The present invention concerns novel materials and methods to detect low frequency surface changes utilizing modified microspheres. Upon exposing the surface to the modified microspheres of the subject invention, the microspheres selectively adhere to any targeted surface marker.

Description of the Invention

BRIEF SUMMARY OF THE INVENTION

The present invention provides materials and methods for evaluating surfaces for low frequency changes. These changes may be caused by, for example, foreign body growth or settlement. Such changes are often accompanied by surface markers that are associated with changes that have occurred, are occurring, or are impending. The subject invention utilizes specifically designed microspheres in a system for easily and accurately detecting these surface changes.

In a particular embodiment of the subject invention, microspheres are modified to comprise species with which a surface marker interacts. The surface suspected of bearing the low-frequency changes (with associated marker(s)) is contacted with the microspheres, and the microspheres adhere to the exposed marker(s), if they are present.

In a specific embodiment, the present invention can be used to detect oral cancer. Oral cancer cells frequently over-express certain receptors, including, for example, the receptor for folic acid. Accordingly, microspheres can be modified with folates attached to, or embedded in, the microsphere surface. These folate-modified microspheres preferentially bind to the cancer cells that over-express receptors for folate.

In a preferred delivery system, the cancer-detecting microspheres of the subject invention are incorporated into a solution for use as mouthwash. When a person rinses the oral cavity with the mouthwash, the folate-modified microspheres adhere preferentially to any cells over-expressing folic acid receptors. Expectorated mouthwash is then collected and analyzed for a decrease in microsphere concentration, which indicates oral cancer.

A further embodiment for the detection of oral cancers comprises an additional rinsing step with a mouthwash containing an ingredient that dislodges the microspheres from the initial mouthwash. After rinsing the oral cavity pursuant to this additional rinsing step, the expectorated mouthwash from this embodiment is collected and analyzed for any dislodged folate modified microspheres. Advantageously, the two washes can be modified to contain two distinct visual indicators, preferably dyes, that are released upon proper stimulation. If the color of the solution matches the visual indicator incorporated into the set of microspheres with the folate-modified surface, the color change indicates a likelihood of oral cancer.

Another aspect of the present invention provides a composition of microspheres in a carrier that are utilized in the method of the present invention. The microspheres can comprise polymers and monomers of methyl methacrylate and N,N-diethylaminoethyl methacrylate. The carrier can be selected for its compatibility with the surface to be tested, and examples include, without limitation, aqueous solutions, organic solvents, powders, aqueous lotions, petroleum lotions, glosses, and films. Advantageously, these microspheres can be further modified to include detectable compounds (such as visual indicators), drugs, flavorings, sweeteners, antiseptic agents, and/or compounds to aid in collection. In one embodiment, multiple sets of microspheres are envisaged, so that ratios between markers (e.g, dyes) can indicate the extent of disease or type of disease.

An additional aspect of the present invention provides a kit for an easy to use, inexpensive home or clinical test for the detection of oral cancers. In one embodiment the kit includes a first vial containing a mouthwash, wherein the mouthwash comprises at least one set of microspheres, wherein one set of microspheres are modified with a species to which an oral cancer marker interacts, wherein any remaining sets of microspheres are not modified with the species; a second vial of mouthwash, wherein the pH of the mouthwash is about 5.5; and a container for collecting expectorated mouthwash from the second vial. The kit can optionally contain at least one swab or sponge attached to a holder, wherein one swab can be used to wipe an oral mucosal surface with the mouthwash of the first vial, and/or another swab can be used to wipe an oral mucosal surface with the mouthwash of the second vial.

DETAILED DESCRIPTION OF THE INVENTION

Low-frequency surface changes are possible indications of surface growth, settlement and/or contamination. Such surface changes are often the first steps to occur in processes that then progress to cause deeper, bulk changes such as tissue invasion or metastasis. Although visual identification and/or verification of such surface alterations may be possible after an extended period of time, the present invention provides a methods for early detection of minute surface changes. Early detection can be used to, for example, minimize any subsequent adverse consequences of the changes.

In a preferred embodiment, the method of the present invention involves detecting low frequency surface changes by exposure to modified microspheres. Preferably, the method provided by the present invention comprises contacting the microspheres with a surface suspected of low-frequency change, collecting any microspheres that do not adhere to the surface, and analyzing the non-adhering microspheres for a decrease in concentration or quantity of the modified microsphere or for a visual indicator. A method of the subject invention is shown in FIG. 1 (see Original Patent).

In accordance with the present invention, many surface changes (and their associated markers) are detectable including, but not limited to, metals (e.g., lead in painted surfaces; scaling in pipes), organisms (e.g., mold on carpeting, walls, or heating and cooling ducts; algae on pipes; bacteria on skin or similar surfaces) and disease indicators (e.g., a cancer cell's increased receptor activity for folic acid, TF, EGF, or VEGF). In specific embodiments, the present invention is used detect disease at an early stage. For example, the present invention can be used to detect cancer cells on mucosal surfaces.

One composition of the present invention utilizes microspheres modified to detect low frequency surface changes. As used herein the term "microspheres" is understood to include microbeads, microparticles, and microcapsules and also the corresponding name-equivalents. In one embodiment, the microspheres comprise materials that swell when the pH of their environment changes.

In a particular embodiment of the subject invention, microspheres are modified to comprise species with which a surface marker interacts. The surface suspected of bearing the low-frequency changes (with associated markers) is contacted with the microspheres, and the microspheres adhere to the exposed marker.

Advantageously, the composition can be specially synthesized to target specific surface markers in particular applications. If the microsphere is expected to come into contact with humans or animals, the indicator and all of the components can be FDA approved to limit harmful interactions with the end user.

In one embodiment, for microspheres utilized for detecting oral cancers, the microspheres are incorporated into an aqueous liquid suspension. Furthermore, the suspension can be designed to minimize settling of the heavier particles. Preferably, less than 25% of the microspheres significantly settle thirty seconds after stirring.

One embodiment of the present invention comprises exposing a surface to a mixture of two or more types of microspheres simultaneously. Each type of microspheres has a distinct characteristic so that it will either adhere or not adhere to the surface marker being sought. The microspheres can then be collected in two stages, so that one stage collects only (or predominantly) one set of microspheres (such as the weakly adhering set), and a second collection collects the remaining set. A subsequent evaluation step identifies the relative amounts of microspheres collected in each step and indicates the relative amount of specific surface markers present. This two step embodiment provides a great increase in sensitivity over methods involving depletion, because much background is removed.

Detection of Oral Cancer

For detecting oral cancer, folic acid or iron-containing transferrin (TF) (or other specie that interacts with a cancer marker) can be attached to the microsphere. Cancer cells generally overexpress receptors for these molecules. Accordingly, the microspheres will preferentially bind to cancer cells.

A first set of microspheres can incorporate a visual indicator. Preferably, the visual indicator is a dye. More preferably, the visual indicator is food coloring. In one preferred embodiment, folic acid is immobilized on the surface of the first set of microspheres. In a specific embodiment, a second set of microspheres is identical to the first except that the second set's surface is not modified with folic acid, and it contains a different visual indicator, for example, a red food coloring dye. In a preferred embodiment, both sets of microspheres incorporate a species to ease collection. Preferably, that species is iron oxide. More preferably, that species is magnetite.

In accordance with this embodiment of the present invention, a patient rinses the oral cavity with the mouthwash containing the first set of microspheres and collects the expectorated mouthwash for analysis. Contacting the mouthwash with the mucosal surface preferentially binds the folate modified microspheres to any cancer cells. If many cancer cells are present, collection of the expectorated mouthwash would be expected to be prominently or completely depleted of the folate modified microspheres. The expectorated mouthwash is analyzed to measure the decrease in concentration of folate modified microspheres.

However, if the number of modified microspheres remaining in the expectorated mouthwash were small, the microsphere concentration could be hard to detect or require specialized analytical equipment. Advantageously, a further embodiment utilizes visual indicators for easy to see indications of oral cancers. It incorporates a second mouthwash that is designed to dislodge the folate-bound microspheres. The second mouthwash can comprise, for example, a second set of microspheres without a folate modified surface but with an incorporated visual indicator, for example, a colored dye.

For dislodgement of the first set of microspheres from the surface of the oral cavity, one embodiment utilizes a slightly acidic pH solution for dislodgement; another embodiment utilizes a free folic acid.

Following rinsing the oral cavity with the second mouthwash (e.g., acidic solution with no microspheres), the second expectorated mouthwash contains any of the first set of microspheres that were bound to the folate receptors without interfering (folate-free) micro spheres.

To determine if any microspheres from the labeled set are present, the microspheres are concentrated. Preferably, a permanent magnet concentrates the microspheres by attracting the magnetite in the microspheres. Visual verification of the cancer cells is accomplished by releasing the visual indicator, preferably dye, encapsulated within the microsphere. Dye release is stimulated, in one embodiment, with solvent. Advantageously, equilibrium for dye release takes place in less than hour. In another embodiment, dye release is stimulated with a change in pH. Released dye is easily detected visually, and a color matching the dye from the first set of microspheres indicates likely oral cancer. A comparison color chart made from mixtures of the two dyes at various levels can aid in analysis. A more sensitive analysis of the color concentration can be made with a simple spectrophotometer.

Microspheres

The microspheres utilized according to the present invention can be manufactured with techniques known in the art. Accordingly, the microspheres can be synthesized using, for example, suspension polymerization, dispersion polymerization, and/or seeded polymerization. The selection of the polymerization method can be used to control the particle size of the individual microsphere. In a preferred embodiment, the particle size of the microsphere is large enough to be easily captured by a magnet, yet small enough not to be easily discharged from the target surface. Preferably, the particle size of the microsphere is within the range of about 1 to about 400 .mu.m. More preferably, the particle size is about 4 to about 50 .mu.m. Most preferably, the particle size is about 4.5 to about 5.5 .mu.m.

In a specific embodiment, methyl methacrylate microspheres can be produced using a suspension polymerization method, yielding a broad particle size distribution with ranges centered from about 1 to 400 .mu.m. Smaller microspheres (single .mu.m size) with a narrower size distribution can be made via dispersion polymerization. Yet smaller (submicron sized) microspheres can be produced via emulsion polymerization. Acrylate based microspheres can also been produced using seeded polymerization mechanisms to yield a variety of monodisperse size ranges. PH-sensitive microspheres, with activation pH typically around 5.5, but adjustable above or below that value, can also be formulated (Batich, et al., "Swelling Behavior of pH-Sensitive Copolymers Based on Styrene and 4- (or -2) Vinylpyridine," Macromolecules, 126, 4675-4681 (1993)).

In one embodiment, the microspheres comprise methyl methacrylate, N,N-diethylaminoethyl methacrylate, and/or polymers of each, and a species to which a surface marker preferentially binds or otherwise detectably interacts. Preferably, when the surface change is head and neck squamous cell carcinoma (HNSCC), the species that will bind to an associated marker is selected from the group consisting of folates, TF, EGF, and VEGF. The species can be, for example, attached to the surface of the microsphere or embedded within.

For microspheres comprising polymethylmethacrylate (PMMA), an active functional group can first be formed on the microsphere by either hydrolysis or aminolysis. For example, first the microspheres can be immersed in a dilute (1 N) solution of ethylene diamine in hexane. The ester bonds on the surface can react with the amino group and form amide bonds with a free primary amino group. Folic acid can be reacted with dicyclohexyl carbodiimide, and the solution added to amino derivatized microspheres.

To aid in surface attachment, a spacer species can be attached to the microsphere by reaction with the microsphere surface. Preferably, the spacer species is a difunctional polyethylene glycol (PEG). More preferably, the spacer species is a t-BOC protected NH.sub.2-PEG. Advantageously, t-BOC protected NH.sub.2-PEG has one free amino group to react with folate in DMSO solvent. After deprotection with trifluoroacetic acid, the reagent can be reacted directly with the methacrylate surface. In a specific embodiment, a PEG800 is useful as a linker. (Gabizon et al., "Targeting Folate Receptor with Folate Linked to Extremities of PEG-grafted Liposomes: in vitro Studies" Bioconjugate Chem. 10, 289-98 (1999)). Alternatively, other difunctional PEG's can be attached as a spacer to folate and have been used to target boron-containing molecules (for boron neutron capture therapy, i.e., BNCT) to the folate receptor expressed on human KB cancer cells (ATCC #CCL 17), a line derived from an epidermal carcinoma of the oral cavity. (Shukla, et al., "Synthesis and Biological Evaluation of Folate Receptor-Targeted Boronated PAMM Dendrimers as Potential Agents for Neutron Capture Therapy" Bioconjugate Chem., 14, 158-167 (2003)).

Microsphere Loading

The microspheres utilized according to the present invention can comprise a visual indicator encapsulated within the microsphere. Optionally, the visual indicator can be incorporated as a nanoparticle within the microsphere. Preferably, the visual indicator is a dye. More preferably, the visual indicator is a food colorant. Under proper stimulation, the microsphere releases the visual indicator. A pH change or a solvent change are typical stimulants.

The method of loading visual indicators into the subject microspheres can comprise a multi-step process. In one embodiment, dried microspheres are first washed in methanol and then permitted to air dry. Washed microspheres are immersed in a 10 wt % solution of dye in alcohol (usually ethanol or methanol) for 24 hours, removed from solvent liquid, and the alcohol permitted to evaporate. The microspheres are then repeatedly washed in fresh water, collected by centrifugation, and finally dried in a vacuum oven at 60.degree. C. under a modest vacuum of about 100 Torr. Solvents for the swelling can be chosen based on the solubility parameters of the solvents and the polymers. Mixed solvents can also be used to maintain the use of materials generally recognized as safe for human exposure in small quantities: PMMA: .delta.=9.5 (cal/cm.sup.3).sup.1/2 Isopropanol: .delta.=8.8 (cal/cm.sup.3).sup.1/2 Water: .delta.=23.4 (cal/cm.sup.3).sup.1/2

To match the solubility parameter of the polymer almost exactly, a 95 v/v % isopropanol and 5 v/v % water solution will yield .delta.=9.5 (cal/cm.sup.3).sup.1/2, as determined by a rule of mixtures calculation. Adjustment of the solvent composition will allow swelling, but not dissolution. Preferable dyes include 9-amino acridine and fluorescein. Other dyes can also be used including, for example, indigostine, quinoline yellow, red 2 g, and mixture thereofs.

In the case of microspheres to be used for cancer cell detection, a mix of folate surface-labeled particles can be loaded with dye "B" and can be mixed with an equal amount of microspheres without surface folate, but containing dye "A." If there is no selective binding, then collected microspheres will show equal amounts of dye "A" and dye "B." If folate binding is occurring, then recovery of the bound microspheres (after rinsing off unbound ones) show an excess of dye "B" relative to dye "A." This difference can be determined by, for example, visual inspection, optionally with comparison to a color chart, or with an analysis by a spectrophotometer.

Formulations and Uses

The microsphere composition of the present invention can be transported by varying the delivery system in which the composition contacts any location. Examples of delivery systems include, but are not limited to, aqueous solutions, for example, mouthwashes; organic solvents; powders; aqueous or petroleum based lotions; and glosses or films.

The composition can further comprise flavoring to improve taste. Optionally, the flavoring can be incorporated into the microsphere or added to the delivery system.

Advantageously, in one embodiment the present invention provides an easy to use beneficial screening test for oral cancer that can be administered by physicians and other health care professionals or less highly trained laypersons. Another aspect of the invention involves a kit for administering a simple, possibly home-based test for likelihood of oral cancer.

The system of the subject invention can also be used as a vehicle for the in situ delivery of biologically active agents. The biologically active agents incorporated into, or included as an additive within, the microspheres of the subject invention can include, without limitation, medicaments, vitamins, mineral supplements, substances used for the treatment, prevention, diagnosis, cure or mitigation of disease or illness, substances which affect the structure or function of the body, or drugs. The active agents include, but are not limited to, antifungal agents, antibacterial agents, anti-viral agents, anti-parasitic agents, growth factors, factors affecting angiogenesis, anesthetics, mucopolysaccharides, metals, cells, and other wound healing agents.
 

Claim 1 of 12 Claims

1. A method for detecting the presence of a marker associated with oral cancer on an oral cavity mucosal surface wherein said method comprises: a) contacting the oral cavity of a patient with a composition, wherein the composition comprises at least one set of microparticles and a carrier, wherein the microparticles comprises a specie that is capable of adhering to the oral cavity mucosal surface marker; and b) determining whether any microparticles have adhered to the surface marker, wherein adherence of the microparticles to the surface indicates the presence of the marker, wherein the determining step comprises either or both of the following: i) recovering the composition from the surface and determining whether the contacting of the composition with the surface reduced the concentration of microparticles in the composition; and ii) dislodging any microparticles that adhere to the surface with a second composition, and analyzing the second composition to determine if any microparticles are present, wherein the presence of microparticles indicates the presence of the marker; wherein the specie is selected from the group consisting of folic acid, folate, and a folate conjugate; and wherein the carrier is a neutral mouthwash aqueous solution.

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