Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
  • C08G73/02—Polyamines
  • C08G73/0233—Polyamines derived from (poly)oxazolines, (poly)oxazines or having pendant acyl groups
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
  • C08G73/02—Polyamines
  • C08G73/0206—Polyalkylene(poly)amines
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
  • C08G73/02—Polyamines
  • C08G73/0206—Polyalkylene(poly)amines
  • C08G73/0213—Preparatory process
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers

Definitions

• New poly (ethyleneimine) based copolymers for attachment and release of genetic material, in particular DNA / RNA, as well as methods for their preparation and use
• the invention relates to novel poly (ethyleneimine) -based copolymers which consist of ethyleneimine and 2-oxazoline units, but have a surprisingly high functionality in comparison to known poly (ethyleneimine) -based copolymers (PEI).
• PEIs of this type are known for binding and release of DNA / RNA.
• the invention also includes the process for their preparation and function-specific uses of the proposed copolymers.
• the proposed poly (ethyleneimine) based copolymers can be used, for example, for functionalizing surfaces, for developing DNA chip systems for mobile analytics and as materials for antifouling coatings of sensors.
• Poly (ethyleneimine) based copolymers are also known (Tian H, F. Li, J. Chen, Y. Huang, X. Chen: N-Isopropylacrylamide-Modified Polyethylenimines as Effective Gene Carriers, Macromolecular, Bioscience 12, 2012, 1680- 1688), in which the functionalization with N-isopropylacrylamide by a so-called Michael addition takes place.
• Michael addition shows binding affinity to plasmid DNA.
• the functionalization of the PEI by means of said Michael addition leads to side chains without possible multiple bonds, whereby the functionalization is limited.
• substrates with a functionalized surface or structured hydrogels, for example for beads, particles, etc., for binding and release of genetic material can not be produced in this way.
• N-hydroxysuccinimide or the slightly more polar N-hydroxysulfosuccinimide (NHSS) reacts under mild conditions with carboxyl-containing components (eg butanoic acid) to form so-called “aminoacyl esters” (D. Sehgal, IK Vijay: A method for the high efficiency of water-soluble carbodiimide mediated amidation, Anal. Biochem 218, 1994, 87-91).
• carboxyl-containing components eg butanoic acid
• Amino groups can be functionalized with these aminoacyl esters, but this functionalization is limited to primary amine groups only. No mention is also made of the introduction of functionalized side chains in polymers. As mentioned above, no multiple bonds are likewise possible here, so that once again substrates with a functionalized surface or structured hydrogels, for example for beads, particles, etc., for binding and release of genetic material, in particular DNA / RNA, can be produced.
• Polyalkyleneimine hydrogels with adjustable degradation rates are also known (M. Carnahan, J. Butlin: Crosslinked Polyalkylenimine Hydrogels with Tunable Degradation Council, WO 2009/102952 A2).
• differently functionalized polyalkyleneimines whose reaction path is not shown, or branched polyethyleneimine, cross-linked by means of activated polyethylene glycol, so that only a part of the original amine groups of the PEI is available for a subsequent biological application.
• About the ratio of the amounts of the starting polymer and the crosslinker the control of the degree of crosslinking takes place only indirectly. The focus here is on the synthesis of hydrogel networks.
• the invention is therefore based on the object to provide novel poly (ethyleneimine) based copolymers which are as low as possible and tailor-made to produce and have high functionality for universal effective uses, but without limiting their binding ability to genetic material, in particular DNA / RNA.
• hydrogels of the poly (ethyleneimine) -based copolymers with high binding affinity to DNA / RNA should be presentable.
• Copolymer chain length between 2 and 1,000,000 units
• R H or organic radical (eg alkyl and aryl radical),
• Copolymer chain length between 2 and 1,000,000 units
• the composition of the tailored copolymers can be determined easily and quickly by means of nuclear magnetic resonance spectroscopy.
• the structure of the copolymers of the invention allows multiple bonds in the copolymer backbone and thus creates conditions for functionalities, but without limiting the binding affinity for genetic material compared to the known prior art.
• the said functionalities thus enable further applications in which the binding / release in particular of DNA and RNA is fully supported.
• a further component can be added to the described copolymer by means of per se known click chemistry (thiol-en photoaddition, azide-click).
• the great advantage of the copolymer produced is the property that existing and exactly quantified amino groups remain unaffected by this reaction.
• the copolymers can be applied to functionalized surfaces and allow attachment and release of genetic material to them. Due to incomplete surface bonding, there are free multiple bonds that can be used for the step-by-step construction of hydrogel layers on the desired surface.
• Another advantage is the possibility of structuring three-dimensional polymer networks, so-called hydrogels. With the aid of a bifunctional linker (dithiol), the copolymers can be crosslinked to form such hydrogels.
• a major disadvantage of such polymer networks is the property of their absolute insolubility in any solvents.
• an activating reagent in particular EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) or DCC (dicyclohexylcarbodiimide) is introduced as acid.
• the functional side chain can also be introduced via a subsequent functionalization by means of unsaturated acid anhydrides or halides (-C1, -Br, -I).
• copolymers according to the invention have both unsaturated functionalities and free amino groups.
• copolymers of the formula I or II are based on a subsequent functionalization of the homopolymer polyethyleneimine (formula III).
• the synthesis method allows a low-cost and procedurally simple implementation, which is possible both on a small scale and in the high-throughput process.
• the preparation of custom-made copolymers leads to substances with precisely defined compositions, which can be varied according to the application. Their exact composition can be determined easily and quickly by means of nuclear magnetic resonance spectroscopy. This allows extensive libraries of copolymers of different composition can be created by simple variation of the starting material quantities.
• the copolymer P (ButEnOx-co-EI) was dissolved in ethanol.
• the combined, clear solutions (10 wt%) were degassed with nitrogen for 30 minutes and then exposed to UV light (365 nm) for about 24 hours.
• the onset of gel formation demonstrated successful hydrogel synthesis.
• the resulting gel was repeatedly washed with methanol and water. This was followed by freeze-drying.
• a library of different hydrogels could be prepared starting from corresponding copolymers.
• the synthesized substances could be extensively characterized (source value, vapor TGA, FT-IR, EA, solid 'H- 13 C-NMR, SEM).
• hydrogels were swollen for 24 hours in HBG buffer (pH 7). Subsequently, a genomic DNA-ethidium bromide solution was added. At defined times an aliquot was taken and after that Fluorescence measurement pipetted back. The targeted release of the previously bound DNA could be achieved with the addition of a heparin solution and a temperature increase to 70 ° C within a very short time.
• Diagram 1 Schematic representation of the binding (A) and release (B) of genomic herring DNA from hydrogels with different PEI content by means of EB tests
• the hydrogels show, in addition to their expected insolubility, a typical swelling behavior in water, which strongly depends on the PEI content and the degree of crosslinking (amount of dithiol) of the gels.
• the DNA binding and release capacity is dependent on the PEI content within the hydrogel structure. For targeted release in addition to an increase in temperature and the presence of heparin is necessary. Within a very short time ( ⁇ 60 min), the DNA can be almost completely released again.
• Example 2 Formulation of hydrogel beads via a thiol-ene photoaddition analogous to a suspension polymerization.
• stepwise hydrogel layers can be built up (Table 1), which have different DNA affinity depending on the layer thickness.
• This application of the binding and release of genetic material, in particular DNA / RNA, from coated surfaces is to be used, for example, in mobile analytics (chip diagnostics).
• the copolymer P (ButEnOx-co-EI 50 o / o ) was dissolved in ethanol together with the photoinitiator 2,2-dimethoxy-2-phenylacetophenone.
• the vial was degassed with nitrogen for 30 minutes and then exposed to UV light (365 nm) for about 24 hours.
• the coated glass surface (1st click) was repeatedly washed with water and methanol.
• the second click was carried out analogously by adding the crosslinker 2,2 '- (ethylenedioxy) diethanethiol instead of the copolymer. Again, the washing steps were carried out. The repeated reaction with the copolymer described (3rd click) led to the formal formation of the hydrogel layer. By free existing double bonds, the layered structure could be carried out up to the 7th click, whereby also far more layers are possible.
• the successful coating of the glass surfaces could be demonstrated by fluorescence microscopy.
• the described copolymer was labeled with the dye fluorescein ( ⁇ 1%) and added to the surface at the click step to be detected.
• the treated surface showed a successful covalent binding by a washing even after intensive washing increased fluorescence of the labeled sample compared to a dye-free coating.
• Measurements of a scratch on the coated surfaces by means of an atomic force microscope resulted in a thickness of 15 nm for the first layer (1st click).
• a height of 35 nm could be determined.
• a successful connection with the help of infrared spectroscopy could be shown.

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• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Medicinal Chemistry (AREA)
• Polymers & Plastics (AREA)
• Organic Chemistry (AREA)
• Macromolecular Compounds Obtained By Forming Nitrogen-Containing Linkages In General (AREA)
• Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
• Medicinal Preparation (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

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• 2013
  • 2013-10-02 DE DE102013016750.7A patent/DE102013016750A1/de active Granted
• 2014
  • 2014-10-02 DK DK14803038.0T patent/DK3052545T3/en active
  • 2014-10-02 CA CA2926154A patent/CA2926154C/en active Active
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