TR202009223A2 - GENETIC MOLECULAR ENCAPSULATION IN MESO-POORED SILICA / POLYETHYLENE GLYCOL HYBRID STRUCTURE - Google Patents
GENETIC MOLECULAR ENCAPSULATION IN MESO-POORED SILICA / POLYETHYLENE GLYCOL HYBRID STRUCTURE Download PDFInfo
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- TR202009223A2 TR202009223A2 TR2020/09223A TR202009223A TR202009223A2 TR 202009223 A2 TR202009223 A2 TR 202009223A2 TR 2020/09223 A TR2020/09223 A TR 2020/09223A TR 202009223 A TR202009223 A TR 202009223A TR 202009223 A2 TR202009223 A2 TR 202009223A2
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- silica
- polyethylene glycol
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 title claims abstract description 115
- 229920001223 polyethylene glycol Polymers 0.000 title claims abstract description 95
- 239000002202 Polyethylene glycol Substances 0.000 title claims abstract description 84
- 239000000377 silicon dioxide Substances 0.000 title claims abstract description 56
- 238000005538 encapsulation Methods 0.000 title claims abstract description 32
- 230000002068 genetic effect Effects 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 52
- 239000000243 solution Substances 0.000 claims abstract description 47
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 30
- 229920002873 Polyethylenimine Polymers 0.000 claims abstract description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 20
- 239000000463 material Substances 0.000 claims abstract description 19
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- 108091034117 Oligonucleotide Proteins 0.000 claims description 61
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- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical group CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims description 23
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 14
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- 230000007062 hydrolysis Effects 0.000 claims description 12
- 238000006460 hydrolysis reaction Methods 0.000 claims description 12
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 9
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical group [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 7
- 102000053602 DNA Human genes 0.000 claims description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 7
- 125000002091 cationic group Chemical group 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 6
- 108020004459 Small interfering RNA Proteins 0.000 claims description 5
- LFQCEHFDDXELDD-UHFFFAOYSA-N tetramethyl orthosilicate Chemical compound CO[Si](OC)(OC)OC LFQCEHFDDXELDD-UHFFFAOYSA-N 0.000 claims description 5
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 4
- 229910052910 alkali metal silicate Inorganic materials 0.000 claims description 4
- 102000040650 (ribonucleotides)n+m Human genes 0.000 claims description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 3
- 239000000908 ammonium hydroxide Substances 0.000 claims description 3
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- IXCSERBJSXMMFS-UHFFFAOYSA-N hcl hcl Chemical group Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 claims 1
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- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
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- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
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- 108010024636 Glutathione Proteins 0.000 description 2
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 2
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- 150000007513 acids Chemical class 0.000 description 2
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
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- 229960001259 diclofenac Drugs 0.000 description 2
- DCOPUUMXTXDBNB-UHFFFAOYSA-N diclofenac Chemical compound OC(=O)CC1=CC=CC=C1NC1=C(Cl)C=CC=C1Cl DCOPUUMXTXDBNB-UHFFFAOYSA-N 0.000 description 2
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- 229960005466 diclofenac diethylammonium Drugs 0.000 description 1
- KPHWPUGNDIVLNH-UHFFFAOYSA-M diclofenac sodium Chemical compound [Na+].[O-]C(=O)CC1=CC=CC=C1NC1=C(Cl)C=CC=C1Cl KPHWPUGNDIVLNH-UHFFFAOYSA-M 0.000 description 1
- KPUWHANPEXNPJT-UHFFFAOYSA-N disiloxane Chemical class [SiH3]O[SiH3] KPUWHANPEXNPJT-UHFFFAOYSA-N 0.000 description 1
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- 229910001950 potassium oxide Inorganic materials 0.000 description 1
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- RMAQACBXLXPBSY-UHFFFAOYSA-N silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 235000015424 sodium Nutrition 0.000 description 1
- GUGNSJAORJLKGP-UHFFFAOYSA-K sodium 8-methoxypyrene-1,3,6-trisulfonate Chemical compound [Na+].[Na+].[Na+].C1=C2C(OC)=CC(S([O-])(=O)=O)=C(C=C3)C2=C2C3=C(S([O-])(=O)=O)C=C(S([O-])(=O)=O)C2=C1 GUGNSJAORJLKGP-UHFFFAOYSA-K 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
- A61K9/143—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with inorganic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
- A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B33/00—Silicon; Compounds thereof
- C01B33/113—Silicon oxides; Hydrates thereof
- C01B33/12—Silica; Hydrates thereof, e.g. lepidoic silicic acid
- C01B33/18—Preparation of finely divided silica neither in sol nor in gel form; After-treatment thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
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Abstract
Buluş genetik molekülün koruyucu bir malzeme içinde taşınması için bir enkapsülasyon yöntemi olup, en genel haliyle, bir silika kaynağına ve polietilen glikol kaynağına alkol eklenerek karıştırılması ve iki ayrı çözeltinin elde edilmesi, silika çözeltisine, polietilen glikol çözeltisinin eklenerek karıştırılması ve çözeltinin hidrolize edilmesi için su ilave edilmesi, kondenzasyon sırasında, jelleşmenin hemen öncesinde, oluşmakta olan amorf yapının gözenekleri içinde taşınması için, hidrolize olan çözeltiye kendi tampon çözeltisi içinde olan genetik molekülün eklenmesi veya hidrolize olan çözeltinin kendi tampon çözeltisi içinde olan genetik moleküle eklenmesi ve sonra jel elde edilmesi, jele kurutma/yaşlandırma yapılarak kuru jelin elde edilmesi ve toz haline getirilmesi işlem basamaklarını içermektedir. Bu yöntemle elde edilen içinde genetik molekül taşıyan mezo- gözenekli silika/polietilen glikol ve silika/polietilen glikol/polietilen imin hibrit malzeme de buluşun kapsamındadır.The invention is an encapsulation method for carrying the genetic molecule in a protective material, in its most general form, by adding alcohol to a source of silica and polyethylene glycol to obtain two separate solutions, mixing the silica solution by adding the polyethylene glycol solution, and adding water to hydrolyze the solution. during condensation, just before gelation, in order to transport the amorphous structure in its pores, adding the genetic molecule in its own buffer solution to the hydrolyzed solution or adding the hydrolyzed solution to the genetic molecule in its own buffer solution, and then obtaining a gel, drying the gel / It includes the process steps of obtaining the dry gel by aging and making it into powder. Mesoporous silica / polyethylene glycol and silica / polyethylene glycol / polyethylene imine hybrid material obtained by this method are also within the scope of the invention.
Description
TARIFNAME MEZO-GÖZENEKLI SILIKA/POLIETILEN GLIKOL HIBRIT YAPI IÇINDE GENETIK MOLEKÜL ENKAPSÜLASYONU Bulusun Konusu Bulus, meze-gözenekli silika/polietilen glikol hibrit yapi içinde çift Zincirli oligonükleotit gibi genetik moleküllerin enkapsülasyonu için gelistirilen bir yöntemi konu almaktadir. Gelistirilen yöntem ile oligonükleotitlerin parçacik içinde tasinarak kan dolasiminda enzim/protein degradasyonundan korunmasi ve tek seferde ve kontrollü salinabileoek oligonükleotit dozunun arttirilmasi saglanmaktadir. DESCRIPTION MESO-POROUS SILICA/POLYETHYLENE GLYCOL IN HYBRID STRUCTURE GENETIC MOLECULE ENCAPSULATION Subject of the Invention The invention is a meso-porous silica/polyethylene glycol hybrid structure. genetics such as double-stranded oligonucleotide A method developed for the encapsulation of molecules is on the subject. With the developed method, oligonucleotides enzyme/protein in the blood circulation by being carried in the particle protection from degradation and in a single and controlled increasing the dose of salinable additional oligonucleotide is provided.
Teknigin Bilinen Durumu Teknikte mezo-gözenekli silika (MP8), genellikle biyolojik/tip uygulamalarinda nem tutma, kromatografik molekül ayrimi gibi çesitli amaçlar için kullanilmaktadir. State of the Art In the art, mesoporous silica (MP8) is usually moisture retention in biological/type applications, chromatographic It is used for various purposes such as molecular separation.
Bunun yani sira sol-jel yöntemiyle silika içinde molekül enkapsülasyonu ile ilgili de birçok çalisma bulunmaktadir. In addition, the molecule in silica using the sol-gel method. There are also many studies on encapsulation.
Yapilan çalismalar genellikle biyosensör uygulamalarina yönelik olmakla birlikte nükleik asit, protein, enzim, bakteri ve hatta hücre enkapsülasyonlarini içermektedir. Studies are generally applied to biosensor applications. Nucleic acid, protein, enzyme, bacteria and even cell encapsulations.
Sol-jel yönteminde genellikle organik kaynaklardan baslayarak, ortam, sicakliginda amorf(kristalin yapida olmayan) silika (Si-O-Si) yapisi olusturulur. Basitçe organik silika kaynagi (örn. TEOS: tetraetil ortosilikat, Si(OC2Hm4) alkol/su ortaminda çözünür ve hidrolize olur (sol Hidroliz sonrasinda olusan Si-OH gruplari baglanarak Si-O-Si amorf yapisini meydana getirir (kondenzasyon). Kondenzasyon ilerledikçe tüm yapi içinde mezo-gözenekler (2-50nm) ESi-OH + HO-SiE a ESi-O-SiE + HzO (Su kondenzasyonu, Su/TEOS ESi-OCýh + HO-SiE a ESi-O-SiE + CýkOH (Alkol kondenzasyonu, Hidroliz ve kondenzasyon reaksiyonlari sirasiyla asit ve baz katalizörler kullanilarak hizlandirilabilir. Genellikle, hidrolizle baslayan reaksiyon zincirinde katalizör çesidi ve miktarina bagli olarak, bir süre geçtikten sonra hidroliz ve kondenzasyon birlikte devam eder. Tüm açik Si-OH gruplari baglanarak Si-O-Si agi olustugunda jellesme gerçeklesir ve gözle görülebilir. Elde edilen jel oda sicakliginda bir süre bekletildiginde (aging: yaslandirma) içeride hapsolan su/alkol buharlasir ve jel kurur. Kuruyan jel parçalanarak Silika parçacik halinde kullanilabilir. Ayrica, jellesme öncesinde - sol formunda iken- cam Vb. yüzeylere sürüldügünde son ürün kaplama olarak da kullanilabilir. In the sol-gel method, it is generally obtained from organic sources. Beginning, the ambient temperature is amorphous (crystalline in nature) non-) silica (Si-O-Si) structure is formed. Simply organic silica source (eg TEOS: tetraethyl orthosilicate, Si(OC2Hm4) dissolves and hydrolyzes in alcohol/water (left Si-O-Si by binding Si-OH groups formed after hydrolysis It creates an amorphous structure (condensation). condensation as it progresses, mesopores (2-50nm) in the entire structure ESi-OH + HO-SiE a ESi-O-SiE + HzO (Water condensation, Water/TEOS ESi-OCýh + HO-SiE a ESi-O-SiE + CýkOH (Alcohol condensation, Acid and base, hydrolysis and condensation reactions, respectively can be accelerated using catalysts. Generally, catalyst type in the reaction chain starting with hydrolysis and Depending on the amount of hydrolysis and condensation continues. All open Si-OH groups When a Si-O-Si network is formed by bonding, gelling occurs and visible to the naked eye. The gel obtained was at room temperature for a while. trapped inside when kept (aging) the water/alcohol evaporates and the gel dries. The dried gel crumbles Silica can be used in the form of particles. Also, gelling before - while in left form- glass Etc. to surfaces When applied, the final product can also be used as a coating.
Son yillarda, RNA interferans yönteminin kanser gibi tedavisi zor ve genetik hastaliklarin tedavisi için tasidigi potansiyelin farkedilmesiyle MP8 parçaciklarinin genetik molekül tasiniminda kullanimi yayginlasmistir. Hasta hücrelerin genetik transkripsiyonunu durdurabilecek DNA/RNA zincirleri çiplak. halde hücrelere gönderildiginde on bes dakika içinde sindirilmektedir. Bu nedenle hedef hücrelere ulasana dek koruyucu bir malzeme(vektör) içinde tasinmalari gerekir. Vektör malzemenin biyouyumlu olmasi ve gen salimi yapabilmesinin yani sira kan dolasiminda tasinirken protein/enzim bozunumuna ugramamasi, hücre zarindan geçebilmesi ve endozomdan kaçabilmesi gereklidir. Yüzeyleri fonksiyonlandirildigindai MP5 bu gerekliliklerinr çogunu karsilamaktadir. Bu parametreler MPS parçacik yüzeylerinin polimerlerle modifikasyonunu gerektirmektedir. Fakat, polimer ve diger fonksiyonlandiriçi gruplarin yüzeyde birlikte kullanimi zamanla daha da kompleks hale gelmekte ve yüzey gözeneklerinden oligonükleotit salimi yapan vektör performansini olumsuz etkilemektedir. In recent years, RNA interference method has been used as cancer. for the treatment of difficult-to-treat and genetic diseases genetics of MP8 particles with the realization of the potential Its use in molecule transport has become widespread. Patient DNA/RNA that can stop the genetic transcription of cells chains are bare. fifteen when sent to cells digested within minutes. Therefore, target cells to be transported in a protective material (vector) until they reach must. Biocompatibility and gene release of vector material while being transported in the bloodstream, as well as being able to inability to degrade protein/enzyme, from cell membrane must be able to pass through and escape the endosome. surfaces When functional, the MP5 fulfills most of these requirements. meets. These parameters are equivalent to MPS particle surfaces. requires modification with polymers. But, polymer and other functional groups on the surface use together becomes more complex over time and vector that releases oligonucleotides from surface pores negatively affect its performance.
MP8 içinde biyomolekül enkapsülasyonuna dair yapilmis çalismalar bulunmaktadir. Bu çalismalar genel olarak farkli silika kaynaklarinin enzim/protein/ilaç enkapsülasyonu için kullanimini içermekte ve deneysel sonuçlar vererek enkapsülasyonun verimini, ilaç ve moleküllerin salinimini göstermektedir. Studies on biomolecule encapsulation in MP8 there are studies. These studies are generally different for enzyme/protein/drug encapsulation of silica sources includes the use of and giving experimental results efficiency of encapsulation, release of drugs and molecules shows.
Teknigin bilinen durumuna örnek olarak US6495352B1 sayili patent dokümani verilebilir. Bahsedilen doküman, organik moleküllerin ve özellikle biyomoleküllerin enkapsülasyonu için gelistirilen, sol-jel yöntemine dayali bir yöntemi konu almaktadir. Söz konusu yöntemde ilk olarak, su içerisinde silisyum dioksit (silika:Si02) ve sodyum/potasyum oksit karisimi gibi bir sulu alkali metal silikat çözeltisinden silika sol hazirlanmaktadir. pH, siloksan yogunlasma oraninin en aza indirilerek solün stabilize edilmesi için uygun düsük bir degere ayarlanmakta ve böylece solün jellesmeden önce depolama stabilitesi saglanmaktadir. As an example of the state of the art, US6495352B1 patent document can be granted. The mentioned document is organic encapsulation of molecules and especially biomolecules The subject is a method based on the sol-gel method developed for takes. In this method, firstly, in water silicon dioxide (silica:SiO2) and sodium/potassium oxide from an aqueous alkali metal silicate solution such as a mixture of silica sol is being prepared. pH, siloxane condensation to stabilize the left by minimizing the being set to an appropriately low value so that the left Storage stability is ensured before gelling.
Enkapsülasyonu yapilacak organik moleküller, uygun bir tampon çözeltisi içinde sole eklenmekte ve sol uygun. bir sicaklikta bekletilerek bir ince film veya jel elde edilmektedir. Söz konusu doküman, temelde enzim (HRP, G6PDH) enkapsülasyonu ve biyosensör uygulamalarinda kullanimi üzerinedir. Söz konusu dokümanda bir“ polietilen glikol (PEG) kaynagi kullanilmamaktadir. Ayrica alkol kullanilmadan enkapsülasyon yapildigi görülmektedir. Solün stabilize edilmesi için iyon degistirici reçine eklenmesi ve sonra bu reçinenin filtrelenmesi gibi ekstra islemler gerektirmektedir. Bahsedilen bulusta sol 48 saate kadar stabilize edildikten sonra enzimler eklenmekte ve 5 dakika içinde jellesen örnek hemen kullanilmaktadir. Jel içinde suyun gözeneklerde enzim ile birlikte kalmasinin enzim aktivitesini düsürecegi düsünülmektedir. Organic molecules to be encapsulated in the buffer solution the sole is added and the left is appropriate. a A thin film or gel is obtained by keeping it at temperature. is being done. The document in question is basically enzyme (HRP, G6PDH) encapsulation and biosensor applications on its use. In the document in question, a “polyethylene glycol (PEG) source is not used. Also alcohol It is seen that encapsulation is done without using it. your left adding ion exchange resin to stabilize it and then extra operations such as filtering this resin requires. Up to 48 hours left in the mentioned invention After stabilization, enzymes are added and The sample that gels in it is used immediately. in gel the fact that water stays with the enzyme in the pores It is thought to decrease its activity.
Method and Evaluation for Diolofenac Diethyloammonium Release” adli patent disi doküman teknigin bilinen durumuna baska bir örnek olarak verilebilir. Bahsedilen doküman, PEG- 600 içeren bir silika jel matrisini ve bu matrisin bir agri kesici olan Diclofenak'in kontrollü salinimi için bir tasiyici olarak kullanimini konu almaktadir. Söz konusu dokümandaki yöntemde, etanol ve PEG karisimina TEOS, saf su ve asetik asit karisimi eklenmekte ve daha sonra bu karisima sulu Diklofenak dietilamonyum çözeltisi eklenmektedir. Elde edilen jelin kurutulmasindan/yaslandirilmasindan sonra kuru jel (xerogel) elde edilmektedir. Bahsedilen doküman bir NSAID olan diclofenak ilacinin silika-PEG içinde enkapsülasyonu ve salimi üzerine olup, çift zincirli nükleik asit enkapsülasyonunda kullanilmamaktadir. verilebilir. Bahsedilen doküman mikron boyutunda konkatemerik (concatemeric) DNA aptamerlerinin yakalanmasi için gelistirilen bir makro-gözenekli biyo/inorganik hibrit yapiya iliskindir. Uzun-zincirli DNA aptamerlerinin yakalanmasi ile bu aptamerler silika malzemesinin makro gözenekleri içerisinde tutulmakta ve böylece aptamerler protein gibi yüksek molekül agirligina sahip hedeflerle ile etkilesime girebilmektedir. Bahsedilen bulus, aptamer enkapsülasyonuna iliskin olup, aptamerler sentetik. olarak üretilmis, bazlarinr fosfodiester baglariylai baglanarak spesifik dizilimde olusturuldugu tek Zincirli nükleik asitlerdir. Bahsedilen tek Zincirli aptamerlerin florosan isikla isaretlenip(analyte) biyosensör uygulamalarina yönelik olarak jel disi bir hedefe (target) baglanmasi saglanmaktir. Bunun yani sira dokümanda üretimi verilen makro-gözenekli yapi genetik molekül vektorü olarak kullanilirsa erken salima neden olacagi düsünülmektedir. Method and Evaluation for Diolofenac Release” non-patent document to the state of the art can be given as an example. The mentioned document, PEG- a silica gel matrix containing 600 and a for the controlled release of Diclofenac, which is It discusses its use as a carrier. Aforementioned In the method in the document, TEOS to a mixture of ethanol and PEG, purified water and acetic acid mixture is added and then this mixture is aqueous Diclofenac diethylammonium solution is added. in hand dry after drying/aging of the gel gel (xerogel) is obtained. The mentioned document is a Silica-PEG of the NSAID diclofenac on encapsulation and release, double chain It is not used in nucleic acid encapsulation. can be given. The mentioned document is in micron size Capture of concatemeric DNA aptamers A macroporous bio/inorganic hybrid developed for is related to the structure. Long-chain DNA aptamers With the capture of these aptamers, the macro content of the silica material aptamers are retained in their pores with high molecular weight targets such as protein can interact. The mentioned invention is aptamer. Concerning encapsulation, aptamers are synthetic. aspect produced by binding the bases with phosphodiester bonds single Strand nucleic are acids. The fluorescence of the aforementioned single-chain aptamers light-marked (analyte) biosensor applications to a non-gel target (target) for is to be provided. In addition to this, the production given in the document macroporous structure as a genetic molecule vector It is thought that if used, it will cause premature release.
Yukaridaki dokümanlar biyosensörlerde kullanima yönelik olarak meze-gözenekli silika içinde sol-jel ile biyomolekül enkapsülasyonu ve nezo-gözenekli silika/hibrit yapi içinde sol-jel ile tek Zincirli nükleik asit enkapsülasyonu ve Vücutta ani salinan ilaçlarda salim hizini kontrol etmeye yönelik olarak da makro-gözenekli silika/hibrit yapi içinde sol-jel ile ilaç enkapsülasyonunu konu almaktadir. Bu cells” isimli makale teknikte benzer alanda bulunan diger dokümanlardir. The above documents are intended for use in biosensors. biomolecule with sol-gel in meso-porous silica encapsulation and in a nesoporous silica/hybrid structure Single-Strand nucleic acid encapsulation with sol-gel and To control the release rate of immediate release drugs in the body in a macroporous silica/hybrid structure. It deals with drug encapsulation with sol-gel. This cells” is another article in the similar field of technique. are documents.
Teknikteki bahsedilen bu dokümanlar vektör olarak kullanima yönelik çift Zincirli nükleik asit enkapsülasyonunda meydana gelen problemlere çözüm sunmamaktadir. Geleneksel yöntemlerde oligonükleotitler, parçacik yüzeylerinde tasindiklari için kan dolasiminda dogrudan maruz kaldiklari enzim/protein degradasyonundan korunamamaktadir. These documents mentioned in the technique are used as vectors. occur in double-stranded nucleic acid encapsulation for It does not offer solutions to emerging problems. Traditional oligonucleotides on particle surfaces are directly exposed in the bloodstream because they are transported cannot be protected from enzyme/protein degradation.
Geleneksel yöntemlerde negatif yüklü oligonükleotitlerin yine negatif yüklü MPS parçacik yüzeylerine adsorplanabilmesi için MPS yüzeyleri Önce katyonik gruplarla fonksiyonlandirilir (Elektrostatik çekim geregi). Aksi takdirde silika yüzeyleri oligonükleotitleri iter. Daha fazla oligonükleotit enkapsülasyonu için daha fazla katyonik grup gerekmektedir. Bu durumda parçacik yüzeyindeki pozitif yük miktari artar ve neredeyse tüm Si-OH gruplari ve diger fonksiyonel gruplar baglanma için kullanilir. Traditionally, negatively charged oligonucleotides to negatively charged MPS particle surfaces. MPS surfaces to be adsorbed first with cationic groups. is functional (Electrostatic attraction requirement). Opposite otherwise the silica surfaces will repel the oligonucleotides. More more cationic for more oligonucleotide encapsulation group is required. In this case, the positive surface of the particle charge increases and almost all Si-OH groups and other functional groups are used for binding.
Güçlü pozitif yüzey sarjinin kan toksisitesini artirici etkisi bulunmaktadir. Bu tip sistemler gen tasiyici olarak kullanildigindai bagisiklik tepkisinir uyandirabilecegi için tek seferde salinabilecek, güvenli azami oligonükleotit dozu sinirlanmis olur. Ek olarak, tüm açik Si-OH gruplari kullanildigi için hücre hedefleme ve kontrollü salinim için yüzeyin fonksiyonlandirilmasi sinirlanmis olur. Bunlara ek olarak oligonükleotitler yüzeye adsorplandigi için proteinler/enzimler tarafindan bozunmasa bile pH degisikligi Vb. Vücut ortami degisikliklerinden dolayi hedef hücrelere ulasmadan salinabilir (prematüre release)veya çift zincirli heliks yapilari bozularak aktivitelerini kaybedebilirler. Increase blood toxicity of strong positive surface charge has an effect. Such systems are used as gene carriers. because it can provoke an immune response when used maximum safe dose of oligonucleotides that can be released in one go gets angry. In addition, all open Si-OH groups for cell targeting and controlled release functionalization of the surface is limited. In addition to these As the oligonucleotides are adsorbed on the surface pH change even if not degraded by proteins/enzymes Etc. target cells due to body environment changes can be released without reaching (premature release) or double chain their helix structure may deteriorate and they may lose their activity.
Yukarida bahsedilen dokümanlarda anlatilan yöntemlerde salinim. hizi kontrol edilememektedir. Yüklenen oligonükleotit miktarinin ve salinimin kontrolü genetik molekül tasiyici sistemler için basta Vücut bagisiklik tepkisini uyandirmamak için olmak üzere kritik onem tasimaktadir. Öte yandan hücre içine girmeleri söz konusu oldugunda, negatif yüzeyli hücre zarindan/endozomdan yine negatif yüzeyli parçaciklar ve/veya oligonükleotitler gecemez. Bu nedenle hem PEG hem diger fonksiyonel gruplar (hedefleyici, PEI, Vb.) hem de oligonükleotitler yüzeyde iken heterojen bir dagilim olur ve verim düser. In the methods described in the above-mentioned documents, oscillation. speed cannot be controlled. uploaded control of oligonucleotide amount and release is genetic Body immunity, mainly for molecule transporter systems critically important to avoid arousing his reaction carries. On the other hand, it is possible for them to enter the cell. reappear from the negative surface cell membrane/endosome negative surface particles and/or oligonucleotides can't sleep. Therefore, both PEG and other functional groups (targeter, PEI, etc.) as well as oligonucleotides on the surface while there is a heterogeneous distribution and the yield decreases.
Yukaridaki problemlere cözümr sunulmasi amaciyla çift zincirli DNA, RNAV nükleikr asitlerin koruyucu bir hibrit malzemeyle enkapsüle edilmesi ve bu koruyucu malzemeden kontrollü salinimi için parçacik üretimi ile ayni anda gerçeklestirilen bir enkapsulasyon yöntemi gelistirilmistir. In order to provide solutions to the above problems, double stranded DNA, a protective hybrid of RNAV nucleic acids encapsulation with material and from this protective material Simultaneous with particle generation for controlled release An encapsulation method has been developed.
Bulusun Detayli Açiklamasi Bulus en genel haliyle mezo-gözenekli silika/polietilen glikol hibrit yapi içinde çift Zincirli oligonukleotit enkapsülasyonuna iliskin bir yöntemi açiklamaktadir. Detailed Description of the Invention In its most general form, mesoporous silica/polyethylene Double Stranded oligonucleotide in glycol hybrid structure describes a method of encapsulation.
Bulus biyomedikal alanina aittir. Akilli biyomalzemeler alaninda degerlendirilebilir. The invention belongs to the field of biomedicine. smart biomaterials can be evaluated in the field.
Bulusun bir amaci, nükleik asitlerin kendilerine has çift Zincirli heliks yapilarinin korunmasi ve dolayisiyla da serbest kaldiklarinda aktif kalmalari için koruyucu bir hibrit (seramik-polimer) malzeme sentez yönteminin gelistirilmesidir. It is an object of the invention that nucleic acids are self-contained pairs. The protection of chained helix structures and therefore also a protective layer to keep them active when they are released. hybrid (ceramic-polymer) material synthesis method is to be developed.
Bulus, genetik molekulün koruyucu bir malzeme içinde tasinmasi için bir enkapsülasyon yöntemi olup, en genel haliyle, - bir silika kaynagina ve polietilen glikol kaynagina alkol eklenerek karistirilmasi ve iki ayri çözeltinin elde edilmesi, - silika çözeltisine, polietilen glikol çözeltisinin eklenerek karistirilmasi ve çözeltinin hidrolize edilmesi için su ilave edilmesi, - kondenzasyon sirasinda, jellesmenin hemen öncesinde, olusmakta olan amorf yapinin gözenekleri içinde tasinmasi için, hidrolize olan çözeltiye kendi tampon çözeltisi içinde olan genetik. molekülün. eklenmesi veya hidrolize olan çözeltinin kendi tampon çözeltisi içinde olan genetik moleküle eklenmesi ve sonra jel elde edilmesi, - jele kurutma/yaslandirma yapilarak kuru jelin elde edilmesi ve toz haline getirilmesi isleni basamaklarini içermektedir. The invention shows the genetic molecule in a protective material. It is an encapsulation method for transporting as it stands, - alcohol to a silica source and a polyethylene glycol source by adding and mixing and obtaining two separate solutions. to be made, - silica solution, polyethylene glycol solution mixing by adding and hydrolyzing the solution adding water for - during condensation, just before gelling, transport of the amorphous structure that is being formed in its pores to the hydrolyzed solution its own buffer solution. in genetics. your molecule. addition or hydrolysis the solution that is in its own buffer solution adding it to the genetic molecule and then obtaining a gel, - Dry gel is obtained by drying/ aging the gel. process steps of grinding and pulverizing contains.
Bulusun bir yapilandirmasinda kondense olmakta olan hibrit yapiya polietilen imin (PEI) eklenmektedir. In one embodiment of the invention, the condensing hybrid polyethylene imine (PEI) is added to the structure.
Yukaridaki yöntemle elde edilen içinde genetik molekül tasiyan mezo-gözenekli silika/polietilen glikol veya silika/polietilen glikol/polietilen imin hibrit malzeme de bulusun kapsamindadir. Genetic molecule in the obtained by the above method mesoporous silica/polyethylene glycol or silica/polyethylene glycol/polyethylene imine hybrid material is within the scope of the invention.
Bulusunr bir yapilandirmasinda, silika kaynagi olarak tetraetil ortosilikat (TEOS) veya tetrametil ortosilikat (TMOS) veya alkali silikatlar kullanilmaktadir. In one embodiment of the invention, as a source of silica tetraethyl orthosilicate (TEOS) or tetramethyl orthosilicate (TMOS) or alkali silicates are used.
Bulusun bir yapilandirmasinda, polietilen glikol kaynagi olarak PEG-4OO kullanilmaktadir. Sivi formda. olmak. üzere orta düzeyde ya da daha yüksek molekül agirligina sahip PEG kullanilabilmektedir. Molekül agirligi lOOO g/mol'e kadar çesitli PEG kaynaklari kullanilabilir. In one embodiment of the invention, the polyethylene glycol source PEG-4OO is used as In liquid form. be. about to medium or higher molecular weight PEG can be used. Molecular weight up to 100o g/mol Various PEG sources can be used.
Bulusun bir yapilandirmasinda polimer olarak baslica polietilen glikol (PEG) ve polietilenimin (PEI) kullanilmaktadir. PEG parçaciklarin topaklanmasini Önler (vücuda verildiginde parçaciklarin hangi organa gidecegi büyük oranda boyutlarina baglidir), MPS yüzeyinde sterik bariyer olusturarak protein/enzim temasini keser. PEI ise katyonik bir polimerdir. Parçaciklarin hücre zarindan geçisini ve endozomdan kaçarak sitoplazmada serbest kalabilmesine olanak tanir. Sitoplazmada serbest kalan vektör hidrolize olur ve serbest kalan oligonükleotit zincirleri hücre transkripsiyonunu saglayan mRNA fonksiyonunu durdurur. Dolayisiyla, RNA interferans ile tedavide kullanilacak MPS için bu temel polimerlerin yapida homojen dagitilmasi, ayni zamanda oligonükleotitlerin de parçacik yüzeylerinde degil içlerinde korunmasi saglanmaktadir. In one embodiment of the invention, the polymer is principally polyethylene glycol (PEG) and polyethyleneimine (PEI) is used. Prevents clumping of PEG particles (Which organ will the particles go to when given to the body? largely depends on their size), steric on the MPS surface It creates a barrier and breaks the protein/enzyme contact. If PEI It is a cationic polymer. From the cell membrane of particles pass through and escape from the endosome to be released in the cytoplasm. allows it to stay. released in the cytoplasm the vector hydrolyzes and the released oligonucleotide mRNA whose chains enable cell transcription stops its function. Therefore, with RNA interference The structure of these basic polymers for MPS to be used in therapy homogeneous distribution of the oligonucleotides protection inside particles rather than on their surfaces is provided.
Bulusun bir yapilandirmasinda alkol, etanol (EtOH), izopropanol veya metanol (MEtOH) olmaktadir. Alkoksitler MPTS gibi silika kaynaklari ile organo-yapilandirilabilir. In one embodiment of the invention, alcohol is ethanol (EtOH), isopropanol or methanol (MEtOH). alkoxides It can be organo-configured with silica sources such as MPTS.
Bulusun bir yapilandirmasinda, molar suzsilika kaynagi orani 4'ten büyüktür. Tam hidroliz için her bir HhH5) grubunun hidrolize olmasi gereklidir (TEOS `ta 4 tane bulunur) Bulusun bir yapilandirmasinda silika kaynagi:alkol orani ve polietilen glikol kaynag12alkol orani hacimsel olarak tercihen en fazla l:2'dir. In one embodiment of the invention, the molar source ratio of suzsilica It is greater than 4. For complete hydrolysis, each HhH5) group It needs to be hydrolyzed (TEOS has 4 of them) In one embodiment of the invention, the silica source:alcohol ratio and polyethylene glycol source12 alcohol ratio by volume preferably at most 1:2.
Bulusun bir yapilandirmasinda polietilen glikol kaynagizsilika kaynagi orani hacimsel olarak tercihen en fazla 1:2'dir. In one embodiment of the invention, polyethylene glycol Welding silica source ratio by volume preferably the most more than 1:2.
Bulusun bir yapilandirmasinda, polietilen glikol kaynagizsilika kaynagi agirlik orani 0,4 olmaktadir. In one embodiment of the invention, polyethylene glycol welded to silica weld weight ratio is 0.4.
Bulusun bir yapilandirmasinda katyonik yüzey sarjini artirmamak için tercihen polietilen imin kaynagizpolietilen glikol kaynagi orani agirlikça ( Bulusun bir yapilandirmasinda hibrit yapi içine katildiginda polietilen imin kaynagi:polietilen glikol kaynagi orani agirlikça en fazla 2:l olmaktadir. Cationic surface charge in one embodiment of the invention In order not to increase, preferably polyethylene is the source of the imine polyethylene glycol source ratio by weight ( When incorporated into the hybrid structure in one embodiment of the invention polyethylene imine source:polyethylene glycol source ratio 2:1 by weight.
Bulusun bir yapilandirmasinda, hidrolizi hizlandirmak için sulu asit Çözeltisi eklenmekte ve karistirilmaktadir. Sulu asit Çözeltisi tercihen hidroklorik asit (HCl), asetik asit, nitrik asit, vb olmaktadir. Asit katalizör molaritesi <6 M (seyreltik, tercihen, 0.1M) ve Iniktari tercihen 5 nü] nin altinda olmak kaydiyla pH<6.5 olacak sekilde eklenmektedir. In one embodiment of the invention, to speed up the hydrolysis aqueous acid solution is added and mixed. Aqueous Acid Solution preferably hydrochloric acid (HCl), acetic acid, nitric acid, etc. Acid catalyst molarity <6 M (diluted, preferably, 0.1M) and less preferably 5 nucl. It is added in such a way that the pH is <6.5, provided that it is below.
Bulusun bir yapilandirmasinda, kondenzasyon sirasinda - jellesmenin hemen öncesinde - biyomoleküller (nükleik asit parçalari, enzim ve proteinler) ve bakteri ve hücreler hibrit sole kendi tampon çözeltileri içinde eklendiginde olusmakta olan amorf' yapinin gözenekleri içinde sivi ile birlikte hapsolur. In one embodiment of the invention, during condensation - just before gelation - biomolecules (nucleic acid parts, enzymes and proteins) and bacteria and cells when the hybrid sole is added in its buffer solutions with liquid in the pores of the amorphous structure that is being formed. are trapped together.
Tüm malzeme üretim ve biyomolekül enkapsülasyon süreci tek asamada, birlikte gerçeklesir ve mezo-gözeneklerin içindeki sivi biyomoleküllerin bozunmadan tasinmasini saglar. Ek olarak, su esasli siVidan arinmis halde bile bu tür jeller, kondense olmamis -OH gruplari ve/Veya farkli kimyasal gruplarla yapilandirilan gözenek yüzeyleri sayesinde biyomoleküllerin tutunmasi için uygun platformlar olusturur. All material production and biomolecule encapsulation process in one In this phase, it occurs together and occurs within the mesopores. It ensures the transport of liquid biomolecules without degradation. Additional As a result, such gels, even when free from water-based liquid, uncondensed -OH groups and/or different chemical thanks to the pore surfaces structured with groups creates suitable platforms for the attachment of biomolecules.
Bulusun bir yapilandirmasinda, hidrolize olduktan sonra ve kondenzasyon sirasinda silika/PEG veya silika/PEG/PEI hibrit sol'ü uygun miktarda ve konsantrasyonda biyomolekül-tampon çözeltisi içine yavasça eklenerek enkapsülasyon ve jellesme saglanir. In one embodiment of the invention, after hydrolysis and silica/PEG or silica/PEG/PEI hybrid during condensation biomolecule-buffer in the appropriate amount and concentration encapsulation and gelation by slowly adding to the solution is provided.
Bulusun bir yapilandirmasinda genetik molekül, çift Zincirli DNA veya RNA oligonükleotitidir (d.NA). Oligonükleotit zincirindeki baz dagilimi, zincir uzunlugu ve oligonükleotit tipi istege bagli olarak seçilebilmektedir. Ek olarak siRNA zincirleri de eklenebilmektedir. In one embodiment of the invention, the genetic molecule is double Stranded. It is a DNA or RNA oligonucleotide (d.NA). oligonucleotide base distribution in the chain, chain length and oligonucleotide type can be selected optionally. In addition, siRNA chains can be added.
Bulusun bir yapilandirmasinda kondenzasyon devam ederken hibrit (Si-O-PEG) yapisinin oligonükleotitler etrafinda olusmasini hizlandirmak için bazik katalizör çözeltisi eklenmektedir. Bazik katalizör olarak tercihen sulu amonyak, amonyum hidroksit, sodyum/potasyum hidroksit ve/veya tris- edta (TE) Vb. kullanilmaktadir. While condensation continues in one embodiment of the invention around the oligonucleotides of the hybrid (Si-O-PEG) structure basic catalyst solution to accelerate the formation is added. Preferably aqueous ammonia as basic catalyst, ammonium hydroxide, sodium/potassium hydroxide and/or tris- edta (TE) Etc. is used.
Bulusun bir yapilandirmasinda, asit katalizör olarak hidroklorik asit (HCl), baz katalizör olarak amonyum hidroksit (NquH) kullanilmaktadir. Bulusa konu yöntemde, HCl ve NH4OH katalizörleri yerine diger zayif asit ve bazlar miktarlari optimize edilerek kullanilabilmektedir. In one embodiment of the invention, the acid is used as the catalyst. hydrochloric acid (HCl), ammonium as base catalyst hydroxide (NquH) is used. In the method of the invention, Other weak acids and bases instead of HCl and NH4OH catalysts quantities can be used by optimizing.
Bulusun bir yapilandirmasinda, oligonükleotit çözelti konsantrasyonu 1, mg/mL'nin. altindadir. Tercihen 500 ug/mL' nin altindadir. In one embodiment of the invention, the oligonucleotide solution concentration of 1, mg/mL. is below. Preferably 500 ug/mL' is below.
Bulusun bir yapilandirmasinda, yüklenecek nükleik asit miktarina bagli olarak, oligonükleotit:silika kaynagi orani agirlikça 1:1'e kadar artirilabilir. In one embodiment of the invention, the nucleic acid to be loaded based on the amount of oligonucleotide:silica source ratio Can be increased up to 1:1 by weight.
Bulusun bir yapilandirmasinda, silika kaynagi olarak TEOS, PEG kaynagi olarak PEG-400, asit katalizör olarak hidroklorik asit (HCl), baz katalizör olarak amonyum hidroksit @MhOH), oligonukleotit olarak Çift zincirli DNA kullanilmaktadir. In one embodiment of the invention, TEOS as the silica source, PEG-400 as source of PEG, acid catalyst hydrochloric acid (HCl), ammonium as base catalyst hydroxide @MhOH), Double-stranded DNA as oligonucleotide is used.
Bulusunr bir yapilandirmasinda. balik. DNA'si (Sigma Aldrich 31149), yosun RNA'si (çift zincirli, Sigma Roche Yeast RNA Bulusun özgün degerini belirleyen unsurlar PEG'in moleküler agirliginin orta düzeyde olmasi, tercihen sivi formda olmasi, hidroliz öncesi TEOS-alkol çözeltisinde homojen bir sekilde dagitilmis olmasi, kimyasallarin eklenme sirasi ve PEG/TEOS (ag.) oranidir. In a configuration of the invention. the fish. DNA (Sigma Aldrich 31149), algae RNA (double-stranded, Sigma Roche Yeast RNA What determines the unique value of the invention is the molecular composition of PEG. Medium in weight, preferably in liquid form homogeneity in TEOS-alcohol solution before hydrolysis. the way it is distributed, the order in which the chemicals are added, and It is the PEG/TEOS (ag.) ratio.
Bulusun. bir yapilandirmasinda. daha detayli olarak MPS-PEG hibritinin üretim basamaklari su sekildedir. Ilk olarak TEOS ve PEG ayri beherlerde etanol (EtOH) ile 10 dakika karistirilir. Her ikisi de etanol içinde çözünür (EtOH'ün zararli olmamasi için çok az miktarda eklenir: tercihen 2,5 mL ama hacimsel olarak TEOS:EtOH=1:2 ye kadar artirilabilir), Daha sonra PEG/EtOH çözeltisi TEOS/EtOH çözeltisine eklenir ve 10 dakika daha karistirilir. (EtOH'ün zararli olmamasi için Çok az miktarda eklenir: tercihen . Find out. in a configuration. in more detail MPS-PEG The production steps of the hybrid are as follows. TEOS first. and PEG in separate beakers with ethanol (EtOH) for 10 minutes. is mixed. Both are soluble in ethanol (EtOH It is added in a very small amount so that it is not harmful: preferably 2.5 mL but up to TEOS:EtOH=1:2 by volume can be increased), then PEG/EtOH solution TEOS/EtOH added to the solution and stirred for another 10 minutes. (EtOH Very little is added so as not to be harmful: preferably .
Kullanilacak PEG miktari PEGzTEOS =l:2 `e kadar artirilabilir. Molekül agirligi lOOO g/mol'a kadar çesitli PEG kaynaklari kullanilabilir. Yalniz EtOH miktari PEG molekül agirligi ve Haktarina bagli olarak artirilmalidir. Amount of PEG to be used up to PEGzTEOS = 1:2 can be increased. Molecular weight various up to 1000 g/mol PEG sources can be used. Only the amount of EtOH PEG it should be increased depending on the molecular weight and Haktar.
TEOS/PEG/EtOH çözeltisine hidrolize olmasi için önce su daha sonra hidrolizi hizlandirmak için sulu asit çözeltisi (HCl, asetik asit, nitrik asit, vb) eklenir ve 5 dakika karistirilir. Eklenecek asit katalizör` molaritesi <6 M (seyreltik, tercihen 0.1M) ve miktari tercihen 5 nüi nin altinda olmak kaydiyla pH<6.5 saglayacak sekilde ayarlanabilir. Hidrolize olan çözeltiye (Si-(OH)n-PEG) tampon çözeltisi içinde oligonükleotitler eklenir (istenen konsantrasyonda; tercihen <5OO ug/mL) ve karistirmaya devam edilir (5 dakika). Kullanilan oligonükleotit Çözelti konsantrasyonu genellikle 1 mg/mL'ninr altindadir ama yüklenecek nükleik asit miktarina bagli olarak, oligonükleotit: TEOS (ag.)=1:1 kadar artirilabilir. Son olarak - kondenzasyon devam ederken- Si-O-PEG hibrit yapisinin oligonükleotitler etrafinda olusmasini hizlandirmakr için bazikr katalizör (sulu amonyak, amonyum hidroksit, sodyum/potasyum hidroksit Vb. ) çözeltisi eklenir ve karisim oda kosullarinda jellesmeye birakilir. Asit ve bazlarin eklenisi zaruri degildir. Jellesme hizinin kontrolü için degistirilebilirler. En hizli jellesme hidrolizi hizlandiran asit ve kondenzasyonu hizlandiran bazlarin birlikte kullanildigi durumda elde edilir. Jellesme öncesi uzun süre isteniyorsa baz katalizör eklenmeyebilir. Bulusa konu olan üretim yontemi hepsini kapsamaktadir. Belirli bir süre (katalizör miktarina bagli olarak; 1-5 gün) ayni kosullarda bekletilip tamamen kuruma/yaslandirma gerçeklestikten sonra kuru jel (xero-gel) nazikçe parçalanir ve toz haline getirilir. Öte yandan, daha önce yapilan çalismalar nükleik asitlerde tampon olarak kullanilan tris- edta (TE) çözeltisinin bazik katalizör etkisi yaptigini göstermistir. Bu amaçla TE de kullanilabilir. Water first to hydrolyze into the TEOS/PEG/EtOH solution. then an aqueous acid solution (HCl, acetic acid, nitric acid, etc.) is mixed. Acid catalyst` molarity to be added <6 M (dilute, preferably 0.1M) and the amount preferably 5 nui provided that it is below pH<6.5 adjustable. To the hydrolyzed solution (Si-(OH)n-PEG) Oligonucleotides are added in the buffer solution (desired in concentration; preferably <500 µg/mL) and continue mixing (5 minutes). Oligonucleotide Solution Used concentration is usually below 1 mg/mL, but depending on the amount of nucleic acid to be loaded, oligonucleotide: TEOS (wt.) can be increased by=1:1. End as - while condensation continues - Si-O-PEG hybrid its structure around oligonucleotides basic catalyst (aqueous ammonia, ammonium hydroxide, sodium/potassium hydroxide Etc. ) solution is added and the mixture is left to gel under room conditions. acid and The addition of bases is not necessary. Control of gelling speed can be changed for Fastest gelling hydrolysis accelerating acid and condensation accelerating bases obtained when used together. Before gelling If desired for a long time, base catalyst may not be added. find it The production method that is the subject covers all of them. Specific time (depending on the amount of catalyst; 1-5 days) same conditions and completely dry / aging dry gel (xero-gel) breaks down gently and powdered. On the other hand, previously Studies have shown that tris- edta (TE) solution has a basic catalyst effect. has shown. TE can also be used for this purpose.
Anlatilan yapilandirmadaki yöntemin islem basamaklari sekil 2'de verilmektedir. Sekil 1'de ise geleneksel MPS parçacik vektör üretimi gösterilmektedir. Geleneksel yöntemde önce parçaciklar üretilir, sonra yüzeylerine oligonükleotitler ve yapilandiriçi moleküller (PEG, PEI, hedefleyiciler, Vb.) eklenir. Gelistirilen bulusta ise silika-PEG hibrit yapisi nükleik asit zincirleri etrafinda jellesir ve hem parçacik içlerinde hem yüzeylerinde PEG bulunur. Oligonükleotitler de sadece yüzeyde kalmaz, yapi içine dagilir. The steps of the method in the configuration described are shown in the figure. It is given in 2. In Figure 1, the traditional MPS particle vector generation is shown. Before the traditional method particles are produced, then onto their surface with oligonucleotides and structuring molecules (PEG, PEI, targeters, Etc.) is added. In the developed invention, silica-PEG hybrid structure It gels around nucleic acid chains and They contain PEG on both surfaces. Oligonucleotides too It does not just stay on the surface, it disperses into the structure.
Sekil Z'ye göre TEOS:EtOH çözeltisine PEG: EtOH çözeltisi eklenmektedir. TEOS yerine TMOS, EtOH yerine MetOH kullanilabilmekte veya alkali silikat çözeltisi kullanilmaktadir. Elde edilen TEOS:PEG:EtOH çözeltisine ultra saf su, su/TEOS molar orani (R) 4'ten çok olacak sekilde eklenmektedir. Elde edilen silika: PEG solüne asit çözeltisi eklenmektedir. Daha sonra çift Zincirli DNA veya RNA oligonükleotitlerini (d.NA), DNA, RNA. veya spesifik siRNA. dizilimleri Vb., içeren tampon çözeltisi eklenmektedir. Burada DNA, RNA parçalarina ek olarak özellikle RNA interferans'ta kullanilan ve spesifik baz dizilimine sahip olan siRNA zincirleri de eklenebilmektedir. PEG: EtOH solution to TEOS:EtOH solution according to Figure Z is added. TMOS instead of TEOS, MetOH instead of EtOH can be used or alkali silicate solution is used. into the resulting TEOS:PEG:EtOH solution. ultrapure water, the water/TEOS molar ratio (R) will be more than 4 is added in the . Obtained silica: acid to PEG sol solution is added. Double Stranded DNA or RNA oligonucleotides (d.NA), DNA, RNA. or specific siRNA. buffer solution containing sequences Etc. is added. Here, in addition to DNA, RNA fragments specific base used especially in RNA interference siRNA chains having the same sequence can also be added.
Daha sonra silika:PEG:[d.NA] solüne baz çözeltisi eklenmektedir. Elde edilen mezo gözenekli silika:PEG:[d.NA]cam Vb. yüzeylere kaplanarak veya dogrudan jele kurutma ve yaslandirma uygulanarak çift zincirli DNA veya RNA oligonükleotitleri (d.NA) enkapsüle edilmis hibrit kuru jel veya kaplama elde edilmektedir. Daha sonra toz haline getirme ve hücre hedefleyici nwleküllerin, PEI vb. koruyucu/yapilandiricir moleküllerinr (*), yüzeye Ibaglanmasi saglanmaktadir. Sonuç olarak tüm yapisinda çift Zincirli DNA veya RNA oligonükleotitleri(d.NA) enkapsüle edilmis hibrit kuru jel parçaciklarinin vektör olarak kullanimi saglanmaktadir. Then silica:PEG:[d.NA] sol into the base solution. is added. The resulting mesoporous silica:PEG:[d.NA]glass Etc. by coating on surfaces or directly double-stranded DNA by applying drying and aging to the gel or hybrid with RNA oligonucleotides (d.NA) encapsulated dry gel or coating is obtained. then powder and cell targeting molecules, PEI etc. Bonding of protective/structuring molecules (*) to the surface is provided. As a result, double-stranded DNA in its entire structure or hybrid with RNA oligonucleotides (d.NA) encapsulated use of dry gel particles as vectors is provided.
Bulusa konu olan hibrit malzeme (silika:PEG) üretildikten sonra istenildigi takdirde farkli yüzey modifikasyonlari yapilabilir. Örnegin, PEI, hibrit sol'e PEG eklenisinin sonrasindar eklenip çift Zincirli DNAV veya RNA oligonükleotitleri (d.NA) enkapsüle eden silika/PEG/PEI hibritleri olusturulabilecegi gibi fosfat tampon çözeltisi (PBS) içinde çözüldükten sonra içerisine parçaciklar eklenir, parçacik miktarina bagli olarak 12-48 saat karistirilir ve parçaciklar filtrelenir. After the hybrid material (silica:PEG) of the invention is produced, different surface modifications if desired can be done. For example, the addition of PEG to PEI, hybrid sol double Stranded DNAV or RNA silica/PEG/PEI encapsulating oligonucleotides (d.NA) Hybrids can be formed as well as phosphate buffer solution. After dissolving in (PBS) particles is added, 12-48 hours depending on the amount of particles. mixed and particles are filtered out.
Kullanilan kimyasallar degerlendirildiginde PEG'in amorf silikaya baglanarak hibrit ve gözenekli bir yapi olusturmasinin farklilik yarattigi görülmektedir. Var olan yöntemlerde PEG zincirleri MPS parçacik yüzeylerine kaplanir (grafting, PEGylation). Bulusta ise PEG eteral oksijen üzerinden Si-O- agina baglanir ve amorf, hibrit bir ag yapisi meydana gelir. PEG in eklendigi parçaciklarda küresel parçacikr seklinin daha belirgin oldugu ve parçacik boyutlarinin küçüldügü (topaklanma azalir) görülmüstür. When the chemicals used are evaluated, PEG is amorphous. a hybrid and porous structure by bonding to silica appears to make a difference. Existing In these methods, PEG chains are coated on MPS particle surfaces. (grafting, PEGylation). In the invention, PEG ethereal oxygen It is connected to the Si-O- network via the amorphous hybrid network. structure occurs. Spherical particles in which PEG is added the particle shape is more distinct and the particle It has been observed that their size is reduced (clumping decreases).
Ayrica, bu yaklasim sayesinde PEG yüzeydeki açik gruplari baglamadan hem tüm yapiya dagilmis olur` hem de parçacik yüzeyleri vektör performansini etkileyebilecek herhangi bir modifikasyona maruz kalmaz. In addition, thanks to this approach, open groups on the PEG surface can be removed. without binding, it will be dispersed both in the whole structure and the particle surfaces that may affect vector performance. not subject to modification.
Bulus sayesinde oligonükleotit vektörü olarak kullanilan MPS parçaciklarinin oligonükleotitleri erken salinimi ve protein degradasyonundan yeterince koruyamamasi problemlerine çözüm saglanmaktadir. Teknikte protein degradasyonundan koruyabilmesi için yapilan tüm modifikasyonlar yüzeyden yapilmakta ve bagisiklik tepkisini uyandirdigi için (yüksek oranda pozitif yüzey modifikasyonu toksisiteyi artirir) tek seferde ;verilebilecek(parçaciklara yüklenebilecek)azami oligonükleotit limitini düsük tutmaktadir. Thanks to the invention, MPS used as an oligonucleotide vector early release of oligonucleotides of particles and protein solution to the problems of not being able to adequately protect it from degradation is provided. from protein degradation in the art All modifications made to protect It is made and because it stimulates the immune response (high positive surface modification increases toxicity) only at a time ;maximum that can be exported (loaded into particles) It keeps the oligonucleotide limit low.
Bulus ile oligonükleotit yükleme ve parçacik üretim süreci tek asamaya düsürülmüs (hibrit sol-jel metodu), hem PEG hem oligonükleotitler parçacik yüzeylerinde degil içlerinde tasinabilmis ve protein degradasyondan korunmustur. Oligonucleotide loading and particle generation process with the invention reduced to a single stage (hybrid sol-gel method), both PEG and oligonucleotides are inside particles, not on their surfaces. transported and protected from protein degradation.
Yukarida bulusun getirdigi çözümler belirtilmistir. Kisaca özetlemek gerekirse; diger oligonükleotit tasinim yöntemlerinde(RNAi de kullanilmak. üzere) oligonükleotitler parçacik üretimi sonrasinda silika yüzeyine adsorplanip PEG ve PEI sadece yüzeye baglanirken, bu bulusta silika/PEG amorf agi olustururken tek basamakta, hibrit yapinin içinde (sadece yüzeyde degil) enkapsülasyon gerçeklestirilmektedir. The solutions brought by the invention are stated above. Briefly To summarize; other oligonucleotide transport oligonucleotides (for use in RNAi) PEG adsorbed onto the silica surface after particle production. and while PEI binds only to the surface, in this invention silica/PEG while creating the amorphous mesh, in a single step, inside the hybrid structure (not only on the surface) encapsulation is carried out.
Tek. asamada, homojen, ve zahmetsizce üretini yapilmaktadir. Only. At this stage, it is produced homogeneously and effortlessly.
Oligonükleotitler' malzeme içinde oldugundan erken salinim riski ortadan kalkar. Oligonükleotitleri baglamak için yüzeyleri toksisite yaratacak sekilde katyonik fonksiyonlandirmaya gerek kalmaz. Enkapsülasyon tamamen fizikseldir. Tampon çözelti içindeki oligonükleotit konsantrasyonu ayarlanarak yükleme verimi kontrol edilebilir. Yapiya giren PEG zincirleri glutatyon (GSH) tarafindan redüklendiginde parçaciklar hidrolize olur ve salinim kolaylasir (Tümör hücrelerinde GSH saglikli hücrelere oranla. 4 kat fazla. bulunur). PEG zincirlerinin malzeme içinde olmasi sayesinde yüzeyde daha fazla açik Si- OH grubu kalir. Diger fonksiyonlandiricilarin (hedefleyici peptitler, ilaçlar Vb.) baglanmasi kolaylasir. PEI miktari optimize edilerek yapi yüksek oranda katyonik grup bulunmadigindan parçaciklar Vücut bagisiklik tepkisini uyandirmaz. Çift zincirli DNA veya RNA oligonükleotitlerinin (d.NA) tümü bu süreçle enkapsüle edilip kontrollü salinabilir. Özellikle RNA interferans tedavilerinde kullanilan siRNA (small interference RNA) tercih. edilebilir. Zincir içi baz dizilimleri çift zincirli oligonükleotitlerinde izole durumda oldugundan sol-jel kimyasinda farklilik yaratmaz. Early release as the oligonucleotides are in the material risk is eliminated. To bind the oligonucleotides cationic surfaces causing toxicity No need for functionality. Encapsulation is completely is physical. Oligonucleotide in buffer solution control the loading efficiency by adjusting the concentration can be done. PEG chains that enter the structure glutathione (GSH) When reduced by the particles hydrolyzed and release is facilitated (GSH in tumor cells is healthy compared to cells. 4 times more. found). PEG chains more open Si- The OH group remains. Other functionalizers (targeting peptides, drugs etc.) becomes easier to bind. PEI amount By optimizing the structure, the cationic group is highly Since particles are not present, the body's immune response it doesn't wake up. All double-stranded DNA or RNA oligonucleotides (d.NA) It can be encapsulated and released in a controlled manner by this process. Especially siRNA (small) used in RNA interference treatments interference RNA) preferred. can be done. In-chain base isolate sequences in double-stranded oligonucleotides It does not make any difference in sol-gel chemistry since it is in the same condition.
Silika ve polimer gruplarla temasta sadece oligonükleotit Zincirlerini meydana getiren seker-fosfat gruplaridir. Only oligonucleotide in contact with silica and polymer groups It is the sugar-phosphate groups that make up the chains.
Oligonükleotit enkapsülasyonu, silika-PEG agi olusurken olusan gözeneklerin içinde gerçeklesir. Böylelikle hem PEG hem oligonükleotitler geleneksel yöntemde oldugu sekilde sadece parçacik yüzeylerine adsorplanmaz, parçacik içlerindeki gözeneklerde enkapsüle edilir. Kan dolasimina girdiginde silika/PEG parçacik yüzeyleri proteinler (örn. albumin, hemoglobin) ile oligonükleotitler arasinda bir ara yüz (sterik bariyer) olusturarak oligonükleotitleri korur. Oligonucleotide encapsulation, forming a silica-PEG network It takes place inside the formed pores. Thus, both PEG both the oligonucleotides as in the conventional method. not only adsorbed on particle surfaces, the particle encapsulated in the pores within them. blood circulation silica/PEG particle surfaces become proteins (eg. albumin, hemoglobin) and oligonucleotides It protects the oligonucleotides by forming a face (steric barrier).
Bulusta oligonükleotit enkapsülasyonu fiziksel bir süreçtir. In the invention, oligonucleotide encapsulation is a physical process.
MP8 sisteminde (daha önceki çalisma ve patentlerde mevcuttur) kondenzasyon ilerledikçe olusmakta olan Si-O-Si ve/Veya Si-O-PEG kolloidleri biyomolekülleri su ile birlikte çevreler. Kolloidlerde bulunan Si-OH gruplari ve PEG zincirleri su ile baglanmaya açiktir. Oligonükleotitler ise sulu tampon çözelti içinde, heliks yapinin polaritesi sayesinde su tabakalarina baglanir. Böylelikle su tabakasi oligonükleotit ve kolloidler arasinda baglanmayi engellerken heliks yapiyi korumus olur. In the MP8 system (in previous work and patents) available) as the condensation progresses, the Si-O-Si and/or Si-O-PEG colloids biomolecules together with water circles. Si-OH groups and PEG in colloids chains are open to bonding with water. If the oligonucleotides polarity of helix structure in aqueous buffer solution It binds to the water layers. Thus, the water layer while preventing binding between oligonucleotides and colloids the helix structure is preserved.
Silika/PEG ag yapisinda ise PEG yapisinda bulunan eter oksijen (CýhO) üzerinden baglanarak amorf silika ag yapisina katilir. Hidrofilik (su sever) ve nötr yüzeylere sahip oldugundan PEG zincirleri oligonükleotitlere zarar vermez, yük dengesini bozmaz. Olusan kuru jel monolitler nazikçe parçalanip toz haline getirildiginde, PEG zincirleri hem yüzeyde hem yapi içinde bulunur. Yüzeydeki Si-OH gruplari fonksiyonlandirma için (PEI, peptit vb.) baglanmaya hazir vaziyette kalir. In the silica/PEG network structure, the ether in the PEG structure amorphous silica network by connecting via oxygen (CıhO) participates in its construction. Hydrophilic (water-loving) and neutral surfaces PEG chains damage oligonucleotides because they have It does not give a load, does not disturb the balance of the load. Formed dry gel monoliths When gently crushed and pulverized, PEG chains It is found both on the surface and in the structure. Si-OH on the surface groups to bind (PEI, peptide, etc.) for functionalization remains ready.
Bulusta oligonükleotitler parçaciklarin yüzeyinde degil içindedir. Bu nedenle salinim saglayacak bir uyarici etken (pH, bag kirici molekül, vb.) olmadikça salinim gerçeklesmez. PEG ve oligonükleotitler parçacik içlerinde, PEI ise miktari optimize edilerek hibrit yapiya eklenebilecegi gibi bir ara yüz olusturacak sekilde tüm yüzeye sonradan kaplanabilir (Literatürde varolan bir yüzey olusturulur ve parçaciklarin zar geçisi kolay Bulus ile silika/PEG hibrit yapisinin RNA interferans gibi tedavi yaklasimlarinda vektör olarak kullanimi ve bu amaç dogrultusunda çift zincirli nükleik asitlerini (DNA/RNA) üretim sirasinda enkapsüle eden genetik molekül yükleme ve koruyarak tasima yöntemi saglanmaktadir. In the invention, the oligonucleotides are not on the surface of the particles. is in it. Therefore, a stimulating factor that will provide release (pH, bond breaking molecule, etc.) it doesn't happen. PEG and oligonucleotides are inside the particle, PEI, on the other hand, is converted to hybrid structure by optimizing the amount. can be added to create an interface as well as all the can be coated on the surface afterwards (there are a surface is created and particles are easy to pass through the membrane. RNA interference of the silica/PEG hybrid structure with the invention its use as a vector in treatment approaches and for this purpose double-stranded nucleic acids (DNA/RNA) genetic molecule loading that encapsulates during production and Preserving transport method is provided.
Bulus biyosensör uygulamalarinda saglayacagi avantaja ek olarak, mezo-gözenekli silika türevi malzemelerin son zamanlarda oldukça popüler olan ve umut verici tedavi arastirmalari olan RNA interferans (RNAi) yaklasimina hitap etmektedir. Bu tedavilerde kullanilan nükleik asit zincirlerinin hasta hücrelerde serbest kaldiklarinda hücre cogalmasini engelleme mekanizmalari birbirinden farkli olsa da hasta hücrelere tasinmalarini saglayacak malzemelerden (vektör) beklenen Özellikler aynidir. In addition to the advantage that the invention will provide in biosensor applications, Finally, the end of mesoporous silica-derived materials very popular and promising treatment at the time addressing the RNA interference (RNAi) approach with research is doing. The nucleic acid used in these treatments cell chains when released in diseased cells Although the mechanisms of preventing proliferation are different from each other, and materials that will enable them to be transported to the sick cells. (vector) expected Properties are the same.
Bulusun bu alandaki avantajlari, sekil 1 ve 2'de görüldügü gibi nükleik asit yüklenmis malzeme üretim sürecini oldukça kisaltmasi; PEG'in geleneksel yöntemden farkli olarak sadece yüzeylere degil tüm yapiya dagitilarak kanda vektörün maruz kalacagi protein/enzim ataklarina karsi korumayi daha verimli hale getirmesi, yüzeyde hedefleyici vb. moleküllerin baglanabilecegi daha fazla açik Si-OH gruplari birakmasi; nükleik asitlerin sadece yüzeylere tutunmasi degil, tüm iç yapiya dagitilmasi sayesinde bagisiklik tepkisini uyandirmadan (vektör miktari artirilmadan yüklenen nükleik asit miktari artirilmaktadir), yüksek miktarda nükleik asit yüklemesine olanak tanimasi ve silika-PEG hibrit yapisinin nükleik asitleri sadece yüksek glutatyon ve/veya asidite ortaminda(hidroliz ve/veya PEG baglari redüklenerek vb. gibi mekanizmalarla) salabilmesi ve dolayisiyla nükleik asitleri kanda erken salimdan korumasi olarak özetlenmektedir. The advantages of the invention in this field are illustrated in figures 1 and 2. The production process of nucleic acid loaded material such as abbreviation; Unlike the traditional method, PEG only exposure of the vector in the blood by being distributed over the entire structure, not on the surfaces. better protect against protein/enzyme attacks. making it efficient, targeting on the surface, etc. of molecules leaving more open Si-OH groups to which it can bind; not only the attachment of nucleic acids to surfaces, but also all internal immune response thanks to its distribution in the structure without awakening (without increasing the amount of vector loaded nucleic acid) the amount of acid is increased), a high amount of nucleic acid loading and silica-PEG hybrid structure. nucleic acids only with high glutathione and/or acidity in the environment (such as by hydrolysis and/or reducing PEG bonds, etc.) by mechanisms) and thus nucleic acids It is summarized as early release protection in blood.
Bulusta, sol-jel ile üretilmis silika-PEG hibrit yapisi gen tasiyici vektör olarak kullanilmaktadir. Gelistirilen yöntem, teknigin bilinen. durumundan farkli olarak. nükleik asit zincirlerinin ve protein/enzim degradasyonu ile topaklanmayi önlemek için kullanilan PEG zincirlerinin sadece malzeme yüzeylerinde - sonradan emdirilerek- degil, tüm yapi içinde birlikte dagilimini saglamaktadir. In the invention, silica-PEG hybrid structure produced with sol-gel used as a carrier vector. developed method known in the art. unlike the situation. nucleic by degradation of acid chains and protein/enzyme PEG chains used to prevent clumping not only on material surfaces - by post-impregnation, It ensures its distribution in the whole structure.
Bulus gen tasiyici sistemlerde vektör olarak üretilebilir ve kullanilabilir. The invention can be produced as vectors in gene carrier systems and can be used.
Bu bulusu öncekilerden farkli kilan üretim sonrasinda parçacik topaklanmasini, kan dolasiminda parçacik ve içerdikleri biyomoleküllerin protein ve enzim degradasyonundan korunmasini saglayan PEG polimerinin, halihazirdaki yöntemlerle önceden üretilmis MPS yüzeylerine kaplanmasi ile degil, sol-jel üretimi sirasinda amorf silika yapisina baglanarak hibrit bir yapi olusturmasi ve ilk kez bu hibrit yapi içinde sol-jel yaklasimi kullanilarak oligonükleotit enkapsülasyonunun yapilmis olmasidir. What makes this invention different from the previous ones is the post-production particle aggregation, particle and the biomolecules they contain, proteins and enzymes PEG polymer, which provides protection from degradation, on MPS surfaces prefabricated by current methods. amorphous silica during sol-gel production, not by coating to form a hybrid structure by connecting to its structure and for the first time using the sol-gel approach in this hybrid structure. oligonucleotide encapsulation is done.
Teknikteki dokümanlarin aksine gelistirilen bulusta› pH'in düsük tutulmasina ihtiyaç yoktur. Bulusta baslangiçta çok az miktarda etil alkol (EtOH), TEOS ve PEG çözücü ortami olarak kullanilmakta, sonrasinda su/TEOS molar orani (R) 4'ten büyük tutularak su kondenzasyonu yapilmakta, alkol kondenzasyonu engellenmektedir. Böylelikle, yine sulu tampon çözeltisi içinde kondense olmakta olan yapiya eklenen nükleik asitler korunmaktadir. Bulusta polimer ekleme ve filtreleme gibi ekstra islemlerin uygulanmasina ihtiyaç yoktur. Contrary to the documents in the technique, in the invention developed, pH it doesn't need to be kept low. Very little in the beginning amount of ethyl alcohol (EtOH), TEOS and PEG as solvent media is used, then the water/TEOS molar ratio (R) is less than 4 water condensation is made by keeping it large, alcohol condensation is prevented. Thus, the aqueous buffer added to the structure that is condensing in its solution nucleic acids are conserved. Addition of polymer and need to apply extra operations such as filtering there is none.
Teknikteki dokümanlarin aksine Bulusta PEG basta olmak üzere yapi jellesme süresi, gözenek boyutu olmak üzere çesitli Özellikleri kontrol etmek için TEOS, EtOH, asit/baz katalizör miktarlari genis aralikta degistirilebilir. Contrary to the documents in the art, the Invention, primarily PEG structure, gelling time, pore size TEOS, EtOH, acid/base to control properties catalyst amounts can be varied over a wide range.
Bulusun bir yapilandirmasi biyosensörlerde kullanilmaktadir. One embodiment of the invention is used in biosensors.
Bulusun bir yapilandirmasi dogrudan RNAi gen tedavisinde kullanilabilecek tasiyici malzeme (vektör) sentezi yöntemidir.One embodiment of the invention is directly in RNAi gene therapy. carrier material (vector) synthesis that can be used method.
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| TR2020/09223A TR202009223A2 (en) | 2020-06-15 | 2020-06-15 | GENETIC MOLECULAR ENCAPSULATION IN MESO-POORED SILICA / POLYETHYLENE GLYCOL HYBRID STRUCTURE |
| PCT/TR2021/050584 WO2021257033A1 (en) | 2020-06-15 | 2021-06-14 | Genetic molecule encapsulation in mesoporous silica/polyethylene glycol hybrid structure |
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