Patent Application
20100254914
The disclosure provides elongated nanostructures useful for biological imaging, measurements, drug delivery and therapeutics. More particularly the disclosure provides nanoworms having an increased bioavailability compared to nanospheres.
Tissue imaging, disease diagnostics and drug delivery are important for effective treatments.
The disclosure provides a composition comprising a plurality of nanostructure or particles conjugated or encapsulated to form an elongated structure having a first principle axis longer than the other two principle axes, with at least one dimension, such as length or diameter, between 1 and 200 nanometers (e.g., 1-100 nm, 1-50 nm, 5-50 nm etc.). In one embodiment, the nanoparticles are magnetic nanoparticles. In another embodiment, the nanostructure or particles comprise an iron oxide. The plurality of nanostructures can be encapsulated in a biocompatible material or conjugated to one another. In one embodiment, the biocompatible material is a dextran, polyethylene glycol, polyvinyl pyrrolidone or chitosan. In yet another embodiment, the nanostructure further comprises a targeting moiety linked to the nanostructure. For example, the targeting moiety can be a receptor ligand, an antibody, an antibody fragment or peptide comprising 2 or more amino acids.
The disclosure also provides a method of making an elongated nanostructure comprising precipitating a metal-containing nanoparticle in a high molecular weight dextran. In one embodiment, the method comprises precipitating iron oxide nanoparticles from a solution containing Fe2+(aq), Fe3+(aq), ammonia or other alkali solution (e.g., NaOH, KOH and the like), and a relatively low concentration of dextran.
The disclosure also provides methods for using a nanostructure of the disclosure for imaging a cell or tissue in vitro or in vivo. For example, the compositions and methods of the disclosure can be used for the imaging of cancer cells or tumors.
The disclosure provides worm-shaped dextran-coated iron oxide (magnetite or maghemite) nanoparticles. This disclosure provides materials comprising a chain-like aggregation of iron oxide (IO) cores (magnetic nanoworms; NW). The disclosure also demonstrates that such nanoworms can improve magnetic resonance contrast and in vivo tumor targeting properties over the well-known monocrystalline (spherical) dextran-coated IO nanoparticles. The chain-like aggregation of iron oxide cores also increases MRI sensitivity, demonstrating that NWs offer an improved ability to image very small tumors. The NW should be able to more efficiently deliver drugs to biological targets due to their large surface area, multiple attachment points, and long blood half-life.
The disclosure also provide methods for synthesis of worm-shaped dextran-coated iron oxide nanoparticles (nanoworms, NW) exhibiting substantial in vivo circulation times and significant tumor targeting when coated with tumor-homing peptides. Such worm-shaped nanoparticles home to tumors more efficiently than spherical both in vitro and in vivo. In one embodiment, the multivalent interactions between the targeting peptide-coated NW and their target molecules in tumor vasculature improves targeting compared to previous nanostructures. The surface chemistry, charge, and number of homing peptides are also found to be factors for improved homing and targeting. Additionally, NWs are found to display a greater magnetic resonance response than the spherical nanoparticles.
In one embodiment, the NW's of the disclosure comprise an increased surface area (compared to nanospheres) that can carry more homing peptides that more effectively interact with their tumor-based targets. The fact that the NW materials display a similar half-life in circulation compared to the smaller nanospheres (NS), even though they have comparable surface charge and chemistries is notable. The factors that lead to the highest circulation times and most effective targeting include: a neutral surface charge; complete and tight coverage of the iron oxide substructure with dextran and other hydrophilic polymers (e.g., PEG), and loading of targeting peptides. The chain-like aggregation of iron oxide cores also increases MRI sensitivity, suggesting that NW may offer an improved ability to image very small tumors. The NW should be able to more efficiently deliver drugs to biological targets due to their large surface area, multiple attachment points, and long blood half-life.
The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and drawings, and from the claims.
As used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a nanostructure” may include a plurality of such nanostructures and reference to “the nanoworm” may include reference to one or more nanoworms, and so forth.
Also, the use of “or” means “and/or” unless stated otherwise. Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting.
It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.”
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although any methods and reagents similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods and materials are now described.
All publications mentioned herein are incorporated herein by reference in full for the purpose of describing and disclosing the methodologies, which are described in the publications, which might be used in connection with the description herein. The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure.
Tumorigenesis is a multi-step process that requires expression of tumor-associated proteins and suppression of proteins controlling normal cell growth. Many of the identified tumor-specific proteins have been exploited to develop powerful antibody, aptamer, peptide, and small molecule-based ligands for targeting of diagnostic or therapeutic agents. Ultrasensitive in vivo imaging for early detection of cancers and efficient delivery of therapeutics to malignant tumors are two primary goals in cancer bionanotechnology. However, the development of non-toxic, functional nanoparticles that can successfully home to tumors presents some significant challenges. Dextran-coated magnetic iron oxide (IO) nanoparticles are of particular interest because they show relatively low toxicity and long in vivo circulation (˜10 hrs) and they dramatically enhance hydrogen T2 relaxation in magnetic resonance imaging (MRI). The clinical power of these materials may be amplified by improving MRI relaxivity, blood circulation times, and the homing of such nanoparticles to tumors.
Ligand-directed targeting of therapeutic nanomaterials has been widely pursued to improve therapeutic efficacy, although limitations imposed by the tumor microenvironment, such as restricted transvascular transport and receptor accessibility and clearance of targeted nanoparticles comprising such antibody, aptamer, peptide and small molecule have prevented realization of their full capabilities. Although the porous microstructure of tumor blood vessels is favorable for non-specific infiltration of circulating nanomaterials into the extravascular region of the tumor, extravasated nanomaterials are generally deposited close to the vessels, resulting in a highly heterogeneous distribution of therapeutic agents in the tumor.
Hyperthermia has been reported to not only improve nanoparticle extravasation in tumors, but it also can selectively damage neoplastic cells to activate immunological processes and induce expression of particular proteins. Use in the clinical setting in concert with chemotherapy and radiotherapy, tumor-specific hyperthermia would be a powerful tool to manipulate tumor microenvironments in order to enhance the interactions between cancerous tissues and therapeutic agents. However, hyperthermia methods in clinical practice lack intrinsic specificity for tumor tissues, requiring complex implementation strategies and frequently resulting in exposure of large volumes of normal tissues to hyperthermic temperatures alongside tumors. Gold nanorods (NRs), for example, passively accumulated in tumors via their fenestrated blood vessels. The accumulated NRs can be used to precisely heat tumor tissues by amplifying their absorption of otherwise benign near-infrared energy and allow the recruitment and more effective penetration of a second, specifically targeted nanoparticle. As discussed more fully below, the disclosure provides not only a single therapy comprising a nanoworm, but also a cooperative nanomaterials system, wherein NWs accumulated in a tumor photothermally activate the local microenvironment to amplify the targeting efficacy of two types of targeted, circulating nanoparticles: magnetic nanoworms (NWs) and liposomes (LPs) loaded with the anti-cancer drug doxorubicin (DOX). Other liposomal or micellar formulation may be used with any number of different chemotherapeutic agent. For example, the chemotherapeutic agent can be doxorubicin, taxol, combretastatin or any combination thereof.
Efforts to increase MRI sensitivity have focused on development of new magnetic core materials or improvements in nanoparticle size or clustering. However, most efforts to improve the morphological characteristics of such nanoparticles have resulted in materials with relatively short blood half-life (1˜2 hrs) due to incomplete additional hydrophilic coatings. While decoy particles that bind to plasma opsonins can be used to improve the circulation time of nanoparticles by blocking uptake by the mononuclear phagocytic system (MPS), it is more desirable to incorporate an inherent ability to avoid the MPS in the nanoparticle itself.
At the micro scale, particle shape plays a dominant role in particle uptake by phagocytes. A study on the uptake of gold nanoparticles into cultured tumor cells concluded that spherically shaped particles have a higher probability of cell internalization than rod-shaped particles. When nanoparticles are used in vivo, one of the most important issues is to avoid clearance by the MPS, which is primarily located in the liver.
The disclosure demonstrates that nanoparticles with elongated shapes exhibit unique in vivo behavior such as low liver uptake and, as a result, prolonged blood half-life. The disclosure demonstrates that a nanostructure with an elongated assembly of metallic cores such as iron oxide (IO) cores provides long in vivo circulation times and that this improves homing of the particles to tumors. High aspect ratio nanomaterials such as carbon nanotubes and worm micelles have been found to circulate in vivo long enough to enable homing to biological targets despite their micron-sized length. In addition, pseudo one-dimensional assemblies of nanocrystals can display desirable optical or magnetic properties not found in the isodimensional materials. The disclosure demonstrates that a chain-like aggregation of metallic cores such as iron oxide (IO) cores (magnetic nanoworms; NW) can improve magnetic resonance contrast and in vivo tumor targeting properties over the well-known monocrystalline dextran-coated IO nanoparticles. The improved targeting characteristics can be attributed, in part, to an increased surface area that can carry more homing peptides that more effectively interact with their tumor-based targets. The fact that the NW materials display a similar half-life in circulation compared to the smaller nanospheres (NS), even though they have comparable surface charge and chemistries is notable. Some of the factors that lead to the highest circulation times and most effective targeting include, but are not limited to: a neutral surface charge; complete and tight coverage of the iron oxide substructure with dextran and other hydrophilic polymers (e.g., PEG), and an optimal loading of targeting peptides. The data presented here indicates that these factors are important for long blood half-life. The chain-like aggregation of the magnetic cores (e.g., the iron oxide cores) also increases MRI sensitivity, suggesting that NW may offer an improved ability to image very small tumors. Several methods to construct one-dimensional assemblies of nanocrystals are known in the art, for example, methods that involve the use of molecular coatings or biotemplates. These approaches appear to provide means to control the chain-like nanostructures fairly precisely.
The disclosure demonstrates that tailoring the shape, as well as the size and charge, can improve in vivo tumor targeting capability of a nanomaterial. This elongated assembly of metals such as iron oxide cores coated by a polymeric material such as dextran is analogous to certain strand-like viruses or biomolecules that display long residence times in the blood stream. The NW should be able to more efficiently deliver drugs to biological targets due to their large surface area, multiple attachment points, and long blood half-life.
The disclosure provides conjugated beads or elongated metallic nanostructures (generally referred to herein as “elongated nanostructure”. As used herein, the term “elongated nanostructure” refers to various materials having one principle axis longer than the other two principle axes, such as a cylindrical or tubular configuration, with at least one dimension, such as length or diameter, between 1 and 100 nanometers. Such elongated nanostructures are capable of MRI detection and imaging as well as photothermal activation. The metallic nanostructure can comprise any metal or alloys thereof.
Metals, alloys and materials useful for the formation of a nanostructure of the disclosure can be obtained based upon a functional layer or thermal bias layer. The material can be selected from the group of noble metal and transition metal including, but not limited to, Ag, Au, Cu, Al, Fe, Co, Ni, Ru, Rh, Pd, and Pt. In another embodiment, the material comprises Fe. A further surface functional layer can be added or formed in combination with the noble or transition metal core material. Such functional layers can include, but are not limited to, Ag oxide, Au oxide, SiO2, Al2O3, Si3N4, Ta2O5, TiO2, ZnO, ZrO2, HfO2, Y2O3, Tin oxide, antimony oxide, and other oxides; Ag doped with chlorine or chloride, Au doped chlorine or chloride, Ethylene and Chlorotrifluoroethylene (ECTFE), Poly(ethylene-co-butyl acrylate-co-carbon monoxide) (PEBA), Poly(allylamine hydrochloride) (PAH), Polystyrene sulfonate (PSS), Polytetrafluoroethylene (PTFE), Polyvinyl alcohol (PVA), Polyvinyl chloride (PVC), Polyvinyldene fluoride (PVDF), Polyvinylprorolidone (PVP), and other polymers; stacked multiple layers at least two layers including above listed metal layers and non-metal layers, and the like. A typical material is a metal such as Au, Ag, Fe, Ti, Ni, Cr, Pt, Ru, Ni—Cr alloy, NiCrN, Pt—Rh alloy, Cu—Au—Co alloy, Ir—Rh alloy or/and W—Re alloy. The material used should be biocompatible.
The geometry or structure of the nanomaterial can incorporate the functional capabilities of nanotip, nanosphere, and nanoring geometries. Other geometries can include spherical, circular, triangle, quasi-triangle, square, rectangular, hexagonal, oval, elliptical, rectangular with semi-circles or triangles and the like. However, the structure(s) should be linked or have an elongated structure. The nanostructures of the materials and geometries ideally have an absorbance or excitation wavelength in the near infrared range. Selection of suitable materials and geometries are known in the art. Excitation at longer wavelengths provides deeper penetration into tissue with minimal photothermal damage.
Various nanostructure geometries are capable of near-infrared (NIR) excitation. For example, crescents, bowls, hollow spheres and the like have a higher local field-enhancement factor in the near-infrared wavelength region due to the simultaneous incorporation of SERS hot spots, leading to the strong hybrid resonance modes from nanocavity resonance modes.
One of skill in the art will recognize that the size, shape, and thickness or where multi-layers are present layer thickness can all be individually controlled by modifying the size of a sacrificial nanostructure template, the deposition angle, the deposited layer thickness, and the material of each layer. In one embodiment, the nanostructure comprises a spherical or semi-spherical structure commonly produced in the art.
The metallic composition of composite nanostructures of the disclosure are biocompatible, and thus can be bio-functionalized.
The term “functionalized” is meant to include structures with one, two or more layers of different metals, structures with functional groups attached thereto, and the like. For example, to form a linkage to a peptide, oligonucleotide or other biomaterial, to prolong or target analyte interaction with a noble metal nanostructure, a binding agent/targeting domain can be used to promote interaction of a nanostructure with a desired target. An alkanethiol, such as 1-decanethiol, can be used to form the capture layer on the noble metal (Blanco Gomis et al., J. Anal. Chim. Acta 436:173 [2001]; Yang et al., Anal. Chem. 34:1326 [1995]). Other exemplary capture molecules include longer-chained alkanethiols, cyclohexyl mercaptan, glucosamine, boronic acid and mercapto carboxylic acids (e.g., 11-mercaptoundecanoic acid).
In one embodiment, the elongated or conjugated beads are encapsulated or linked to dextran or other polymeric materials that are useful for increasing the circulating half-life or stability in vivo. Other polymeric materials can be selected from the group, but are not limited to, polyethylene glycol (PEG), a lipid, chitosan, zein, polylactic acid, polyglycolic acid, collagen, fibrin, co-polymers of polylactic acid and polyglycolic acid, and co-polymers of dextran and polylactic acid. In a further embodiment, the elongated nanostructure is linked to a targeting moiety or plurality of targeting moieties (e.g., a peptide ligand, antibody, antibody domain, receptor, receptor fragment and the like) to target the nanostructure to a particular cell type or tissue. In some embodiments, the ligand is a peptide. In another embodiment, the peptide has a density that maintains free amine groups at a minimum and maintains the coverage of the underlying nanoparticle. In a further embodiment, the peptide is an F3 peptide or a CREKA (SEQ ID NO:2) peptide. In yet another embodiment, the CREKA peptide comprises less than about 60 peptides per elongated nanostructure. Table 1, for example, depicts certain elongated nanodevice characteristics:
[a]The number following the letter identifier designates the number of amine groups per particle. The letter P indicates that a PEG spacer is used. The -F or -C suffix denotes an F3- or CREKA-conjugated particle, respectively. For example, NW-P175-C denotes a NW with 175 amines to which CREKA is conjugated through a PEG spacer. MM = aminated Micromod. Micromod is a commercially available IO nanoparticle preparation.
[b]Mean hydrodynamic size based on dynamic light scattering measurements.
[c]Number of targeting peptides per single NW or NS.
[d]Number of targeting peptides (×1020) per gram of Fe.
Other conjugate moieties include proteins, peptides, and peptide mimetics. In one aspect, members from this group of moieties are selected based on their binding specificity to a ligand expressed in or on a target cell type or a target organ. Alternatively, moieties of this type include a receptor for a ligand on a target cell (instead of the ligand itself), and in still other aspects, both a receptor and its ligand are contemplated in those instances wherein a target cell expresses both the receptor and the ligand. In other embodiments, members from this group are selected based on their biological activity, including for example enzymatic activity, agonist properties, antagonist properties, multimerization capacity (including homo-multimers and hetero-multimers). With regard to proteins, conjugate moieties contemplated include full length protein and fragments thereof which retain the desired property of the full length proteins. Fusion proteins, including fusion proteins wherein one fusion component is a fragment or a mimetic, are also contemplated. This group also includes antibodies along with fragments and derivatives thereof, including but not limited to Fab′ fragments, F(ab)2 fragments, Fv fragments, Fc fragments, one or more complementarity determining regions (CDR) fragments, individual heavy chains, individual light chain, dimeric heavy and light chains (as opposed to heterotetrameric heavy and light chains found in an intact antibody, single chain antibodies (scAb), humanized antibodies (as well as antibodies modified in the manner of humanized antibodies but with the resulting antibody more closely resembling an antibody in a non-human species), chelating recombinant antibodies (CRABs), bispecific antibodies and multispecific antibodies, and other antibody derivative or fragments known in the art.
Cell receptor ligands useful for targeting include ligands that are able to bind to cell surface receptors (e.g., insulin receptor, EPO receptor, G-protein coupled receptors, receptors with tyrosine kinase activity, cytokine receptors, growth factor receptors, etc.), to modulate (e.g., inhibit, activate, etc.) the physiological pathway that the receptor is involved in (e.g., glucose level modulation, blood cell development, mitogenesis, etc.). Examples of cell receptor ligands include, but are not limited to, cytokines, growth factors, interleukins, interferons, erythropoietin (EPO), insulin, glucagon, G-protein coupled receptor ligands, etc.
Accordingly any number of targeting ligands can be conjugated to the nanostructure (e.g., a receptor bound to the surface of a nanostructure that interacts reversibly or irreversibly with a specific analyte). Alternatively or in addition an uptake moiety can be linked to the nanoparticle (e.g., a TAT moiety, see, for example, International Patent Publ. No. WO/2007/095152). Examples of targeting ligands include antigen-antibody pairs, receptor-ligand pairs, and carbohydrates and their binding partners. Binding ligands to a wide variety of analytes are known or can be readily identified using known techniques. As will be appreciated by those in the art, any two molecules that will associate, may be used, either as the analyte or the functional group (e.g., targeting/binding ligand). Suitable analyte/binding ligand pairs include, but are not limited to, antibodies/antigens, receptors/ligand, proteins/nucleic acids; nucleic acids/nucleic acids, enzymes/substrates and/or inhibitors, carbohydrates (including glycoproteins and glycolipids)/lectins, carbohydrates and other binding partners, proteins/proteins; and protein/small molecules. In one embodiment, the binding ligands are portions (e.g., the extracellular portions) of cell surface receptors.
The disclosure thus provides elongated nanostructures comprising a plurality of individual nanostructures linked to one another. Typically the nanostructure comprise a metallic nanosphere. The nanosphere may comprise any number of different metals or allows such as, but not limited to, gold, silver, copper, iron and alloys and combinations thereof. The nanostructure may be further coated in a polymeric material that improves circulatory time in vivo. The elongated nanostructure my further comprise a targeting ligand or plurality of targeting ligands. The targeting ligands may be identical or different. Furthermore, In one embodiment, the disclosure provides synthesis and biological application of worm-shaped dextran-coated nanoparticles (nanoworms, NW) exhibiting prolonged in vivo circulation times and improved tumor targeting when coated with tumor-homing peptides compared to spherical nanostructures. The synthesis of NW was based on the observation that nanostructures such as magnetic nanoparticles can become aligned along strands of high molecular weight dextran, and the nanoparticle geometry (worm-shaped vs spherical) affects their efficacy both in vitro and in vivo. This can be attributed to the improvement in tumor homing to prolonged in vivo circulation and enhanced multivalent interactions between the targeting peptide-coated NW and their target molecules in tumor vasculature.
In one embodiment, nanoparticles comprising iron oxide are produced from a mixture of iron (II) chloride and iron (III) chloride with a polysaccharide (e.g., Ficoll™) in water, by treatment with base (e.g., NaOH or NH4OH) and heating under an inert atmosphere.
Furthermore, the disclosure demonstrates that for a constant ratio of attached targeting peptides per iron atom, NW display a greater ability to be taken up by cultured tumor cells than NS. These results suggest that NW will also facilitate the in vivo homing of multivalent ligands to biological targets. Two peptides (F3 and CREKA) were chosen for in vivo tumor targeting study, because they are known to recognize different tumor targets. F3-conjugated NW exhibited rapid MPS clearance regardless of the protecting or attachment chemistries used. This rapid clearance is attributed to the large number of positively charged residues on the relatively large F3 peptide. Other peptides that can be used for targeting including antibodies, antibody fragments, receptor proteins and fragments, ligand binding proteins and moieties (e.g., including soluble polypeptide/peptide domains derived from transmembrane proteins) and the like.
The short peptide CREKA endows superior targeting capability to the NW. PEGylated NW conjugated with the appropriate number of CREKA targeting moieties circulate in vivo for a long period (blood half-life of over 12 hrs), and prominent tumor uptake is observed in both MDA-MB-435 and HT1080 tumors. Other targeting-molecules can be used in place of or in addition to CREKA, however, it is suggested that the blood half-life of the targeting molecule-nanomaterial ensemble must be considered when selecting the appropriate ligand for in vivo tumor targeting when several ligand candidates with similar targeting affinity are available. CREKA, a short linear peptide which is likely non-immunogenic and is neutrally charged, maintains its binding to blood clots (fibrin(ogen)) when coupled to PEGylated NW. Furthermore, it displays the same self-amplifying homing behavior seen previously with CREKA-conjugated IO nanoparticles.
The methods of the disclosure are useful for treating diseases or disorders comprising cell proliferative diseases or disorders, inflammation, tissue damage and the like. For example, the methods and compositions of the disclosure are useful for treating or studying cell proliferative disorder such as cancer, inflammatory disorder and autoimmune disorders to name a few.
The nanoworms and elongated nanostructures of the disclosure can also be used in a combination therapy comprising hyperthermia and drug delivery. For example, as described herein, the elongated nanostructures (including nanoworms) have improved circulatory times, and reduced clearance. In addition, the structures can be effectively targeted to tumors and other tissues by conjugating the elongated nanostructure to a targeting ligand (e.g., a peptide etc.). The elongated nanostructures can then be localized by magnetic forces (where the nanostructure comprises a magnetic metal) and/or through ligand binding at a desired site. The nanostructures can then be excited to thermally modify the tissue, increasing vasculature and damaging the desired tissue. Following and simultaneously with the delivery of the elongated nanostructure and targeted chemotherapeutic agent (e.g., a chemotherapeutic small molecule, antibody, peptide or the like) can be administered to the subject. The chemotherapeutic, for example, may be formulated in a targeted liposome. In one embodiment, the disclosure demonstrates the doxorubicin liposomes can be used. The liposomes may further comprise a targeting ligand (e.g., the same targeting ligand used on the elongated nanostructures) to cause the targeted delivery of the liposome's payload to the desired tissue.
Accordingly, the disclosure provides a method for treating a cell proliferative disorder comprising a tumor by administering a targeted nanoworm to the subject, thermally treating the tumor site by thermally activating the nanoworm and contacting the subject with a chemotherapeutic agent. In one embodiment, the chemotherapeutic agent comprises a targeted liposome containing the chemotherapeutic agent.
A nanostructure comprising a NW or elongated structure can be formulated in pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers useful for administration to a cell, tissue or subject are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters. A pharmaceutically acceptable carrier can contain physiologically acceptable compounds that act, for example, to stabilize or to increase the absorption of the conjugate. Such physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the physico-chemical characteristics of the therapeutic agent and on the route of administration of the composition, which can be, for example, orally or parenterally such as intravenously, and by injection, intubation, or other such method known in the art. The pharmaceutical composition also can contain a second (or more) compound(s) such as a diagnostic reagent, nutritional substance, toxin, or therapeutic agent, for example, a cancer chemotherapeutic agent and/or vitamin(s).
In some embodiments, the disclosure provides kits and systems for tissue imaging and drug delivery.
Excitation of the nanostructures of the disclosure can be performed by contacting the nanostructure with appropriate electromagnetic radiation (e.g., an excitation wavelength). Wavelengths in the visible spectrum comprise light radiation that contains wavelengths from approximately 360 ran to approximately 800 run. Ultraviolet radiation comprises wavelengths less than that of visible light, but greater than that of X-rays, and the term “infrared spectrum” refers to radiation with wavelengths of greater 800 nm. Typically, the desired wavelength can be provided through standard laser and electromagnetic radiation techniques.
The following examples are intended to illustrate but not limit the disclosure. While they are typical of those that might be used, other procedures known to those skilled in the art may alternatively be used.
Preparation and physical characterization of magnetic nanoworms. The synthesis of magnetic nanoworms (NW) involves precipitation of IO nanoparticles from a solution containing Fe2+(aq), Fe3+(aq), ammonia or other alkali solution (e.g., NaOH, KOH and the like), and a relatively low concentration of dextran. The dextran used was of a greater molecular mass than typically employed in such preparations (20 kDa vs. 10 kDa). Suitable dextran for use can be from 10 to 30 kDa. Nanoworms (NW) were synthesized using a modification of the published preparation of dextran-coated iron oxide nanoparticles. For the NW synthesis, a higher concentration of iron salts and a higher molecular weight dextran (MW 20,000 or 40,000, Sigma) were used. In one preparation 0.63 g of FeCl3.6H2O and 0.25 g FeCl2.4H2O were mixed with 4.5 g dextran in 10 mL of Millipore water at room temperature. This acidic solution was neutralized by the dropwise addition of 1 mL concentrated aqueous ammonia under vigorous stirring and a steady purge of nitrogen, and it was then heated at ˜70° C. for 1 hr. After purification by centrifuge filtering column (100,000MWCO, Millipore), the magnetic colloid was cross linked in strong base (5M aqueous NaOH solution) with epichlorohydrin (Sigma) and filtered through a 0.1 μm pore diameter membrane (Millipore). NW with a size range of 5080 nm were separated using a MACS® Midi magnetic separation column (Miltenyi Biotec). Nanosphere (NS) with a size range of 25˜35 nm were prepared using techniques known in the art. NW or NS with different numbers of free amines were prepared for peptide conjugation by reacting them with different concentrations of aqueous ammonia at room temperature for 48 hrs. The amine number per NS was measured using the SPDP assay. The amine number per NW was calculated assuming that the molecular weight of a NW is 7 times higher than a NS, based on the mean value of aggregated iron oxide cores for one NW observed in the TEM images and supported by the light scattering (DLS) data. Negatively charged NW (NW-N) were prepared by reacting non-aminated NW with 1 M chloroacetic acid in strong base (5M aqueous NaOH solution) for 2 hrs at room temperature. Micromod IO nanoparticles (50 nm nanomag-D-SPIO with amines) were obtained from Micromod Partikeltechnologie GmbH, Rostock, Germany. Feridex IO nanoparticles were obtained from Berlex, N.J., USA.
The nanostructure appears as linearly aggregated IO cores with a mean hydrodynamic size (in the long dimension) of 65 nm (
aNone of the particles in this table contain targeting peptides.
bNS = dextran-coated magnetic nanosphere. NW = dextran-coated magnetic nanoworm. NW-P = dextran-coated magnetic nanoworm with 5 kDa PEG linkers attached to the dextran. NW-N = dextran-coated magnetic nanoworm with pendant negatively charged carboxyl groups. MM = commercially obtained Micromod particles that have been aminated. The dextran coatings have been cross-linked and modified with free amines; the number after the letter identifier designates the approximate number of free amino groups per particle. For example, NW-P175 denotes a magnetic nanoworm with 175 amines to which PEG(5 kDa)-succinimidyl -methylbutanoate is conjugated.
cHydrodynamic size based on DLS measurement
dBlood half-life (mouse, tail-vein injection) determined by magnetization or fluorescence measurement as described in the text. Relative error in these measurements is ±10%
eR2 is longitudinal relaxation rate equal to reciprocal of T2 relaxation time (R2 = 1/T2) and is calculated with T2-weighted MRI map.
fNumber of peptides per single nanoworm or nanosphere.
Targeting peptide conjugation. One of two targeting peptides were used with the NW or NS samples: KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (F3) (SEQ ID NO:1), which preferentially binds to blood vessels and tumor cells in various tumors, and CREKA (SEQ ID NO:2), which recognizes clotted plasma proteins in the blood vessels and stroma of tumors. The fluorescein (FITC)-conjugated peptides were synthesized using Fmoc chemistry in a solid-phase synthesizer, and purified by preparative HPLC. Their sequence and composition were confirmed by mass spectrometry. For the F3 peptide, an extra cysteine residue was added to the N-terminus to allow conjugation with NW or NS. For near-infrared (NIR) fluorescence imaging, Cy7-labeled NW or NS were prepared by reacting aminated NW (500 μg Fe) or NS (900 μg Fe) in PBS buffer with 6 μg of Cy7-NHS ester (GE Healthcare Bio-Sciences) in DMSO (Sigma) for 1 hr to have same fluorescence per iron atom for both NW and NS (one Cy7 dye per one iron oxide core). The remaining free amines were used for conjugation with the targeting peptides. 500 μg Fe of Cy7-labed NW or NS were first reacted with 200 μg of Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC, Pierce Chemicals) or 2 mg NHS-PEG(5 kDa)-MAL (Nektar) in PBS solution for 1 hr and then purified using a desalting column (GE Healthcare Bio-Sciences). 200 μg of targeting peptide with a free terminal cysteine was then added to the 500 μg Fe NW or NS sample in PBS solution. After incubation for 2 h with mild shaking at room temperature, the sample was purified on a desalting column (GE Healthcare Bio-Sciences) for the CREKA samples or with a centrifuge filter (100,000 MWCO, Millipore) for the F3 samples, and then re-suspended in PBS solution. The FITC-peptide or Cy7 dye number per one NW or NS was determined with their absorbance spectra.
Conjugation of targeting peptides to magnetic nanoworms: in vitro cell internalization. The efficiency of peptide-targeted cellular internalization of NW compared with NS was tested in vitro. Conceptually, the elongated shape of the NW is expected to provide a larger number of interactions between the targeting ligands and their cell-surface receptors compared with spherical nanoparticles (
Magnetic measurement. A solution of the NW or NS sample was frozen and lyophilized to dryness in gelatin capsules. The capsules were inserted into the middle of transparent plastic straws. The measurements were performed at 298 K using a Quantum Design (CA, USA) MPMS2 superconducting quantum interference device (SQUID) magnetometer. The samples were exposed to direct current magnetic fields in stepwise increments up to one Tesla. Further corrections were made for the diamagnetic contribution of the capsule and straw.
Zeta potential measurements. Zeta potentials of NW or NS were measured using a Malvern (Worcestershire, UK) Zetasizer ZS90 equipped with an autotitration system. Zeta potentials were plotted in the pH range 3-9. The surface charge of the NW or NS samples is reported for the value of the zeta potential at pH 7 to simulate physiological conditions.
MRI T2 mapping. MRI T2 mapping of NW or NS samples was performed using a 7 cm bore, Bruker (Karlsruhe, Germany) 4.7 T magnet. Samples were serially diluted with aqueous PBS (Mediatech) in a 384-well plate, containing 95 μl total sample/well.
In vitro cell internalization. MDA-MB-435 human breast carcinoma cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 μg/ml penicillin-streptomycin. For cell internalization tests, the 10,000 cells were seeded into each well of 24-well plates and cultured overnight. The cells were then incubated with 40 μg (total Fe content) of Cy7-labeled peptide-conjugated NW or NS per well for 30 min, 1 h, or 2 h at 37° C. in the presence of 10% FBS (triplicate per NW or NS formulation). The wells were rinsed three times with cell media and then imaged in the Cy7 channel (762 nm excitation/800 nm emission) with a NIR fluorescent scanner (LI-COR biosciences). The relative fluorescence of the images (each well) was analyzed using the ImageJ (NIH) or OsiriX (Apple) programs. To quantify the internalized amount of NW or NS, the cells were carefully detached from each well using trypsin-EDTA, and centrifuged into a pellet. The pellets were freeze-dried in gelatin capsules, and then analyzed for magnetization using SQUID. For fluorescence microscopy, the cells (3000 cells per well) were seeded into 8-well chamber slides (Lab-Tek) overnight. The cells were then incubated with 10 μg (total Fe content) of Cy7-labeled peptide-conjugated NW or NS per well for 3 h at 37° C. in the presence of 10% FBS. After incubation, the slides were rinsed three times with PBS, fixed with 4% paraformaldehyde, and then washed three times with PBS and mounted in Vectashield Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, Calif.). The slides were observed with a fluorescence microscope (Nikon, Tokyo, Japan).
Cell internalization was used to probe the multivalent effect provided by the compositions and materials of the disclosure. An F3 peptide was used, which selectively targets cell surface nucleolin in tumor cells and tumor endothelial cells, and is known to have cell-penetrating properties. FITC-tagged F3 peptides were conjugated to dextran-coated, aminated NW or NS through the sulfhydryl group of a cysteine residue that had been added to the peptide. The peptides were conjugated via a short crosslinker (sulfo-SMCC) or a 5 kDa polyethylene glycol) (PEG) chain. Absorbance assays of the F3-conjugates showed that the numbers of peptides coupled to the particles could be controlled (Table 3). Additionally, F3 conjugation to the particles through PEG chains resulted in fewer peptides per particle.
aThe particles in this table contain targeting peptides.
bSample numbers are as defined in Table 2. The -F or -C suffix denotes F3 or CREKA-conjugated particle, respectively. These targeting peptides have been conjugated to the free amines on the dextran coatings via sulfo-SMCC crosslinker or via PEG crosslinker. F3 peptide selectively targets cell surface nucleolin in tumor cells and tumor endothelial cells, and CREKA peptide recognizes clotted plasma proteins, which accumulate in tumors but not in normal tissues.
cNumber of peptides per single nanoworm or nanosphere.
Fluorescently labeled (Cy7) NW and NS were used to determine the efficiency of cell internalization. The total number of attached Cy7 dye molecules was controlled to yield the same fluorescence intensity on a per-iron basis for both types of particles. The various formulations of Cy7-labeled, F3-conjugated particles were incubated with MDA-MB-435 human breast cancer cells for 2 hrs. The cells were imaged with a NIR fluorescence scanner. The nanoworms NW-42-F, NW-175-F, and NW-350-F display similar numbers of F3 targeting peptides per iron atom compared with NS-7-F, NS-30-F, and NS-59-F, respectively (Table 3). No internalization of NS or NW into the tumor cells was seen without F3 coating of the particles, and internalization increased with the number of F3 peptides attached per NW/NS (
The SQUID (Superconducting Quantum Interference Device) magnetometry provides a direct measure of the total number of magnetic IO nanoparticles in a sample, as it measures the magnetization of a sample rather than the total iron content or the fluorescence intensity from a molecular tag. The SQUID measurements are relevant to the MRI imaging applications, because the magnetization data correlates with T2. The SQUID technique has the additional advantage that it can be performed on cells, cell extracts, or on whole organs, and little sample workup is needed. The SQUID data confirmed that NW were more effectively taken up by the cells; for F3-conjugated particles incubated for 2 h prior to analysis, ˜65 pg of Fe from NW was internalized per cell, whereas only ˜16 pg of Fe was internalized per cell from NS, (samples NW-175-F and NS-30-F,
Short PEG chains are often used to avoid MPS uptake minimizing interactions of blood proteins with nanomaterials. Using a PEG linker to attach the F3 peptide to the NW resulted in less cellular uptake, even when a large number of F3 targeting peptides where attached to the particles (NW-P175-F and NW-P350-F,
In vivo behavior of peptide-conjugated magnetic nanoworms. Circulation in the blood stream for a long period of time is a factor for in vivo target-specific reporting and drug delivery with nanomaterials. In vivo circulation of unmodified NW using doses of 3 mg Fe/kg and 10 mg Fe/kg body mass was tested in mouse.
Blood half-life and biodistribution. To quantify the in vivo circulation times of NW or NS samples in Nude BALB/c mice (n=3-4 for each formulation), heparinized capillary tubes (Fisher) were used to draw 15 μL (for fluorescence) or 70 μl (for magnetization) of blood from the periorbital plexus at different times after intravenous injection of the NW or NS samples (1, 3, or 10 mg Fe/kg body mass). The extracted blood samples were immediately mixed with 10 mM EDTA to prevent coagulation. For Cy7-labeled NW or NS formulations, blood extracted at different times was imaged in a 96-well plate in Cy7 channel (762 nm excitation/800 nm emission) with a NIR fluorescence scanner (LI-COR biosciences, NE, USA). The images were analyzed using the ImageJ (NIH) or Osirix (Apple) programs. For non-labeled NW or NS samples, blood samples extracted at different times were immediately freeze-dried in gelatin capsules, and then analyzed for magnetization using SQUID. The blood half-life was calculated by fitting the blood fluorescence or magnetization data to a single-exponential equation used in a one-compartment open pharmacokinetic model. Additionally, the NW were extracted from the blood stream 24 hrs after intravenous injection and rinsed completely 5 times on a magnetic column (Miltenyi Biotec) with PBS solution, and their size was analyzed using DLS. For the mouse biodistribution studies, unmodified NW in PBS (100 μL) were intravenously injected into Nude BALB/c mice at a dose of 3 mg Fe/kg body mass (n=3 for both the PBS controls and the NW samples). The animals were sacrificed 24 hrs after injection by cardiac perfusion with PBS under anesthesia, and the blood, brain, heart, kidney, liver, lung, lymph node, skin and spleen were collected. Organs and blood were immediately weighed, freeze-dried in gelatin capsules, and then analyzed for magnetization using SQUID.
In vivo tumor homing. MDA-MB-435 human breast carcinoma cells or HT1080 human fibrosarcoma cells (1×106) were injected into the mammary fat pad or subcutaneously injected into Nude BALB/c mice, respectively. Tumors were used when they reached ˜0.5 cm in size. Some 0.2 cm or 1 cm tumors were used to compare the dependence of tumor size on NW homing. Cy7-labed or non-labeled NW or NS were intravenously injected into mice (n=3˜8 for each formulation) with a dose of 1 mg Fe/kg body mass (for fluorescence studies) and 3 mg Fe/kg body mass (for magnetization studies). For real-time observation of tumor/liver uptake, animals were imaged under anesthesia in Cy7 channel using the BonSai fluorescence-imaging system (Siemens, Pa., USA) 6 hrs, 24 hrs or 48 hrs after injection. For NIR fluorescence imaging of organs, animals were sacrificed 24 hrs after the injection by cardiac perfusion with PBS under anesthesia, and organs were dissected and imaged in Cy7 channel with a NIR fluorescence scanner (LI-COR biosciences, NE, USA). All the NIR images for animals or organs were taken at the same exposure time. To quantify the amount of NW or NS homing, collected tumors were weighed, freeze-dried in gelatin capsules, and then analyzed for magnetization using SQUID. For histologic analysis, frozen sections of tumors were prepared. The sections were fixed with 4% paraformaldehyde and stained with DAPI for observation of NW or NS only. The rat anti-mouse CD-31 (1:50, BD PharMingen) and biotinylated mouse fibrin(ogen) antiserum (1:50, Nordic) were used for immunochemicostaining of tumor tissue sections. The corresponding secondary antibodies were added and incubated for 1 hour at room temperature: AlexaFluor-594 goat anti-rat or rabbit IgG (1:1,000; Molecular Probes), streptavidin Alexa Fluor 594 (1:1000; Molecular Probes). The slides were washed three times with PBS and mounted in Vectashield Mounting Medium with DAPI. At least three images from representative microscopic fields were analyzed for each tumor sample.
For both injection doses, the NW exhibited long circulation times (
The biodistribution of NW 24 hrs post injection was similar to that reported previously for CLIO. These particles both display a tendency to undergo MPS clearance in the liver, spleen, and lymph nodes (
The NW passively accumulate in tumors, and they appear to display long residence times once they get in. The reason is believed to be that tumor vessels are generally found to be more permeable to nanoparticles than the vessels of healthy tissues. To test the role of this passive tumor targeting, intravenously injected mice bearing MDA-MB-435 tumors with Cy7-labeled NW or NS (1 mg Fe/kg) were imaged at various intervals after injection with an NIR fluorescence-imaging system. Passive tumor uptake of NW was slightly greater than NS, although the difference was not statistically significant (
The efficiency of NW and NS in homing peptide-directed targeting into tumors was analyzed. Two tumor-homing peptides were used to target the particles: F3 and a pentapeptide with the sequence CREKA. The CREKA peptide recognizes clotted plasma proteins, which accumulate in tumors but not in normal tissues. CREKA-conjugated 10 nanoparticles accumulate in tumors, but do so effectively only after pre-injection with Ni-liposomes designed to inhibit MPS uptake. This inability of nanoparticles to evade the MPS poses a significant limitation to nanoparticle targeting in vivo. To test the susceptibility of peptide-conjugated NW to MPS uptake, NW preparations containing different numbers of peptides, different degrees of amination, and the peptide conjugated either through a short linker or PEG (Table 2) were examined. The MDA-MB-435 human breast cancer xenograft and HT1080 human fibrosarcoma xenograft tumor models were chosen for these studies.
The in vivo targeting capability of Cy7-labeled, F3-conjugated NW (NW-F) in mice bearing MDA-MB-435 tumors was tested. The NW-F preparations were essentially cleared by the liver from the blood within 30 min of intravenous injection, regardless of the peptide number or the presence of a PEG layer (
Next, the ability of CREKA-conjugated NW (NW-C) to home to tumor targets in vivo was tested. The NW-C preparations that maintained the longest circulation times contain ≦60 CREKA homing peptides per NW and a PEG layer (
CREKA-conjugated NW displayed somewhat improved uptake in MDA-MB-435 tumors compared with CREKA-conjugated NS. (
Histological analysis showed that most of the PEGylated CREKA-conjugated NW colocalize with large blood vessels in the MDA-MB-435 tumor, whereas most of the NW in the HT1080 tumor appeared to have extravasated into the tumor tissue along the smaller vessels (
In contrast to the in vivo behavior of F3-modified NW, when CREKA is used as the targeting peptide (NW-C), the NW effectively home to their tumor targets. CREKA is a short linear peptide that is neutrally charged and most likely non-immunogenic. A tradeoff between the number of attached peptides and the efficiency of tumor targeting is observed for the NW-C preparations; the most effective in vivo tumor targeting is observed with ˜60 CREKA peptides per NW. This number correlates with a substantial decrease in blood half-life that is observed when >60 CREKA peptides are attached to a NW. The trend is observed for both HT1080 and MDA-MB-435 tumor types, although the overall targeting efficiency of NW-C is greater for HT1080 tumors. Additionally, in contrast to the NW-F preparations, significantly long circulation times (>10 h) are observed with some of the NW-C preparations.
For both HT1080 and MDA-MB-435 tumors, greater targeting efficiency is observed for nanoworms comprising CREKA (NW-C) when a PEG linker is used to attach the CREKA targeting group. It is postulated that the PEG linker facilitates CREKA homing by providing a less restrictive environment (relative to the short sulfo-SMCC linker), improving the peptide's ability to bind to clotted plasma proteins associated with the tumor. Additionally, the PEG linker increases residence time of the nanostructure in the blood stream.
The decrease in circulation time observed for NW containing >60 CREKA peptides is possibly attributable to the presence of unreacted amines and damage to the dextran coating (exposing bare IO cores) that occurs during preparation of the more extensively functionalized nanoparticles. The data indicate that the blood half-life of a targeting molecule/nanoparticle ensemble must be considered when selecting the appropriate ligand to target a tumor. As also observed with NW-F, a dramatic decrease in circulation time and a corresponding decrease in targeting efficiency can occur when targeting ligands are linked to nanomaterials.
A control experiment using KAREC, a scrambled version of CREKA, was performed in mice bearing MDA-MB-435 tumors. KAREC was attached to the NW using a PEG linker, and the formulations displayed similar circulation times to the PEGylated NW-C formulations. Significantly lower tumor targeting efficiency was observed with the scrambled peptide (
NIR fluorescence images of mice injected with NW-C confirm the tumor uptake results obtained by magnetic (SQUID) measurements (
Histological analysis revealed that most of the PEGylated NW-C localize with large blood vessels in the MDA-MB-435 tumor, whereas they extravasate into the tumor tissue along the smaller vessels in the HT1080 tumor. In addition, NW in the MDA-MB-435 tumor colocalize with fibrin(ogen) in the blood vessels, indicative of the self-amplifying homing. NW in the HT1080 tumor localize with fibrin(ogen) in blood vessels as well as in tumor stroma. These results suggest that HT1080 tumors, like other tumors contain clotted plasma proteins that provide initial binding sites for the CREKA peptide, and that the nanoparticles induce additional clotting within the tumor. Thus, the larger uptake of NW-C observed in the HT1080 tumor relative to MDA-MB-435 tumor is attributed to passive transport across a highly vascularized and porous microstructure, active peptide-mediated binding, and self-amplifying homing due to clotting induced by the CREKA-coated particles.
NW are more effectively attach to tumor cells in vitro while exhibiting comparable blood circulation times relative to spherical NS. The superior in vitro targeting efficiency was attributed to multivalent interactions between the elongated NW and receptors on the tumor cell surface. Similar improvement were seen in tumor targeting by NW in vivo. The optimized NW-C formulation (NW-P175-C) displays significantly higher levels of uptake in MDA-MB-435 tumors relative to NS-C (NS-P30-C). Targeting efficacy was also compared between NW-C and CREKA-conjugated commercial 10 nanoparticles (MM-500-C, blood half-life: ˜30 min). This inability of the nanoparticles to evade the MPS by themselves highlighted a significant limitation to the practical application of nanoparticle therapies that is overcome using the elongated nanostructures of the disclosure.
Preparation of gold nanorod, magnetic nanoworm, and doxorubicin liposomes. Gold nanorods (NRs) were purchased from Nanopartz with a peak plasmon resonance at 800 nm and coated with polyethelene glycol (PEG) molecules [HS-PEG(5k)]. Superparamagnetic, dextran-coated iron oxide nanoworms (NWs) with a longitudinal size of ˜70 nm were synthesized, and derivatized with near-infrared (NIR) fluorophore, Cy5.5-NHS. For control NWs, partially Cy5.5-labeled aminated NWs were coated with a PEG molecule [NHS-PEG(5k)]. For LyP1-conjugated NWs (LyP1NWs), LyP1 peptides with extra cysteine were attached to partially Cy5.5-labeled aminated NWs via a PEG crosslinker [NHS-PEG(5k)-MAL]. Control liposomes (LPs), with no functional group were prepared from hydrogenated soy sn-glycero-3-phosphocholine (HSPC), cholesterol, and 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-polyethylene glycol 2000 [DSPE-PEG(2k)] (75:50:6 mol ratio) by lipid film hydration and membrane (100 nm) extrusion. Incorporation of DOX was achieved using the pH gradient-driven protocol. For LyP1-conjugated LPs (LyP1LPs), LPs with maleimide groups were prepared from HSPC, cholesterol, DSPE-PEG(2k), and DSPE-PEG(2k)-MAL (75:50:6:6 mol ratio). LyP1 peptides with an extra cysteine were attached to maleimide-terminated LPs in PBS. LPs were intravenously injected in vivo to ensure control LPs and LyP1LPs exhibited similar circulation times (blood half-lives for both: ˜3 hrs).
In vitro cellular fluorescence imaging. The cells were treated with 80 ug Fe/mL of Cy5.5 labeled control NWs or LyP1NWs per well for 20 min at 37° C. or 45° C. in the presence of 10% FBS and incubated for an additional 2 hr at 37° C. in the presence of 10% FBS. The cells were then rinsed three times with cell medium, fixed, stained, and imaged by fluorescence microscopy.
In vivo temperature-induced tumor targeting of magnetic nanoworms. Mice bearing bilateral MDA-MB-435 human carcinoma tumors were intravenously injected with Cy7-labeled LyP1NWs or NWs and one flank of the mouse (containing one of the tumors) was immediately heated at 45° C. for 30 min in a temperature-controlled water bath. At 24 hrs post-injection, the tissues were harvested and the Cy7 fluorescence in tissues were imaged using NIR fluorescence imaging system (LI-COR Odyssey).
In vitro temperature-induced cytotoxicty of therapeutic nanoparticles. Cells were treated with free DOX, control LPs, or LyP1LPs with different concentrations at 37° C. or 45° C. for 20 min (in cell incubator) and then incubated for an additional 4 hrs at 37° C. The cells were rinsed with cell medium three times, and then further incubated for 44 hrs at 37° C. The cytotoxicity of free DOX, control LPs, or LyP1LPs was evaluated using MTT assay (Invitrogen). Cell viability was expressed as the percentage of viable cells compared to controls (cells treated with PBS).
In vivo tumor targeting of therapeutic nanoparticles by NR-mediated photothermal heating. Mice bearing bilateral MDA-MB-435 human carcinoma tumors were intravenously injected with NRs (10 mg Au/kg). At 72 hrs post-injection of NR, control LPs or LyP1LPs (3 mg DOX/kg) were systemically administered and the tumor in one flank was irradiated with NIR-light (˜0.75 W/cm2 and 810 nm) for 30 min, maintaining an average tumor surface temperature at ˜45° C. under infrared thermographic observation. At 24 hrs post-injection of liposomes, doxorubicin fluorescence in the homogenized tumors was analyzed.
In vivo therapeutic studies. To study the effect of photothermal treatment on tumor volumes, mice bearing bilateral MDA-MB-435 human carcinoma tumors were intravenously injected with NRs (10 mgAu/kg). At 72 hrs post-injection of NR, control LPs, or LyP1LPs (3 mg DOX/kg) were systemically administered and the tumor in one flank was irradiated with NIR-light (˜0.70 or 0.75 W/cm2 and 810 nm) for 30 min, maintaining average tumor surface temperature at 45° C. Each therapeutic cohort included 4˜6 mice. Tumor volume and mouse mass was measured every 3 days after the single treatment for a period of 3-4 weeks by an investigator blinded to the treatments administered. Survival rates (Kaplan Meier analyses) for the photothermal treatments were quantified using mice bearing single MDA-MB-435 human carcinoma tumors, intravenously injected with NRs (10 mgAu/kg). Control LPs or LyP1LPs (3 mg DOX/kg) were systemically administered 72 hrs post-injection and one of the tumor-bearing flanks was irradiated with NIR-light (˜0.75 W/cm2 and 810 nm) for 30 min, maintaining average tumor surface temperature at ˜45° C. Each therapeutic cohort included 6 mice. Tumor volume and mouse mass was measured every 3 days after the single treatment for a period of 9 weeks by an investigator blinded to the treatments administered. Mice were sacrificed when tumors exceeded 500 mm3. Student's t test was used for statistical analysis of the results.
In a first stage of the cooperative nanoparticle system, the photothermally-heated gold nanorods are administered. Polyethylene glycol (PEG)-coated NRs with a maximum optical absorption of 800 nm are found to accumulate passively in a MDA-MB-435 xenograft tumor. Effective in vivo photothermal heating of the tumor is achieved by application of NIR irradiation (810 nm, ˜0.75 W/cm2) from a diode laser (
A cyclic nine-amino acid peptide (Cys-Gly-Asn-Lys-Arg-Thr-Arg-Gly-Cys (SEQ ID NO:3), referred to as LyP-1, was chosen as the targeting ligand based on a screen of several tumor targeting peptides in MDA-MB-435 xenograft tumors, which showed enhanced LyP1 accumulation in the heated tumors. The LyP-1 peptide has been reported to selectively recognize lymphatics and tumor cells in certain tumor types and subsequently inhibit tumor growth. Recently, it was found that the p32 or gC1qR receptor, whose expression is elevated on the surface of tumor-associated cells undergoing stress, is the target molecule for the LyP-1peptide. Thus, the targeting of LyP-1 was investigated as it relates to up regulation of p32 receptors in the heated tumor.
The level of p32 expression in MDA-MB-435 xenografts was examined as a function of time post-heat treatment. An externally measured temperature of 45° C. was chosen for the laser heat treatment based on a preliminary screen of temperature dependent nanoparticle accumulation. It has been reported that cancer cells are most vulnerable to hyperthermia, chemotherapeutics or a combined therapy above temperatures of 43° C. Expression of p32 on the MDA-MD-435 tumors was slightly up regulated 6 hrs after heat treatment, which then returned to almost normal levels 24 hrs post-treatment (
The interaction of nanoparticles decorated with LyP-1 peptides with cancer cells was then examined upon heat treatment. An optimized formulation of NWs was prepared, and coated with LyP-1 peptides via PEG linkers (˜40 peptides per nanoworm). Significant quantities of the LyP-1 peptide-conjugated NWs (LyP1NWs) were internalized into heated MDA-MB-435 cells relative to unheated cells. In contrast, the C8161 cells displayed lower heat-mediated internalization than the MDA-MB-435 cells (
The possibility of selective homing of LyP1NWs to heated xenograft tumors in vivo was then tested. Similar to the in vitro results, targeting of LyP1NWs to heated MDA-MB-435 tumors was prominent relative to unheated tumors, while the ability of LyP1NWs to home to heated C8161 tumors was not significantly different relative to the unheated tumors (
Having verified temperature-induced amplification of nanoparticle targeting to tumor cells in vitro and to xenografted tumors in vivo, in vitro photothermal-assisted cytotoxicity of targeted therapeutic carriers was evaluated. Liposomes constructed from lipids that are not thermally sensitive were prepared and loaded with the anti-cancer drug doxorubicin (DOX). The LyP1 peptide-conjugated DOX liposomes (LyP1LPs) displayed greater levels of cytotoxicity toward MDA-MB-435 cells relative to control DOX liposomes (DOX concentration >10 ug DOX/mL in both experiments). Enhanced cytotoxicity was observed for heat-treated (45° C.) samples, whereas the measured difference in cytotoxicity at 37° C. was insignificant (
The therapeutic efficacy of the complete cooperative nanomaterials system was tested on a xenograft mouse cancer model. Twenty-four hrs post-treatment, targeting efficacy of LyP1LPs was significantly larger in the photothermally engineered tumors than in the normal tumors and than that of control LPs (
As mentioned above, hyperthermia in the temperature range ˜43° C. has been shown to selectively damage malignant cells relative to normal cells. Similarly, the increased temperature in the tumor produced by NR-mediated photothermal heating slows tumor growth in vivo, although it does not reduce tumor volume. However, tumors (or tumor cells) whose local microenvironment has been engineered by NR-mediated heating are more vulnerable to attack by therapeutic nanoparticles (
The data demonstrates that the appropriate combination of nanomaterials currently under investigation in cancer therapy can significantly enhance therapeutic efficacy relative to the individual components. Site-specific photothermal heating of NRs can engineer the local tumor microenvironment to enhance the accumulation of therapeutic targeted liposomes, which increases the overall hyperthermal and chemotherapeutic tumor-destroying effects. This cooperative nanosystem holds clinical relevance because gold salts (for rheumatoid arthritis therapies) and doxorubicin-containing liposomes (Doxil®) have been approved for clinical use, and local hyperthemia is a well-established means of destroying diseased tissues in the human body. Although the liposomes in this study are similar to Doxil®, it should be pointed out that the gold nanorod and iron oxide nanoworm formulations used in the study are distinct from clinically approved gold or iron oxide materials. Accordingly cooperative, synergistic therapies using dual or multiple nanomaterials can significantly reduce the required dose of anti-cancer drugs, mitigating toxic side effects and more effectively eradiating drug-resistant cancers.
A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, other embodiments are within the scope of the following claims.
This application claims priority under 35 U.S.C. §119 from Provisional Application Ser. No. 61/155,415, filed Feb. 25, 2009, the disclosure of which is incorporated herein by reference.
The U.S. Government has certain rights in this invention pursuant to Grant Nos. CA119335, CA0124427, and N01-CO-37117 awarded by the National Institutes of Health.
| Number | Date | Country | |
|---|---|---|---|
| 61155415 | Feb 2009 | US |
