier according to the following algorithm : weighed portion of lyophilizated signaling protein preliminarily mixed with fine powder of polylactide ( average particle size 10 - 20 μm ) was loaded into the high pressure chamber with further co 2 ballooning . the chamber temperature was 50 ° c ., nozzle temperature was about 30 ° c . the pressure of supercritical co 2 made 10 mpa . the content of autoclave was thoroughly mixed with magnetic mixer ( 150 revolutions per minute ) for 60 minutes with further output of received mixture and carbon dioxide through the nozzle in suction chamber . after three hours exposure to atmospheric conditions ( requisite for complete co 2 removal and final consolidation of polymer ) gathered particles were dispersed and hydrated till average size of nanocapsules did not exceed 10 nm . ultradispersion was done in t18 basic ultra - turrax disperser . dispersing element was s18n - 19g ( ika - werke gmbh & amp ; co . kg , germany ). preferable methods of production of mixed liposomes from non phospholipid amphiphilic membrane mimetics and phospholipids are based on phase behavior of amphiphilic lipids and phospholipids . the ratio of aqueous solution of amphiphilic membrane mimetic and aqueous solution of phospholipid varied from 5 : 1 to 20 : 1 . mixing temperature makes to 20page at times amphiphilic membrane mimetic and other components have to be warmed in order to obtain homogeneous aqueous solution before adding phospholipids . protein or pharmaceutical of signaling agent was added to the ingredients during mixing . the ingredients were added in certain order , intensively mixed till multilamellar vesicles were obtained . then the particles were minced till single lamellar or small multilamellar liposomes . by atomizers : a ) injection of phospholipid solution at a high speed through one nozzle into water phase of amphiphilic membrane mimetic ; b ) injection of amphiphilic membrane mimetic minimum through one nozzle in water phase of phospholipid ; c ) injection of phospholipid solution at a high speed minimum through one nozzle simultaneously with injection of amphiphilic membrane mimetic solution minimum through one nozzle in a joint box . ultramicroemulsions ( mot more than 10 μm ) were received basing on spontaneously dispersing concentrates of pharmacologically active compound ethers ( ace ), such as undecanoates , laureates , palmitates and stearates . spontaneously dispersing concentrate is diluted by phosphate buffer with signaling protein , and thermodynamically stable microemulsion with 1 . 5 - 3 μm radius micelles is obtained . emulsifiers , stabilizators etc . are added to extend life period of “ water in oil ” emulsion . signaling proteins are in aqueous phase . determining period of biodegradation of nanocapsules in a cell special attention should be given to the type of substance to encapsulate , as well as the method of production of nanocapsules that will permit to program the time of exit of signaling substance from nanocapsule into an autologous sc in rigidly set time . biocompatible properties of the proposed nanocapsules are experimentally proved and confirmed . the nanocontainers ( nanocapsules ) in sc of the basic preparation were implanted by means of exposure of suspension or emulsion of nanocapsules with molecules of signaling protein to the basic preparation of sc for 60 minutes or by culturing of autologous sc with the solution of nanocapsules with signaling protein molecules for 24 hours in standard conditions . moreover implantation of nanocapsules according to the claimed invention implies an opportunity to apply standard methods of gene therapy , such as direct intracellular injections of suspension or emulsion of nanocapsules in membrane , cytoplasm or nucleus of sc of basic preparation , as well as use of nanocapsules conjugates with cholera toxin , cholesterol , polyethylen glycol and asialo mucoprotein , use of cationic lipids with nanocapsules , covalent lypophilic chemicals with nanocapsules , hydrocarbon enhancers of cell permeability and membrane fusion gene components . in certain cases composite nanocapsules were prepared with the coating consisting of biodegradable biopolymer and proteins of the coating of herpetic virus . use of neurotropic proteins of herpes virus for nanocapsule coating imitate pseudoviral capsid . in this case external coating of a nanocapsule can merge with the plasmatic membrane of an autologous sc , hence nanocapsules with molecules of signaling protein easily penetrate sc by active endocytosis . the described methods of encapsulating of protein water soluble preparations in nanocontainers can be also used for superselective transfer of conventional chemotherapeutic agents used for cytotoxic and cytoreductive therapy . the claimed preparation of autologous sc with nanocontainers permits target delivery of nanocontainers with pharmaceutics to the site of tumor through migration mechanisms of homing to the lesion site . the preparation of sc with reprogrammed cell signaling obtained according to the claimed invention can be administered to patients by available methods , for example , by intracerebral , intramedullar , intraventricular or intravascular transplantation depending on the objective of the therapy , as well by implantation into tissue - engineering constructions used to fill the defects of brain and spinal cord . according to the above said the claimed invention can be viewed as new direction in the therapy of tumors , ischemic , degenerative and traumatic injuries of cns and be qualified as highly technological method of cell cytocorrective or cytoregulatory therapy of cns tumors , as well as the therapy of other solid tumors and somatic disorders . the concrete examples of the application of the claimed invention and the methods used at that are brought in the present description only in explicatory aims and do not limit the range and essence of the invention , determined solely by the invention formula . the experts in the field will understand various versions and modifications of the claimed invention that do not go beyond the limits of the invention formula . 1 . bryukhovetskiy a . s . transplantation of neural cells and tissue engineering of brain in nervous diseases .— moscow . : zao klinika neurovita , 2003 . 2 . kobayashi n ., navarro - 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