WO2008105902A2 - Nouvelles nanoparticules pour ciblage de biofilm - Google Patents

Nouvelles nanoparticules pour ciblage de biofilm Download PDF

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WO2008105902A2
WO2008105902A2 PCT/US2007/073529 US2007073529W WO2008105902A2 WO 2008105902 A2 WO2008105902 A2 WO 2008105902A2 US 2007073529 W US2007073529 W US 2007073529W WO 2008105902 A2 WO2008105902 A2 WO 2008105902A2
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biofilm
protein cage
protein
ccmv
cage comprises
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WO2008105902A3 (fr
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Trevor Douglas
Mark J. Young
Peter Suci
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Montana State University Bozeman
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/38011—Tombusviridae
    • C12N2770/38022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/38011—Tombusviridae
    • C12N2770/38023—Virus like particles [VLP]

Definitions

  • Proposed treatments include development of agents that will inactivate biofilm subpopulations that are tolerant to antimicrobial agents as a result of genetic programming [Lewis 2001] and induction of biofilm detachment by introduction of signaling molecules [Davies 2003]. Practical application of any of these strategies will require diagnosis of the infection as a biofilm infection, identification of the pathogen, and methods for monitoring the success of the treatment program. Non-invasive imaging techniques will play a prominent role in this overall treatment process. [0006] One relatively simple strategy for controlling biofilm infections may be early detection followed by appropriate treatment with a conventional antimicrobial agent.
  • Figure 2 shows a) the hexamers and pentamers of a CCMV subunit arranged with icosahedral symmetry, b) a cryoimage of a CCMV cage, and c) the location of sulfhydryls of cys residues incorporated into CCMV; and d) carboxyl groups on surface exposed glu residues of CCMV.
  • Figure 3 is an antibody titration curve representing the murine immune response to genetically modified CCMV (right) compared to keyhole limpet hemocyanine (left).
  • Figure 6 is a schematic showing three possible binding configurations for antibody-conjugated CCMV in the cell wall of S. aureus.
  • the present invention is based, in part, on the discovery that certain materials, such as nanoparticles, are useful as therapeutic agents against, or for the imaging of, biofilm infections. Accordingly, the present invention provides compositions and methods useful for the targeting and/or imaging of biofilm infections.
  • 0023J According to one aspect of the present invention, it provides a method of targeting a biofilm. The method comprises contacting a biofilm with a composition comprising a protein cage or a protein cage aggregate.
  • the protein cages or protein cage aggregates can be any cage suitable for targeting a biofilm.
  • the protein cage comprises a viral, non-viral or bacterial protein.
  • the protein cage is based on robust platform nanotechnology which uses the self-assembled protein cage architecture derived from viruses and other cage-like architectures for targeted delivery of imaging and antimicrobial agents to biofilm forming bacterial colonies.
  • the size and monodispersity of many protein cages make them ideal platforms for specific targeting of biofilms with image contrast agents. Advances in nano- engineering have greatly increased our ability to finely tune the presentation of functional groups on selected cages. This expanding capability provides new opportunities to optimize targeting specificity, incorporate antimicrobials into cages and conjugate imaging agents to cages.
  • Antibody coupling increases the mean diameter to approximately 52 nm, which falls within the ideal size range to achieve the optimal balance between efficient clearance and vascular exudation.
  • CCMV is used for targeted delivery of contrast agents to both more accessible biofilm infections (e.g., vascular graft infections) and those that are more sequestered from the blood pool (osteomyelitis).
  • MR molecular imaging e.g., MR molecular imaging
  • the monodispersity should substantially enhance the predictability of hematogenous transport and clearance behavior; and 6) Genetically modified and multiply labeled CCMV preparations maintain their integrity over long periods of storage.
  • the protein cage comprises at least one modified subunit. In another embodiment, it comprises at least two modified subunits. In yet another embodiment, the protein cage comprises more than one type of modified subunit, for example, a chemically modified subunit or a genetically modified subunit.
  • the protein cage comprises one or more targeting moieties.
  • the protein cage comprises at least two targeting moieties. Examples of targeting moieties include, but are not limited to, polypeptide targeting moieties or antibody targeting moieties.
  • the protein cage comprises CCMV, the nanoscale dimensions of which are used for engineering optimal multiple binding functionalities onto a particle that can deliver desired levels, for example, high concentrations, of desired moieties to sites of biofilm infection.
  • the protein cage comprises CCMV to which antibodies are conjugated and/or a paramagnetic contrast agent is loaded. Such a protein cage can, for example, deliver high concentrations of paramagnetic material to sites of biofilm infection.
  • the protein cage comprises a guest material.
  • the inner surface of the assembled protein cage architecture provides an ideal interface for either covalent or electrostatic attachment of therapeutic and medical imaging agents, which are then encapsulated within the protein cage and therefore sequestered from the exterior environment (e.g. not bioactive against non-targeted cells and tissues).
  • Antimicrobial agents are covalently attached to functional groups uniquely located on the interior surface through cleavable linkers. Antimicrobial agents can be packaged within the cage architecture through interactions (electrostatic, hydrophobic) with the interior surface, using diffusion as a release mechanism. Alternatively, a catalyst capable of producing an antimicrobial agent such as reactive oxygen species (ROS) can be incorporated within the cage either through covalent or electrostatic interactions. Photodynamic therapy agents which are effective for the light driven destruction of microbial biofilms can also be incorporated within the cage.
  • ROS reactive oxygen species
  • the Ab-conjugate presentation can be manipulated to optimize the binding specificity of cages to a model biofilm. Affinity of functionalized protein cages for biofilm associated epitopes is enhanced by engineering optimal antigen binding site presentation, while affinity associated with non-specific interactions remains unaffected. As a consequence, it is possible to obtain a high level of specific labeling of biofilms by the cage-bound contrast agent for MR imaging of biofilm infections at a relatively low dosing level.
  • Ab presentation can be manipulated in two ways: by varying the density of presentation of a single Ab, thus altering the possibilities for multivalent binding; and by conjugating two different Ab to the cage, which introduces the possibility of dual valent binding.
  • the invention further provides a protein cage that comprises a reversible switch.
  • the switch can be any switch that controls the structure of the protein cage and operation of the switch changes the physical or chemical nature of the protein cage.
  • the reversible switch switches the protein cage between a static open state, in which, for example, the protein cage exists in an open or swollen form that allows external material access to its cavity, and a static closed state, in which, for example, the protein cage exists in a closed form that prevents external material from accessing its cavity.
  • the reversible switch is a pH-dependent switch.
  • the protein cages of the invention can additionally comprises at least one hydrolase cleavage site.
  • the hydrolase cleavage site can be located in any subunit of the protein cage, or on the interior or the exterior of the protein cage.
  • the hydrolase can be any hydrolase known to one of skill in the art and generally classified as an EC 3 enzyme.
  • the hydrolase is a protease, e.g., trypsin or cathepsin.
  • the biofilm infection targeted by the methods of the invention can be any biofilm infection arising from or associated with any now known, or later discovered source.
  • the biofilm comprises Staphylococcus aureus (Sa) bacteria.
  • Sa is a human pathogen that plays a prominent role in nosocomial infections [USDHHS 1996; Central Public Health Laboratory 2000]. Sa forms biofilms as part of its adaptation to life in the host. It is well adapted to adhere to both tissues and biomaterials coated with blood proteins via a set of adhesins known as microbial surface components recognizing adhesive matrix molecules (MSCRAMM) [Harris 2002; Navarre 1999].
  • Sa biofilms form readily in vitro and in vivo on biomaterials [Williams 1997; Gracia 1998; Luppens 2002; Kadurugamuwa 2003; Wu 2003]. Compared to other bacterial biofilm-formers, Sa biofilms exhibit resistance to antimicrobial agents that is extraordinary both in terms of the spectrum of antimicrobial agents and the doses that can be tolerated [Olson 2002]. Biomaterial- centered infections are a clear example of biofilm pathogenesis and historically Staphylococcus epidermidis and Sa have been prominent players [Dankert 1986; Gristina 1987].
  • the biofilm arises from a nosocomial or HIV- related infection.
  • the biofilm is associated with an endocarditis- related or osteomyelitis-related infection.
  • the biofilm is adhered to tissues or biomaterials, in vitro or in vivo, or the biofilm is adhered to surgical implants or grafted biomaterial. Further, the biofilm can be an antibiotic-resistant biofilm.
  • the present invention provides a method of imaging a cell, tissue, or biofilm.
  • the method comprises contacting a cell, tissue, or biofilm with a medical imaging composition comprising a protein cage or a protein cage aggregate.
  • the medical imaging composition penetrates the cell, tissue, or biofilm.
  • the medical imaging composition further comprises a medical imaging agent such as those described herein.
  • the method further comprising rendering an image of the cell, tissue, or biofilm.
  • the image can be rendered by any imaging technique known to one of skill in the art. Exemplary methods of rendering an image include, but are not limited to, MRJ, NMR, x-ray, optical ultrasound and neutron capture therapy.
  • ATR-FTIR attenuated total reflection Fourier transform infrared spectroscopy
  • Figure 6 shows schematic representations drawn approximately to scale showing possible binding configurations for Ab conjugated CCMV (Ab-CCMV) onto the cell wall of Sa. StAv, streptavidin; SpA, protein A; Fc and Fab segments of the Ab are indicated; Figure 6a shows indirect conjugation via StAv; Figure 6b shows direct Ab conjugation (whole Ab); and Figure 6c shows direct Ab conjugation (Fab' or Fab).
  • ATCC Se strains 29213, 12598 and 10832 and Pseudomonas aeruginosa PAOl (CBE collection) will be used for experiments.
  • ATCC 29213 is SpA (protein A) positive [Bernardo 2002] and a biofilm former [Harrison 2004].
  • Cowan I (ATCC 12598) and Wood 46 (ATCC 10832) strains will serve as positive and negative controls for SpA expression, respectively [Warm 1999].
  • P. aeruginosa PAOl will be used as a negative control in some experiments.
  • TSB tryptic soy broth
  • FIG. 7a shows indirect (StAv mediated) conjugation between biotinylated CCMV-SH (CCMV-S- B) and biotinylated SpA-Ab or Pg-Ab.
  • Figures 7b, c, d show alternative methods for direct conjugation in anticipated order of increasing complexity;
  • Figure 7b shows broken squares indicate desirable direct conjugation options for constructing and purifying CCMV presenting dual valency.
  • SMCC (Pierce product 22322) and sulfo-SMCC (Pierce product 22360) (water soluble) contain maleimide (mal) and succinimidyl ester (SE) groups that link free sulfhydryls with primary amines; EDC and NHS react with carboxyl groups to create an active ester intermediate that reacts with primary amines; if reaction with lysines of Ab does not interfere with the antigen binding site (ABS) then this is probably the simplest direct conjugation scheme for both SpA-Ab (to CCMV-SH) and Pg-Ab (to CCMV carboxyl groups); CCMV carboxyl groups were previously activated with EDC/NHS and reacted with primary amines of functional groups with no cross-linking between CCMV particles [Gillitzer 2002].
  • CCMV-SH will be pre-labeled with an amine reactive group (either a fluorescent label or the Gd-chelating agent), then reacted with aminoethyl-8 (N-(iodoethyl) trifluoroacetamide) (Pierce product 23010) that converts free sulfhydryls to amines.
  • EDC activates carboxyl groups to create an active ester intermediate that will react with the hydrazide group of KMUH to form an imide bond; the maleimide of KMUH will react with free sulfhydryls.
  • Figure 8 shows construction of CCMV with different densities of multivalent presentation ( Figure 8a) and with dual valent presentation ( Figure 8b). Symbols are essentially the same as in Fig. 7. (+) refers to "wild type" CCMV.
  • Mixed reassembly (Fig. 8a) will be used to construct CCMV having different densities of exposed free sulfhydryls where the mean density is controlled by the input ratio (m:n) of CCMV-SH (S 102C) and CCMV(+) monomer subunits.
  • any successful direct conjugation method (Figure 7b-d) can be used. Purification of excess CCMV from Ab-CCMV will be done using a Protein A affinity column (Pierce product 20356); Size exclusion chromatography (SEC) will be used to remove excess Ab from the purified preparation if necessary.
  • SEC Size exclusion chromatography
  • Figure 8b shows CCMV with dual valent presentation will be prepared by using CCMV multivalent SpA-Fab' preparations as starting material; then Pg-Ab (either 1/2 Ab or whole Ab) will be conjugated to the carboxyl groups using a successful method presented in Fig. 7 b-d.
  • a nickel-chelate affinity column (Pierce product 44920) will be used to purify excess SpA-Ab-CCMV (produced by steps outlined above) from CCMV conjugated to both SpA-Ab and Pg-Ab.
  • CCMV particles remain assembled under elution conditions for the protein A affinity column (0.15 M NaCl, pH 2.8) and nickel-chelate affinity column (0.1 M sodium acetate, pH 5.0) (verified by DLS and TEM) and we previously used a similar method to confer asymmetry on intact CCMV-SH particles (Al 63C) [Klem 2003]. If the elution conditions for the protein A affinity column disrupt the SpA-Ab ABS the ImmunoPure Gentle Ag/Ab Elution Buffer (Pierce product #21013) will serve as an alternative. [0072] Fluorescent labels and the Gd chelating agent will both be attached via
  • Fluorescence per cell conferred by binding of fluorescein labeled CCMV-S-B and fluorescein labeled StAv (StAv-F) from a commercial source (e.g., streptavidin, fluorescein conjugate Molecular Probes, S869) will be compared. Labeling ratio of the StAv-F will be obtained spectroscopically.
  • CCMV-S-B will be labeled using published methods [Gillitzer 2002]. Side scattering will be used to quantify cells.
  • Cell DNA will be labeled with a cell permeant fluorescent nuclear stain (SYTO 62, Molecular Probes, Sl 1344, 637/660) [Yarwood 2004; Strathmann 2004].
  • Example 11 Optimize binding of Gd chelating agent to CCMV-S-B
  • Gd will be coupled to CCMV-S-B and loading will be optimized. This has been mostly completed using wild type CCMV.
  • DOTA clinically relevant chelating agent
  • Approximately 520 lysines can be labeled using the NHS ester ([Gillitzer 2002]).
  • Gd-DOTA to a lysine decamer and then coupling the peptide to CCMV via the N- terminus using the EDC/NHS reaction [Hermanson 1996; Gillitzer 2002]. This will increase the contrast enhancement by 5 to 10 times depending on the rigidity of the CCMV coupled peptide. There is precedent for this chemical modification [Uzgiris 2004].
  • Example 13 Compare MRI contrast of biofilms obtained with Gd-CCMV-S-B and a commercially available targeted contrast agent using SpA-Ab-B/StAv mediated cell binding [0079] We will assess the performance of CCMV as a delivery vehicle for
  • MRI contrast agent to biofilms Coupling via DOTA will be used for Gd loading onto CCMV-S-B (Example 11).
  • indirect conjugation StAv mediated
  • Biofilms will be cultured in a flow cell compatible with MRI measurements [Seymour 2004] (Fig. 4c).
  • a custom made accessory allows the flow cell (1 mm square glass capillaries) to be placed in the magnet with attached tubing so that biofilms can be exposed to flowing medium in situ.
  • a T- valve regulates flow from either of two sources enabling acquisition of MR images of biofilms before and during exposure to Gd- CCMV-S-B.
  • This methodology will be used to compare MRI contrast of biofilms obtained with Gd-CCMV (non-biotinylated control), Gd- CCMV-S-B and commercially available 50 nm diameter superparamagnetic biotinylated nanoparticles designed to allow targeted StAv mediated delivery of a T 2 MRI contrast agents to cells (mMACSTM Streptavidin Kit, Invitrogen) [Artemov 2003]. Similar experiments have been performed using Gd coupled to wild type CCMV.
  • SpA-Ab (not biotinylated) will be activated by attaching a maleimide group to lysines using SMCC or sulfo-SMCC (conjugation to amines via NHS ester).
  • Amount of Ab covalently bound to CCMV will be assessed by SDS- polyacrylamide gel electrophoresis (SDS-PAGE) (4-15% Tris-HCI precast gel, Bio-Rad Laboratories) and size exclusion chromatography (SEC) (Bio-Silect 125-5 column, Bio-Rad Laboratories) under conditions in which CCMV dissociates into monomer subunits (100 niM TRIS, pH 7.6).
  • SDS-PAGE SDS- polyacrylamide gel electrophoresis
  • SEC size exclusion chromatography
  • Example 15 Construct and characterize CCMV-S with different densities of multivalent presentation
  • CCMV-SH and CCMV wild type will be dissembled, mixed in known ratios and reassembled.
  • CCMV wild type or -SH
  • TRIS pH 7.6
  • CaCl 2 300 mM
  • TCEP reducing agent
  • RNase A 5 ⁇ l
  • SpA-Ab will be conjugated to the mixed reassembly products using a successful direct conjugation method ( Figures 7b-d). Excess cage will be removed from the reaction mixture by protein A affinity chromatography as in Example 14 and excess SpA-Ab will be removed by SEC if necessary. Amount of Ab covalently bound to CCMV will be determined as in Example 14 for each multivalent preparation. Flow cytometry will be used to assess SpA-Ab mediated CCMV binding to Sa cells using methods outlined in Example 10.
  • Example 16 Characterize biotinylated Pg-Ab TPg-Ab-B) StAv mediated binding of fluorescently labeled CCMV-S-B to Sa planktonic cells using flow cytometry
  • Example 10 CCMV-S-B. Analogous to Example 10, this will confirm that the simplest binding scheme confers Pg-Ab mediated CCMV binding to planktonic ATCC 29213, providing a starting point for assessment of the influence of dual valency on Ab-CCMV binding to biofilms. Methods are as described in Example 10. According to the supplier, the Pg-Ab binds to Se cells that are SpA negative and is species specific. Thus the positive control will be the SpA negative Wood 46 strain and the negative control with be P. aeruginosa PAOl .
  • Fluorescent labeling of Ab-CCMV preparations and biofilm culturing in microwells will be the same as in Example 12.
  • Preparation of biofilms for CSLM will be the same as in Example 12 except that biofilms will not be exposed to StAv.
  • binding time courses having 5 time points in triplicate for 6 different preparations of Ab-CCMV having different densities of Ab (multivalent) presentation. The reasonable time points will be 5, 10, 30, 60 and 120 min.
  • binding studies different wells in the 96 microtiter well plate will be used to test the effect of different bulk concentrations of multivalent preparations of SpA- Ab-CCMV on binding to biofilms.
  • Example 18 Obtain relationship between bulk concentration and binding to immobilized ECM for SpA-Ab-CCMV possessing various densities of SpA-Ab
  • Example 19 Characterize MRI contrast of biofilms obtained with multivalent SpA-Ab- CCMV exhibiting the best binding characteristics
  • Example 1 The methods for MRI measurement to biofilms were presented in Example 13.
  • Example 20 Obtain relation ship between bulk concentration and binding to biofilms cultured in an annular reactor for SpA-Ab-CCMV exhibiting the best binding characteristics
  • the biofilm annular reactor provides a means to grow biofilms under conditions that contrast sharply with those in the microwells [Goeres 2005].
  • the annular (CTC) reactor is similar to the rotating disk reactor [Yarwood 2004; Lin 2004] with the advantage that coupons can be inserted or removed during the course of biofilm development. Whereas microwells provide a low shear batch growth environment, the annular reactor creates a high shear, continuous culture environment in which residence time can be controlled independently of shear rate.
  • Polycarbonate coupons (1 cm diameter) colonized with Sa bio film will be removed into 24 well microtiter wells and exposed to Ab-CCMV preparations (as in Examples 12 or 17), and removed into new wells sequentially for rinsing, fixing, and staining with SYTO 62, the nuclear stain, similar to a previous Sa biofilm study [Lin 2004].
  • Analysis using CSLM will be similar to Examples 12 and 17 except that biofilms will be viewed from the bulk liquid side instead of the base.
  • SpA-Fab' will be produced by the standard protocol of: 1) digestion of SpA-Ab with immobilized pepsin (Pierce 20343) to obtain F(ab') 2 [Hermanson 1996] followed by 2) reduction with 2-mercaptoethyliamine to obtain Fab' [Hermanson 19961.
  • the strategy for conjugation of SpA-Fab' (or Fab produced by papain digestion) to CCMV will be essentially the same as for the whole SpA-Ab (Example 14).
  • Example 24 Obtain relationship between bulk concentration and binding to biofilms and ECM for CCMV presenting dual SpA-Ab/Pg-Ab valency
  • Example 25 Characterize MRI contrast of biofilms obtained with dual and multivalent SpA-Ab-CCMV exhibiting the best binding characteristics
  • Example 19 This example is similar to Example 19 - we will accumulate data indicating how the different Ab-CCMV preparations perform in an in vitro system that is closest to the actual clinically relevant system. As for Example 19, since the MRJ bio film measurements are relatively time consuming, selected Ab-CCMV exhibiting the best binding characteristics will be chosen from among all the preparations tested thus far.
  • Example 26 Compare MRI contrast potential of mineralized subE and a commercially available targeted contrast agent
  • CCMV loaded with superparamagnetic iron oxide [Artemov 2003] as an MRI contrast agent.
  • SPIO superparamagnetic iron oxide
  • a genetic construct of CCMV offers the possibility for loading with SPIO.
  • SPIO loaded subE it would be possible to conjugate biotin or Ab to the cage via lysines.
  • Ti and T 2 values for bulk preparations of SPIO a mineralized subE will be compared with bulk preparations of optimally loaded Gd-CCMV obtained in Example 1 1 and a commercial product (Example 13).
  • SubE will be mineralized by air oxidation of 0.5 mg/mL protein with 25 mM ferrous ammonium sulfate (pH 6.5) [Douglas 2002].
  • Example 27 Alternate approach for characterization of the influence of multivalent Ab presentation on binding of Ab-CCMV to Sa biofilms
  • Characterization of the influence of multivalent Ab presentation on binding of Ab-CCMV to Sa biofilms can be achieved even without a suitable direct conjugation by using indirect (StAv mediated) conjugation, we have the tools to proceed with characterization of the influence of multivalent Ab presentation on binding of Ab- CCMV to Sa biofilms (analogous to Examples 16 and 17 above).
  • CCMV-S-B having different densities of biotin will be used as starting material.
  • Her-21neu receptor in breast cancer cells using targeted iron oxide nanoparticles were targeted.
  • Hybrid virus-polymer materials 1. Synthesis and properties of PEG-decorated cowpea mosaic virus. Biomacromolecules 4:472-6.
  • Tyson GW Chapman J, Hugenholtz P, Allen EE, Ram RJ, Richardson PM, Solovyev W, Rubin EM, Rokhsar DS, Banfield JF. (2004) Community structure and metabolism through reconstruction of microbial genomes from the environment. Nature 428:37-43. USDHHS. (1996) Centers for Disease Control and Prevention. National Nosocomial Infection Survielance System report: data summary from October 1986- April 1996. Atlanta. (GA)
  • Vancraeynest D Hermans K, Haesebrouck F. (2004) Genotypic and phenotypic screening of high and low virulence Staphylococcus aureus isolates from rabbits for biofilm formation and MSCRAMMs. Vet Microbiol 103:241-7.

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Abstract

La présente invention concerne de nouvelles compositions et des procédés utilisant des nanoparticules comprenant des cages protéiques pour la délivrance d'agents antimicrobiens et d'imagerie à des colonies bactériennes de formation de biofilm.
PCT/US2007/073529 2006-07-14 2007-07-13 Nouvelles nanoparticules pour ciblage de biofilm Ceased WO2008105902A2 (fr)

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WO2010028013A3 (fr) * 2008-09-02 2010-06-24 University Of Maryland, Baltimore Diagnostic d'infection de biofilm in vivo et traitement
US8541006B2 (en) 2007-07-30 2013-09-24 University Of Maryland, Baltimore Methods and devices for the detection of biofilm
US8865857B2 (en) 2010-07-01 2014-10-21 Sofradim Production Medical device with predefined activated cellular integration
US9247931B2 (en) 2010-06-29 2016-02-02 Covidien Lp Microwave-powered reactor and method for in situ forming implants
US9510810B2 (en) 2009-02-21 2016-12-06 Sofradim Production Medical devices incorporating functional adhesives
US9775928B2 (en) 2013-06-18 2017-10-03 Covidien Lp Adhesive barbed filament

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US6759229B2 (en) * 2001-12-18 2004-07-06 President & Fellows Of Harvard College Toxin-phage bacteriocide antibiotic and uses thereof
WO2003096990A2 (fr) * 2002-05-17 2003-11-27 Montana State University Cages proteiques pour l'administration d'agents therapeutiques et d'imagerie medicale
US20060014136A1 (en) * 2004-07-14 2006-01-19 Inimex Pharmaceuticals, Inc. Method of screening for protection from microbial infection

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US8541006B2 (en) 2007-07-30 2013-09-24 University Of Maryland, Baltimore Methods and devices for the detection of biofilm
WO2010028013A3 (fr) * 2008-09-02 2010-06-24 University Of Maryland, Baltimore Diagnostic d'infection de biofilm in vivo et traitement
US8697375B2 (en) 2008-09-02 2014-04-15 University Of Maryland, Baltimore In vivo biofilm infection diagnosis and treatment
WO2010035009A1 (fr) * 2008-09-24 2010-04-01 Ucl Business Plc Cages protéiques constituées de douze pentamères
US9510810B2 (en) 2009-02-21 2016-12-06 Sofradim Production Medical devices incorporating functional adhesives
US9247931B2 (en) 2010-06-29 2016-02-02 Covidien Lp Microwave-powered reactor and method for in situ forming implants
US8865857B2 (en) 2010-07-01 2014-10-21 Sofradim Production Medical device with predefined activated cellular integration
US9775928B2 (en) 2013-06-18 2017-10-03 Covidien Lp Adhesive barbed filament

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