Patent Application
20090093700
The invention relates generally to the field of imaging systems, and more particularly to the imaging of objects. More specifically, the invention relates to an apparatus and method enabling analytical imaging of objects (for example, small animals and tissue) in differing modes, including bright-field, dark-field (e.g., luminescence and fluorescence), x-ray and radioactive isotopes, and enhanced magnetic resonance imaging (MRI), by the use of injectable diagnostic agents for infrared and multimodal medical imaging.
Reference is made to commonly assigned, co-pending (a) regular U.S. patent application Ser. No. 11/400,935 (Docket 91687) filed Apr. 10, 2006 by Harder et al entitled “FUNCTIONALIZED POLY(ETHYLENE GLYCOL)”; (b) regular U.S. patent application Ser. No. 11/165,849 (Docket 88835CIP) filed Jun. 24, 2006 by Bringley et al entitled “NANOPARTICLE BASED SUBSTRATE FOR IMAGE CONTRAST AGENT FABRICATION”; and (c) regular U.S. patent application Ser. No. 11/872,866 (Docket 92735) filed Oct. 16, 2007 by Zheng et al entitled “ACTIVATABLE IMAGING PROBE USING NANOPARTICLES”, all of which are incorporated by reference in this application. Reference also is made to commonly assigned U.S. Pat. Nos. 6,444,988 and 7,031,084, which are incorporated by reference in this application.
Electronic imaging systems are well known for enabling molecular imaging. The electronic imaging system 10 shown in
U.S. Pat. No. 6,495,812 discloses an apparatus used in the analysis of fluorescent markers attached to biological materials. In the apparatus disclosed the light source, a laser, and detector are mounted on the same movable device to allow the light beam and the focal point of the detector to intersect at the object of interest carried on a separate stage compensating for the variations in the thickness and material used to hold the sample on the stage. This device is limited to biological materials on a slide (e.g., strands of DNA). In addition the light source is of a fixed wavelength, that of the laser. U.S. Patent Publication No. 2004/0004193 discloses a fluorescent image capture device with a filter wheel disposed in the illumination path. The device disclosed is limited to the capture of only a fluorescent image. U.S. Patent Publication No. 2000/148846 discloses a fluorescent image capture device with a filter wheel disposed between the object being illuminated and the capture device. The device disclosed is limited to the capture of only a fluorescent image. U.S. Patent Publication No. 2005/0175538 discloses a device for collecting light emitted from an animal where a luminescent reporter has been injected into the animal. The device disclosed is limited to the capture of only a luminescent image.
To increase the effectiveness of these electronic imaging systems, efforts have been focused upon developing nanoparticulate systems capable of delivering imaging agents directly to the cells of interest. These nanoparticles can carry biological, pharmaceutical or diagnostic components within living systems. These nanoparticulate systems typically comprise drugs, therapeutics, diagnostics, biocompatibilization functionalities, contrast agents, and targeting moieties attached to or contained within a nanoparticulate carrier. Work in this field has the goals of affording imaging and therapeutic agents with such profound advantages as greater circulatory lifetimes, higher specificity, lower toxicity and greater therapeutic effectiveness. Work in the field of nanoparticulate assemblies has promised to significantly improve the treatment of cancers and other life threatening diseases and may revolutionize their clinical diagnosis and treatment.
Certain nanoparticles have been proposed as carriers for certain pharmaceutical agents. See for example: Sharma et al. Oncology Research 8, 281 (1996); Zobel et al. Antisense Nucl. Acid Drug Dev., 7:483 (1997); de Verdiere et al. Br. J. Cancer 76, 198 (1997); Hussein et al., Pharm. Res., 14, 613 (1997); Alyautdin et al. Pharm. Res. 14, 325 (1997); Hrkach et al., Biomaterials, 18, 27 (1997); Torchilin, J. Microencapsulation 15, 1 (1988); and literature cited therein.
The nanoparticle chemistries provide for a spectrum of rigid polymer structures, which are suitable for the encapsulation of drugs, drug delivery and controlled release. Some problems of these carriers include aggregation, colloidal instability under physiological conditions, low loading capacity, restricted control of the drug release kinetics, and synthetic preparations which are tedious and afford low yields of product.
The size of the nanoparticulate assemblies is one parameter determining their usefulness in biological compositions. After administration in the body, large particles are eliminated by the reticuloendothelial system and cannot be easily transported to the disease site (see for example, Volkheimer, Pathologe 14:247 (1993); Kwon and Kataoka, Adv. Drug. Del. Rev. 16:295 (1995).
Moghimi et al (Moghimi, S. M.; Hunter, A. C.; Murray, J. C. “Nanomedicine: Current Status and Future Prospects.” FASEB Journal 2005, 19, 311-330.) reports that particles larger than 100 nm are susceptible to clearance by interstitial macrophages while particles of 150 nm or larger are susceptible to accumulation in the liver. Also, the transport of large particles in the cell and intracellular delivery is limited or insignificant. See for example, Labhasetwar et al. Adv. Drug Del. Res. 24:63 (1997). It is suggested that an aggregated cationic species with a size from 500 nm to over 1 micron are ineffective in cell transfection. Large particles, particularly, those positively charged exhibit high toxicity in the body, in part due to adverse effects on liver and embolism. See for example Volkheimer, previously cited; Khopade et al Pharmazie 51:558 (1996); Yamashita et al., Vet. Hum. Toxicol, 39:71 (1997).
Specific nanoparticles have been found to be nontoxic, and are capable of entry into small capillaries in the body, transport in the body to a disease site, crossing biological barriers (including but not limited to the blood-brain barrier and intestinal epithelium), absorption into cell endocytic vesicles, crossing cell membranes and transportation to the target site inside the cell. The particles in that size range are believed to be more efficiently transferred across the arterial wall compared to larger size microparticles, see for example, Labhasetwar et al., previously cited. Without being bound by any particular theory, it may be that a high surface to volume ratio enables more successful targeting.
U.S. Patent Publication No. 2003/0211158 discloses microgels, microparticles, typically 0.1-10 microns in size, and related polymeric materials capable of delivering bioactive materials to cells for use as vaccines or therapeutic agents. The materials are made using a crosslinker molecule that contains a linkage cleavable under mild acidic conditions. The crosslinker molecule is exemplified by a bisacryloyl acetal crosslinker. The materials have the common characteristic of being able to degrade by acid hydrolysis under conditions commonly found within the endosomal or lysosomal compartments of cells thereby releasing their payload within the cell. The materials can also be used for the delivery of therapeutics to the acidic regions of tumors and sites of inflammation. These particles, however, are of a large enough size range that uptake by the reticuloendothelial system can be expected to be a problem. In addition, the degree of PEGylation is low and in-vivo agglomeration has been identified as a problem (see for example, Kwon, Y. J.; Standley, S. M.; Goh, S. L.; Frechet, J. M. J. Journal of Controlled Release 2005, 105, 199-212).
Some authors have described the difficulty of making stable dispersions of surface modified particles. Achieving stability under physiological conditions (e.g., pH 7.4 and 137 mM NaCl) is yet even more difficult. Burke and Barret (Langmuir, 19, 3297 (2003)) describe the adsorption of the amine-containing polyelectrolyte, polyallylamine hydrochloride, onto 70-100 nm silica particles in the presence of salt. The authors state (p. 3299) “the concentration of NaCl in the solutions was maintained at 1.0 mM because higher salt concentrations lead to flocculation of the suspension.”
WO 2004/108902 discloses using a biocompatible fluorescent nanoparticle imaging probe as part of a method using the Kodak 1D v.3.6.3 software (Kodak Imaging System) for dual modality imaging. The probes disclosed are single imaging probes, for example a fluorescent imaging probe and a different probe used for X-ray imaging. Rather than having both imaging modalities on one probe, the two different probes are injected into the subject at the same time or in quick succession.
Some fluorescent dyes have small separation between the energy of the absorption and emission, (often 10-30 nm at maximum commonly referred to as Stokes shift) so there is significant overlap in the spectral curves for absorption and emission. Filters can be used to insure that the excitation energy is not being detected during the measure of emission energy. The filters are often broad band pass filters (for example plus or minus 20 nm) or narrow band pass filters (for example plus or minus 10 nm). Because of this limitation, dyes are often excited at a higher energy than is optimum and the emission is measured at a lower energy than is optimum, which results in the need for more excitation energy from the light source and greater sensitivity from the detector or CCD. Some dyes have large bandwidths (>100 nm) and others have narrow bandwidths (<50 nm). For dyes with small Stokes shifts and narrow bandwidths, it is difficult to get efficient excitation and emission. Typical instruments have limited capability to choose the wavelength of excitation through the energy output of the light source and choice of the filter, which can create situations where fluorescent dyes cannot be used because too little energy is absorbed or too much energy must be filtered from emission. The combination of an instrument that will allow the variable selection of the excitation energy with fluorophores that absorb from 300 nm to 900 nm is desirable.
An embodiment of an apparatus for multimodal imaging of an object in accordance with the invention may include a support stage for receiving an object to be imaged; an object supported on the stage, a light source for producing a beam to illuminate the object; a filter positioned to receive and pass the beam toward the object; and a lens and camera system for capturing a first image of the object. The filter may be tiltable relative to the beam and may be an interference filter or an acousto-optic tunable filter. A pair of AOTFs may be cascaded, whereby the beam is doubly filtered. An interference filter may be cascaded with an AOTF. In the latter embodiment, the interference filter may one in which tilt relative to the beam affects the spectral band that the interference filter passes and may further comprise means for tilting the filter relative to the beam.
Another embodiment of an apparatus for multimodal imaging of an object in accordance with the invention may include a light source; a support stage for receiving an object to be imaged; means for selectively directing light from the light source through a first filter assembly to produce a first beam of light of a first frequency range for illuminating an object on the stage in a first imaging mode or through a second filter assembly to produce a second beam of light of a second frequency range for illuminating an object on the stage in a second imaging mode; and a lens and camera system for capturing light from the object illuminated by either the first or second beam of light to produce a first image in response to the first beam and a second image, different from the first image, in response to the second beam. A third imaging mode may be provided by including an x-ray source and phosphor plate for producing light for capture by the camera and lens system.
The foregoing and other objects, features, and advantages of the invention will be apparent from the following more particular description of the embodiments of the invention, as illustrated in the accompanying drawings. The elements of the drawings are not necessarily to scale relative to each other.
The invention will described in detail with particular reference to certain embodiments thereof, but it will be understood that variations and modifications can be effected within the spirit and scope of the invention.
In the complex pharmaceutical analyses of images of small objects or subjects, such as small animals and small volumes of tissue in larger animals, it is advantageous to obtain images, which are particularly enhanced by using different in-vivo, imaging modalities. Using the current practices of bright-field, dark-field and radiographic imaging for the analysis of small objects or subjects (such as a mouse) can be expensive and may not provide the precision of co-registered images that is desired.
By treating the animals or tissues with imaging probes which are configured to be the multimodal biological targeting units and using an apparatus and a method of the present invention, precisely co-registered dual modality imaging units comprised of nanoparticles within a subject animal or tissue can be localized; and multiple images from various imaging modalities can be obtained and accurately overlaid onto a simple bright-field reflected image of the same animal or tissue within minutes of animal immobilization or placement of a tissue sample.
The present invention uses an integrated imaging system to capture images using differing imaging modes including multi-spectral illumination, thereby enabling simplified multi-modal imaging. More particularly, using the imaging system of the present invention, an immobilized object, such as an animal or tissue sample, can be imaged in several imaging modes without changing or moving the immobilized object. These acquired multi-modal images can then be merged to provide one or more co-registered images for analysis.
Imaging modes supported by the method, apparatus and probes of the present invention include: x-ray imaging, bright-field imaging, dark-field imaging (including luminescence imaging, fluorescence imaging) and radioactive isotope imaging. Images acquired in these modes can be merged in various combinations for analysis. For example, an x-ray image of the object can be merged with a near infrared (NIR) fluorescence image of the object to provide a new image for analysis.
An apparatus for multi-modal imaging, suited for use in accordance with the present invention, is shown in
Continuing with regard to
Phosphor plate 125 is mounted slideably for motion toward and away from sample object stage 104, such as on guide rails or rollers, not illustrated. While those skilled in the art might recognize other configurations, in one embodiment, phosphor plate 125 is mounted for sliding translation in the direction of arrow A relative to frame 120, beneath the sample, and in intimate contact with the underside of support sheet 122, as illustrated. As will be more particularly described below, in first imaging position P1 shown in
Plate 125 further comprises a phosphor layer 130 that transduces ionizing radiation to visible light practically captured and managed by a lens and a camera system 18, such as a CCD camera. Phosphor layer 130 can have a thickness ranging from about 0.01 mm to about 0.1 mm, depending upon the application (i.e., soft x-ray, gamma-ray or fast electron imaging). On the underside of phosphor layer 130, as illustrated, an optical layer 132 is provided for conditioning emitted light from phosphor layer 130. Optical layer 132 can have a thickness in the range of less than about 0.001 mm. Particular information about phosphor layer 130 and optical layer 132 is disclosed in commonly assigned U.S. Pat. No. 6,444,988. A supporting glass plate 134 is provided for plate 125. Glass plate 134 is spaced at a suitable mechanical clearance from an optical platen 126, for example, by an air gap/void 136. In one embodiment, the surfaces of clear optical media (e.g., a lower surface of glass plate 134 and both surfaces of optical platen 126) are provided with anti-reflective coating to minimize reflections that may confuse the image of the object.
In the multi-spectral light source shown in
Referring again to
Communication/computer control system 20 positions appropriate bandpass interference filter 235a, b, c, or d into the path of light beam 240 depending on what spectral band of light is chosen for a first beam to illuminate the subject/object 112 on the sample object stage 104. The wavelengths of the bandpass interference filters in bandpass interference filter assembly 230 can range from 400 to 800 nm, each having a spectral bandpass of between 10 to 20 nm, for example. Portions of the spectral bandpass of the filters can overlap if desired. Light beam 240a, now filtered to the desired spectral band, is directed via a first angled mirror 260 and a second angled mirror 265 to lens 275, which focuses light beam 240a onto an optical fiber input 280 that transmits the light to illuminate a the subject or object 112 on the sample object stage 104 (as shown schematically in
Acousto-optic tunable filters (AOTF) have previously been investigated as alternatives to spectrometers and for spectral selection of fluorescent emission, but have not been used or proposed as a method or device to selectively tune within a wide wavelength range of excitation light, for example from a xenon lamp, for fluorescence imaging. The following description concerns an embodiment of an illumination system suitable for exciting fluorescent contrast agents for optical molecular imaging.
Now referring to
Because the light generated by xenon lamps is substantially unpolarized, the light from the xenon lamp input to the AOTF crystal in the selected spectral band results in both the +1st diffracted order and −1st diffracted order beams being output. Both the −1st diffracted order and the +1st diffracted order beams can be used by recombining them after the undiffracted 0th order beam is dumped or trapped, thus providing the advantage of maximal light intensity in the selected spectral band relayed to the object/subject as needed for high-speed fluorescence imaging. Alternatively, the input light may be linearly polarized either horizontally or vertically with respect to the AOTF crystal, for example by a polarizer, if only a simplified optical relay geometry is desired; in this case, vertical linearly-polarized light would be desirable due to the constant deflection angle over the tuning range of the device which enables the most simplified optical relay geometry.
For this embodiment, optimum spectral purity of the tuned light was desired. In order to maximize spectral purity, the −1st diffracted order and +1st diffracted order beams, which contain the light in the selected spectral band, must be spatially separated from the undiffracted 0th order beam that contains the light outside the selected spectral band. The maximum allowable divergence (full angle) was determined by the diffraction angle of the AOTF as Δθi=Δθ. If Δi is larger than Δθ, the diffracted and undiffracted beams will overlap and efficient separation will be impossible. All three of the output beams have the same divergence as the input beam up to the acceptance angle of the crystal, which in this exemplary system was greater than 8°. In order to separate the undiffracted 0th order beam from the −1st and +1st diffracted order beams, the divergence half-angle of the input light preferably should be no greater than half the separation angle between the undiffracted 0th order beam and the −1st and +1st diffracted order beams. The beams begin to diffract and separate angularly shortly before exiting the crystal. Depending on the cross-sectional area and the divergence of the beams, the beams need to propagate some distance before the undiffracted 1th order beam and the −1st and +1st diffracted order beams are spatially separated. To ensure that the divergence half-angle of the input light will be no greater than half the separation angle between the undiffracted 0th order beam and the −1st and +1st diffracted order beams, first the separation angle of the undiffracted 0th order beam is maximized by appropriate selection of the AOTF, and then as necessary the divergence of the source is reduced. For example, a xenon lamp having a divergence half-angle of 4° and an AOTF having a separation angle of 4.2°, hence half of the separation angle 2.1°, between the undiffracted 0th order beam and the −1st and +1st diffracted order beams requires the divergence of the light from the xenon lamp needs to be reduced before entering the AOTF. This can be achieved by using a pair of lenses 215 and 225 with a spatial filter 222 at their common focus. The diameter of the spatial filter in combination with the ratio of the focal lengths of the lenses may be used to select the half-angle of the divergence of the resulting beam, preferably to be only slightly less than half of the separation angle between the undiffracted 0th order beam and the −1st and +1st diffracted order beams to avoid excessive rejection of useful light input to the AOTF.
Furthermore, it is often desirable to reduce Δθi as λ is a function of Δθi as shown in the equation
meaning that a Δθi larger than several degrees will result in a range of λ, or equivalently, a larger bandwidth as shown in the equation
In small-animal fluorescence imaging, spectral resolution is not weighted as highly as illumination power or IBOBR defined as in-band to out-of-band ratio, which characterizes the quality of the illumination spectrum, and thus the spatial filter diameter is narrowed only enough to achieve spatial separation of the diffracted beams and no further. The output beam from the xenon lamp is for example 25 mm in diameter. In order to pass as much light as possible through the AOTF, the entire 10 mm×10 mm active aperture of the AOTF is used.
Referring again to
Referring now to the flow chart shown in
The configuration implemented in the embodiment of
In another embodiment shown in
Communication/computer control system 20 positions appropriate filter 235a, b, c, or d into the path of light beam 240 depending on what wavelength of light is chosen to illuminate the subject/object 112 on the sample object stage 104. Light beam 240a now filtered to the desired spectral band is directed via the first angled mirror 260 and the second angled mirror 265 both mounted on the moveable assembly 250. Light beam 240a is then directed through a lens 275 and onto an optical fiber input 280 of the imaging system 10.
With movable assembly 250 located in position “B” of
In the embodiment shown in
In the embodiment shown in
In the lens/camera system 18 shown in
In all of the aforementioned tuning of wavelength by tilting an interference filter, the effectiveness of a desired rejection or transmission is measured by the extent to which a desired wavelength is transmitted or an undesired wavelength is rejected. The efficacy with which the methodology meets a desirable endpoint depends upon the extent of light management needed to support a fluorescent imaging application peculiar to the application, and must be determined by those familiar to the art of so doing.
In one embodiment, a rotatable filter assembly 500, shown in
The apparatus of
A variety of nanoparticles are useful in imaging probes suitable for imaging in accordance with the present invention. In multimodal imaging probes, the nanoparticle preferably has one or more targeting moieties and one or more diagnostic imaging components capable of being imaged by one or more imaging modes such as luminescence or fluorescent imaging component, X-ray and MRI.
As a first example, discussed subsequently in further detail, nanoparticles may be used that have the form of a nanolatex nanogel comprising a water-compatible, swollen, branched polymer network of repetitive, cross-linked, ethylenically unsaturated monomers as described in commonly assigned, copending U.S. patent application Ser. No. 11/732,424 filed Apr. 3, 2007 by Leon et al entitled “LOADED LATEX OPTICAL MOLECULAR IMAGING PROBES.” For an imaging probe using such a nanolatex nanogel particle to be multimodal the nanoparticle making up the probe must carry two or more imaging components, for example a near IR dye for fluorescent imaging and gadolinium for x-ray imaging.
As a further example, discussed subsequently in further detail, nanoparticles may be used that are derived from self-assembly of amphiphilic block or graft copolymers to form crosslink particles with imaging dye immobilized in the particle via covalent chemical bond in the core of the nanoparticles and alkoxy silane cross-linking resulting in organic/inorganic hybrid materials as described in commonly assigned, copending U.S. patent application Ser. No. 11/738,558 filed Apr. 23, 2007 by Shiying Zheng et al entitled “IMAGING CONTRAST AGENTS USING NANOPARTICLES.” In such nanoparticles derived from self-assembly, the imaging dyes contain functional groups that can react with the cross-linkable groups of the hydrophobic component and are immobilized in the core of the nanoparticles by covalent bonding. More specifically the imaging dyes contain alkoxy silane groups. Since the imaging dyes are immobilized in the nanoparticles, the quantum efficiency is enhanced.
As another example, discussed subsequently in detail, an amine-modified silica nanoparticle having a polymer shell comprising amine functionalities may be used as described in commonly assigned, copending U.S. patent application Ser. No. 11/872,866 filed Oct. 16, 2007 by Zheng et al entitled “SILICA-CORED CARRIER PARTICLE” and its Continuation application Ser. No. 11/930,417 filed Oct. 31, 2007 by Zheng et al. The core/shell particle has attached one or more fluorescent groups, polymer groups such as polyethylene glycol, targeting molecules, antibodies or peptides. Suitable particles are described in the U.S. Patent Application of Bringley et al, previously mentioned. Especially useful are silica nanoparticles having a near infrared fluorescent core and having attached to their surface, amine groups and/or polyethylene glycol. Suitable particles also are described in the U.S. Patent Application of Bringley et al.
Such exemplary nanoparticles have been found to be nontoxic, and are capable of entry into small capillaries in the body, transport in the body to a disease site, crossing biological barriers (including but not limited to the blood-brain barrier and intestinal epithelium), absorption into cell endocytic vesicles, crossing cell membranes and transportation to the target site inside the cell. The particles in that size range are believed to be efficiently transferred across the arterial wall compared to larger size microparticles, see Labhasetwar et al., previously cited. Without being bound by any particular theory, it is also believed that because of high surface to volume ratio, the small size provides successful targeting of such particles using targeting molecules.
Whenever used in the specification the terms set forth shall have the following meaning:
The term “nanoparticle” or “nanoparticulate” refers to a particle with a size of less than 100 nm.
The term “colloid” refers to a mixture of small particulates dispersed in a liquid, such as water.
The term “biocompatible” means that a composition does not disrupt the normal function of the bio-system into which it is introduced. Typically, a biocompatible composition will be compatible with blood and does not otherwise cause an adverse reaction in the body. For example, to be biocompatible, the material should not be toxic, immunogenic or thrombogenic.
The term “biodegradable” means that the material can be degraded either enzymatically or hydrolytically under physiological conditions to smaller molecules that can be eliminated from the body through normal processes.
The term “targeting molecule” refers to any molecule, atom, or ion linked to the polymer networks or surface of the current invention that enhance binding, transport, accumulation, residence time, bioavailability or modify biological activity of the polymer networks or biologically active compositions of the current invention in the body or cell. The targeting molecule will frequently comprise an antibody, fragment of antibody or chimeric antibody molecules typically with specificity for a certain cell surface antigen. It could also be, for instance, a hormone having a specific interaction with a cell surface receptor, or a drug having a cell surface receptor. For example, glycolipids could serve to target a polysaccharide receptor. It could also be, for instance, enzymes, lectins, and polysaccharides. Low molecular mass ligands, such as folic acid and derivatives thereof are also useful in the context of the current invention. The targeting molecules can also be polynucleotide, polypeptide, peptidomimetic, carbohydrates including polysaccharides, derivatives thereof or other chemical entities obtained by means of combinatorial chemistry and biology. Targeting molecules can be used to facilitate intracellular transport of the nanoparticles of the invention, for instance transport to the nucleus, by using, for example, fusogenic peptides as targeting molecules described by Soukchareun et al., Bioconjugate Chem., 6, 43, (1995) or Arar et al., Bioconjugate Chem., 6, 43 (1995), caryotypic peptides, or other biospecific groups providing site-directed transport into a cell (in particular, exit from endosomic compartments into cytoplasm, or delivery to the nucleus).
The described composition can comprise a biological, pharmaceutical or diagnostic component that includes a targeting moiety that recognizes the specific target cell. Recognition and binding of a cell surface receptor through a targeting moiety associated with a described nanoparticle used as a carrier can be a feature of the described compositions. For purposes of the present invention, a compound carried by the nanoparticle may be referred to as a “carried” compound. For example, the biological, pharmaceutical or diagnostic component that includes a targeting moiety that recognizes the specific target cell described above is a “carried” compound. This feature takes advantage of the understanding that a cell surface binding event is often the initiating step in a cellular cascade leading to a range of events, notably receptor-mediated endocytosis. The term “Receptor Mediated Endocytosis” (“RME”) generally describes a mechanism by which, catalyzed by the binding of a ligand to a receptor disposed on the surface of a cell, a receptor-bound ligand is internalized within a cell. Many proteins and other structures enter cells via receptor mediated endocytosis, including insulin, epidermal growth factor, growth hormone, thyroid stimulating hormone, nerve growth factor, calcitonin, glucagon and many others.
Receptor Mediated Endocytosis affords a convenient mechanism for transporting a described nanoparticle, possibly containing other biological, pharmaceutical or diagnostic components, to the interior of a cell. In RME, the binding of a ligand by a receptor disposed on the surface of a cell can initiate an intracellular signal, which can include an endocytosis response. Thus, a nanoparticle used as a carrier with an associated targeting moiety, can bind on the surface of a cell and subsequently be invaginated and internalized within the cell. A representative, but non-limiting, list of moieties that can be employed as targeting agents useful with the present compositions includes proteins, peptides, aptomers, small organic molecules, toxins, diphtheria toxin, pseudomonas toxin, cholera toxin, ricin, concanavalin A, Rous sarcoma virus, Semliki forest virus, vesicular stomatitis virus, adenovirus, transferrin, low density lipoprotein, transcobalamin, yolk proteins, epidermal growth factor, growth hormone, thyroid stimulating hormone, nerve growth factor, calcitonin, glucagon, prolactin, luteinizing hormone, thyroid hormone, platelet derived growth factor, interferon, catecholamines, peptidomimetrics, glycolipids, glycoproteins and polysaccharides. Homologs or fragments of the presented moieties can also be employed. These targeting moieties can be associated with a nanoparticle and be used to direct the nanoparticle to a target cell, where it can subsequently be internalized. There is no requirement that the entire moiety be used as a targeting moiety. Smaller fragments of these moieties known to interact with a specific receptor or other structure can also be used as a targeting moiety.
An antibody or an antibody fragment represents a class of most universally used targeting moiety that can be utilized to enhance the uptake of nanoparticles into a cell. Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. Antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies. In one technique, an immunogen comprising the polypeptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats). A superior immune response may be elicited if the polypeptide is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.
Monoclonal antibodies specific for an antigenic polypeptide of interest may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto.
Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies. In addition, various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. The polypeptides of this invention may be used in the purification process in, for example, an affinity chromatography step.
A number of “humanized” antibody molecules comprising an antigen-binding site derived from a non-human immunoglobulin have been described Winter et al. (1991) Nature 349:293-299; Lobuglio et al. (1989) Proc. Nat. Acad. Sci. USA 86:4220-4224. These “humanized” molecules are designed to minimize unwanted immunological response toward rodent antihuman antibody molecules that limits the duration and effectiveness of therapeutic applications of those moieties in human recipients.
Vitamins and other essential minerals and nutrients can be utilized as targeting moiety to enhance the uptake of nanoparticle by a cell. In particular, a vitamin ligand can be selected from the group consisting of folate, folate receptor-binding analogs of folate, and other folate receptor-binding ligands, biotin, biotin receptor-binding analogs of biotin and other biotin receptor-binding ligands, riboflavin, riboflavin receptor-binding analogs of riboflavin and other riboflavin receptor-binding ligands, and thiamin, thiamin receptor-binding analogs of thiamin and other thiamin receptor-binding ligands. Additional nutrients believed to trigger receptor mediated endocytosis, and thus also having application in accordance with the presently disclosed method, are carnitine, inositol, lipoic acid, niacin, pantothenic acid, pyridoxal, and ascorbic acid, and the lipid soluble vitamins A, D, E and K. Furthermore, any of the “immunoliposomes” (liposomes having an antibody linked to the surface of the liposome) described in the prior art are suitable for use with the described compositions.
Since not all natural cell membranes possess biologically active biotin or folate receptors, use of the described compositions in-vitro on a particular cell line can involve altering or otherwise modifying that cell line first to ensure the presence of biologically active biotin or folate receptors. Thus, the number of biotin or folate receptors on a cell membrane can be increased by growing a cell line on biotin or folate deficient substrates to promote biotin and folate receptor production, or by expression of an inserted foreign gene for the protein or apoprotein corresponding to the biotin or folate receptor.
RME is not the exclusive method by which the described nanoparticle can be translocated into a cell. Other methods of uptake that can be exploited by attaching the appropriate entity to a nanoparticle include the advantageous use of membrane pores. Phagocytotic and pinocytotic mechanisms also offer advantageous mechanisms by which a nanoparticle can be internalized inside a cell.
The recognition moiety can further comprise a sequence that is subject to enzymatic or electrochemical cleavage. The recognition moiety can thus comprise a sequence that is susceptible to cleavage by enzymes present at various locations inside a cell, such as proteases or restriction endonucleases (e.g. DNAse or RNAse).
A cell surface recognition sequence is not a requirement. Thus, although a cell surface receptor targeting moiety can be useful for targeting a given cell type, or for inducing the association of a described nanoparticle with a cell surface, there is no requirement that a cell surface receptor targeting moiety be present on the surface of a nanoparticle.
To assemble the biological, pharmaceutical or diagnostic components to a described nanoparticle used as a carrier, the components can be associated with the nanoparticle carrier through a linkage. By “associated with”, it is meant that the component is carried by the nanoparticle. The component can be dissolved and incorporated in the nanoparticle non-covalently.
Generally, any manner of forming a linkage between a biological, pharmaceutical or diagnostic component of interest and a nanoparticle used as a carrier can be utilized. This can include covalent, ionic, or hydrogen bonding of the ligand to the exogenous molecule, either directly or indirectly via a linking group. The linkage is typically formed by covalent bonding of the biological, pharmaceutical or diagnostic component to the nanoparticle used as a carrier through the formation of amide, ester or imino bonds between acid, aldehyde, hydroxy, amino, or hydrozoa groups on the respective components of the complex. Art-recognized biologically labile covalent linkages such as imino bonds and so-called “active” esters having the linkage —COOCH, —O—O— or —COOCH are preferred. The biological, pharmaceutical or diagnostic component of interest may be attached to the pre-formed nanoparticle or alternately the component of interest may be pre-attached to a polymerizeable unit and polymerized directly into the nanoparticle during the nanoparticle preparation. Hydrogen bonding, e.g., that occurring between complementary strands of nucleic acids, can also be used for linkage formation.
In the imaging probe as described in the previously mentioned U.S. patent application Ser. No. 11/732,424, the nanoparticle may be in the form of a nanolatex nanogel comprising a water-compatible, swollen, branched polymer network of repetitive, crosslinked, ethylenically unsaturated monomers of Formula I:
(X)m—(Y)n-(Z)o Formula I
wherein X is a water-soluble monomer containing ionic or hydrogen bonding moieties; Y is a water-soluble macromonomer containing repetitive hydrophilic units bound to a polymerizeable ethylenically unsaturated group; Z is a multifunctional cross-linking monomer; m ranges from 50-90 mol %; n ranges from 2-30 mol %; and o range from 1-15 mol %. The present invention also relates to a method for preparing a nanogel comprising preparing a header composition of a mixture of monomers X, Y, and Z, and a first portion of initiators in water, wherein X is a water-soluble monomer containing ionic or hydrogen bonding moieties, Y is a water-soluble macromonomer containing repetitive hydrophilic units bound to a polymerizeable ethylenically unsaturated group, and Z is a multifunctional cross-linking monomer; preparing a reactor composition of a second portion initiators, surfactant, and water sufficient to afford a composition of 1-10% w/w of monomers X, Y, and Z; bringing the reactor composition to the polymerization temperature; holding the reactor composition at the polymerization temperature for the duration of the reaction, and adding the header composition to the reactor composition over time to form a reaction mixture, wherein the nanogel comprises a water-compatible, swollen, branched polymer network of repetitive, crosslinked, ethylenically unsaturated monomers of Formula I:
(X)m—(Y)n-(Z)o Formula I
wherein m ranges from 50-90 mol %; n ranges from 2-30 mol %; and o range from 1-15 mol %. For the imaging probe to be multimodal the nanoparticle making up the probe must carry two or more imaging components for example a near IR dye for fluorescent imaging and gadolinium for x-ray imaging.
In the imaging probe as described in the previously mentioned U.S. patent application Ser. No. 11/738,558, the nanoparticles are derived from self-assembly of amphiphilic block or graft copolymers to form crosslink particles with imaging dye immobilized in the particle, more specifically the imaging dye is immobilized via covalent chemical bond in the core of the nanoparticles and alkoxy silane cross-linking results in organic/inorganic hybrid materials.
It is well known that, in the presence of a solvent or solvent mixture that is selective for on block, amphiphilic block or graft copolymers have the ability to assemble into colloidal aggregates of various morphologies. In particular, significant interest has been focused on the formation of polymeric micelles and nanoparticles from amphiphilic block or graft copolymers in aqueous media. This organized association occurs as polymer chains reorganize to minimize interactions between the insoluble hydrophobic blocks and water. The resulting nanoparticles possess cores composed of hydrophobic block segments surrounded by outer shells of hydrophilic block segments. The core-shell structures of amphiphilic micellar assemblies have been utilized as carrier systems in the filed of drug delivery.
The amphiphilic copolymers that are useful in the present invention have a hydrophilic water soluble component and a hydrophobic component. Useful water soluble components include poly(alkylene oxide), poly(saccharides), dextrans, and poly(2-ethyloxazolines), preferably poly(ethylene oxide). Hydrophobic components useful in the present invention include but are not limited to styrenics, acrylamides, (meth)acrylates, lactones, lactic acid, and amino acids. Preferably, the hydrophobic components derived from styrenics and (meth)acrylates containing cross-linkable alkoxy silane groups. The imaging dyes contain functional groups that can react with the cross-linkable groups of the hydrophobic component and are immobilized in the core of the nanoparticles by covalent bonding. More specifically the imaging dyes contain alkoxy silane groups. Since the imaging dyes are immobilized in the nanoparticles, the quantum efficiency is enhanced. Suitable particles are described in the U.S. Patent Application of Zheng et al, previously mentioned.
In the imaging probe as described in the previously mentioned U.S. patent applications Ser. No. 11,872,866 and Ser. No. 11/930,417, the nanoparticle may be in the form of an amine-modified silica nanoparticle, having a polymer shell comprising amine functionalities. The core/shell particle has attached one or more fluorescent groups, polymer groups such as polyethylene glycol, targeting molecules, antibodies or peptides. Suitable particles are described in the U.S. Patent Application of Bringley et al, previously mentioned. Especially preferred are silica nanoparticles having a near infrared fluorescent core and having attached to their surface, amine groups and/or polyethylene glycol. Suitable particles also are described in the U.S. Patent Application of Bringley et al, previously mentioned.
In multimodal imaging probes the nanoparticle has one or more imaging components capable of being imaged by one or more imaging modes such as luminescence or fluorescent imaging component, X-ray and MRI.
The luminescence or fluorescent imaging component can be a near IR dye. Fluorophores include organic, inorganic or metallic materials that luminesce with including phosphorescence, fluorescence and chemo luminescence and bioluminescence. Examples of fluorophores include organic dyes such as those belonging to the class of naphthalocyanines, phthalocyanines, porphyrins, coumarins, oxanols, flouresceins, rhodamines, cyanines, dipyrromethanes, azadipyrromethanes, squaraines, phenoxazines; metals which include gold, cadmium selenides, cadmium telerides; and proteins such as green fluorescent protein and phycobiliprotein, and chemo luminescence by oxidation of luminal, substituted benzidines, substituted carbazoles, substituted naphthols, substituted benzthiazolines, and substituted acridans.
Where Dye is represented by the structure
Where dye is represented by
Where dye is represented by:
The invention has been described in detail with particular reference to certain preferred embodiments thereof, but it will be understood that variations and modifications can be effected within the spirit and scope of the invention.
Priority is claimed from commonly assigned, copending provisional U.S. Patent Application Ser. No. 60/970,623 filed Sep. 7, 2007 by Harder et al, entitled “APPARATUS AND METHOD FOR MULTI-MODAL IMAGING USING NANOPARTICLE MULTI-MODAL IMAGING PROBES,” the disclosure of which is incorporated by reference in this specification. This application is a continuation-in-part of commonly assigned, copending (a) regular U.S. patent application Ser. No. 11/221,530 filed Sep. 8, 2005 by Vizard et al entitled “APPARATUS AND METHOD FOR MULTI-MODAL IMAGING”; and (b) provisional U.S. Patent Application Ser. No. 61/024,621 filed Jan. 30, 2008 by Feke et al entitled “APPARATUS AND METHOD FOR MULTI-MODAL IMAGING”, the disclosures of each of which are incorporated by reference in this specification.
| Number | Date | Country | |
|---|---|---|---|
| 60970623 | Sep 2007 | US | |
| 61024621 | Jan 2008 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11221530 | Sep 2005 | US |
| Child | 12201204 | US |
