WO2011070402A1 - Liaison acide aminé/monomère photosensible et applications de bioconjugaison en sciences biologiques et en biotechnologie - Google Patents

Liaison acide aminé/monomère photosensible et applications de bioconjugaison en sciences biologiques et en biotechnologie Download PDF

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WO2011070402A1
WO2011070402A1 PCT/IB2009/055707 IB2009055707W WO2011070402A1 WO 2011070402 A1 WO2011070402 A1 WO 2011070402A1 IB 2009055707 W IB2009055707 W IB 2009055707W WO 2011070402 A1 WO2011070402 A1 WO 2011070402A1
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protein
monomer
nano
preparing
aminoacid
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PCT/IB2009/055707
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English (en)
Inventor
Ridvan Say
Arzu Ersoz
Deniz Hur
Filiz Yilmaz
Adil Denizli
Ayca Atilir Ozcan
Sibel Emir Diltemiz
Suzan Yazar
Ozlem Bicen
Sibel Buyuktiryaki
Rustem Kecili
Guner Saka
Tugba Findik
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Ridvan Say
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Priority to PCT/IB2009/055707 priority Critical patent/WO2011070402A1/fr
Priority to US13/203,833 priority patent/US20110311505A1/en
Publication of WO2011070402A1 publication Critical patent/WO2011070402A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6873—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting an immunoglobulin; the antibody being an anti-idiotypic antibody
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0065—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the luminescent/fluorescent agent having itself a special physical form, e.g. gold nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0065—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the luminescent/fluorescent agent having itself a special physical form, e.g. gold nanoparticle
    • A61K49/0067—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the luminescent/fluorescent agent having itself a special physical form, e.g. gold nanoparticle quantum dots, fluorescent nanocrystals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
    • A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
    • A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
    • A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
    • A61K49/1833—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with a small organic molecule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
    • A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
    • A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
    • A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
    • A61K49/1833—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with a small organic molecule
    • A61K49/1848—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with a small organic molecule the small organic molecule being a silane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
    • A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
    • A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
    • A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
    • A61K49/1866—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle the nanoparticle having a (super)(para)magnetic core coated or functionalised with a peptide, e.g. protein, polyamino acid
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material
    • G01N2458/40—Rare earth chelates

Definitions

  • This invention is related to preparation of photosensitive ruthenium based aminoacid monomers and oligomers, protein conjugating method and their use in life sciences and biotechnology.
  • nanobioconjugates can make a worthy contribution to the development of new diagnostic and delivery systems. That is to say that, they offer optical, magnetic, biocompatible, biostabil and orientation properties, for sensitive detection, drug targeting and therapy, isolation and purification plus biocatalysis.
  • a major step toward the applicability of the nanobioconjugates for sensorics, drugs, separators and catalysators is therefore the design and enrichment of adequate conjugation of their nanoparticle surface. This conjugation step should lead to a chemical and physical stabilization of the nanostructures as well as the ability.
  • immobilization may lead to partial or complete loss of protein activity, due to random orientation and structural deformation.
  • Oriented immobilization of active proteins are good steric accessibilities of active binding sites and increased stability.
  • Protein inactivation starts with the unfolding of the protein molecule through the contact of water with hydrophobic clusters located on the surface of protein molecules.
  • carbohydrate moieties may stabilize proteins (Turkova, J., J. Chromatog. B, 722, 1999, 11).
  • Antibodies have been used in the design of separation and biosensors materials for decades due to their extremely high specificity.
  • the antibody needs to be attached to a solid matrix, and the antibody immobilization may be in many cases a critical step in the design of separation and biosensor materials.
  • the first requirement of the system requires to have a final inert surface that prevents the unspecific adsorption on the matrix of other components of the samples or of the molecules used to report the signal. If this is not exercised, the specificity of the systems may be greatly reduced.
  • Another important feature that the immobilization systems should accomplish is to maintain the antibody functionality. That is to say that, it should not produce severe distortions of the antibody structure. However, in some instances, the immobilisation of antibodies presents a further complication.
  • analyte is a protein or a cell
  • antibodies properly oriented regarding the surface of the support will be able to recognize it.
  • antibodies oriented toward the support surface can not bind antigen.
  • the balancing of both features will produce the final synergie between producebility and sensitivity of the biosensor and bioseparation process (Batalla, P. Et. AL, Biomacromolecules, 2008,9, 719).
  • polyvalent interactions are characterized by the simultaneous binding of multiple ligands on a biomolecule or a nano-surface to multiple receptors. Polyvalent interactions can be collectively much stronger than corresponding monovalent interactions.
  • Photoimmobilization demands the presence of mediating photosensitive reagents, generally activated by incident light of an appropriate wavelength. After light activation, the reagents undergo distinct chemical processes that finally lead to the formation of covalent bonds between the photogenerated intermediates and the biomolecules (Rusmini, F. et.al, Biomacromolecules, 8, 2007, 1775).
  • the underlying chemistry is proposed in the literature to involve the formation of radicals allowing tyrosine residues to give covalent bonds with another tyrosine and to use a strategy to oxidative protein- protein crosslinking using photosensitization (Fancy, D. A., and Kodadek, T. Proc. Natl. Acad. Sci. USA, 96, 1999, 6020; Brown, K. C, and Kodadek, T., Met. Ion. Biol. Sys., 38, 2001, 351; Duroux-Richard, I. et al, Chem. & Biol. 12, 2005, 15).
  • Certain nanomaterials are ideal probe candidates because of their (i) small size (1-100 nm) and correspondingly large surface-to-volume ratio, (ii) chemically tailorable physical properties, (iii) unusual target binding properties, and (iv) overall structural robustness. Tailorable physical properties and oriented surface modification are very important aspects of nanomaterials. Indeed, in this regard, nanomaterials and biology have a sustained history as nanoparticles have been used bio-conjugation and cellular labeling agents for the past four decades (Rosi, N. L., Mirkin, C. A., Chem. Rew., 105, 2005, 1547).
  • nanobio-conjugates for life sciences and biotechnology applications is one of the fastest moving fields of nanobiotechnology.
  • conjugation technologies they can not only be rendered biocompatible, but also, to fulfill tasks.
  • monomeric or polymeric coatings are applied to provide biocompatibility and additional bio conjugation (also for multivalent interaction) for targeting those particular drugs which prevent the disease spreeding and other applications (Hezinger A. F. E., Tessmar J., and Gopferich, A., Eur. J. Pharm. Biopharm., 68, 2008, 132).
  • WO03065888 relates to novel tumor specific phototherapeutic and photodiagnostic agents.
  • the compounds consist of a carbocyanine dye for visualization, photosensitizer for photodynamic treatment, and tumor receptor-avid peptide for site-specific delivery of the probe and phototoxic agent to diseased tissues.
  • WO0012575 relates to a method for synthesizing continuous, polymeric solid phase supporting materials with spatially defined and interspaced reaction sites.
  • the solid phase supporting material is comprised of a supporting matrix and graft copolymer chains with reactive groups.
  • the surface of the supporting matrix is coated with a photoinitiator.
  • the supporting matrix and the photoinitiator are exposed in the presence of an unsaturated functional monomer.
  • the objective of this invention is to preparation of photosensitive ruthenium based aminoacid monomers and oligomers, aminoacid monomer-protein cross-linking using photosensitation and conjugation approach on micro and nano-structures by ruthenium- chelate based monomers. Its vast range applications of multifunctional, biocompatible, stabil and specific micro and nanobio-conjugates, which will stand-alone or simultaneously enable ; both purification and determination, both targeting and imaging and theranostics and catalysis and determination.
  • the construction and method of preparation is applicable to silica materials, superparamagnetic particles, QDs, CNTs, Ag/Au nanoparticles and Au surfaces and polymeric materials.
  • the photosensitive aminoacid monomer linkers can react via chemically and biocompatible to a lot of different micro and nano-surface and then to the protein when they act as a single-step cross-linking reaction using irradiation.
  • the conjugation based on click biochemistry can be carried out at mild conditions, independent of pH and temperature, without affecting conformation and function of protein.
  • Figure 1 shows tyrptophan aminoacid monomer having Chlorobis(2-2 '-bipyridyl) MATrp- ruthenium(II) photosensitive chelate (preferably, the monomer linker moiety is selected from the methacryloyl)
  • Figure 2 shows two tyrptophan amino acid monomers having Bis(2-2 '-bipyridyl) bis(MATrp)-ruthenium(II) photosensitive chelate (preferably, the monomer linker moiety is selected from the methacryloyl)
  • Figure 3 shows tyrptophane aminoacid monomer and MUABt ligands having Bis(2-2 '- bipyridyl)-MATrp-MUABt ruthenium(II) photosensitive chelate (preferably, the monomer linker moiety is selected from the methacryloyl)
  • Figure 4 shows tyrosine aminoacid monomer having Chlorobis(2-2 '-bipyridyl) MATyr- ruthenium(II) photosensitive chelate (preferably, the monomer linker moiety is selected from the methacryloyl)
  • Figure 5 shows two tyrosine aminoacid monomer having Bis (2-2 '-bipyridyl) bis(MATyr) - ruthenium(II)photosensitive chelate (preferably, the monomer linker moiety is selected from the methacryloyl)
  • Figure 6 shows tyrosine and tryptophan aminoacid monomers having bis(2-2 '-bipyridyl) MATyrMATrp-ruthenium(II)photosensitive chelate (preferably, the monomer linker moiety is selected from the methacryloyl)
  • Figure 7 shows tyrosine aminoacid monomer and MUABt having Bis(2-2 '- bipyridyl)MATyrMUABt-ruthenium(II)photosensitive chelate (preferably, the monomer linker moiety is selected from the methacryloyl)
  • Figure 8 shows cysteine aminoacid monomer having Chlorobis (2-2 '-bipyridyl) MACys- ruthenium(II) photosensitive chelate (preferably, the monomer linker moiety is selected from the methacryloyl)
  • Figure 9 shows two cysteine aminoacid monomers having bis (2-2 '-bipyridyl) bis(MACys) ruthenium(II) photosensitive chelate (preferably, the monomer linker moiety is selected from the methacryloyl)
  • Figure 10 shows MALDI-TOF/MS spectrum of ([Ru (bpy) 2 MATrp-MATyr] n ) oligomer.
  • Figure 11 shows transmission emission microscope (TEM) images of Nano BSA particles according to an example of the invention.
  • Figure 12 shows comparison of photolummescence spectrum of nano BSA, HRP conjugated BSA and HRP conjugated BSA with DAB according to this example of the invention.
  • Figure 13 shows photolummescence spectrum of nanotransferrin and antitransferrin affinity interaction according to an example of the invention.
  • Figure 14 shows fluorescence spectroscopy analyses of 4, 8 and 16 ppm antitransferrin solution according to this example of the invention.
  • Figure 15 shows fluorescence spectroscopy analysis of 16 ppm transferrin solution, 16 ppm antitransferrin solution and their mixture according to this example of the invention.
  • Figure 16 shows Fluorescence spectroscopy analysis of transferrin-antitransferrin mixture (above peak) and nanotransferrin-antitransferrin mixture (below peak) according to this example of the invention.
  • Figure 17 shows flow cytometry analyses of methotrexate conjugated nanotransferrin for killing the cancer cells according to an example of the invention.
  • Figure 18 shows flow cytometry analyses of nanoTNF-a for killing the cancer cells.
  • Ql ratio (necrotic cells) was 8,5 %
  • Q3 ratio (viable cells) was 69,8 %
  • Figure 19 shows comparison of photolummescence spectrum of Gold-Protein A interaction with Nano IgG for staphylococcal targeting.
  • Figure 20 shows comparison of photolummescence spectrum of Anti IgG Antibody Interaction with Nano IgG conjugated Gold-Protein A.
  • Figure 21 shows comparison of photo luminescence spectrum of Anti IgM Antibody Interaction With [Anti IgG-Nano IgG] Conjugated Gold-protein A.
  • Figure 22 shows comparison of photoluminescence spectrum of IgM Interaction With Anti IgM-conjugated onto [Anti IgG-Nano IgG] Conjugated Gold-protein A.
  • Figure 23 shows Schematic representation to IgM interaction with anti IgM antibody conjugated onto [anti IgG antibody-nano IgG] conjugated gold-protein A.
  • Figure 24 shows transmission emission microscope (TEM) image of Nano lipase particles according to an example of the invention.
  • Figure 25 shows Effect of pH on p-NPP hydrolysis using lipase and nanoparticles.
  • Figure 26 shows effect of temperature on p-NPP hydrolysis using lipase and nanoparticles.
  • Figure 27 shows Effect of mannose concentration on mannose adsorption.
  • Figure 28 shows Effect of IgM concentration on IgM adsorption.
  • Figure 29 shows comparison of fluorescence changing of nanoavidin nanoparticles interaction with biotin at 620 nm for 20°C.
  • Figure 30 shows comparison of fluorescence changing of antitubulin antibody nanoparticles interaction with cell fraction at 650 nm for 0 °C.
  • Figure 31 shows the quenching of emission by interaction of antiSOD-SPN and SOD fraction.
  • Figure 32 shows effect of concentration of TNFa on the TNFa antibody functionalized SPN.
  • Figure 33 shows Scatchart plot of Tnfa rebinding to Tnfa antibody functionalized SPN
  • Figure 34 shows flow-cytometry analyses for (Transferrin-folic acid)- iron oxide particle conjugate and HL60 cells interaction.
  • Figure 35 shows TEM images of transferrin and folic acid modified silanized ironoxide nanoparticles and 5RP7 cancer cells interaction.
  • Figure 36 shows fluorescence spectra of IgG functionalized QDs with the increasing of anti IgG antibody functionalized Au/Ag nanoclusters.
  • concentration of QDs were 7.7 x 10 "4 M and that of anti IgG antibody functionalized Au/Ag nanoclusters were 0.0, 5.5 x 10 "5 , 1.64 x 10 "4 , 2.73 x 10 "4 , 5.46 x 10 "4 M, respectively.
  • Figure 37 shows Biotin recognition by biotin imprinted Au/Ag nanoclusters : The effect of concentration of biotin on the biotin imprinted Au/Ag nanoclusters.
  • Figure 38 shows flow cyctometry analysis based on ambush and cell targeting prodrug therapy of transferin decorated and P450 conjugated QD particles and IFA conjugated and anti-transferrin decorated Au-Ag nanoclusters
  • Figure 39 shows TEM image of ambush and cell targeting prodrug therapy and HL-60 cancer cells interaction.
  • Figure 40 shows SEM images of (A) Lys-MAT doped acrylamide cryogel and (B) Lys- MAT doped acrylamide cryogel with M.Luteus fixation.
  • Figure 41 shows TEM image of photosensitive polymer doped silica nanoparticles
  • Figure 42 shows QCM Binding curve for 100 ⁇ g mL "1 biotin.
  • Figure 43 shows Response of the QCM sensor to biotin concentration
  • Figure 44 shows Adsorption isotherms of sialic acid on SPR sensors Unless defined otherwise, all technical ve scientific terms used herein have the same meaning as commonly understood by one of the familiar skills in the art to which the invention pertains. The following definitions supplement those in the art and are directed to the current applications and are not be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials, platforms and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be confined.
  • An "amino acid monomer” is monomer linker moiety, i.e. methacryloyl residue having (L)-tyrosine, trytophane or cysteine.
  • a “a residue” is a methacryloyl residue.
  • a "photosensitive ruthenium based aminoacid monomer chelate” is a hapten coordination compound that has at least one amino acid monomer as ligand and provides light induced covalent conjugation of biomolecules.
  • a “photosensitive oligomer (polymer)” is a sequence of ruthenium chelate based amino acid monomers and dissolve in aqua and provides light induced covalent conjugation of biomolecules on to micro and nanoplatforms.
  • a “biomolecule” is a biological macromolecule and typically a protein (an enzyme, an antibody) that has at least one tyrosine, tyrptophan or cysteine aminoacid.
  • a “platform” is a micro and nano template for biomolecule as sensor or carrier.
  • Common platforms in the context of the present invention include silica nanoparticles, carbon nanotues, Ag/Au nanoclusters, cadmium sulphide quantum dots, gold surfaces based on quartz crystal or surface plasmon resonanans, super paramagnetic nanoparticules, nanolayers of organoclay and the like.
  • ANADOLUCA is abbreviation of AmiNoAcid Decorated and Light Underpining Conjugation Approach. It is a novel concept approach which contains the novel syntetic materials and their applications.
  • Nano-enzyme is an enzyme developed in ANADOLUCA concept. This nano-enzyme is not soluble in solvents where it was activated and reusabled.
  • a nano-protein is a new generation polymeric material prepared in ANADOLUCA concept. This nano-protein can be use directly as a monomer (enzymes, antibodies or similar proteins) without using any other platforms. Additionally, different kind of drugs and biomolecules can be conjugated to this nano-protein.
  • Conjugation can be defined as the attachment of biomolecules to a micro and nano- surface without affecting conformation and function of protein. In some cases, conjugation may lead to partial or complete loss of protein activity, due to random orientation and structural deformation.
  • the present invention provides a covalent and photosensitive crosslinking conjugation of the antibodies and other proteins on micro- and nano-structures without affecting conformation and function of proteins through the aminoacid-monomer and/or ruthenium- chelate based aminoacid monomers linkages seems to be ANADOLUCA: the aminoacid- monomer linkages to give covalent bonds with tyrosine, cysteine or a tryptophane of antibodies and other biomolecules and may act as novel nanobioconjugate systems in the field of ligand-receptor interaction investigations for biosensors, synergie between diagnosis and therapie (theranostics) and biocatalysis applications.
  • a monomer having the function of a photosensitive bio-conjugator is provided, the monomer is oligo (or poly)-merized to prepare photosensitive oligo (poly)mer and/or the photosensive polymer is used to conjugate the proteins on to micro and nano-platforms.
  • the monomer according to the present invention is a novel methacryloyl and ruthenium based monomer having at least one aminoacid monomer ligand which conjugates proteins to a platforms.
  • the ruthenium-chelate based monomers include methacryloyl incorporating tyrosine, or trytophane as ligand and/or chlor or MUABt and other ligands, which can be presented by the following Formula 1-9.
  • the reaction mixture was stirred 3 h. After this perion white solid was filtered and washed with CH 2 CI 2 . The collected filtrate was extracted with 2 M NaOH (3x50 mL) to remove excess of Bt-H. The organic layer was dried with MgS0 4 , the solvent was removed under vacuum. The crude product was purified using ethyl acetate-hexane mixture over silicagel column to get MUA-Bt as white microcrystals in %87 yield.
  • MALDI-TOF-MS The ion peaks at 79, 128 and 155 m/e relating to bipyridyl. m/e 136, 293, 413 and 448 relating to RuCl, RubpyCl, Ru(bpy) 2 , and Ru(bpy) 2 Cl respectively, m/e 272, 408, 529 and 564 relating to MATrp, RuMATrpCl, Rubpy MATrp, RubpyMATrpCl. This data confirm that MATrpRu(bpy) 2 Cl structure was produced exactly.
  • MALDI-TOF-MS The ion peaks at 79 and 155 m/e relating to bipyridyl. m/e 272, 373, 413, 529 and 685 relating to MATrp, Ru-MATrp, Ru(bpy) 2 , Ru(bpy)-MATrp and Ru(bpy) 2 MATrp respectively.
  • MALDI-TOF-MS The ion peaks at 79, 128 and 155 m/e relating to bipyridyl.
  • m e 101, 413 shows Ru and Ru(bpy) 2 respectively
  • m/e 272 and 318 peaks show MATrp and MUABt monomers
  • m/e 419, 529 and 1003.1 peaks show Ru -MUABt
  • Ru(bpy) -MATrp and Ru(bpy) 2 MATrp- MUABt Chlorobis(2-2 '-bipyridyl) MATyr- ruthenium (II) (4- Figure 4): 1 eq RuCl 2 (bipyr) 2 was dissolved in methanol.
  • m/e 101 , 413 and 448 data show Ru, Ru(bpy) 2 and Ru(bpy) 2 Cl respectively, m/e 250 and 351 peaks show MATyr monomer and Ru- MATyr respectively.
  • MALDI-TOF-MS The ion peaks at 79, 128 and 155 m/e relating to bipyridyl. m e 101 , 413 peaks show Ru and Ru(bpy) 2 respectively, m/e 250, 599 and 755 data show MATyr monomer, Ru-(MATyr) 2 and -Ru(bpy)- MATyr complex.
  • MALDI-TOF-MS The ion peaks at 79 and 155 m/e relating to bipyridyl.
  • m e 101, 413 data show Ru and Ru(bpy) 2 respectively and m/e 250 and 575 data show MATyr monomer and MUABt-Ru(bpy) complex.
  • the photosensitive oligo(poly) mer according to the present invention is the oligomer including the repeating unit produced from photosensitive ruthenium based aminoacid- monomers and can be represented by the following Reaction 1.
  • the phosensitive oligomer of Reaction 1 can be prepared by a conventional polymerization reaction.
  • the photosensitive oligomer of Reaction 1 can be prepared according to the following polymerization Reaction 1.
  • the monomers for preparing the photosensitive oligomer such as Bis(2-2'-bipyridyl)-MATrp-MATyr ruthenium(II) monomer of Formula ( Figure 6) and other kinds of ruthenium based aminoacid monomers, are mixed by necessary amounts in organic solvent, and subjected to a polymerization reaction to obtain a reaction product.
  • organic solvent for the polymerization reaction includes acetone, dimethyl sulfoxide, dimethyl formamide, dioxane, acetonitrile, methanol, and so on.
  • the obtained reaction product can be crystalized in an organic solvent such as tetrahydrofurane (THF) to produce the photosensitive oligomer (water dissolve) of the present invention.
  • THF tetrahydrofurane
  • the polymerization reaction is preferably carried out in the presence of an initiator.
  • exemplary initiator includes 2-2'-azobisisobutyronitrile (AIBN), benzoyl peroxide (BPO), APS, and so on.
  • AIBN 2-2'-azobisisobutyronitrile
  • BPO benzoyl peroxide
  • APS APS
  • polymers contain about 15- 24 repeating units according to MALDI-TOF-MS analyses.
  • oligomer of Bis(2-2'-bipyridyl)-MATrp-MATyr ruthenium(II) has 19 monomer units ( Figure 10).
  • the acceleration voltage was set to 20 kV, the delay time was 400 ns, grid voltage % 70, laser intensity 2092 and at reflector mode.
  • nanocarrier platforms nanocargoes
  • This invention describes preparation and use of protein nanocarrier platforms (nanocargoes) that are designed to have low immunogenicity on their own to minimize the potential for antibody against them and to have cell receptors for targeting, drugs for therapie and imaging molecule and/or photosensitive hapten conjugates.
  • the resultant conjugates are important in bioconjugation research, nanotheranostic technology, drug targeting research, antibody purification and in immune research and creation of vaccines.
  • BSA carrier protein
  • small molecules like tyrosine, tyrotophane, cysteine or photosensitive ruthenium based aminoacid monomer chelates in the invention, as they are called, haptens can be made immunogenic by coupling them to nanocargoes and at the same time, photosensitive cross-linking and bioconjugation of other proteins like transferrin, HRP.
  • Polymeric particles can be constructed from a number of different monomers or copolymer combinations.
  • polymeric protein nanoparticles through photosensitive emulsion polymerization consist of polyaminoacid or ruthenium based polyaminoacid copolymers of protein like /MATyr/protein, MATrp/protein and MATyr-Ru (bpyr)2-MATyr/protein and its copolymer forms having ethylenglycol dimethacrylate (EDMA).
  • EDMA ethylenglycol dimethacrylate
  • the example describes the synthesis of the BSA nanocargo having BSA and aminoacid monomers like MATyr (or MACys and/or MATrp) by photosensitive microemulsion polymerization technique.
  • 0,5 g polyvinyl alcohol (PVA) was dispersed in 45 mL deionized water to form microemulsion system. In the synthesis, 25 mL of this dispersed solution of PVA was used.
  • 0,5 g MATyr was dissolved in 2 mL dimethylsulfoxide to prepare MATyr solution. 4 mg BSA was dissolved in 2 mL deionized water and then added into MATyr solution.
  • HRP-conjugated nano BSA cargoes for imaging and sensing BSA is a single polypeptide chain consisting of about 583 amino acid residues and no carbohydrates.
  • Carbodiimides are used to mediate the formation of amide linkages between carboxylates and amines (Hoare, D.G., Koshland, D.E., (1966) J. Am. Chem. Soc, 88, 2007).
  • the single-step method using EDC alone is appropriate for use in coupling molecules having one or more amines present without any carboxylates. If the molecule being coupled has both amines and carboxylates, such as proteins, then it best the two-step method.
  • Carboylate or carboxylate having particles can be coupled to amine-containing molecules using a number of reaction strategies.
  • amines of nanoBSA aminoacids can be activated by carboxylate of photosensitive monomer haptens like MATyr, MATrp, MACys, MATyr-Ru(bpyr) 2 - MATyr, MATrp-Ru(bpyr) 2 -MATrp, MACys-Ru(bpyr) 2 -MACys through amide bond formation in presence of EDC which reacts carboxylic groups of these aminoacid monomer haptens and the photosensitive aminoacid monomer hapten labeled BSA nanocargo to results in bio conjugation and cross-linking of HRP,
  • EDC horseradish peroxidase
  • HRP horseradish peroxidase
  • BSA which has been used like a nanocargo
  • HRP was added to give covalent bonds with tyrosine, cysteine or a tryptophane of transferrin through photopolymerization in presence of APS for targeting and labeling.
  • DAB working solution was prepared by dissolving 0,002 g DAB in distilled water and diluted to 2.0 mL (5 mM).
  • the solution of HRP (from Sigma) was prepared by dissolving 2 mg HRP in 5 ml distilled water, and then was stored at 4°C. Deionized water was added to 10 mg BSA nanocargoes for 200 ppm preparation of disperse solution. 250 ⁇ EDC 0,2 M was added to BSA nanocargo dispersed solution.
  • FIG. 12 shows fluorescence spectroscopy analysis of quenching of BSA nanoparticles with attach of HRP molecules.
  • 100 of HRP conjugated Nano BSA in 2 mL deionized water was excited at 310 nm, and emission was recorded at 621.01 nm. Fluorescence intensity value was measured as 288.422 (above peak).
  • HRP conjugated BSA was excited at 310 nm and emission was recorded at 621.01 nm. Fluorescence intensity value of it was measured as 72.8 (below peak).
  • Figure 12 shows increasing fluorescence intensity of HRP conjugated BSA nanoparticles with attach of HRP/DAB reaction. It was excited at 310 nm, and emission was recorded at 621.01 nm. Fluorescence intensity value was measured as 155.318 (middle peak).
  • DAB-based HRP reaction fistly, 2 mg DAB was dispersed in 5 ml dionized water and then it was filtered with a 0,2 ⁇ pore fitler unit and 200 was taken in it. After that 500 hydrogen peroxide was added into DAB solution and mixed in the sonicator for 30 min.
  • HRP was correctly conjugated to BSA nanocargoes, 10 ⁇ , DAB-H2O2 solution was added into 1 mg BSA-EDC/NHS-HRP nanoparticles and brownish reaction product was seen. Then nanoparticles were dispersed in 500 ⁇ , dionized water and then half amount of 50 ⁇ , DAB solution was added and brownish reaction product was seen again so HRP conjugated BSA process was succeed.
  • microemulsion media was prepared by dispersing 0,5 g of polyvinyl alcohol in 45 mL deionized water.
  • 2000 ppm of BSA, lipase and HRP and MATyr solutions (0,2 g mL "1 ) was prepared and mixed.
  • oligomer of ruthenium based hapten was added to mixture and mixed for 10 minute additionally. This mixture was added into 25 mL of polyvinylalcohol microemulsion media.
  • the activity of the heterobifunctional having BSAnanocargoes were analyzed spectrophotometrically measuring the increment in the absorption at 410 nm promoted by the hydrolysis of pNPP (Winkler and Stuckmann, 1979). For this purpose, firstly stock solution of subtrate containing 20 mM p-nitrophenyl palmitate (p-NPP) in isopropanol was prepared.
  • p-NPP p-nitrophenyl palmitate
  • working substrate was prepared by diluting the p-NPP stock solution (1 :20) using 20 mM Tris HCl buffer (pH 8) containing 0.1 mL Triton X-100 and activity of heterobifunctional BSA nanoparticles were measured by mixing 0.9 mL of working substrate and 60 of suspended nanoparticles in water. Activity of heterobifunctional nano BSA was found 9,5x10 "3 ⁇ paranitrophenol (product) / min (International Unit (IU)).
  • Nano transferrin particles was prepared by microemulsion polymerization technique by dispersing 0.5 g polyvinyl alcohol in 45 mL deionized water.
  • 200 ppm transferin solution was prepared by dissolving transferin in pH 7 phosphate buffer.
  • 100 ⁇ , of 0,2-1,0 g mL "1 MATyr-Ru(bipyr)2-MATyr complex was added into 200 ppm transferin solution and mixed for 2 h. This solution was added into 25 mL of dispersed polyvinyl alcohol media.
  • APS solution was prepared by 0.02 g APS in 45 mL deionized water as an initiator solution.
  • Nanotransferrin in 2,0 mL deionized water was excited at 260 nm, and emission was recorded at 521 ,04 nm. Fluorescence intensity value was measured as 168,390 (below peak). 16 ppm antitransferrin solution was excited at 260 nm, and emission was recorded at 521 ,04 nm. The fluorescence intensity value was measured as 431 ,005 (middle peak) and nanotransferrin-antitransferrin mixture was excited at 260 nm, and emission was recorded at 521 ,04 nm. Fluorescence intensity value was measured as 545,874 (above peak).
  • Antitransferrin was diluted in different concentration for displaying diluted antitransferin effect to change of fluorescence spectrum.
  • Figure 14 shows fluorescence spectroscopy analyses of 4 ppm (below peak), 8 ppm (middle peak) and 16 ppm (above peak) antitransferrin solution. Both of them were excited at 260 nm, and emission was recorded at 521,04 nm.
  • 16 ppm of the transferrin solution was prepared to interact with antitransferrin for comparing nanotransferrin-antitransferrin interaction and trans ferin-antitransferrin interaction.
  • Figure 15 shows fluorescence spectroscopy analysis of 16 ppm transferrin solution, 16 ppm antitransferrin solution and their mixture. Both of them were excited at 260 nm, and emission was recorded at 521 ,04 nm. Fluorescence intensity value of transferin was measured as 156,235 nm (below peak). Fluorescence intensity value of antitransferrin was measured as 431 ,005 nm (middle peak) and fluorescence intensity value of transferin-antitransferrin mixture was measured as 807.889 nm (above peak).
  • 0,06 g matrix media of nanotransferrin particles in [83] were dispersed in 500 ⁇ deionized water and then 100 ⁇ 0,1 M NHS and 100 ⁇ 0,4 M EDC were added into it and mixture was mixed for 2 h. 1 ⁇ g methotrexate was dissolved in DMSO and deionized water mixture (5: 1) and it was added into mixture and they were mixed for 24 h. Afterwards, solution was centrifuged and sediment was washed with deionized water for three times.
  • Figure 17 showed necrotic cells ratio (PI positive Annexin V negative presented in the left-upper quadrant (Ql) was 3,5 %, early apoptotic cells ratio (Annexin V positive PI negative in the right-lower of the Fig 17 (Q4) and late apoptotic cells ratio (Annexin V and PI positive in the right-upper quadrant (Q2) were respectively 4,2 % and 29,6 % and viable cells ratio (Annexin V and PI negative in the left-lower quadrant (Q3) was 62,7 %. In this manner Fig. 17 was demostrated these specific targeted nanotransferrin approach was killed the cancer cells and viable cells ratio was decreased.
  • Nano TNF-a was prepared by microemulsion polymerization technique.
  • Microemulsion system was prepared by dispersing 0,5 g polyvinyl alcohol in 45 m viable water. 25 mL of this mixture was used for synthesis.
  • 1000 ppm TNF-a solution was prepared in pH 7.0 PBS buffer. 40 ⁇ . of 0,2-1 ,0 g mL "1 MACys was added into TNF-a solution and that were stirred together for 4 h. TNF-a and MACys were added directly into PVA dispersion media. Then 50 ⁇ , of 1.0 mg mL "1 aqueous solution of P(MATrp-Ru(bipyr) 2 - MATyr) monomer hapten was added into the mixture.
  • initiator solution was prepared by dissolving 0.02 g of ammonium persulfate in 45 mL water. And 20 mL of this initiator solution was added into reaction media and mixed for 36 h at room temperature at daylight under nitrogen atmosphere. Nanoparticles were seperated from reaction media by centrifugation after mixing 20 h and washed with deionized water for three times to remove unreacted materials from nanoparticles occured. Nanoparticles were stored at 0°C until use. 0,05 g matrix media of nano was dispersed in 1 mL DMSO and 25 solution was taken in it and added into cell culture test plate that had 50 000 5RP7 cells/mL.
  • 25 DMSO was added into another section of cell culture test plate that had 50 000 5RP7 cells/ml to be DMSO control. Also 50 000 5RP7 cells/ml were added into another section of cell culture test plate without using any substance to be control. 25 ⁇ DMSO was not toxic in that cell culture condition. 5RP7 cells were stained with Annexin V-FITC and PI Apoptotic Detection Kit for flow cytometry analysis. Figure 18 showed necrotic cells ratio (presented in the left-upper quadrant (Ql) was 8,5 %, early apoptotic cells ratio (in the right-lower of the Fig.
  • NanoIgG carrier with photosensitive ruthenium hapten also be usefull in fluorescence detection and in tracking involves various bio-interactions.
  • Photosensitive ruthenium based aminoacid monomer hapten can be used in preparation of nanocarriers through photosensitive polymerization and conjugation, and in detection and imaging of nanocarriers and its interaction.
  • Nano Immunoglobulin G was prepared by microemulsion polymerization technique.
  • Microemulsion system was prepared by dispersing 0.5 g polyvinyl alcohol in 45 mL water. 25 mL of this mixture was used for synthesis. 2 mg of IgG was dissolved in 2 mL deionized water. 40 of 1.0 mgmL 1 MATyr-Ru(bipyr) 2 -MATrp complex was added to IgG solution and stirred together for 4 h.
  • IgG and MATyr-Ru(bipyr) 2 -MATrp complex was added directly to PVA dispersion media.
  • initiator solution was prepared by dissolving 0.02 g of ammonium persulfate in 45 mL water. And 20 mL of this initiator solution was added to reaction media and mixed for 24 h at room temperature at daylight under nitrogen atmosphere. Nanoparticles was seperated from reaction media by centrifugation and washed with deionized water three times to remove unreacted materials from nanoparticles occured. Nanoparticles was stored at 0°C until use.
  • the most extensive assay application of antibody conjugation using conjugation reagents is for the preparation of antibody-enzyme conjugates.
  • the antibody-enzyme conjugate assay system can be just as sensitive as a radiolabeled antibody system.
  • HRP is one of most popular enzymes used in enzyme-limked immunosorbent assay (ELISA) technology. In the example of the invention will be prepared reusable HRP conjugated nano antibody carriers.
  • IgG nanoparticles were dispersed in 500 deionized water. 250 0,4 M EDC solution was added into the IgG solution and that was mixed for 1 h on magnetic mixture. After that 250 ⁇ ⁇ MATyr solution was added and all that mixed for 24 h. The next day 200 ppm 500 ⁇ , horse radish peroxidase (HRP) solution was prepared and 250 ⁇ , 100 ppm APS, 250 ⁇ , of 2.0 mgmL "1 oligomerized polymer hapten of MACys-Ru(bipyr) 2 -Cl monomer and 500 ⁇ ⁇ HRP solution were added to mixture simultaneously. All of them was mixed for 24 h. And light microscopy image of HRP conjugated nano IgG particles shift brownish after DAB/H 2 0 2 reaction.
  • HRP horse radish peroxidase
  • Gold-Protein A interaction with Nano IgG for staphylococcal targeting 500 ⁇ , of pH 7.4 phosphate buffer solution was added to IgG nanoparticles. Then, 5 ⁇ , gold-protein A was added into 45 ⁇ , IgG nanoparticles and solution was mixed for a few minute.
  • Figure 19 shows quenching of IgG nanoparticles emission with interaction of gold protein A molecules.
  • 500 ⁇ , of Nano IgG in 2,5 mL pH 7.4 phosphate buffer solution was excited at 290 nm, and emission was recorded at 581.02 nm. Fluorescence intensity value was measured as 95.455 (below peak).
  • Protein A is a 40-60 kDa surface protein originally found in the cell wall of the bacteria Staphylococcus aureus. Protein A is a wall-anchored protein with either four or five domains that each bind to the Fc region of IgG [Timothy J. Foster, IMMUNE EVASION BY STAPHYLOCOCCI, Nature Publishing Group, (2005):948: 3]
  • Second phase Anti IgG Antibody Interaction with Nano IgG conjugated Gold-Protein A
  • Nano IgG conjugated Gold-Protein A We want to prove active fab region of nano IgG, in this reason we interact anti IgG antibody with Nano IgG conjugated gold-protein A.
  • 0,0003 g anti IgG antibody was dissolved in 500 ⁇ , pH:7,4 phosphate buffer solution. 50 ⁇ , anti IgG antibody solution was added into the 50 ⁇ , nano IgG conjugated Gold-Protein A and the solution was mixed for a few minute.
  • Figure 20 shows fluorescence spectroscopy analysis of anti IgG antibody interaction with nano IgG conjugated gold-protein A emission.
  • Second phase Anti IgM Antibody Interaction With [Anti IgG-Nano IgG] Conjugated Gold-protein A
  • This example was demostrated multi binding sites region of gold-protein A, firstly anti IgM antibody was interacted with [anti IgG antibody-nano IgG] conjugated gold-protein A.
  • 0,0006 g anti IgM antibody was dissolved in 100 ⁇ , pH:7,4 phosphate buffer solution.
  • 50 anti IgM antibody solution was added into the 100 [anti IgG antibody-nano IgG] conjugated Gold-Protein A. The solution was mixed for a few minute.
  • Figure 21 shows fluorescence spectroscopy analysis of anti IgM antibody interaction with [anti IgG antibody-nano IgG conjugated gold-protein A emission.
  • 500 ⁇ Anti IgM antibody interaction with [anti IgG antibody-nano IgG conjugated Gold-Protein A in 2,5 mL pH 7.4 phosphate buffer solution was excited at 290 nm, and emission was recorded at 338.00 and 581.02 nm. Fluorescence intensity value was measured as 250.723 and 151.840 (below peak in second spectrum) and peak at the 340.00 nm shows increasing and schifting to 338.00 nm. And peak at the 581.02 nm shows quenching through surface of nano IgG conjugated gold protein A after anti IgM interaction (Figure 23).
  • FIG. 22 shows fluorescence spectroscopy analysis of 500 ⁇ ⁇ IgM interaction with anti IgM antibody decorated and [anti IgG antibody-nano IgG] conjugated gold-protein A in 2,5 mL pH 7.4 phosphate buffer solution was excited at 290 nm, and emission was recorded at 336.00 and 581.02 nm.
  • Fluorescence intensity value was measured as 249.635 and 408.166 (above peak) at the 581.02 nm. And peak at 581.02 nm shows increasing as a result of IgM interaction with anti IgM antibody conjugated onto [anti IgG antibody-nano IgG]conjugated gold-protein A complex ( Figure 23). This peak shows increasing of nanoIgG that both anti IgM and anti IgG antibody conjugated to gold protein A after IgM interaction.
  • Nano lipase was synthesised according to microemulsion polymerization technique procedure.
  • Microemulsion system was prepared by dispersing 0.5 g PVA in 45 mL deionized water.
  • 0.5 g MATrp was dissolved in 5 mL of dimethylsulfoxide and added into lipase solution prepared by dissolving 10 mg of lipase in 5 mL deionized water.
  • 250 of 1.0 mg mL "1 of aqueous solution of polymer of MACys-Ru(bipyr)2-Cl ruthenium based monomer hapten was added into the mixture and mixed for 1 h. This mixture was added into 25 mL of PVA dispersing medium.
  • the nano lipase particles are spherical and between 40 -140 nm in diameter ( Figure 24).
  • Enzyme Activity Assay of nano lipase The activities of free lipase and nanoparticles were analyzed spectrophotometrically measuring the increment in the absorption at 410 nm promoted by the hydrolysis of pNPP (Winkler and Stuckmann, 1979). For this purpose, firstly stock solution of subtrate containing 20 mM p-nitrophenyl palmitate (p-NPP) in isopropanol was prepared.
  • p-NPP p-nitrophenyl palmitate
  • working substrate was prepared by diluting the p-NPP stock solution (1 :20) using 20 mM Tris HC1 buffer (pH 8) containing 0.1 mL Triton X-100 and activity of lipase and nanoparticles were measured by mixing 0.9 mL of working substrate and 0.1 mL of diluted enzyme sample (or 25 mg nanoparticles).
  • the H is one of the important parameters cabaple of alterning enzymatic activities in aqueous solution.
  • the optimum pH was determined applying different pH values ranging from 6,0-10,0 using 20 mM p-nitrophenylpalmitate.
  • V Vm. S / ( Km + S )
  • Hydrolytic activity of nanoparticles including lipase was evaluated in the framework of Michealis-Menten kinetics. For this purpose, paranitrophenyl palmitate substrate concentrations in the range of 2 mM-20 mM were used in each case and initial reaction rates (IRR) of hydrolysis were determined. Then, 1/IRR versus reciprocal of the substrate concentration (1/S) for nanoparticles was plotted that is called Lineweaver-Burk plot and the values of Vm and Km were obtained from plots as 0,72 mMmin "1 and 9,3 mM, respectively.
  • Km reflects the affinity of an enzyme for a particular substrate (the lower value of Km the higher affinity).
  • Km represents the affinity of functional groups of nanoparticles including lipase for substrate p- nitrophenyl palmitate.
  • the experimental cycle was repeated 5 times using the same particles.
  • the particles were washed with ethanol.
  • particles were washed with distilled water.
  • Reusability cycles could be repeated during at least 5 cycles without detecting and significant change in the enzyme activity. After 5 cycles, the enzymatic activity had decreased by ca. 5 %.
  • Nano lysozyme was prepared by microemulsion polymerization technique.
  • Microemulsion polymerization system was prepared by dispersing 0.5 g polyvinyl alcohol in 45 mL deionized water. Only 25 mL of this dispersed solution was used in nano lysozyme synthesis.
  • 0.5 g MATyr was dissolved in 5 mL of dimethylsulfoxide and added into lysozyme solution prepared by dissolving 10 mg of lysozyme in 5 mL deionized water.
  • Nano lysozyme was interacted with S. aureus and E. coli bacteria culture to test its antibacterial activity.
  • colony number in the culture was 154xl0 5 before interacted with nanolysozyme particles.
  • colony number of S. aureus bacteria was decreased and it was seen that 45xl0 5 . total decrease is % 71 for S. aureus and nanolysozyme was shown an antibacterial effect on S. aureus bacteria.
  • Lectins like conconavalin can be used as targeting molecules to localize particular glycoconjugates and as detecting molecules.
  • Nano Concanavalin A (ConA) was synthesized acoording to same procedure of microemulsion polymerization technique. First of all, 0.5 g polyvinyl alcohol was dispersed in 45 mL of water to create microemulsion polymerization media. 2 mg of Con A was dissolved in 2 mL of deionized water and 0,1 g mL "1 of MATrp was added into this Con A solution. They are mixed for 2 h. Then, 150 ⁇ xL of 1.0 mg mL "1 aqueous solution of P(MUABT-Ru(bipyr) 2 -MATrp) was added into the mixture.
  • initiator solution was prepared by dissolving 0.02 g of ammonium peroxodisulfate in 45 ml of water. 25 mL of this solution was added to reaction medium. All the reactants were mixed for 48 h at room temperature at daylight under nitrogen atmosphere. At the end of mixing period, nanoparticles was centrifuged and washed 4 times with deionized water to remove unreacted substances. Nano ConA particles was stored at 0°C until use.
  • FTIC conjugated nano concanavalin A 200 ppm
  • 1 m°C of NanoCon A dispersion was prepared by deionized water. 150 0.1 M of NHS and 150 0.4 M of EDC were added into 200 ppm Nano Con A dispersion and stirred.
  • fluorescein isothiocyanate solution was prepared by dissolving 0.0002 g fluorescein isothiocyanate (FTIC) in 1.5 mL of deionized water. 500 of this solution was added into NanoConA- NHS-EDC system and mixed all of them during 6 h. At the end of mixing period, reaction solution was centrifuged and precipitate was washed with deionized water until removing all unreacted fluorescein from the supernatant.
  • FTIC conjugated Nano Con A emission and Nano Con A 500 FTIC conjugated Nano Con A in 1 mL deionized water was excited at 250 nm, and emission was recorded at 500.00 nm. Fluorescence intensity value was measured as 995 and peak at 500.00 nm shows increasing from 790 to 995. And peak at 500.00 nm shows increasing as a result of FTIC conjugaton on to Nano Con A.
  • Mannose is a sugar monomer of the hexose series of carbohydrates and presents in numerous glycoconjugates including N-linked glycosylation of proteins. C-mannosylation is also abundant and can be found in collage-like regions. D-Mannose is a natural occurring simple sugar that appears to be a safe, practical alternative for the treatment of urinary tract infections (UTI's). UTIs start when Escherichia coli (E. Coli) invade the bladder and penetrate a protective coating of the superficial cells that line the bladder. In most cases, urine flow washes out bacteria from the bladder. But the cell wall of E. coli bacteria has tiny finger-like projections that contain complex molecules called lectins on their surface.
  • UTIs start when Escherichia coli (E. Coli) invade the bladder and penetrate a protective coating of the superficial cells that line the bladder. In most cases, urine flow washes out bacteria from the bladder. But the cell wall of E. coli bacteria has tiny finger-like projections that contain complex molecules called lectins on
  • D-Mannose cellular glue that binds the bacteria to the bladder wall so they cannot be easily rinsed out by urination.
  • the chemical structure of D-Mannose causes it to stick to E. coli bacteria, may be even more tenaciously than E.coli adheres to human cells.
  • D-mannose has been shown to reduce bacteria in rats in a dose dependent manner. In fact, D- mannose was found to significantly reduce bacteria in one day (Michaels et al, 1983).
  • Immunoglobulin M plays a central role in the initial response of the immune system to foreign antigens.
  • the serological diagnosis of many human infections is based on the determination of rising pathogen IgM titers during their early appearance (Nigra G, Mattia S, Midulla M. , Serodiagn Immunother Infect Dis 1989;3:355-61.; Punnarugsa V, Mungmee V., J Clin Microbiol 1991 :2209-12.; Chen P-J, Wang J, Hwang L, Yang Y, Hsieh C, Kau J, et al. Proc Natl Acad Sci U S A 1992;89:5971-5).
  • IgM content in human serum can be used to estimate immune function and is an important parameter for diagnosing acute and chronic hepatitis, rheumatoid arthritis, hepatocirrhosis, and malignant plasma cell tumors. Thus, a sensitive estimate of IgM is important in clinical laboratories.
  • IgM is a pentameric molecule in which each monomer consists of 14 immunoglobulin fold domains containing 5 -Asn- linked oligosaccharide sites located on the ⁇ heavy chain at residues 170, 332, 395, 402 and 563.
  • the carbohydrate composition of human IgM shows a high mannose content, being totally different from other serumimmunoglobulins. Because of this, mannose binding proteins (MBPs) (For example Con A) have been used for purification of immunoglobulin M (IgM) (Koppel R.; Solomon B. Journal of Biochemical and Biophysical Methods, Volume 49, Number 1, 30 October 2001, pp. 641- 647 (7) ).
  • nano-Con A was prepared for recognition of IgM.
  • IgM detection measurements were performed by fluorescence spectrophotometer (Fig. 28).
  • the specific IgM adsorption increased with the IgM intial concentration and reached a plateau (at around 5 ppm of IgM concentration), at which we may assumed that all the active points available for IgM adsorption were occupied with IgM molecules.
  • Nano avidin was prepared by microemulsion polymerization technique. Microemulsion system was prepared by dispersing 0.5 g polyvinyl alcohol in 45 mL deionized water. 25 mL of this mixture was used for synthesis. 2 mg of avidin was dissolved in 2 mL pH 7,0 phosphate buffer to prepare avidin solution. 40 ⁇ , MATyr-Ru(bipyr) 2 -Cl complex was added to avidin solution and stirred together for 4 h. Avidin- MATyr-Ru(bipyr) 2 -Cl complex was added directly to PVA dispersion media.
  • Antitubulin antibody nanoparticles were synthesised in micromemulsion polymerisation reaction media. 0.5 g polyvinyl alcohol dispersed in 45 mL deionized water to create the microemulsion polymerisation system. On the other hand, 1 mg antitubulin antibody was dissolved in 1 mL of pH 7 phosphate buffer solution. Beside this, MUABt-Ru(bipyr) 2 -MATrp complex solution was prepared by dissolving 0.0008 g of MUABt-Ru(bipyridyl) 2 -MATrp in 800 methanol. This complex solution was mixed antitubulin antibody during 1 h, then, added into 25 mL of dispersed polyvinyl alcohol dispersed media.
  • Figure 30 shows the emission changing of nano-antitubulin particles interaction with cell fraction at 650 nm.
  • Bradford Assay is a rapid and accurate method commonly used to determine the total protein concentration of a sample.
  • the assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible color change. Within the linear range of the assay (-5-25 mcgmL "1 ), the more protein present, the more Coomassie binds
  • the amount of proteins in the nano structures that were prepared using different photosensitive aminoacid monomer and monomer hapten and having different conjugates were determined by measuring the initial and final concentrations of protein within the adsorption medium using Bradford Assay. A calibration curve was constructed with protein solution of known protein concentration (0.02-0.25 mg mL "1 ) and was used in the calculation of protein amount.
  • SOD Superoxide dismutase separation by anti SOD antibody functionalized superparamagnetic nanoparticles
  • SPN superparamagnetic nanoparticles
  • the activated SPN was washed several times with ethanol and then phosphate buffer and then dried.
  • 200 phosphate buffer and 30 MATyr-Ru-MATyr were added onto silyl-activated SPN and then 50 ⁇ .
  • 200 ppm anti SOD antibody and 100 ⁇ . 0.1 mg mL "1 APS were added into this solution and mixed for three hour.
  • the SOD antibody functionalized SPN (anti SOD-SPN) particles were washed several times.
  • enzyme activity of SOD was controlled by a SOD assay method.
  • TNFa-TNFa antibody functionalized SPN was also controlled with UV spectrophotometry by using of Bradford analysis. Beginning TNFa solution and filtrate solution after the interaction with SPN were measured and a decreasing was observed between these two absorbances. TNFa-bound SPNs were washed with 0.1 M Glycine-HCl to obtain TNFa.
  • iron oxide-TSPM-MATyr solution was dispersed in 5 mL of phospate buffer (pH: 7) and interacted with 5 mL 50 ppm transferrin solutions, APS and 250 P(MATyr-Ru(bipyr) 2 -Cl solution. Mixture was mixed on a magnetic platform for 24 h. After transferrin was bound onto silanized ironoxide nanoparticles, folic acid solution was prepared to bind onto it. 0,0022 g folic acid was resolved in 100 mL of deionized water so 0,05 mM folic acid solution was prepared.
  • transferrin and folic acid modified silanized ironoxide nanoparticles were used to analyse of effect for cancer cells (5RP7 cancer cells) by flow cytometry.
  • 5RP7 cells were stained with Annexin V-FITC and PI Apoptotic Detection Kit for flow cytometry analysis.
  • Figure 34 showed necrotic cells ratio (presented in the left-upper quadrant, Ql) was 39.5%, early apoptotic cells ratio (in the right-lower of the Figure 34 (Q4) and late apoptotic cells ratio (in the right-upper quadrant (Q2) were 0.8 % and 6.7 %, respectively and viable cells ratio (in the left-lower quadrant (Q3) was 53 %.
  • CdS synthesis 0.01M of Cd(OAc) 2 -2H 2 0 solution (24 mL) was prepared with ethanol. Solution was stirred continuously for 30 min in nitrogen ambient. Sodium sulfide (0.01 M, 24 mL) was slowly added, stirred under nitrogen ambient for 30 min and then centrifuged to collect precipitate. It was washed in double distilled water and dried in air. The entire synthesis was carried out at room temperature (Diltemiz, S.E., Say, R., Buyuktiryaki, S., Hur, D., Denizli, A., Ersoz, A., (2008) Talanta 75, 890-896). CdS/QD were excited at 310 nm, and emission was recorded at 620.0 nm. Fluorescence intensity value was measured as 301,9. a. QD's Functionalization with MACys monomer
  • Qunatum dots functionalized with MUABt-Ru(bpy) 2 -MATrp complex 1 mL was taken from CdS solution and added to 1 mL MUABt-Ru(bpy) 2 -MATrp solution (5 mg mL "1 ), diluted with ethanol to 20 mL and mixed during a day. Fluorescence value was measured by 20 times dilution. CdS/QD were excited at 310 nm, and emission was recorded at 621.01 nm. Fluorescence intensity value was measured as 251. MUABt-Ru(bpy) 2 -MATrp modified nanoparticle's fluorescence intensity was decreased according to CdS. c. QD-MACys's Functionalization with MATyr monomer
  • MACys modified qunatum dots functionalized with MATyr monomer 1 mL was taken from CdS-MACys solution and added to MATyr solution which was dissolved in (5,08 mg) 1 mL water, diluted with ethanol to 20 mL and mixed during a day. Fluorescence value was measured by 20 times dilution. CdS/QD were excited at 310 nm, and emission was recorded at 621.01 nm. Fluorescence intensity value was measured as 426.830. MATyr modified nanoparticle's fluorescence intensity was decreased according to CdS- MACys. d. QD-MACys's Functionalization with MATrp monomer
  • MACys modified qunatum dots functionalized with MATrp monomer 1 mL was taken from CdS-MACys solution and added to MATrp solution which was disolved in (5,78 mg) 1 mL water, diluted with ethanol to 20 mL and mixed during a day. Fluorescence value was measured by 20 times dilution. CdS/QD were excited at 310 nm, and emission was recorded at 621.01 nm. Fluorescence intensity value was measured as 319.620. MATrp modified nanoparticle's fluorescence intensity was decreased according to CdS- MAC. e. QD-MAC's Functionalization with MATrp-Ru(bpy) 2 -MATrp complex
  • MACys modified qunatum dots functionalized with MATrp-Ru(bpy) 2 -MATrp complex 1 mL was taken from CdS-MACys solution and added to 1 ml MATrp-Ru(bpy) 2 -MATrp solution which was disolved in (5,00 mg) 1 mL of water, diluted with ethanol to 20 mL and mixed during a day. Fluorescence value was measured by 20 times dilution. CdS/QD were excited at 310 nm, and emission was recorded at 621.01 nm. Fluorescence intensity value was measured as 451.960.
  • MATrp-Ru(bpy) 2 -MATrp complex modified nanoparticle's fluorescence intensity was decreased according to CdS-MACys.
  • MACys modified qunatum dots functionalized with MUABt-Ru(bpy) 2 -MATrp complex 1 mL was taken from CdS-MACys solution and added to 1 mL MUABt-Ru(bpy) 2 -MATrp solution which was disolved in (5,00 mg) 1 mL of water, diluted with ethanol to 20 mL and mixed during a day. Fluorescence value was measured by 20 times dilution. CdS/QD were excited at 310 nm, and emission was recorded at 622.02 nm. Fluorescence intensity value was measured as 328.062. MUABt-Ru(bpy) 2 -MATrp complex modified nanoparticle's fluorescence intensity was decreased according to CdS-MACys.
  • Qunatum dots functionalized with MATyr monomer 1,0 mL was taken from CdS- MACys solution and added to MATyr solution which was dissolved (5.08 mg) in 1,0 mL of water and mixed during a day. Fluorescence value was measured by 50 times dilution. CdS/QD were excited at 310 nm, and emission was recorded at 620.0 nm. Fluorescence intensity value was measured as 667.989.
  • these particules were seperated into four parts and 1 ml 10 ⁇ 2 ppm, 10 "1 ppm, 0,2 ppm, 0,5 ppm, 0.8 ppm and 1,0 ppm IgM solutions added to these parts, respectively.
  • the fluorescence intensity values of these interactions were measured in reference to dilution factor.
  • the fluorescence intensity of the Concanavalin A interacted CdS/QD nanoshells can be enhanced by IgM concentration.
  • the enhancement of fluorescence intensity is proportional to IgM concentration.
  • the polymeric shell having QD solution was centrifuged and diluted to 1 mL with pH 7,4 PBS buffer and interacted with 150 ⁇ ⁇ Hepatite B vaccine. These mixtures mixed for one hours, centrifuged and dispersed to 3 mL with PBS and fluorescence intensity values were measured. Fluorescence intensity value was measured as 285.057. When the Anti Hepatit B conjugated and polymerised shell having QDs interacted with Hepatite B vaccine, fluorescence intensity was decreased.
  • IgG functionalized QDs 2 mg MACys activated QDs were dispersed in 3 mL phosphate buffer and 3 mg MATyr was added to this solution and mixed for 3 h. End of this period, 100 ⁇ , of ruthenium based hapten oligomer, 0.8 mg IgG and finally 100 ⁇ , 0.1 mg mL "1 APS were added into this solution and mixed for 3 h. Then the particles were isolated with centrifigation and washed several times with de-ionized water, and alcohol.
  • Au-Ag nanocluster was synthesized according to Ref. (Gultekin A, Diltemiz SE, Ersoz A, et al, TALANTA (2009) 78, 4-5, 1332-1338). 50 mg Au-Ag nanocluster was dispersed in 1 mL toluen and 1,0 mM MACys was added. This solution was mixed 24 h for methacryloyl activation. The activated Au-Ag nanostructure was washed several times with toluen and then dried.
  • biotinylated TNF-a antibody functionalized Au-Ag nanoparticles 5.0 mg mL "1 , 20 MACys-Ru(bipyr) 2 -MACys was added onto methacryoyl-activated Au-Ag nanoparticles and then 200 ⁇ , 1.0 mg mL "1 biotinylated TNF-a antibody and 50 100 mM APS were added into this solution and mixed for 3 h.
  • the biotinylated TNF-a antibody functionalized Au-Ag nanoparticles was washed with phosphate buffer several times.
  • the particles were isolated with centrifguation and mixed with 5 mL 0.1 M NaOH for 24 h for removal of biotin template and then washed several times with deionized water, and alcohol. Finally, the biotin imprinted nanostructures were dispersed in 3 mL phosphate buffer and stored.
  • Ag-Au particles were modified for prodrug therapy.
  • Ag-Au particles were modified with MACys solution (as explained in Interaction between avidin and biotinylated TNF-a antibody conjugated Au-Ag nanoclusters).
  • 1 mL MATyr solution was added onto 1 mL Ag-Au-MACys solution and mixed all of them for 2 h.
  • 50 ppm antitransferrin solution was prepared in phosphate buffer and 1 mL antitransferrin solution, 100 ⁇ ⁇ of ruthenium based polymer solution and 100 ⁇ ⁇ 100 mM APS were added onto MATyr modified Ag-Au-MACys solution. All of them were mixed on magnetic mixer for 24 h.
  • prodrug solution was prepared to add onto solution. Ifosfamide (IF A) was used like a prodrug and 0,007275 g IFA was resolved in 25 ⁇ , deionized water. Then, IFA solution, 1 mL of 0,1 M NHS and 1 mL of 0,4 M EDC solutions were added onto antitransferrin conjugated Ag-Au nanostructure and all of them were mixed on magnetic mixer for 24 h.
  • IF A Ifosfamide
  • necrotic cells PI positive
  • early apoptotic cells annexin V positive and PI negative
  • late apoptotic and/or dead cells annexin V positive and PI positive
  • viable cells no staining.
  • Figure 38 showed necrotic cells ratio (presented in the left-upper quadrant, Ql) was 0.1 %
  • early apoptotic cells ratio in the right-lower of the Figure 38, Q4
  • late apoptotic cells ratio in the right-upper quadrant (Q2) were respectively 34.4 % and 15.2 %
  • viable cells ratio in the left-lower quadrant, Q3 was 50.3 %.
  • Lysozyme-MATyr (Lys-MATyr) doped acrylamide cryogels were prepared by free radical cryo polymerization of monomer solution of Acrylamide with crosslinker N, N'- Methylene bis acrylamide (MBAAm) initiated by N, N, N', N'- Tetramethyl- ethylenediamine (TEMED) and Ammonium per sulfate (APS) in plastic petri dish.
  • Lys-MATyr Lysozyme-MATyr
  • TEMED N, N', N'- Tetramethyl- ethylenediamine
  • APS Ammonium per sulfate
  • the same amount of lysozyme was dissolved in 5 mL of deionized water. 3 mL of each solution were taken and mixed 5 h at 250 rpm at room temrepature.
  • AAm monomer and cross - linker (MBAAm) were dissolved in deionized water with stirring. The monomer concentration was 7% (w/v) with an AAm/MBAAm molar ratio of 10: 1. Then, Lys-MATyr mixture (2 % (w/v) of the total mass of AAm+MBAAm) added the solution and mixed at 150 rpm during 30 min. Then, solution was cooled for 3-4 min. in an ice bath.
  • antibacterial cryogel material was weight and then devoted to two equal weights in the sterile fresh tubes. 5 mL nutrient broth was added to all tubes. And then one serial of these tubes were inoculated with Escherichia coli from the overnight culture and the other serials were inoculated with S. aureus from the overnight culture. An uninoculated tube (5 m inoculated Nutrient broth) was observed for blank. For the positive control, S.aureus and E.coli were inoculated tubes. After incubation at 37°C for 24 hours, all tubes were mixed by vortex. Supernatant were taken and measured at 540 nm together negative controls (blank) and positive controls (Absorbance of S.
  • Figure 40 shows SEM images of (A) Lys-MAT doped acrylamide cryogel and (B) Lys-MAT doped acrylamide cryogel with M. Luteus fixation.
  • Annexin V-FITC modification For the Annexin V-FITC modification, MAArg and photosensitive hapten functionalized CNT was dispersed with 1 mL of deionized water and mixed with 10 Annexin V- FITC, 20 binding buffer and 100 mM 50 APS. Then solution was mixed for 24 h. At the end of 24 h solution was centrifuged at 10000 rpm for 10 min and sediment was washed with deionized water. After that sediment was dispersed in 10 mL PBS pH 7.4. Activity of Annexin V-FITC modified CNT was tested with human cheek cells by using fluorescence microscope. The inner lining of human cheek was gently scraped. The cells were transfered into a eppendorf tube that had 50 ⁇ ⁇ PBS pH 7.3.
  • Annexin V-FITC modified CNT was added into the eppendorf tube and cells were incubated for 15 min. Afterward 25 ⁇ ⁇ sample was taken to observe activity of Annexin V-FITC modified CNT with using Leica fluorescence microscope. According to cell images demostrated living cell was not labeled with Annexin V-FITC modified CNT, only apoptotic cells was labeled. In conclusion, Annexin V-FITC modified CNT was active to interacted with phosphatidylserine side on the apoptotic cell surface.
  • organosmectite For the preparation of organosmectite, 20 g of the simectite was dispersed in 500 mL deionized water at 80°C. Then, concentrated 5 mL of HC1 in 100 mL deionized water was added, and the solution was heated and stirred for 3 h. The suspension was filtered, and the solid residue was washed with hot distilled water. The product was dried at 55°C for several days in a fan oven, then dried under vacum for 24 h.
  • working substrate was prepared by diluting the p-NPP stock solution (1 :20) using 20 mM Tris HC1 buffer (pH 8) containing 0.1 mL Triton X-100 and activity of lipase in the layers of smectite measured by mixing 0.9 mL of working substrate and 60 ⁇ , of suspended nanoparticles in water.
  • the experimental cycle was repeated 5 times using the same particles.
  • the particles were washed with ethanol.
  • lipase conjugated organoclay were washed with distilled water. Reusability cycles could be repeated during at least 5 cycles without detecting and significant change in the enzyme activity. After 5 cycles, the enzymatic activity had decreased by ca.7 %.
  • Gold surfaces of QCM electrodes were cleaned in a piranha solution (1 :3 30 % H 2 0 2 / concentrated H 2 S0 4 ) for 3 min before coating.
  • the cleaned gold surfaces were immersed into MACys solution (10 mM) for 24 h, in order to introduce thiol groups on to gold surface of QCM sensors.
  • the sensors were then washed with ethanol and deionized water for 10 min. to remove the excess of thiols. A stable self-assembled monolayer of thiol was formed on to electrode surfaces after all these steps.
  • the reaction mixture containing the (MATyr-Ru(bipyr)2-MATyr) monomer, avidin (100 ⁇ g) and APS (2.5 mmol) in HBS buffer was degassed and dropped on QCM sensors. Polymerisation was carried out at room temparature for 4 h.
  • the crystal was mounted in the holder / flow cell, rinsed with pH 7.4 HBS buffer (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA) and brought to resonant frequency.
  • Biotin was dissolved in HBS buffer (pH 7.4) to have a concentration from 0 - 40 ⁇ g mL "1 and pumped through the flow cell at 0.1 mL min "1 .
  • the frequency of the sensor was monitored until it became stable. The frequency shift for each concentration of biotin was determined and the evaluation was performed in triplicate.
  • biotin was removed from the coating by washing with 0.1 M glycine-HCl (pH: 1.8) (0.5 mL min "1 , 60 min) and then three times with HBS buffer. The frequency of the sensor approximately recovered to the value of beginning resonant frequency.
  • the avidin coated sensor is expected to bind the biotin sensing.
  • the frequency of the sensor decreased after pumping the biotin solution, then reached the constant value in 30 min ( Figure 42). It can be seen that the reaction reached equilibrium quickly. These frequency changes strongly indicated that the biotin molecules were bounded to the polymer on the quartz crystal.
  • the sensors were located in SPR system and HEPES buffer was injected to baseline.
  • reaction mixture containing the sialic acid (1 mmol), 0.4 M EDC and 0.1 M NHS was injected with flow rate of 5 ⁇ mm 1 .
  • the sensor surface was rinsed with HEPES buffer solution and regenerated with 0.1 M glycine-HCl buffer solution (pH: 2.0) for removal of sialic acid, then washed again with water.
  • the adsorption isotherm of sialic acid on polymer coated sensor can be obtained by plotting the observed reflectivity as a function of the time (Figure 44). As seen in figure reflectivity increased at approximately 15 th min. because of interaction between concanavalin A with sialic acid.

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Abstract

La présente invention concerne une préparation de monomères et d'oligomères d'acides aminés à base de ruthénium photosensibles, la réticulation de monomères d'acides aminés et de protéines au moyen de la photosensibilisation et la conjugaison sur micro- et nanostructures par des monomères à base de chélate de ruthénium. Sa large gamme d'applications biotechnologiques concerne des micro- et nano-bioconjugués multifonctionnels, biocompatibles, stables et spécifiques, qui seront autonomes ou permettront simultanément (i) purification et détermination, (ii) ciblage, imagerie, et théranostique et (iii) catalyse et détermination. La construction et le procédé de préparation sont applicables aux matériaux siliceux, aux particules superparamagnétiques, aux points quantiques (QD), aux nanotubes de carbone (CNT), aux nanoparticules Ag/ Au, aux surfaces Au et aux matériaux polymères. Les coupleurs d'acides aminés photosensibles peuvent réagir de façon chimique et biocompatible à beaucoup de micro- et nano-surfaces différentes puis à la protéine lorsqu'ils servent de réaction de réticulation à étape unique par rayonnement. La conjugaison photosensible basée sur la biochimie « click » peut être effectuée dans des conditions légères, indépendantes du Ph et de la température, sans affecter la conformation et la fonction de la protéine.
PCT/IB2009/055707 2009-12-11 2009-12-11 Liaison acide aminé/monomère photosensible et applications de bioconjugaison en sciences biologiques et en biotechnologie WO2011070402A1 (fr)

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