Devices, compositions, and methods for in vivo fluorescence imaging

Abstract

The present inventions provide improved solution compositions and methods for detecting cancerous and dysplastic lesions in mucosal tissue in vivo. Specifically, the disclosed pretreatment solution results in enhanced removal of mucous and tissue debris. The disclosed detection solution compositions (nuclear stain-based and lectin-based) provide enhanced disease detection, when used individually or sequentially, tissue permeation, fluorescent signal, and shelf stability. Lectin fragmentation methods and processes of manufacturing are also disclosed. Additionally, various methods utilizes the disclosed inventive solution compositions are disclosed for accurate disease detection in vivo yielding high sensitivity and specificity values.

Description

[0001] Atty. Docket No.: 1249-11470-PCT

[0002]

[0003] PCT

[0004] Devices, Compositions, and Methods for In Vivo Fluorescence Imaging

[0005] Field of the Invention

[0006] The present invention relates to devices, compositions and methods for use in screening and testing patients to detect for the possibility of having precancerous, dysplastic and cancerous lesions in mucosal tissues. In particular, the application to the esophagus and oral cavity are discussed in detail. However, the compositions and methods are translatable to other mucosal tissues (cervical, gastrointestinal, bile duct, colon, anus, nasal, etc.).

[0007] Background

[0008] Esophageal Cancer and Barrett’s Esophagus Background

[0009] Barrett’ s Esophagus (BE) is a common premalignant condition that affects 1 ,2M Americans and is characterized by abnormal tissue changes in the lower part of the esophagus as a result of chronic acid reflux. If left untreated or unmonitored, BE may progress to dysplasia and ultimately to cancer. Once BE transitions to high-grade dysplasia, approximately 60% will transform to esophageal cancer (EC) within five years. Although the exact causes are unknown, the incidence of EC has increased rapidly in the US and Europe over the past few decades. Unfortunately, esophageal cancer has a very low 5-year survival rate of only 19.9%, which is Atty. Docket No.: 1249-11470-PCT

[0010] -2 -mainly due to the fact that >70% of ECs are diagnosed after spreading to local lymph nodes or metastasizing. At these late stages, treatment is significantly more difficult, invasive, and dangerous for the patient, leading to poor patient prognosis, outcomes, and survival rates. However, when detected early in a localized area, the 5 -year survival rate of EC significantly increases to almost 50%. If detected in the BE stage or low-grade dysplasia stage, survival rates are >99% as long as monitoring is maintained. Therefore the early detection of BE, esophageal dysplasia, and EC is imperative to downstage the magnitude of esophageal disease and associated morbidity and mortality.

[0011] More than seven million (7,000,000) upper endoscopies are performed each year in the US, and 1,200,000 (17%) of those surveyed are diagnosed with BE and require continual monitoring to observe disease progression. Overall, the prevalence of BE in the US is 0.4% (interestingly, the prevalence in Europe is higher at 1.1%). Each year, approximately 18,000 Americans are diagnosed with EC, which is more than the number of annual cervical or testicular cancer diagnoses.

[0012] Current Practice for BE and EC Screening and Detection

[0013] Current screening and surveillance for Barrett’s Esophagus (BE) and esophageal cancer (EC) primarily rely on white-light endoscopic visual inspection by gastroenterologists, followed by tissue sampling via the Seattle protocol. This protocol involves taking four-quadrant biopsies at 1-2 cm intervals along the length of any suspected BE segment. Despite being the standard of care, this approach is inherently limited by its reliance on random sampling, which leads to significant diagnostic inaccuracy. Reported false negative rates range from 33% to 56%, while false positive rates can reach up to 31%, resulting in both missed pathology and unnecessary interventions.

[0014] Moreover, the Seattle protocol typically requires 6 to over 30 biopsies per patient, significantly increasing procedure duration, anesthesia exposure, pathology workload, and overall healthcare costs. Patients are also subjected to greater postoperative discomfort, an elevated risk of bleeding, infection, and longer recovery times. These inefficiencies place a substantial burden on healthcare systems and reduce screening accessibility.

[0015] Due to these limitations, BE and EC have not experienced the improvements in early detection seen in other cancers such as breast, cervical, or prostate cancer, where Atty. Docket No.: 1249-11470-PCT

[0016] - 3 -screening tools are more targeted, standardized, and effective. A recent survey of practicing gastroenterologists revealed widespread dissatisfaction with the current approach — 92% expressed the need for stronger evidence to support surveillance biopsy protocols, while 90% simultaneously felt they were not collecting enough biopsies to confidently rule out disease. This diagnostic gap underscores the critical need for more accurate, real-time, and minimally invasive technologies to enhance early detection and risk stratification in patients with suspected BE or EC.

[0017] Oral Cancer and Dysplasia Background

[0018] Oral cancer includes cancers of the lip, tongue, cheeks, floor of the mouth, hard and soft palate, sinuses and pharynx. It is the sixth most common cancer in the U.S. and the third most prevalent in developing countries. The current five-year survival rate for oral cancer is 65%. This is much lower than other more common cancers such as cervical, breast, testicular, and melanoma, which is surprising as oral tissue is so easily accessible. The five-year survival rate is even worse in third world countries. For example, India exhibits the highest prevalence of oral cancer and a low survival rate of 30-39%. The main inhibitor to increasing these survival rates is the fact that many oral cancers are diagnosed and treated at advanced stages with poor prognoses. A quick and ergonomically friendly method that allows for the early detection of oral cancer would help greatly augment patient prognosis and survival rates worldwide.

[0019] Most oral cancers are preceded by precancerous lesions, which typically present as visible white and / or red lesions (e.g. leukoplakia, erythroplakia, speckled leukoplakia, etc.). Precancerous lesions usually progress from varying grades of dysplasia (mild, moderate, and severe) to carcinoma-in-situ / cancer. Oral precancer affects approximately 2% of Americans. The presence and progression of these lesions directly correlates to frequency of tobacco usage, and many early-stage dysplastic lesions can fully heal after tobacco cessation. Conversely, if tobacco habits are maintained, and lesions left unexamined and untreated, oral precancerous lesions have a cancer transformation rate of approximately 17.5%. As such, it is imperative to screen for precancerous lesions during early stages and treat them before they progress to cancer.

[0020] Current Practices for Oral Cancer Screening and Detection Atty. Docket No.: 1249-11470-PCT

[0021] -4 - Currently, most clinicians visually screen patients for precancerous or cancerous lesions; however, visual screening relies heavily on the lesion’s clinical appearance. Unfortunately, many lesions are difficult to visualize, examine, and diagnose at early stages. As such, many of the lesions detected visibility are typically late-stage cancers. To circumvent this issue, researchers and clinicians have developed procedures to screen for precancerous and cancerous sites in the oral mucosa. Typically, these procedures use a colored dye that tends to localize in and stain precancerous and cancerous lesions. U.S. Pat. No. 4,321,251 to Mashberg discloses such a procedure, which utilizes sequential washes, rinses and applications of water, diluted acetic acid and a solution of toluidine blue-0 dye. This procedure results in precancerous / cancerous tissue being stained darker blue.

[0022] Another patent, U.S. Pat. No. 6,417,003 to Cipriani and assigned to Zila, Inc., describes a kit using a similar method for oral cancer screening. There, the dye toluidine blue-O and a rinse are provided to the clinician at preselected concentrations and volumes. This kit is marketed by Zila, Inc. under the tradename ViziLite™ Oral Screening System. The Vizilte™ system initially has patients rinse with acetic acid for 60 seconds, which dehydrates the oral mucosa, resulting in cancerous tissue dehydrating differently than normal tissue. At this point the clinician inspects the oral mucosa using an included light stick or other illuminated device; if a suspected lesion is apparent, then toluidine blue-0 dye is used to further test for cancerous tissue, with cancerous tissue becoming increasingly stained blue due to increased DNA content. However, this procedure is very subjective as it is based upon the clinician’s perspective of “the hue of blue.” Further, under normal oral health, nucleated scales covering the papillae on the dorsum of the tongue and the pores of seromucinous glands in hard palate are often stained with TBlue, further decreasing the efficacy and specificity of this treatment.

[0023] Another device called the VELScope™ was released by LED Dental (W02005099563A1). This device is unique in the fact that it does not require a solution or rinse; it utilizes tissue autofluorescence from endogenous fluorophores that naturally vary in concentration between precancerous / cancerous tissue and normal tissue. More specifically, this device produces light in the range of 400 to 460 nm that mainly excites the flavin adenine dinucleotide (FAD) molecules within cells. As oral cancer cells have less FAD than normal cells, cancerous cells exhibit decreased fluorescence than the surrounding normal mucosa. To Atty. Docket No.: 1249-11470-PCT

[0024] -5 -effectively use the VELScope™, a practitioner is required to look through the VELScope™ as filtering of FAD light emission is necessary for visualization; emitted light is first filtered through a 475 nm long-pass filter then through a notch filter of undisclosed bandwidth. Additional tissue characteristics affecting autofluorescence for the VELScope™ are decreased collagen fluorescence and increased light absorbance in cancerous tissue due to less collagen and increased hemoglobin and vasculature, respectively.

[0025] Unfortunately, the aforementioned devices, methods and kits have yielded inconsistent results during clinical evaluations, and do not provide a definitive diagnosing procedure capable of distinguishing normal / benign tissue from precancerous / cancerous tissue. Additionally, many methods have not achieved routine use, partly because the necessary sequential steps are too time consuming. Furthermore, these devices can be cost prohibitive for many clinicians. Lastly, the use of staining dyes results in an ergonomically unfriendly procedure as patient compliance is lowered due to visible oral staining. Therefore, there is a need for a quick, compliant and accurate system to screen every patient for oral cancer during routine office visits, such as dental visits for cleaning, etc.

[0026] Summary of the Invention

[0027] The present invention provides improved devices, compositions and methods for distinguishing mucosal precancerous conditions, dysplasia and cancer from normal and benign tissues. It should be noted that all devices, compositions, and methods are anticipated to be used in the human body, i.e. in vivo, without necessitating removal of tissue from the human. In particular, devices are described which allow the user to screen for potentially cancerous, precancerous, and / or dysplastic lesions in vivo. Additionally, the composition of a pretreatment solution is described, as well as the composition of two different detection solutions. One detection solution comprises at least one fluorescent nuclear stain while the other detection solution comprises at least one fluorescently-labeled lectin. The described method is a multistep process using the aforementioned solutions, as well as light sources and viewing devices, to screen and distinguish precancerous / cancerous / diseased lesions from normal / benign tissue. Lastly, various kits are inferred by this invention by combining various inventive aspects (e.g. various solution compositions as part of a multi-step process) with other materials optionally including but not limited to: syringes, swabs, brush biopsy components, punch Atty. Docket No.: 1249-11470-PCT

[0028] - 6 -biopsies, gauze, filters, electronic equipment for visualizing fluorescence, isotonic saline, sterile water, tubes, instructions for use, amongst others.

[0029] The application of the invention to esophageal cancer and oral cancer are described in detail. However, the compositions and methods are translatable to other mucosal tissues (cervical, gastrointestinal, nasal, bile duct, colon, anal, etc.). The application of the invention to cancer is described in detail. However, the compositions and methods are translatable to other diseases characterized by aberrant glycosylation or nucleic acid content or expression.

[0030] The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the description, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the reside list serves only as a representative group and should not be interpreted as an exclusive list.

[0031] Brief Description of the Drawings

[0032] Figure l is a block diagram of a tissue pretreatment protocol, wherein a sample of porcine tongue tissue (A) was 50% embedded in agar and the free end was marked with green tissue ink (B). The embedded tissue of each sample served as a control while the exposed tissue was subjected to the pretreatment solution (C).

[0033] Figure 2 is a bar graph displaying the effectiveness of 11 pretreatment solutions at removing surface epithelium of oral tissue biopsies. The asterisk designates statistically significant results (p < 0.05), whereas the caret designates results suggestive of significance (p = 0.06), when compared to the PBS control. AVE ± SD; n = 3.

[0034] Figure 3 is a scatter plot of DAPI fluorescence from stained tissue phantoms versus DAPI concentration. An optimal concentration for fluorescence was observed to be ImM. AVE + SD; n = 3.

[0035] Figure 4 is a schematic representation of tissue processing and imaging. Two sections of tissue, one normal and one tumor (1) are placed on the same slide (2) and incubated in a lectin-containing solution for 30 minutes at 37° C (3 and 4). Tissue sections are then covered with a coverslip (5) and imaged using the optical system (6). Atty. Docket No.: 1249-11470-PCT

[0036] - 7 - Figure 5 is a schematic representation of fluorescence quantification from tissue. The mean fluorescent intensities (MFI) of ten regions-of-interest were measured from the epithelial layer of normal tissue and from squamous cell carcinoma nests found in tumor tissue. The signal -to-noise ratio (SNR) was then calculated as the MFI of the cancerous tissue divided by the MFI of the normal tissue epithelium.

[0037] Figure 6 is a scatter plot of the signal -to-noise ratio versus lectins for patients 1-5. All lectins exhibited binding to the tissue sections; however, only seven lectins (Jacalin, LcH, UEA-I, VVA, MAA, UDA, and SNA) demonstrated favorable binding (SNR > 1.5 or SNR < 0.67; p < 0.05) for at least 3 / 5 patients. The blue box designates cutoff SNR values. Concentrations tested are lOug / mL unless noted differently.

[0038] Figure 7 shows the binding of MAA (20ug / mL) to cancerous and normal tissue sections from patient 2 which provided a SNR of 2.5. Overall, MAA (20ug / mL) demonstrated the greatest average SNR of 1.97 ±0.71. White boxes indicate where fluorescent measurements were taken.

[0039] Figure 8 shows the binding of (lOug / mL) to cancerous and normal tissue sections from patient 5. UEA-I yielded a high SNR for this patient and was able to distinguish unexpected cancer in the normal biopsy section, although this section looked visibly normal. White boxes indicate where fluorescent measurements were taken.

[0040] Figure 9 shows the bindings of inhibited UEA-I (lOug / mL) to cancerous and normal tissue section from patient 5. Inhibition was performed using L-a-fucose. Inhibition yielded a 2.61 and 1.79-fold decrease in fluorescence on cancerous and normal tissue, respectively, when compared to the uninhibited lectin results shown in Fig. 8. This helps illustrate the specificity of the lectin towards its target. White boxes indicate where fluorescent measurements were taken.

[0041] Figure 10 shows images of tissue phantoms after applying various WGA-FITC concentrations (100, 50, 20, 10, 5, 1 and 0 uM). The top row shows the negative control tissue phantom (2w / w% agar, 4w / w% pectin) did not result in any staining with WGA-FITC solutions. The bottom row shows the positive control tissue phantom (2w / w% agar, 4w / w% chitosan) was stained with WGA-FITC solutions as it contains binding sites for WGA-FITC.

[0042] Figure 11 is a scatter plot showing normalized fluorescence intensity of WGA- Atty. Docket No.: 1249-11470-PCT

[0043] - 8 - FITC solutions versus concentration after applying to tissue phantoms (AVE ± SD; n =3). A scientific camera mounted in an optical system was used to obtained fluorescence measurements. A maximal fluorescence intensity was observed at lOOuM WGA.

[0044] Figure 12 is a scatter plot showing intensity increase per additional luM of WGA-FITC. Data demonstrates diminishing returns for increases in concentration to measured fluorescence. A clear maximum is observed between 5-20uM concentrations. A concentration of 20uM is preferred as it maximizes fluorescent gains and safety (less active ingredient) while minimizing product cost and allowing for potential degradation / loss of activity during shelf life. AVE ± SD; n = 3.

[0045] Figure 13 shows PE’s compatibility with the WGA lectin. All tested samples in Table 9 were compared to the PBS control (A). 1% Pluronic F-68 resulted in no inhibition of the WGA lectin (B), while 1% propylene glycol (C) and DMSO (D) resulted in intermediate and significant inhibition of the lectin’s bioactivity, respectively. PEs that did not inhibit WGA’s activity were used during the remainder of the study.

[0046] Figures 14A and B show the compatible PEs’ effect on tissue penetration depth of WGA. The results for all 18 samples are shown in (A). The asterisk designates statistically significant results (p < 0.05) whereas the caret designates results suggestive of significance (0.1 > p > 0.05). Sample 13 (C) offered the greatest penetration enhancement, 340um compared to 91um for the PBS control (B), a 3.75-fold increase in depth penetration.

[0047] Figure 15 is a scatter plot and best fit line showing ethanol concentration with in lectin-based detection solution versus WFA-FITC binding to chitin beads within 1 hour.

[0048] Figure 16 is a bar graph showing the results of the combination experiments listed in Table 12. Double asterisks indicate the designated pretreatment solution yielded statistically significantly better lectin penetration than IX PBS, whereas a single asterisk means the designated pretreatment solution yielded statistically significantly worse lectin penetration than PBS (p<0.05). The absence of an asterisk means the pretreatment solution was not statistically different than the PBS control (p>0.05).

[0049] Figure 17 shows penetration depth of WGA-FITC with PEs following PBS and citric acid pretreatments outlined in Table 12 as combinations II and III. Citric acid pretreatment in both combinations II and III (B and D) yielded statistically significantly deeper WGA-FITC Atty. Docket No.: 1249-11470-PCT

[0050] - 9 -penetration than the PBS control (A and C) (p < 0.05).

[0051] Figure 18 shows ex vivo results from UMCG clinical investigation. WGA fluorescent staining directly correlates to dysplasia and cancer with higher grades of disease corresponding to increased WGA fluorescence.

[0052] Figure 19 is a line graph of retention time in minutes vs detection in Au for WGA-800CW at 280nm.

[0053] Figure 20 is a line graph of fluorescence intensity (Au) vs fluorophore to protein label ratio.

[0054] Detailed Description

[0055] Although the disclosure hereof is detailed and exact to enable those skilled in the art to practice the invention, the physical embodiments herein disclosed merely exemplify the invention which may be embodied in other specific structures. While the preferred embodiment has been described, the details may be changed without departing from the invention.

[0056] Device for in vivo fluorescence imaging

[0057] The invented device may have slightly different features, sizes, and / or shapes depending on the application. For example, the invented device is preferably handheld, battery operated and may resemble a flashlight or dental curing light for screening the following mucosal sites: oral cavity, cervix, amongst others. In this embodiment, the battery is preferably removable and replaceable. Conversely, the invented device is preferably an endoscope-like device for screening the following mucosal sites: esophagus, stomach, bile duct, colon, intestines, amongst others. Regardless of the differences in device design, features, sizes, and / or shapes, the invented device will contain characteristics and features as described.

[0058] The device comprises a durable housing made of medical-grade materials such as aluminum, polycarbonate, silicone, or thermoplastics, selected for biocompatibility, sterilizability, and thermal conductivity. The housing encloses the primary electronics, power source, light sources, and optical components. The device also includes at least one single-color light emitting diode (LED) that is tuned to the excitation wavelength of the target fluorophore (i.e. the nuclear stain or fluorescently-labeled lectin within the detection solutions) to cause fluorescence which can be visualized via the human eye (naked eye or with filter glasses) or a Atty. Docket No.: 1249-11470-PCT

[0059] - 10 -camera. As an example, preferably the peak emission wavelength of the single-color LED is between 340-385nm (UV LED), 460-500nm (Blue LED), or 730nm-790nm (Far-red LED) for 4’, 6-diamidino-2-phenyllindole (DAPI), fluorescein isothiocyanate (FITC) or IR800CW fluorophores, respectively, which matches the various fluorophores’ peak excitation wavelength. Each LED is mounted distally within the housing to provide direct illumination to the target tissue. This eliminates the need for fiber-optic bundles or light pipes, simplifying construction and minimizing optical losses. Thermal management of the at least one distally mounted LED(s) is accomplished by mating the at least one distally mounted LED to a primary heat sink consisting of a printed circuit board (PCB; more preferably a metal-clad PCB (mcPCB) and even more preferably a copper-clad PCB). If additional thermal management is needed for proper operation and longevity of the at least one LED, then the primary heat is preferably mated to a secondary heat sink which may or may not be the device housing material (if metal). A tertiary heat sink, which may or may not be the device housing material, may further be utilized and mated with the secondary heat sink for additional thermal management of the at least one LED. Preferably the secondary heat sink, and tertiary heat sink, if applicable, consist of materials with a high thermal conductivity of at least 100 W / m*K and preferably at least 200 W / m*K. Mating between the different heat sinks is preferably accomplished using a thermal epoxy, thermal adhesives, thermal tape, or other material containing an appropriate thermal conductivity of at least 10 W / m*K and preferably at least 100 W / m*K. If more than one LED is utilized, the at least two LEDs can be mounted to separate primary heat sinks which may optionally be mated to the same or different secondary heat sink or tertiary heat sink.

[0060] Optionally, various shortpass, bandpass, or long-pass filters may be incorporated to specifically attune the excitation wavelength from the LED. As an example, preferably a shortpass filter is included at a location in front of the LED with a cutoff between 380-440nm, 480-5 lOnm, or 770-805nm for the UV LED, Blue LED, and Far-red LED, respectively. It should be noted that more than one single-color LED can be incorporated into the device, which will allow for dual spectrum fluorescent imaging. Preferably, the device also includes a white light LED which allows the device to provide white light illumination. In certain uses of the invented device, such as fluorescent endoscopy, the incorporation of white light illumination is vital for simultaneous fluorescence and white light imaging which allow Atty. Docket No.: 1249-11470-PCT

[0061] - 11 -for image overlay of the white light and fluorescence images for accurate tissue registration and device spatial orientation.

[0062] In certain embodiments, at least one camera module is embedded within the device for visualization and photography of the white light and / or fluorescence imaging. Optionally, filters may be incorporated in front of the at least one camera module to visualize specific wavelengths of light. If desired, the camera module may be mated to the primary, secondary, or tertiary heat sink to facilitate thermal management of the camera. Preferably, the at least one camera module and at least one LED are mounted to the same primary heat sink which consists of an mcPCB.

[0063] Pretreatment solution composition

[0064] The pretreatment solution of the present invention is formulated to optimize mucosal tissue for fluorescent imaging by preparing the surface for uniform and effective dye penetration. Specifically, it serves to (1) remove surface debris, mucus, and non-viable cells, (2) dehydrate the epithelial surface, and (3) enhance subsequent uptake of the detection solutions.

[0065] The composition includes at least one organic acid, one dehydrating agent, one solvent, and one surfactant. Optional additives may include preservatives, mucolytic agents, flavorants, sweeteners, and water-free lubricants. The preferred components are outlined below and designed to maintain compatibility with downstream nuclear and lectin-based detection chemistries.

[0066] Preferred Components and Functions

[0067] • Organic acid(s) (e.g., citric acid, glycolic acid, acetic acid): aid in pH modulation, tissue permeabilization, and protein denaturation.

[0068] • Dehydrating agent(s) (e.g., ethanol): reduce surface water content and enhance contrast agent diffusion.

[0069] • Solvent: typically water or water-alcohol mixtures, serving as the carrier.

[0070] • Surfactant(s) (e.g., poloxamers, Triton X-100, fluorosurf actants): lower surface tension and facilitate mucus removal and tissue wetting.

[0071] • Optional mucolytic agents (e.g., N-acetylcysteine): assist in disintegrating mucus layers. Atty. Docket No.: 1249-11470-PCT

[0072] - 12 - • Optional flavorants / sweeteners (e.g., methyl salicylate, xylitol): improve patient tolerability for oral or esophageal use.

[0073] • Optional lubricants (e.g., polyethylene glycol, propylene glycol, glycerin): aid in tissue coating and may reduce friction during application.

[0074] Preferably, the pretreatment solution is comprised of the aforementioned ingredients in the approximate ranges outlined in Table 1. In certain embodiments, the at least one organic acid is comprised of glycolic acid, citric acid, oxalic acid, acetic acid, amongst others. The at least one dehydrating agent is preferably an alcohol, for example, ethanol. In certain instances, the at least one dehydrating agent may also comprise the at least one solvent. Preferably, the at least one solvent is comprised of water. The at least one surfactant may comprise, but is not limited to: sulfonates, sulfates, ethoxylated alcohols, ethoxylated alkylphenols, fatty acid esters, ethoxysulfates, betaines, amine oxides, fluorosurfactants, amongst others. Optionally, mucolytic agents may be incorporated into the composition, such as n-acetylcysteine, or these mucolytic agents may be administered separate from, and prior to the pretreatment solution. Optionally, at least one substantially water free lubricant (e.g. polyethylene glycol, propylene glycol, glycerin) may be added to the pretreatment solution composition at a weight / weight percent concentration between 5% - 35%. Optionally at least one flavorant, which may additionally comprise sweeteners, may be added to the composition at a weight / weight concentration between 0.1% - 20% to enhance the palatability of the pretreatment solution. These flavorants include, but are not limited to, methyl salicylate (wintergreen), spearmint, citrus, cinnamon, sorbitol, xylitol, mannitol, amongst others and combinations thereof. In some embodiments, the pH of the pretreatment solution composition is preferably acidic (pH < 7), more preferably between pH 2 - 6, and most preferably between 3 - 5.5. In certain embodiments, the surface tension of the pretreatment solution is less than 80 dynes / cm. In some embodiments, the surface tension is less than 50 dynes / cm. In certain embodiments, the surface tension is less than 30 dynes / cm. At room temperature, the disclosed pretreatment solution may have a viscosity of about 0.1 cP to 1000 cP. In some embodiments, the viscosity is about 1 cP to 10 cP.

[0075] Table 1: Pretreatment solution composition. Atty. Docket No.: 1249-11470-PCT

[0076] - 13 -

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[0080]

[0081] In a preferred embodiment the pretreatment solution is composed of the following compounds shown in Table 2. These particular components were chosen because they do not inhibit active components in the disclosed detection solutions (nuclear stain and fluorescently-labeled lectin solutions; testing is described in detail later in the specification) and they help solubilize and remove mucosal debris. Preferably the pretreatment solution has a shelf-life of at least one year, and more preferably at least two years.

[0082] Table 2: Pretreatment solution’s composition in the preferred embodiment. More preferred values are listed in parentheses.

[0083]

[0084] Atty. Docket No.: 1249-11470-PCT

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[0089] Nuclear stain-based detection solution composition

[0090] The present invention is comprised of fluorescent nuclear stains that target molecular characteristics of precancer and cancer in vivo. This composition exploits hallmark features of neoplastic transformation — namely, increased DNA content (aneuploidy), elevated cell density, and abnormal nuclear architecture — by targeting nucleic acids with fluorescent stains that preferentially accumulate in dysplastic and malignant tissues. This is due to the fact that optical molecular imaging is sensitive to tissue volume, and there are several commercially available fluorescent stains that bind to DNA.

[0091] In a preferred embodiment, UV excitable nucleic acid dyes are used as these fluorophores emit visible fluorescence upon excitation by specific light wavelengths and do not cause visible staining under ambient lighting, making them suitable for in vivo use. Examples of such compounds are, but not limited to, 4’, 6-diamidino-2-phenyllindole (DAPI), Hoechst 33342, Hoechst 33258, Acridine Orange, dihydroethidium, hydroxystilbamidine, aminocoumarin, and berberine sulphate. In the preferred embodiment of the solution’s composition, DAPI is used; however, the other nuclear fluorophores could be used as they function in a similar manner.

[0092] While fluorescent nuclear stains such as DAPI and Hoechst dyes are widely used in vitro and in cell culture, their use for direct in vivo diagnostic staining of intact mucosal tissue has not been previously disclosed. The present invention overcomes the historic challenges associated with applying these cationic DNA-binding agents to intact epithelial surfaces by optimizing solution composition for tissue penetration, staining efficiency, and patient tolerability. Although DNA is present in all oral cells (except red blood cells), the increased amount of DNA and cell density in cancerous tissue results in a higher DNA density overall. The preferred use of these compounds will result in increased localization of fluorescent compounds within precancerous and cancerous cells due to the increase in DNA Atty. Docket No.: 1249-11470-PCT

[0093] - 15 -density. In turn, this will lead to increased cancerous or precancerous tissue fluorescence upon fluorophore excitement utilizing appropriate light sources and wavelengths. Alternatively, staining of precancerous tissue with this nuclear stain-based detection solution may result in a decrease in visualized fluorescence compared to normal tissue. This phenomenon has not been visualized before but is hypothesized to be due to electrostatic repulsion of the cationic nuclear stain from positively charged tissue proteins.

[0094] The use of DNA binding fluorophores, in conjunction with optical molecular imaging, has yet to be defined and used in vivo to detect or discern between normal / benign tissue and diseased (precancerous and cancerous) tissue. In particular, the decreased staining of precancerous and dysplastic tissue with the nuclear stain-based detection solution has not been shown before

[0095] The nuclear stain detection solution is comprised of at least one nuclear stain, at least one organic acid, at least one conjugate base of the organic acid(s) in salt form, at least one solvent, at least one dehydrating agent, and at least one preservative (Table 3). Optionally, at least one surfactant (preferably non-ionic or cationic surfactants) or other surface modifiers, at least one mucolytic agent, at least one cell membrane disrupter, and at least one flavoring agent may be added. Optionally, at least one substantially water free lubricant (e.g. polyethylene glycol, propylene glycol, glycerin) may be added to the nuclear stain detection solution composition at a weight / weight percent concentration between 5% - 35%. The formulation is constructed to help remove the stratum comeum (dead cell layer), mucus, debris, and aid nuclear fluorophore diffusion into the tissue and fluorophore translocation across epithelial cell membranes. These non-nuclear stain additives may help stabilize the nuclear fluorescent stain and facilitate a prolonged storage / shelf-life of at least one year and preferably at least two years.

[0096] Optionally at least one flavorant, which may additionally comprise sweeteners, may be added to the nuclear stain detection solution composition at a weight / weight concentration between 0.1% - 20% to enhance the palatability of the pretreatment solution. These flavorants include, but are not limited to, methyl salicylate (wintergreen), spearmint, citrus, cinnamon, sorbitol, xylitol, mannitol, amongst others and combinations thereof. In some embodiments, the pH of the nuclear stain detection solution composition is preferably acidic Atty. Docket No.: 1249-11470-PCT

[0097] - 16 - (pH < 7), more preferably between pH 2 - 6, and most preferably between 3.5 - 5.5. In certain embodiments, the surface tension of the nuclear stain detection solution is less than 80 dynes / cm. In some embodiments, the surface tension is less than 50 dynes / cm. In certain embodiments, the surface tension is less than 30 dynes / cm. At room temperature, the disclosed nuclear stain detection solution may have a viscosity of about 0.1 cP to 1000 cP. In some embodiments, the viscosity is about 1 cP to 10 cP.

[0098] In certain embodiments, the at least one organic acid is comprised of glycolic acid, citric acid, oxalic acid, acetic acid, amongst others. The at least one dehydrating agent is preferably an alcohol, for example, ethanol. In certain instances, the at least one dehydrating agent may also comprise the at least one solvent. Preferably, the at least one solvent is comprised of water. In certain embodiments, the at least one preservative agent is comprised of thymol, sodium benzoate, potassium sorbate, at least one paraben, antioxidants, amongst others. The at least one surfactant may comprise, but is not limited to: sulfonates, sulfates, ethoxylated alcohols, ethoxylated alkylphenols, fatty acid esters, ethoxysulfates, betaines, amine oxides, fluorosurfactants, amongst others. Optionally, mucolytic agents may be incorporated into the composition, such as n-acetylcysteine, or these mucolytic agents may be administered separate from, and prior to the nuclear stain solution. Optionally, chelating agents may be incorporated into the composition, such as EDTA, HEDP, PBTC, polyacrylic acid, amongst others.

[0099] Table 3: Nuclear stain detection solution composition.

[0100]

[0101] Atty. Docket No.: 1249-11470-PCT

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[0105] In the preferred embodiment, the nuclear stain detection solution’s composition is described in Table 4.

[0106] Table 4: Nuclear stain detection solution’s composition in the preferred embodiment. More preferred values are listed in parentheses.

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[0109]

[0110] Lectin-based detection solution composition Atty. Docket No.: 1249-11470-PCT

[0111] - 18 - The present invention discloses a lectin-based detection solution comprising at least one fluorescently labeled lectin specifically designed to target aberrant glycosylation patterns characteristic of precancerous, dysplastic, and cancerous mucosal lesions in vivo. Aberrant glycosylation, a hallmark of carcinogenesis, alters the expression and structure of cell surface glycomolecules — such as glycoproteins and glycolipids — resulting in modified glycan presentations that can be selectively bound by lectins. Applying fluorescently-labeled lectins to mucosal tissues in vivo allows for the visualization of lectin binding using fluorescent molecular imaging. This allows for the delineation of precancerous and cancerous tissue from normal and benign tissue during in vivo fluorescence imaging. Although the use of fluorescence is described in detail, the lectin molecules can be tagged with any reporter molecule, such as a radiolabel, colored microsphere, quantum dot, amongst others, that can be visualized, measured and / or recorded utilizing a device.

[0112] The lectin-based detection solution composition is comprised of at least one fluorescently labeled lectin, at least one solvent, at least one dehydrating agent, and at least one surfactant (Table 5). Optionally, at least one preservative agent can be added to the composition. Importantly, the non-lectin ingredients do not significantly inhibit the lectin but aid lectin diffusion into the tissue and binding of the lectin to cell surface glycomolecules. The unique chemistry composition described also allows the lectin solution to be shelf stable in an aqueous solution for at least one year, preferably at least 18 months, and more preferably at least two years.

[0113] Table 5: Lectin-based detection solution composition

[0114] < <

[0115]

[0116] Atty. Docket No.: 1249-11470-PCT

[0117]

[0118]

[0119] In certain embodiments, the at least one fluorescently labeled lectin consists of lectin protein molecules that have been conjugated to at least one fluorophore molecule. Preferably, the lectin is selected from the following non-exhaustive list: ConA, LCA, LcH, GNA, PNA, AIL, VVL, WGA, MAL, MAH, SBA, UEA, AAL, BP A, ECA, Jacalin, LEA, MAA, PHA-E, PHA-L, PHA-M, PHA-P, PSA, TJA, VEA, SNA, UDA, NPA, NPL, amongst others. The at least one fluorescently labeled lectin is at a weight / weight (w / w%) concentration of preferably 0.001% - 0.5%, more preferably 0.005% - 0.1%, and most preferably 0.01% -0.1%. In certain embodiments, the lectin is chosen to bind to a specific glycan, which can include, but is not limited to, mannose, galactose, n-acetylgalactosamine, n-acetylglucosamine, n-acetylneuraminic acid, fucose, sialic acid, amongst others. Preferably, the fluorophore is selected from the following non-exhaustive list: fluoresceine, fluoresceine derivatives (e.g. FITC), rhodamine, cyanine dyes, Alexa Fluor dyes, BODIPY dyes, coumarin, IRDye 800 and conjugates (e.g. IR800CW), quantum dots, amongst others. The at least one dehydrating agent is preferably an alcohol, for example, ethanol. In certain instances, the at least one dehydrating agent may also comprise the at least one solvent. Preferably, the at least one solvent is IX PBS in water, but may comprise other water-based buffers including, but not limited to, MES, TRIS, HEPES, TEA, amongst others and combinations thereof. In certain embodiments, the at least one preservative agent is comprised of thymol, sodium benzoate, potassium sorbate, antioxidants, amongst others, although the at least one preservative is optional. The at least one surfactant may comprise, but is not limited to: sulfonates, sulfates, ethoxylated alcohols, ethoxylated alkylphenols, fatty acid esters, ethoxysulfates, betaines, amine oxides, fluorosurfactants, amongst others. Optionally, mucolytic agents may be incorporated into the composition, such as n-acetylcysteine, or these mucolytic agents may be administered in vivo separate from, and prior to in vivo administration of the lectin-based detection solution composition.

[0120] Optionally, at least one substantially water free lubricant (e.g. polyethylene glycol, propylene glycol, glycerin) may be added to the lectin-based detection solution composition at a weight / weight percent concentration between 5% - 35%. Optionally at least Atty. Docket No.: 1249-11470-PCT

[0121] -20 -one flavorant, which may additionally comprise sweeteners, may be added to the lectin-based detection solution composition at a weight / weight concentration between 0.1% - 20% to enhance the palatability of the pretreatment solution. These flavorants include, but are not limited to, methyl salicylate (wintergreen), spearmint, citrus, cinnamon, sorbitol, xylitol, mannitol, amongst others and combinations thereof. In some embodiments, the pH of the lectin-based detection solution composition is preferably between pH 6 - 8, more preferably between pH 6.5 - 7.5, and most preferably about 7.2. In certain embodiments, the surface tension of the lectin-based detection solution is less than 80 dynes / cm. In some embodiments, the surface tension is less than 50 dynes / cm. In certain embodiments, the surface tension is less than 30 dynes / cm. At room temperature, the disclosed lectin-based detection solution may have a viscosity of about 0.1 cP to 1000 cP. In some embodiments, the viscosity is about 1 cP to 10 cP. In certain embodiments, the lectin-based detection solution has an osmolarity preferably between 250 mOsm / L - 350 mOsm / L, and more preferably between 290 mOsm / L and 310 mOsm / L.

[0122] Depending on the clinical use application, it may be advantageous to use lectins of a specific molecular weight. In certain embodiments, the molecular weight of the at least one fluorescently-labeled lectin within the lectin-based detection solution composition is preferably less than 200kDa, more preferably less than 150kDa, even more preferably less than lOOkDa, even more preferably less than 50kDa, and most preferably less than 20kDa. It should be noted that these molecular weight values are for lectin monomers, so this description is not related to dimers, trimers, or other lectin oligomer molecules. In fact, in certain embodiments, the specific chemistry of the disclosed lectin-based detection solution may not allow for the formation of lectin dimers, trimers or other oligomer molecules that would otherwise form within an aqueous solution, such that only lectin monomers exist in the lectin-based detection solution. Additionally, it is possible, and advantageous in certain embodiments, that a single isolectin (isoform) or at least one isolectin (isoform) is utilized within the lectin-based detection solution composition. In certain embodiments, the at least one lectin agglutinates a 2% suspension of human erythrocytes at a concentration preferably less than 50 ug / mL, more preferably less than 20 ug / mL and most preferably less than 10 ug / mL.

[0123] The ratio between lectin molecular weight (or lectin fragment molecular weight, Atty. Docket No.: 1249-11470-PCT

[0124] -21 -discussed next) and fluorophore molecular weight is important for the composition to ensure the fluorophores do not significantly alter the binding properties of the lectin (or lectin fragment). In certain embodiments, this ratio is preferably at least 3:1, more preferably at least 5:1, even more preferably at least 10:1, and most preferably at least 20:1. As a non-limiting example, WGA’s monomer molecular weight is ~18kDa and IR800CW’s molecular weight is 962Da, so this would yield a lectin to fluorophore ratio of 18.7:1 (18kDa divided by 962Da = 18.7). The labeling ratio (number of fluorophores per protein molecule, which could be a lectin or lectin fragment) is also important and is preferably at least 0.5:1, more preferably at least 1:1, even more preferably at least 2:1, and most preferably at least 3:1. While increasing the fluorophore label ratio may seem advantageous in certain instances, it may result in unwanted consequences such as signal saturation, fluorophore quenching, diminished protein functionality, increased background, decreased solubility, increased material cost, among others. Accordingly, it is generally preferred to maintain a fluorophore to protein (lectin or lectin fragment) label ratio between 0.5:1 and 1.5:1.

[0125] A critical innovation of the present invention lies in the generation and utilization of lectin fragments — smaller molecular weight derivatives of native lectin proteins — that retain specific glycan-binding functionality while exhibiting enhanced biophysical and pharmacokinetic properties favorable for in vivo fluorescence imaging and diagnostic applications. Smaller sized proteins or fragments typically offer improved tissue diffusion, binding to target sizes, and signal to noise ratios when used in vivo for fluorescence imaging. To facilitate improved tissue penetration and fluorescence imaging, the at least one fluorescently labeled lectin within the lectin-based detection solution composition may be replaced with at least one fluorescently labeled lectin fragment.

[0126] Several techniques may be utilized individually or in combination to sufficiently fragment the raw purified lectins. These fragmentation techniques include, but are not limited to, chemical, thermal, and enzymatic methods - as detailed below:

[0127] • Chemical methods: Reduction of disulfide bonds using agents such as dithiothreitol (DTT) or beta-mercaptoethanol (BME), acid or base hydrolysis, or selective cleavage of peptide bonds.

[0128] • Enzymatic digestion: Proteolytic cleavage with specific proteases such as trypsin, Atty. Docket No.: 1249-11470-PCT

[0129] -22 - chymotrypsin, subtilisin, elastase, thermolysin, or proteinase K to generate defined fragments.

[0130] • Physical methods: Application of heat, pressure, or sonication to facilitate partial denaturation and fragmentation.

[0131] In certain embodiments, specific chemical bonds are individually or collectively targeted for fragmentation (e.g. disulfide bonds, specific amide bonds, etc.). For example, reducing agents (e.g. dithiothreitol, beta-mercaptoethanol, etc.) may be used to break disulfide bonds. In other embodiments, the lectin protein is fragmented at specific amino acid bonds, or following specific amino acid sequences. Generally, the fragmentation process and reaction is undertaken at a pH of preferably 3-11, more preferably 5-10, and most preferably 6-9. The fragmentation process can be completed within minutes to days at room temperature although this can easily be accelerated by increasing the temperature or pressure of the reaction. Temperature and pressure changes, considered individually or collectively, may additionally offer benefits to lectin fragmentation. In certain embodiments, at least one cationic surfactant, non-ionic surfactant, or anionic surfactant is added to help facilitate the fragmentation reaction or participate in the fragmentation reaction (for example, by helping initially denature the raw lectin protein to expose targeted locations or amino acid sequences for fragmentation). Through the disclosed lectin fragmentation process, unique and novel lectin fragments are created that offer significant value for in vivo disease detection, as discussed within this application, but these fragments may be valuable for laboratory testing, and in vitro diagnostics, among others.

[0132] The ability to fragment lectins into smaller, biologically active, and shelf stable compounds is unique and novel to this disclosure. Prior to fluorescent labeling, the process of fragmenting the lectins may comprise any of the following steps individually or in any combination: subjecting the lectin monomers to reducing agents (e.g. dithiothreitrol (DTT), beta-mercaptoethanol (BME), amongst others), heat and / or pressure, acids, bases, and proteases (e.g. trypsin, chymotrypsin, subtilisin, elastase, thermolysin, proteinase K, amongst others). In certain embodiments, the fragmentation process reduces the native lectin size / molecular weight preferably by at least 20%, more preferably by at least 35%, even more preferably by at least 50% and most preferably by at least 70%. The fragmentation process is sufficiently efficient in that at least 30% of the raw lectin is fragmented. Atty. Docket No.: 1249-11470-PCT

[0133] -23 - Because lectins are biologically active molecules, fragmentation of these lectins must maintain some level of useful biological activity as continued fragmentation will not yield sufficient protein structure useful for fluorescent labeling and glycan and disease detection. In certain instances of the disclosure, the fragmentation process preferably maintains at least 30% glycan affinity of the native lectin, more preferably maintains at least 50% glycan affinity of the native lectin, and most preferably maintains at least 75% glycan affinity of the native lectin. It may be possible that more than one lectin fragmentation maintains its activity and structure and is therefore useful within the lectin-based detection solution composition. In certain instances, fragmentation of the lectin may expose previously hidden or structurally hindered glycan binding sites that are now capable of binding to cell surface glycans in vivo. In certain embodiments, the fragmentation process is used only on certain isolectins, while in other embodiments, the fragmentation process inhibits the dimerization, trimerization, or oligomerization of lectin monomers that would otherwise normally occur within an aqueous solution. In further embodiments, the fragmentation process is specifically developed and implemented to target and isolate specific domains or subunits of the lectin. In other embodiments, no specific domains or subunits of the lectin are targeted and the lectin is fragmented in other formats.

[0134] In certain embodiments, the at least one lectin fragment agglutinates a 2% suspension of human erythrocytes at a concentration preferably less than 50 ug / mL, more preferably less than 20 ug / mL and most preferably less than 10 ug / mL. Once fragmentation is complete, typical fluorophore labeling protocols can be followed to fluorescently label the at least one lectin fragment for use within the disclosed lectin-based detection solution composition.

[0135] In a preferred embodiment, the lectin-based detection solution’s composition is described in Table 6. As noted, the use of IR800CW fluorescently labeled wheat germ agglutinin (WGA- IR800CW) is a preferred example of the disclosed composition. In this example, the lectin-based detection solution is comprised of: WGA- IR800CW, which is comprised of a mix of WGA isoforms (WGA1, WGA2, and WGA3) fluorescently conjugated to IR800CW dye, and may optionally include a WGA fragment of approximately 13kDa in size that is also fluorescently conjugated to IR800CW dye, wherein WGA-IR800CW has a Atty. Docket No.: 1249-11470-PCT

[0136] -24 -fluorophore to protein label ratio between 1:1 and 1:1.5, and wherein WGA-IR800CW is at a concentration between 0.05% - 0.1%. In certain embodiments, the WGA-IR800CW described in this example may only consist of a WGA fragment of approximately 13kDa in size that is fluorescently conjugated to IR800CW dye. Within this specific embodiment, the lectin fragment to fluorophore ratio is approximately 13.5:1 (13kDa WGA fragment divided by 962Da for IR800CW = 13.5).

[0137] Table 6: Lectin-based detection solution’s composition in the preferred embodiment. More preferred values are listed in parentheses.

[0138]

[0139] Method

[0140] The described inventive method is a multi-step process that helps delineate precancerous, dysplastic, and cancerous tissue from normal / benign tissue using the described inventive solutions / compositions and devices. Initially the pretreatment solution may optionally be applied to the target mucosa. This pretreatment solution may be as described in the embodiment or may separately be a solution already used in the medical community for Atty. Docket No.: 1249-11470-PCT

[0141] -25 -pretreating mucosal tissues (for example, solutions comprising n-acetylcysteine and water). A water rinse may be performed at this point. Next, at least one detection solution will be applied to the target mucosa. In certain instances, it is advantageous to first use the disclosed nuclear stain-based detection solution followed by use of the disclosed lectin-based detection solution, provided fluorescence imaging of the nuclear stain-based detection solution and lectin-based detection solution are performed at different wavelengths. A water rinse may be performed in between, or after, the application of the at least one detection solution. Lastly, the target mucosa will be exposed to specific wavelengths of light, and proper filtering, to excite fluorophores in the at least one detection solution; this may be accomplished using the device inventions within this patent application or commercially available units for fluorescent medical imaging. Visualizing the at least one detection solutions’ fluorescence, either with a device or with the naked eye, will allow doctors to delineate between normal / benign tissue and precancerous, dysplastic and / or cancerous tissue. In certain embodiments, fluorescent imaging of the at least one detection solution, and preferably the lectin based detection solution composition, is preferably performed between 500nm - 2500nm, more preferably between 750nm - 1900nm or lOOOnm - 1300nm or 1400nm - 1900nm, and most preferably between 780nm - 1300nm. More specifically, the sequential application of a pretreatment solution followed by at least one detection solution provides a higher sensitivity and specificity to detect precancerous, dysplastic and cancerous lesions compared to traditional methods.

[0142] Ideally, the solutions will be topically applied as a spray or mouth rinse to cover the entire target mucosa, or as a swab to specifically target areas of interest. This approach allows for a non-invasive method to quickly apply solutions to the oral mucosa or cervix for lesion detection. For harder to reach mucosal sites, a syringe spray, spray catheter, or other methods of topical application can be utilized.

[0143] Testing

[0144] The following paragraphs define in more detail the embodiments of the invention described herein. The following embodiments are not meant to limit the invention or narrow the scope thereof, as it will be readily apparent to one of ordinary skill in the art that suitable modifications and adaptations may be made without departing from the scope of the invention, embodiments, or specific aspects described herein. Atty. Docket No.: 1249-11470-PCT

[0145] -26 -

[0146] Epithelium Removal Testing Using the Disclosed Pretreatment Solution

[0147] Materials and Methods

[0148] Potential pretreatment agents (Table 7) were tested to examine their ability to remove the superficial keratinized epithelial layers of the porcine tongue. Organic acids were primarily examined due to their natural occurrence in many foods in the human diet. However, other agents such as hydrogen peroxide and bleach were included for comparison. Tissue samples were 50% embedded in agar (Sigma Aldrich, St. Louis, MO); the embedded tissue of each sample served as a control while the exposed tissue was subjected to the pretreatment solution (Fig. 1). The exposed end of each tissue sample was inked green to indicate this part of the tissue was tested. Samples were then submerged in lOmL of 37°C pretreatment solution for 5 mins. Tissue sections were then submerged in a PBS bath (50mL) for 1 min, to remove residual pretreatment agents, and then fixed in 10% neutral buffered formalin (NBF) overnight. The following day, tissue samples were sectioned into 5um slices using a microtome and subjected to a standard hematoxylin and eosin (H&E) stain. Epithelial thickness of the treated and embedded tissue was measured at three or more locations for each sample using Aperio ImageScope Software (Leica Biosystems, Richmond, IL). The amount of epithelium removed was then calculated by subtracting the epithelial thickness of the experimental section from the epithelial thickness of the control embedded section, then dividing this value by the epithelial thickness of the control section. All pretreatment solution results were compared to a control consisting of the same test being performed in PBS. Each pretreatment solution was tested in triplicate.

[0149] Table 7: Pretreatment agents used during testing.

[0150]

[0151] Atty. Docket No.: 1249-11470-PCT

[0152] -27 -

[0153]

[0154] Results

[0155] The results from the tested pretreatment solutions (Table 7) can be seen in Fig.

[0156] 2. Interestingly, the control PBS testing yielded 17% ± 8.0% of epithelium removed. As PBS is not expected to have any effect on epithelial removal, the procedure for agarose embedding resulted in some level of epithelium removal. Thus, the results have been normalized to account for the PBS control. The most effective pretreatment solutions were glycolic acid, citric acid, oxalic acid, acetic acid, and sodium hypochlorite, which removed 35.8% ± 23.3%, 27.5% ± 11.9%, 25.4% ± 14.4%, 20.1% ± 13.8%, and 34.61% ± 16.2% more of the epithelium than their embedded control sections, respectively (note - these values are normalized to the PBS control).

[0157] Although sodium hypochlorite removed substantial epithelium, its use in an oral pretreatment solution is not clinically feasible and was included in the study as a positive control. These results support the use of organic acids to help remove superficial dead cell epithelial layers.

[0158] Conclusion

[0159] This testing illustrates that organic acid based solutions are effective at removing the dead cell and superficial cell layers of the epithelium. The described pretreatment solution in the preferred embodiment was validated during a clinical trial (discussed in the clinical trial validation section), further supporting its use and efficacy.

[0160] In a particular embodiment of the invented pretreatment solution, the at least one organic acid is preferably chosen from glycolic acid, citric acid, oxalic acid, or acetic acid. The at least one organic acid is preferably at a concentration equal to or less than 20%, more Atty. Docket No.: 1249-11470-PCT

[0161] -28 -preferably less than or equal to 10%, and even more preferably less than or equal to 5%. In certain embodiments, the pH of the pretreatment solution is preferably between 1-7, more preferably between 2-5, and most preferably between 3-4.5. Within five minutes of in vivo application, the disclosed pretreatment solution preferably removes at least 10% of the surface mucosal epithelium, more preferably removes at least 20% of the surface mucosal epithelium, and most preferably removes at least 25% of the surface mucosal epithelium.

[0162] Optimal Nucleic Acid Concentration Testing

[0163] Testing has shown there is an optimal concentration for DAPI (a nuclear stain), where too low of a concentration isn’t visible after fluorophore excitation, and too high of a concentration quenches the fluorescence after excitation.

[0164] Materials and Methods

[0165] Tissue phantoms were created by sectioning lean chicken breast into 1 x 1 x 0.5” segments. A 20mM DAPI solution was synthesized in deionized water and serial dilutions were performed to obtain concentrations ranging between lOuM and 20mM. 20uL aliquots of the dilutions were spread onto tissue phantoms and incubated for 30 sec. The tissue phantoms were rinsed with excess deionized water to remove any residual DAPI. An optical system was used to image fluorescence of the DAPI stained tissue phantoms. The optical system was comprised of a CoolSnap HQ CCD scientific camera, 365nm UV LEDs, and appropriate filtering for DAPI (435-485nm bandpass filter; Thor Labs, Newton, NJ) all mounted within a light tight box. A digital camera was used for digital photographs to illustrate observed fluorescence to the examiner (Cannon Powershot A3100). ImageJ software was used to measure fluorescence values from the scientific camera images. Each dilution was tested in triplicate. All chemicals were used as received from Sigma Aldrich (Milwaukee, WI).

[0166] Results

[0167] DAPI fluorescence of the stained tissue phantoms versus DAPI concentration can be seen in Fig. 3. An optimal concentration was noted at ImM DAPI; however DAPI fluorescence was visible to the naked eye between O.lmM and lOmM. Below O.lmM, the DAPI fluorescence was hardly visible to the naked eye, yet visible with the CCD Atty. Docket No.: 1249-11470-PCT

[0168] -29 -camera. Above lOmM, the DAPI fluorescence was quenched as a majority of the DAPI was unable to bind to surface DNA. Moreover, at this high of a concentration, the DAPI left a visible orange stain on the tissue.

[0169] Conclusion

[0170] Testing provided an optimal nuclear stain concentration to maximize fluorescence visualization by the “naked” eye (i.e. not assisted by low light imaging components) in the described nuclear stain solution. Moreover, the nuclear stain detection solution was validated during a clinical trial (discussed in the clinical trial validation section).

[0171] In an embodiment of the invented nuclear stain-based detection solution, the nuclear stain is preferably included at a concentration between 0.05 and lOmM, more preferably included at a concentration between 0.1 and 2mM, and most preferably at a concentration of about ImM.

[0172] Lectin Histochemistry Study

[0173] Testing has shown there are several lectins of interest that are able to detect oral cancer within tissue slices.

[0174] Materials and Methods

[0175] Clinical sample set

[0176] Frozen oral squamous cell carcinoma tissue and corresponding normal tissue from human subjects were used to investigate lectin-tissue binding patterns. Tissue was obtained from BioNet / University of Minnesota’s tissue bank and were already histopathologically diagnosed by a board-certified oral pathologist. Tissue was sectioned into five-micron slices using a microtome with normal and disease tissue sections from the same patient placed on the same microscope slide.

[0177] Application of lectin probes Atty. Docket No.: 1249-11470-PCT

[0178] - 30 - Sixteen fluorescein isothiocyanate (FITC) lectin conjugates (EY laboratories, San Mateo, CA; Invitrogen, Carlsbad, CA; Vector Labs, Burlingame, CA), with varying binding profiles, were applied to the slides at a concentration of lOug / mL, a volume of 10 mL, and incubated for 30 minutes at 37°C (Table 8, Fig. 4). This ensures both the normal and cancerous tissue sections are subjected to the same conditions. Following incubation, samples were washed with 10 mL IX phosphate-buffered saline (PBS) three times to remove any unbound lectin. UEA-I, MAA, and Jacalin were tested at the standard lOug / mL and also at 20ug / mL to examine the effect of doubling the lectin concentration.

[0179] Table 8- Lectins used in this study, their sources and their biological targets.

[0180]

[0181] Atty. Docket No.: 1249-11470-PCT

[0182] - 31 -

[0183]

[0184] Optical imaging

[0185] Samples were imaged using a ScanScope FL System (Aperio Technologies, Vista, CA) and a Nikon Eclipse E800 fluorescent microscope equipped with a CoolSnap HQ CCD camera (Photometries, Tucson, AZ). Fluorophore excitation was performed via a 100- watt mercury arc lamp in conjunction with fdter cubes specific to the used fluorophores. The FITC filter cube allowed 466-500nm light to illuminate the sample while emission allowed 510-560nm light to be visualized. Tissue samples were also counterstained with DAPI ProLong Gold antifade (Invitrogen, Carlsbad, CA). The DAPI filter cube allowed for 330-380nm excitation and 435-485nm emission.

[0186] Quantification of imaging data

[0187] Full slide fluorescence images from the ScanScope FL System were quantitatively analyzed using Aperio ImageScope software (Aperio, Vista, CA). Mean fluorescence intensities (MFI) of ten regions-of-interest were measured from the epithelial Atty. Docket No.: 1249-11470-PCT

[0188] -32 -layer of the normal tissue and from squamous cell carcinoma nests within the tumor tissue for each patient and lectin combination (Fig. 5). Exposure times were kept consistent for each pair of patient tissue specimen (i.e. diseased and control tissue from the same patient), which ensures that comparisons can be made between fluorescent results of diseased versus normal tissue. To quantify a measure of lectin binding affinity between normal and cancerous tissue, the signal-to-noise ratio (SNR) was calculated by taking the MFI value of the cancerous tissue and dividing it by the MFI value of normal tissue. Lectins were deemed advantageous if at least 3 / 5 (60%) of patients exhibited a SNR greater than 1.5 (i.e. 3 / 2) or less than 2 / 3, as these SNRs would be deemed distinguishable by the naked eye, camera, or other imaging equipment.

[0189] Statistical analysis

[0190] Average SNR for each lectin is present. Two-tailed, paired Student’s t-tests were used to statistically compare the fluorescent intensities observed between normal and malignant expression of lectin binding. Statistical significance was inferred using a 95% confidence interval, which relates to a p value < 0.05.

[0191] Results

[0192] Fluorescence imaging using FITC-lectin conjugates

[0193] Fluorescent data revealed that all lectins demonstrated binding to the tissue sections (Fig. 6). In particular, seven of fifteen lectins (47%) were deemed advantageous as their SNR response offered acceptable delineation between cancerous and normal tissue slices for at least 3 / 5 patients (60%); these lectins include Jacalin, LcH, UDA, VVA, MAA, UEA-I and SNA. SNA showed the most significant difference between cancerous and normal tissue fluorescence with a p value of 0.002. Fluorescent images of cancerous and normal tissue sections from patient 2 treated with MMA (20ug / mL) can be seen in Fig. 7. Interestingly, testing revealed that some normal tissue sections were actually cancerous (Fig. 8). Here sections from patient 5 were treated with UEA-I (lOug / mL) and unexpected cancer was discovered in the normal biopsy section, which was later then verified by a board- certified oral pathologist.

[0194] Inhibiting the lectins resulted in decreased fluorescence achieved in cancerous and normal tissues; an example of this is shown in Fig. 9 where UEA-1 (lOug / mL) was first Atty. Docket No.: 1249-11470-PCT

[0195] - 33 -inhibited with its inhibitory sugar, L-a-fucose (0.2M), for 10 minutes at 37C before application to tissue sections from patient 5. Inhibition of the lectin resulted in a 2.61 and 1.79 fold decrease in fluorescence observed on cancerous and normal tissue, respectively, when compared to the uninhibited lectin results. The resulting SNR was 1.21, a 2.2 fold decrease in SNR. This result illustrates the specificity of the lectin towards its target H / O blood groups. Similar inhibition results were seen across all lectin conjugates (data not shown). As an optional additional solution of the disclosed invention, a rinse solution comprising the lectin’s inhibitory sugar can be applied after fluorescent imaging to help remove lectin from the tissue.

[0196] There was no significant difference between UEA-I, Jacalin, and MAA tested at lOug / mL and 20ug / mL (p>0.34). This helps show that the lectin was always in abundance during lOug / mL testing and effectively saturated lectin targets on the cell surface, meaning the collected fluorescent data is a true representation of cell surface glycan expression.

[0197] Conclusions

[0198] In certain embodiments of the lectin-based detection solution, the lectin is chosen from the following seven lectins: Jacalin, LcH, UEA-I, UDA, VVA, MAA, and SNA, as these were shown to be advantageous to detect oral cancer from normal tissue. Lectins that have high affinity for the same glycan residues as these seven lectins are also covered within the intellectual property as they will respond similarly since they target the same glycan molecule. As these seven lectins target a variety of glycan structures, synergy between these lectins could be evaluated in a cocktail, which would hopefully help increase SNRs and diagnostic potential.

[0199] Optimal Lectin Concentration Testing

[0200] Testing has shown there is an optimal concentration for WGA-FITC (a fluorescently labeled lectin), where too low of a concentration isn’t visible after fluorophore excitation, and too high of a concentration doesn’t increase fluorescence visualization.

[0201] Materials and Methods

[0202] WGA-FITC (BioWorld, Dublin, OH) solutions were synthesized at lOOum, Atty. Docket No.: 1249-11470-PCT

[0203] -34 - 50um, 20uM, lOuM, 5um, and luM WGA-FITC concentrations using the composition outlined in the preferred embodiment. A 2 w / w% agar, 4w / w% chitosan gel was synthesized as a positive control tissue phantom for testing. Chitosan was chosen as this polymer has abundant n-acetyl glucosamine residues for WGA binding (note WGA binds both to sialic acid and n-acetyl glucosamine sugar residues). As a negative control, chitosan was substituted with pectin, which does not provide any WGA-FITC binding residues. Thus, as the control setup doesn’t have any WGA binding residues, the only fluorescence would be due to WGA diffusion into the tissue phantom, which is not molecularly specific binding of the probe to the target molecule. Briefly, tissue phantom synthesis was accomplished by first heating water to 70°C. Polymers (agar, and chitosan or pectin) were then dissolved under slow stirring for 30 min. The ensuing solution was poured into 2” diameter petri dishes (-0.25” thick) and cooled to room temperature, which formed solid tissue phantoms. Agar, chitosan, and pectin were used as received from Sigma Aldrich (Milwaukee, WI).

[0204] Twenty microliter aliquots of the test solutions were pipetted onto the tissue phantoms, and the solutions sat undisturbed for 30 seconds (20uL pipette, Hach, Loveland, CO). A deionized water rinse ensued, followed by imaging within an optical system, which is comprised of a CoolSnap HQ CCD scientific camera and appropriate filtering for FITC (530nm ± 5nm bandpass filter; Thor Labs, Newton, NJ) mounted within a light tight box. Excitation of the dye 2 solutions was accomplished using collimated blue LEDs (LumiLEDs, Luxeon Rebel Blue, San Jose, CA), filtered through a 500nm short pass filter, from a distance of 15cm. A digital camera was used for digital photographs to illustrate observed fluorescence to the examiner (Cannon Powershot A3100 with FITC emission filtering mounted in front of the camera aperture). ImageJ software was used to measure fluorescence values from the scientific camera images. Each solution was tested in triplicate using three different tissue phantoms.

[0205] Results

[0206] Digital and scientific camera images of the testing performed on the tissue phantoms can be seen in Fig. 10. Zero binding of the WGA-FITC, and likewise zero fluorescence, is seen on the negative control tissue phantoms at all concentration, whereas WGA-FITC binding and fluorescence is seen on the experimental tissue phantom. This result Atty. Docket No.: 1249-11470-PCT

[0207] - 35 -illustrates that WGA-FITC binding, and subsequent fluorescence, is molecularly specific to n-acetylglucosamine and is not simply diffusion into the tissue or tissue phantom. Results showed a maximum fluorescence intensity at lOOuM WGA-FITC concentration (Fig. 11). However, an increase in concentration of WGA-FITC didn’t produce a similar increase in fluorescence, i.e.

[0208] 20uM to lOOuM (5x) increase in concentration only provided approximately a 2x increase in fluorescence, indicating diminishing returns. Fig. 12 shows this relationship by analyzing the increase in fluorescent intensity per luM increase in WGA-FITC concentration. A clear maximum is observed between 5-20uM concentrations. A preferred concentration of 20uM maximizes fluorescent gains and biocompatibility / safety (less active ingredient) while minimizing product cost and allowing for potential degradation / loss of activity. However the preferably concentration of lectin is between 5um and lOOuM which is clearly visible to the naked eye compared to lower concentrations.

[0209] Conclusion

[0210] A lectin-fluorophore conjugate concentration of 20uM is preferred in the embodiment as it maximizes fluorescent gains and safety (less active ingredient) while minimizing product cost and allowing for potential degradation / loss of activity during shelf life. However, in certain embodiments, and depending on the lectin and fluorophore chosen, the lectin fluorophore conjugate is at a concentration preferably between lOnM and ImM, more preferably between lOOnM and 500uM, even more preferably between luM and 50uM, and most preferably about 20uM.

[0211] Permeation Enhancer Compatibility with WGA-FITC Testing

[0212] The goal of this testing was to analyze which penetration enhancers (PEs; Sigma Aldrich, St. Louis, MO; Invitrogen, Carlsbad, CA) do not alter the WGA lectin’s bioactivity.

[0213] Materials and Methods

[0214] Testing was performed by initially incubating lOmL of 20uM of WGA-FITC (Invitrogen, Carlsbad, CA) with concentrations of the PEs outlined in Table 9. These particular compounds were evaluated as they’re known exfoliants, surfactants, and permeation enhancers. Atty. Docket No.: 1249-11470-PCT

[0215] -36 - Next, each WGA-FITC / PE solution was incubated with a 5um section of porcine tongue tissue for 30min at 37°C. Tissue sectioning of the porcine tongue was performed using a microtome. Tissue fluorescence images were obtained using a ScanScope FL System and a Nikon Eclipse E800 fluorescence microscope equipped with a CoolSnap HQ CCD camera (Photometries, Tucson, AZ). FITC excitation was performed via a 100-watt mercury arc lamp in conjunction with a FITC specific filter cube (466-500nm excitation and 510-560nm emission). Tissue sections were also counterstained with DAPI ProLong Gold antifade (Invitrogen, Carlsbad, CA) in conjunction with a DAPI specific filter cube (330-380nm excitation and 435-485 emission). Fluorescent images were quantitatively analyzed using Aperio ImageScope software (Aperio, Vista, CA) and ImageJ (National Institutes of Health, Bethesda, Md). Seven fluorescent measurements were recorded in the epithelial regions of each sample; average fluorescent measurements and standard deviation values (X ± SD) were calculated and normalized to the control (WGA-FITC in 100% lx PBS). Bioactivity of the lectin was inferred from the normalized fluorescent values of stained tissues with “X > 0.7”, “0.4 < X < 0.7”, and “X < 0.4” being indicative of “little to no inhibition”, “intermediate inhibition”, and “significant inhibition”, respectively. Each PE was tested in triplicate.

[0216] Table 9: Penetration enhancers tested for compatibility with lectin bioactivity

[0217]

[0218] Atty. Docket No.: 1249-11470-PCT

[0219] - 37 -

[0220]

[0221] Results

[0222] Compatibility testing results are summarized in Table 10. PBS, Zonyl FS-300, Ethanol (25%), Pluronic F-68, Triton X-100 (1%), propylene glycol (5-10%), and Taurocholic acid did not inhibit the lectin’s binding and / or fluorescence. As an example Fig. 13 shows a comparison between the PBS control (2A), 1% Pluronic F-68 (2B), 1% propylene glycol (2C), and 100% DMSO (2D). The Pluronic F-68 did not inhibit WGA bioactivity whereas the propylene glycol and DMSO intermediately and significantly inhibited WGA bioactivity, respectively. PEs that did not inhibit WGA’s bioactivity were investigated for further testing.

[0223] Table 10: Compatibility of penetration enhancers with WGA-FITC lectin. Bioactivity was inferred by fluorescence of WGA-FITC stained tissue slices following incubation of the lectin with experimental penetration enhancers. Penetration enhancers that did not inhibit the lectin were examined in further testing. N = no inhibition, I = intermediate inhibition, and S = significant inhibition.

[0224]

[0225] Atty. Docket No.: 1249-11470-PCT

[0226] - 38 -

[0227]

[0228] Conclusion

[0229] This testing provided a list of acceptable PEs that don’t affect the bioactivity of lectins. In particular embodiments of the invention, the PEs that don’t significantly decrease the bioactivity of lectins (i.e. “N” or “I” in Table 10) are included in the invented tissue pretreatment solution and / or the invented lectin-based detection solution. These PEs include, but are not limited to, Zonyl FS-300 (or other tradenames including Masurf FS-2825, or other similar fluorosurfactants), ethanol at a preferable concentration of <25% although 50% may be acceptable in certain applications, Pluronic F-68 (Poloxamer 188, 407, or other PEO / PPO copolymers), Triton X- 100 at 1 %, Propylene glycol between 5-10%, taurocholic acid (0.5-1 %), bile salts (0.5-1%), and methylsulfonylmethane <12.5%.

[0230] In particular embodiments of the invention, the lectin-based detection solution contains permeation enhancers that preferably do not decrease lectin bioactivity by more than 50%, more preferably do not decrease lectin bioactivity by more than 30%, even more preferably do not decrease lectin bioactivity by more than 20%, and most preferably do not decrease lectin bioactivity by more than 10%.

[0231] Permeation Enhancer Efficacy Testing

[0232] The PEs that did not inhibit lectin bioactivity (Table 10) were tested to evaluate their efficacy at increasing lectin epithelial penetration; PEs were also tested in combination to Atty. Docket No.: 1249-11470-PCT

[0233] - 39 -examine synergy. Table 11 outlines all experimental samples.

[0234] Materials and Methods

[0235] Fresh porcine tongue sections 50% embedded in agarose (using the protocol illustrated in Fig. 1) was used using tissue specimen sizes of 10mm in diameter. Topical application of 2mL of 20uM WGA-FITC solution was applied to the epithelial surface of tissue sections and incubated for 1 hour at 37°C. Tissue slices were sectioned perpendicular to the surface epithelium, placed on a microscope slide, and imaged as described in the “epithelium removal testing” section. Fluorescence images were analyzed using Aperio ImageScope Software, and three measurements of WGA-FITC penetration into the epithelium were obtained for each sample and compared to the control (WGA-FITC in PBS). Each PE and PE combination were tested in triplicate.

[0236] Table 11- Penetration enhancers tested for tissue penetration.

[0237]

[0238] Atty. Docket No.: 1249-11470-PCT

[0239] -40 -

[0240]

[0241] Results

[0242] Results are shown in Fig. 14. Fig. 14A shows the depth penetration for all samples tested. Sample 13 resulted in the greatest epithelial penetration, a 3.75 fold increase compared to the PBS control (340 um versus 91 um and Fig. 7B and 7C, respectively; p = 0.003), and consisted of 25% ethanol, 1% triton X-100, and 2% Pluronic F-68. Samples 10, 12, and 17 also showed statistically significantly greater penetration than the PBS control (p = 0.02, 0.04, and 0.02, respectively). Samples 11, 16 and 18 were suggestive of significance (0.1 > p > 0.05). Interestingly, no PE was independently effective enough to increase penetration significantly; thus the combination of multiple PEs is unique, inventive, and non-obvious. However, of all PEs tested, Pluronic F-68 was common to all samples with statistically suggestive and significant results.

[0243] Conclusion

[0244] The most advantageous PE combination (25% ethanol, 1% triton X-100, and 2% Pluronic F-68) resulted in ~3X deeper WGA-FITC epithelial penetration than the IX PBS control. Utilizing this PE solution would allow for more cell binding of molecular probes and proteins to help better distinguish precancerous and cancerous tissue from normal / benign tissue. Although this example is specific to the use of lectins, other molecular probes can be used, such as antibodies, lectin fragments, antibody fragments, amongst others.

[0245] In particular embodiments of the invention, the lectin-based detection solution contains permeation enhancers that preferably increase epithelial penetration of the lectin-fluorophore conjugate by 1.5X, more preferably increase epithelial penetration of the lectin-fluorophore conjugate by 2X, even more preferably increase epithelial penetration of the lectin- Atty. Docket No.: 1249-11470-PCT

[0246] -41 -fluorophore conjugate by 2.5X, and most preferably increase epithelial penetration of the lectin-fluorophore conjugate by 3X. In particular embodiments of the invention, the lectin-based detection solution contains permeation enhancers that preferably increase epithelial penetration of the lectin-fluorophore conjugate by at least 50 microns, more preferably increase epithelial penetration of the lectin-fluorophore conjugate by at least 100 microns, even more preferably increase epithelial penetration of the lectin-fluorophore conjugate by at least 250 microns, and most preferably increase epithelial penetration of the lectin-fluorophore conjugate by at least 325 microns.

[0247] Ethanol Concentration Testing

[0248] Further testing was undertaken to elucidate the effect of ethanol on the lectin activity (WGA-FITC) within the lectin-based detection solution.

[0249] Materials and Methods

[0250] The “more preferred” formulation for the lectin-based detection solution provided in Table 6 was used for this experiment, however, the ethanol concentration was varied at 0%, 5%, 10%, 20%, and 25%. Correspondingly, any change in ethanol concentration was compensated for by equal and opposite changes in IX PBS concentration, such that the concentration of all other ingredients (i.e. surfactants, etc) remained the same throughout all testing groups.

[0251] Initially, an absorbance measurement of the various lectin-based detection solutions was obtained on a UV / VIS spectrophotometer between 400-600nm (Vernier, Beaverton, OR). Calibration of the UV / VIS spectrophotometer was performed to a “buffer solution” which consisted of the “more preferred” formulation for the lectin-based detection solution provided in Table 6 but without the lectin-fluorophore conjugate (WGA-FITC). Therefore, any UV / VIS absorbance is solely due to the presence of WGA-FITC, and more specifically, the FITC fluorophore conjugated to the WGA lectin which is highly absorbing in this wavelength range.

[0252] Separopore 4B-CL chitin beads (BioWorld, Dublin, OH) were used as a testing substrate because chitin is composed of n-acetylglucosamine, which is a binding target of Atty. Docket No.: 1249-11470-PCT

[0253] -42 - WGA-FITC. A 0.5mL aliquot of chitin beads was initially washed in 5mL of buffer solution (the Table 6 “more preferred” formulation but without the lectin) on a tube inverter for 15 mins. Afterwards, the beads were centrifuged at 3300X for 5 min followed by disposal of the supernatant. 5mL of the lectin-based detection solution was then added to the washed beads and rotated on a tube inverter for 1 hour. Afterwards, the beads were centrifuged at 3300X for 5 min. A 2mL aliquot of the supernatant was placed in the UV / VIS cuvette and its absorbance was measured between 400-600nm.

[0254] Using the lectin-based detection solution’s max absorbance values measured before and after bead incubation allowed for the calculation of WGA-FITC bound to the beads. In other words, the final absorbance measured represents the amount of WGA-FITC remaining in the lectin-based detection solution after 1 hour that did not bind to the chitin beads. Since the initial absorbance measurement was obtained for the same solution before bead incubation, one can easily calculate the difference in absorbance, which is directly related to WGA-FITC concentration (through Beer’s Law), and attribute any difference in absorbance to WGA-FITC binding to the chitin beads.

[0255] The same procedure was completed in triplicate for all lectin-based detection solutions, containing varying concentrations of ethanol.

[0256] Results

[0257] Fig. 15 shows the results from this experiment. As a baseline, 73% of WGA-FITC within the lectin-based detection solution at 0% ethanol bound to the chitin beads within 1 hour. A direct relationship was seen where increases in ethanol concentration yielded proportional decreases in WGA-FITC binding to the chitin beads. At a higher ethanol concentration of 25% in the lectin-based detection solution, only 28% of WGA-FITC bound to the chitin beads within 1 hour.

[0258] Conclusion

[0259] This data, along with all other data, illustrates that the binding profile of WGA-FITC is very dependent on the composition of the lectin-based detection solution. Possible explanations for the decrease in WGA-FITC binding with progressively higher ethanol Atty. Docket No.: 1249-11470-PCT

[0260] -43 -concentrations could be - the WGA-FITC could become denatured at higher ethanol concentration, the n-acetylglucosamine could become altered at higher ethanol concentrations, or the WGA-FITC binding to n-acetylglucosamine is hampered and slowed due to changes in affinity.

[0261] In particular embodiments of the invention, the dehydrating agent concentration in the lectin-based detection solution is preferably less than 25%, more preferably less than 20%, even more preferably less than 15%, and most preferably less than 10%. In particular embodiments of the invention, the dehydrating agent consists of ethanol wherein the ethanol concentration in the lectin-based detection solution is preferably less than 25%, more preferably less than 20%, even more preferably less than 15%, and most preferably less than 10%. It should be noted that all testing utilized a lectin-based detection solution with an ethanol concentration of 25%; therefore, it is anticipated that lowering the ethanol concentration would further increase lectin-fluorophore conjugate binding and cancer / dysplasia detection.

[0262] Although not explicitly discussed, the same experiment was conducted on “control beads” (Separopore 6B microbeads) that lack chitin binding groups. Negligible binding of WGA-FITC to these control beads was observed supporting that WGA-FITC binding is specific to n-acetylglucosamine.

[0263] Tissue Pretreatment then WGA-FITC Application with PEs Testing

[0264] The goal of this testing was to quantify the increased tissue penetration of WGA-FITC following tissue pretreatment.

[0265] Materials and Methods

[0266] Four different experiments were performed on tissue sections to test the synergistic effects of tissue pretreatment and penetration enhancing chemicals; the protocols for these combination experiments are outlined in Table 12. All these experiments used the optimal PE solution discovered in the aforementioned “PE efficacy” testing section. The pretreatment agents of Table 7 were used during these experiments; although, 0.25% Zonyl FS-300 surfactant (DuPont, Wilmington, DE) was added to each pretreatment agent to help increase penetration. Following pretreatment and lectin incubation, samples were imaged as Atty. Docket No.: 1249-11470-PCT

[0267] -44 -described previously. Aperio ImageScope Software was used to measure the WGA-FITC penetration at three locations within the tissue epithelium. Sample results were compared to the control (WGA-FITC incubated with PBS with no pretreatment). All tests were performed in triplicate.

[0268] Table 12: Testing protocols for combination experiments which include both penetration enhancers and tissue pretreatment. Pretreatment solutions contained 0.25% FS-300 surfactant to enhance tissue penetration. 25% ethanol, 1% Triton X-100 and 2% Pluronic F-68 were added into the lectin solutions during incubation, as this combination of penetration enhancers was discovered to be the most advantageous for lectin penetration.

[0269]

[0270] Results

[0271] Four different combinations of lectin concentration, pretreatment duration and incubation duration were employed in this section of the study and are summarized in Table 12. All trials utilized the most advantageous permeation enhancer combination discovered earlier (25% ethanol, 1% Triton X-100 and 2% Pluronic F-68) and examined lectin penetration depth into tissue epithelium.

[0272] Combination I (20uM lectin, 1 min pretreatment, and 30 min lectin incubation) did not result in any solution having statistically significantly different lectin penetration than PBS (p > 0.05, Fig. 16).

[0273] Combination II (20uM lectin, 5 min pretreatment, and 30 min lectin incubation) resulted in 1% TCA acid, 3% maleic acid, 10% citric acid, 10% glycolic acid, 10% acetic acid, and 5.25% bleach yielding statistically significantly better lectin penetration than PBS (p < Atty. Docket No.: 1249-11470-PCT

[0274] -45 - 0.05). Here 10% citric acid performed the best, resulting in a 3.87 fold increase in lectin penetration compared to PBS (147 ± 20.9 um versus 38 ± 8.3 um, respectively, p = 0.001).

[0275] Combination III (20uM lectin, 5 min pretreatment, and 5 min lectin incubation) only had 10% citric acid offering statistically significantly greater lectin penetration than the PBS control (123 ± 18.0 um versus 90 ± 7.8 um, respectively, p = 0.045, Fig. 16). Interestingly, many pretreatment solutions performed statistically significantly worse than the PBS control in combination III; these include 3% oxalic acid, 3% maleic acid, 10% glycolic acid, 10% lactic acid, 1.5% phenol, 3% H2O2, and 5.25% bleach (p < 0.05).

[0276] Lastly, combination IV (60uM lectin, 5 min pretreatment, and 5 min lectin incubation) resulted in only 3% oxalic acid offering statistically significantly deeper lectin penetration than PBS (124 ± 3.3 um versus 81 ± 4.2 um , p = 0.0002).

[0277] Conclusions

[0278] The most synergistic combination was seen by pretreating the tissue with 10% citric acid for 5 min, then incubating 20uM WGA-FITC with the optimal PE cocktail for either 5 or 30 min with the tissue (combination III and II, respectively). Pretreating the tissue with a unique pretreatment solution resulted in deeper WGA-FITC tissue penetration. Implementing such a procedure allows for more WGA-FITC to bind to tissue markers, which increases the fluorescence of WGA-FITC for easier visualization and interpretation.

[0279] In certain embodiments of the invention, the application of a lectin-based detection solution is preceded by the application of a pretreatment solution comprising citric acid, ethanol, surfactants, and water which helps increase epithelial penetration of the lectin compared to a PBS control. In certain other embodiments, the application of a lectin-based detection solution is preceded by the application of a pretreatment solution comprising citric acid, ethanol, surfactants, and water which preferably increases lectin epithelial penetration by 25 microns compared to a PBS control, more preferably increases lectin epithelial penetration by 50 microns compared to a PBS control, even more preferably increases lectin epithelial penetration by 100 microns compared to a PBS control, and most preferably increases lectin epithelial penetration by 125 microns compared to a PBS control. Atty. Docket No.: 1249-11470-PCT

[0280] -46 - Ex Vivo Validation - Esophagus

[0281] Materials and Methods

[0282] Patients with BE scheduled for endoscopic mucosal resection (EMR) were included. N-acetylcysteine was used as an mucolytic prior to topically applying WGA labelled to AlexaFluor-647 (WGA-AF647, 500ug / mL) on fresh EMR specimens directly after endoscopy and tissue retrieval. WGA-AF647 was solubilized in the more preferred embodiment detailed in Table 6. Fluorescence imaging was performed using the IVIS Spectrum System (Perkin Elmer, Waltham, MA) prior to and one minute after application of 2mL WGA-AF647 solution using a spray catheter. Fluorescence intensities on EMR specimens and subsequent perpendicular bread-loaf slices, imaged using an Odyssey Imaging System (Li -Cor, Lincoln, NE), were correlated with H&E histology ex vivo by board-certified pathologists.

[0283] Results

[0284] Seventy-four EMR tissue specimens were obtained from twenty-three patients. Overall, the described lectin-based detection solution (using WGA-AF647) was able to delineate disease with high sensitivity (92%) and specificity (95%). Identical results were also receiving using WGA-AF680 (AlexaFluor 680 fluorophore conjugated to WGA lectin), although this data is not shown.

[0285] Conclusions

[0286] The clinical trial validated the effectiveness of the described inventive lectin-based detection solution and method in detecting esophageal dysplasia and cancer with high sensitivity and specificity in an ex vivo test environment that is translatable to in vivo application and use.

[0287] High performance liquid chromatography (HPLC) testing on WGA-IR800CW with varying fluorophore to protein label ratios

[0288] Materials and Methods

[0289] To evaluate the potential impact of IR800CW fluorophore labeling ratios on the Atty. Docket No.: 1249-11470-PCT

[0290] -47 - WGA protein, HPLC testing was performed. IR800CW was conjugated to WGA following standard protocols known in the literature at ratios of 1:1, 1.25:1, 1.5:1, 1.75:1, and 2:1 (fluorophore: lectin).

[0291] Results

[0292] The results show peak fronting and shouldering of the chromatogram at higher IR800CW label ratios (i.e. 2:1 and 1.75:1). While this is anticipated to be from the normal distribution of label ratios within each test group, especially as the IR800CW molecule is fairly large (962Da) compared to the WGA monomer (~18kDa), this change is not considered ideal from a clinical testing standpoint as it calls into question potential changes to the protein as a result of labeling.

[0293] Conclusions

[0294] In certain embodiments of the invention, the at least one fluorescently labeled lectin within the lectin-based detection solution composition consists of WGA-IR800CW, wherein the fluorophore to protein ratio is between 1:1 and 1.5:1. While this test is specific to the lectin fluorophore conjugate, WGA-IR800CW, this fluorophore to protein ratio may be useful for other lectin fluorophore conjugates.

[0295] Fluorescence intensity testing on WGA-IR800CW with varying fluorophore to protein label ratios

[0296] Materials and Methods

[0297] A fluorescence intensity experiment was undertaken on various IR800CW to WGA lectin labeling ratios (0.5:1, 1 : 1, 2:1, and 4: 1). Following fluorophore conjugation, stock solutions of each WGA-IR800CW conjugate were made at 0.12mg / mL in IX PBS. IOOUL of each conjugate was aliquoted into a 96 well plate and fluorescence intensity was measured on a Biotek Synergy plate reader (excitation 700nm, emission 789nm).

[0298] Results Atty. Docket No.: 1249-11470-PCT

[0299] -48 - Fluorescence intensity (Au) results are shown in Table 13 and Fig. 20. As is shown, an optimal fluorescence intensity is observed at a fluorophore to protein ratio between 1:1 and 2:1.

[0300] Table 13: Fluorescence intensity results for various WGA-IR800CW conjugates.

[0301]

[0302] Conclusions

[0303] In certain embodiments of the invention, the at least one fluorescently labeled lectin within the lectin-based detection solution composition consists of WGA-IR800CW, wherein the fluorophore to protein ratio is between 1 : 1 and 2:1. While this test is specific to the lectin fluorophore conjugate, WGA-IR800CW, this fluorophore to protein ratio may be useful for other lectin fluorophore conjugates.

[0304] Enzyme-linked immunosorbent assay (ELISA) on WGA-IR800CW with varying fluorophore to protein label ratios

[0305] Materials and Methods

[0306] An indirect ELISA assay was undertaken to evaluate the potential impact of IR800CW fluorophore labeling ratios on the binding affinity of the WGA protein to porcine mucin, which is a glycoprotein that contains WGA’s target glycan (n-acetyl glucosamine). For this test, a 96-well plate was coated with porcine mucin and then the uncoated parts are blocked with bovine serum albumin (BSA) to prevent non-specific bonding. Next, a dilution series of WGA-IR800CW and reference WGA was added to the wells. The bound WGA and WGA-IR800CW were then detected by first adding the primary antibody anti-WGA and then the Atty. Docket No.: 1249-11470-PCT

[0307] -49 -secondary antibody "goat anti -rabbit horseradish peroxidase (HRP)". The coupled HRP is an enzyme that reacts with the dye 3,3’,5,5’-tetramethylbenzidine (TMB) to produce a chromogenic reaction, which was quantitatively measured using a Biotek plate reader at 450nm to determine the amount of bound antibody. Testing was performed on freshly made lectin-based detection solution compositions using WGA-IR800CW (i.e. t = 0 days) and after 60 days of refrigeration storage at 6C.

[0308] Results

[0309] Results are shown in Table 14. Generally, higher IR800CW labeling ratios appear to negatively impact WGA binding affinity to n-acetylglucosamine; however, this decrease in binding affinity is less apparent over time, which may be due to structural stabilization of the WGA-IR800CW conjugate over time.

[0310] Table 14: Indirect ELISA results demonstrating WGA-IR800CW binding affinity to n-acetylglucosamine for various fluorophore to protein labeling ratios and over time.

[0311]

[0312] Conclusions

[0313] In certain embodiments of the invention, the at least one fluorescently labeled lectin within the lectin-based detection solution composition maintains preferably at least 60% binding affinity of the native unconjugated lectin, more preferably maintains at least 80% binding affinity of the native unconjugated lectin, and most preferably maintains at least 90% binding affinity of the native unconjugated lectin. In other words, the fluorophore conjugation does not decrease the lectin-fluorophore conjugate’s gly can binding affinity by more than 40%. Atty. Docket No.: 1249-11470-PCT

[0314] - 50 - In certain embodiments of the invention, the at least one fluorescently labeled lectin within the lectin-based detection solution composition consists of WGA-IR800CW, wherein the fluorophore to protein ratio is between 1 : 1 and 2:1, and wherein the binding affinity of the WGA-IR800CW conjugate maintains preferably at least 60% binding affinity of the native unconjugated WGA, more preferably maintains at least 80% binding affinity of the native unconjugated WGA, and most preferably maintains at least 90% binding affinity of the native unconjugated WGA.

[0315] Definitions

[0316] Bioactivity - In this application, bioactivity generally refers to glycan binding affinity of a lectin.

[0317] Cancer - This term generally refers to the uncontrolled division of abnormal cells in the body that can result in a malignant growth or tumor.

[0318] Detection Solution - This term generally refers to the invented compositions of this patent application, as described, which comprises a nuclear based detection solution and a lectin-based detection solution.

[0319] Dysplasia - Sometimes referred to as precancer, this term generally refers to tissue containing abnormal cellular changes that are not yet cancerous. Dysplasia is diagnosed in various grades (mild, moderate, and severe or low-grade and high-grade) that are associated with a risk level towards cancer progression. Some mild or low-grade dysplasias may revert back to normal tissue, however, severe or high-grade dysplasias have a significant risk of cancer development.

[0320] Endoscope - This term generally refers to an instrument that can be introduced into the body to give a view of its internal parts.

[0321] Endogenous - This term generally refers to something found naturally inside the body. For example, endogenous fluorophores are naturally occurring and present within cells or tissues of the body.

[0322] Exogenous - This term generally refers to something not from the body. For example, exogenous fluorophores are fluorescent molecules that are not naturally found within the human body but are instead introduced for fluorescence visualization purposes.

[0323] Emission Wavelength - This term generally refers to the wavelength emitted from a Atty. Docket No.: 1249-11470-PCT

[0324] - 51 -fluor ophore after the fluorophore has been excited via its excitation wavelength.

[0325] Excitation Wavelength - This term generally refers to the wavelength of electromagnetic radiation that most efficiently excites a fluorophore and typically yields the highest amount of fluorescence emission.

[0326] Fluorescence - This term generally refers to the visible or invisible radiation emitted by certain substances / molecules as a result of incident radiation of a shorter wavelength.

[0327] Fluorophore - A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound, substance, or molecule that can re-emit light upon light excitation.

[0328] Glycosylation - This term generally refers to a post-translational modification of proteins where glycan (sugar) molecules are conjugated to the protein molecule. Glycosylation can also occur as the protein is being translated. Glycosylation can also occur to lipids where glycan molecules are conjugated to lipids.

[0329] Glycomolecule - This term generally refers to a molecule, typically a protein or lipid, that contains glycan (sugar) molecules. In this described invention, cell surface glycomolecules are of importance as the lectin (or other molecular probe) will only bind to glycans presented on the cellular surface.

[0330] In Vivo - This term generally refers to inside the human body; contrasts with ex vivo (performed in or on tissue outside the body but with minimal alterations from natural conditions) or in vitro (performed outside the body in a test tube or culture dish).

[0331] Isolectin - This term is generally synonymous with isoform and generally refers to nearly identical subsets of a lectin that differ slightly in amino acid sequence. For example, WGA lectin is generally composed of three isolectins / isoforms (WGA1, WGA2, and WGA3). Lectin-Based Detection Solution - This term generally refers to the invented composition of this patent application, as described, which comprises a fluorescently-labeled lectins, or lectin fragments, that targets aberrant glycosylation associated with precancer and cancer.

[0332] Molecular probe - This term generally refers to a molecule that has a high affinity for a target molecule. Throughout the embodiment, it is assumed the molecular probe is conjugated to a reporter molecule, which allows for the detection of the molecular probe’s binding. In a preferred embodiment, the reporter molecule is a fluorophore (unless stated otherwise), which allows for the fluorescent visualization of the molecular probe’s binding patterns using a Atty. Docket No.: 1249-11470-PCT

[0333] - 52 -camera or the human eye. Additionally, in another embodiment of the invention, the molecular probe may be itself fluorescent, such as a nuclear fluorescent stain (e.g. DAPI) which has an affinity for nucleic acids and acidic molecules and is also fluorescent.

[0334] Molecular imaging - This term generally refers to the visualization, characterization, and measurement of biological processes at the molecular and cellular levels in humans and other living systems. Molecular imaging typically consists of 2- or 3 -dimensional imaging as well as quantification over time. The techniques used include radiotracer imaging / nuclear medicine, MR imaging, MR spectroscopy, optical imaging, fluorescence-based imaging, ultrasound, and others. One important variable during molecular imaging is ensuring a molecular probe and / or reporter molecule’s signal is obtained that exceeds the noise floor or background signal. Typically researchers discuss this as the “signal to noise” or “signal to background” ratio. The disclosed invention relates to fluorescence imaging.

[0335] Mucosal tissue - This term generally refers to the tissue that lines the surface of various cavities in the body and that covers the surfaces of internal organs. Mucosal tissue consists of one or more layers of epithelial cells overlying a layer of connective tissue. Examples of mucosal tissue include: endometrium, esophageal mucosa, gastric mucosa, intestinal mucosa, nasal mucosa, olfactory mucosa, oral mucosa, penile mucosa, vaginal mucosa, tongue, anal, cervical mucosa. In other words, mucosal tissue can be found in the following bodily sites: oral cavity, lips, nasal passage, vagina, cervix, urethra, anus, uterus, bile duct, colon, intestines, ears, penis, and stomach, amongst other areas.

[0336] Nuclear Stain-Based Detection Solution - This term generally refers to the invented composition of this patent application, as described, which comprises a fluorescent nuclear stain that targets molecular characteristics of precancer and cancer.

[0337] PBS - Also noted as “IX PBS” both of which refer to a phosphate buffered saline solution at IX concentration, which is understood in the scientific and medical community to mean a distilled water solution containing the following salt concentrations: 8.0 g / L sodium chloride (NaCl), 0.2 g / L potassium chloride (KC1), 1.44 g / L sodium phosphate dibasic (Na2HPO4), and 0.24 g / L monobasic potassium phosphate (KH2PO4).

[0338] Permeation Enhancer (PE) - This term generally refers to chemicals that help facilitate molecule penetration into the epithelium. PEs include, but are not limited to, surfactants, Atty. Docket No.: 1249-11470-PCT

[0339] - 53 -dehydrating agents, alcohols, fatty acids, peptides, nanoparticles, among others. This term is sometimes used interchangeably with penetration enhancers.

[0340] Precancer - See dysplasia. May also refer to precancerous conditions that are not yet dysplastic, for example, Barrett’s esophagus is a known precancerous condition in the esophagus.

[0341] Pretreatment solution - This term generally refers to the invented pretreatment solution is engineered to 1) remove debris, mucus, and dead cells, 2) dehydrate the mucosa, and 3) prepare the mucosa for application of the detection solutions. The pretreatment solution is comprised of organic acids, desiccants, solvents, preservatives, surfactants and flavorants as described.

[0342] Reporter molecule - This term generally refers to a molecule that is chemically bonded to a molecular probe to provide a means of measuring or quantifying the molecular probe’s binding to a target. Reporter molecules may comprise quantum dots, fluorophores, colored microspheres, CT contrast agents, MRI contrast agents, or radiolabels. The disclosed invention generally relates to fluorophores or fluorescent molecules; however, in certain instances fluorophores can be substituted with other reporter molecules. For example, WGA-IR800CW could be replaced with WGA-radiolabel for other modality imaging.

[0343] Sensitivity - This term generally refers to the proportion of actual positives that are correctly identified by a test / procedure (i.e. the percentage of people with disease who are correctly identified as having the disease).

[0344] Specificity - This term generally refers to the proportion of actual negatives that are correctly identified by a test / procedure (i.e. the percentage of people without a disease who are correctly identified as not having the disease).

[0345] Surfactant - This term generally refers to a chemical that tends to reduce the surface tension of the liquid it is dissolved in. In this invention, surfactant(s) can refer to any commonly known and commercially available surfactant including but not limited to: sulfonates, sulfates, ethoxylated alcohols, ethoxylated alkylphenols, fatty acid esters, ethoxysulfates, betaines, amine oxides, fluorosurfactants, quaternary ammonium salts, alkylbenzene sulfonates, fatty acid soaps, lauryl sulfates, amongst others.

[0346] Target mucosa - This term generally refers to mucosal areas within the body that may be subjected to the disclosed inventions for disease detection. Examples of mucosal tissue include: endometrium, esophageal mucosa, gastric mucosa, intestinal mucosa, nasal mucosa, olfactory Atty. Docket No.: 1249-11470-PCT

[0347] - 54 -mucosa, oral mucosa, penile mucosa, vaginal mucosa, tongue, anal, cervical mucosa. In other words, mucosal tissue can be found in the following bodily sites: oral cavity, lips, nasal passage, vagina, cervix, urethra, anus, uterus, bile duct, colon, intestines, ears, penis, and stomach, amongst other areas.

[0348] Tissue autofluorescence - This term generally refers to the natural emission of light by biological structures within tissue when they have absorbed light of a lower wavelength. Typically, tissue autofluorescence is accomplished by illuminating the tissue with blue light and observing the resulting green light emitted by biological structures within tissue. Usually a loss of autofluorescence is indicative of non-normal tissue due to inflammation, or diseases such as cancer. Tissue autofluorescence is different than fluorescent light originating from artificially added fluorescent markers (fluorophores).

[0349] Tracer - This term is generally synonymous with molecular probe; see molecular probe.

[0350] As used herein, “a”, “an”, “the”, “at least one”, and “one or more” are used interchangeably.

[0351] The term “comprises,” and variations thereof, do not have a limiting meaning where these terms appear in the description and claims.

[0352] The foregoing is considered as illustrative only of the principles of the invention. Furthermore, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation shown and described. While the preferred embodiment has been described, the details may be changed without departing from the invention.

Claims

Atty. Docket No.: 1249-11470-PCT- 55 - What is claimed is:

1. A lectin-based detection solution composition comprising:at least one fluorescently-labeled lectin;at least one dehydrating agent;at least one non-ionic surfactant; andat least one solvent,wherein the lectin-based detection solution is applied in vivo, wherein the fluorescently-labeled lectin molecules are excited such that they fluoresce,wherein this fluorescence is perceivable by the human eye or a camera, and wherein the fluorescently-labeled lectin molecules detect mucosal -based tissue cancers through visible staining differentiation of diseased tissue.

2. The composition of claim 1, wherein the at least one fluorescently-labeled lectin consists of WGA-IR800CW, the at least one dehydrating agent consists of ethanol, the at least one non-ionic surfactant consists of three surfactants: poloxamer 188, triton x- 100, and Masurf FS-2825, and the at least one solvent is IX phosphate buffered saline (PBS).

3. The composition of claim 2, wherein the at least one fluorescently-labeled lectin consists of WGA-IR800CW at a concentration of 0.01%, the at least one dehydrating agent consists of ethanol at a concentration of 25%, the at least one non-ionic surfactant consists of three surfactants: poloxamer 188 at a concentration of 2%, triton x-100 at a concentration of 1%, and Masurf FS-2825 at a concentration of 0.25%, and the at least one solvent consists of phosphate buffered saline at a concentration of at least 71.74%.

4. The composition of claim 3, wherein the phosphate buffered saline is at a concentration of 91.74%.

5. The composition of claim 1, wherein the at least one fluorescently-labeled lectin targets at least one glycan selected from a group consisting of: mannose, glucose, sialic acid, a-L-fucose, blood group H / O, T antigen, Tn antigen, N-acetyl galactosamine, or N-acetyl glucosamine.Atty. Docket No.: 1249-11470-PCT- 56 - 6. The composition of claim 1, wherein the at least one fluorescently-labeled lectin is at a concentration between luM and 50uM.

7. The composition of claim 1, wherein the at least one fluorescently-labeled lectin is at a weight / weight (w / w%) concentration between 0.001% - 0.5%.

8. The composition of claim 1, wherein the at least one non-ionic surfactant is a permeation enhancer that yields an epithelial penetration of the fluorescently-labeled lectin of at least 250 microns.

9. The composition of claim 1, further comprising one or more of the following: at least one lubricant, at least one at least one flavorant, at least one sweetener, at least one preservative, at least one mucolytic agent, and at least one permeation enhancer.

10. The composition of claim 9, further comprising at least one lubricant at a concentration between 5% - 35%.

11. The composition of claim 9, further comprising at least one flavorant at a concentration between 0.1% - 20%.

12. The composition of claim 1, wherein the pH of the lectin-based detection solution composition is between 6.5 - 7.5.

13. The composition of claim 1, wherein the surface tension of the lectin-based detection solution is less than 50 dynes / cm.

14. The composition of claim 1, wherein the osmolarity of the lectin-based detection solution is preferably between 290 mOsm / L and 310 mOsm / L.

15. The composition of claim 1, wherein the at least one fluorescently-labeled lectin is comprised of a lectin selected from the following list: ConA, LCA, LcH, GNA, PNA, AIL, VVL, WGA, MAL, MAH, SB A, UEA, AAL, BP A, ECA, Jacalin, LEA, MAA, PHA-E, PHA-L, PHA-M, PHA-P, PSA, TJA, VEA, SNA, UDA, NPA, or NPL.

16. The composition of claim 1, wherein the at least one fluorescently-labeled lectin is comprised of a fluorophore selected from the following list: fluoresceine, fluoresceine derivatives, rhodamine, cyanine dyes, Alexa Fluor dyes, BODIPY dyes, coumarin, IRDye 800 and conjugates, or quantum dots.

17. The composition of claim 1, wherein the solvent is preferably a water-based buffer selected from the following list: MES, TRIS, HEPES, TEA.Atty. Docket No.: 1249-11470-PCT- 57 - 18. The composition of claim 1, wherein the at least one fluorescently-labeled lectin is comprised of a lectin with a molecular weight less than 50kDa.

19. The composition of claim 1, wherein the at least one fluorescently-labeled lectin is comprised of only lectin monomers.

20. The composition of claim 1, wherein the at least one fluorescently-labeled lectin is comprised of a specific isolectin or lectin isoform.

21. The composition of claim 1, wherein the at least one fluorescently-labeled lectin is comprised of a lectin that agglutinates a 2% suspension of human erythrocytes at a concentration less than 50 ug / mL.

22. The composition of claim 1, wherein the at least one fluorescently-labeled lectin is comprised of a lectin and fluorophore conjugate, wherein the molecular weight of the lectin is preferably at least 10 times greater than the molecular weight of the fluorophore conjugate.

23. The composition of claim 22, wherein the labeling ratio (fluorophores to protein) is preferably between 1 : 1 and 2:1.

24. The composition of claim 23, wherein the labeling ratio (fluorophores to protein) is preferably about 1.5:1.

25. The composition of claim 1, wherein the at least one fluorescently-labeled lectin is replaced by a fluorescently-labeled lectin fragment.

26. The composition of claim 1, wherein the at least one fluorescently labeled-lectin is replaced by a fluorescently-labeled WGA fragment of about 13kDa.

27. The composition of claim 1, wherein the at least one fluorescently-labeled lectin consists of WGA-IR800CW and wherein the WGA component of WGA-IR800CW is a 13kDa fragment of the native WGA lectin.

28. A tissue pretreatment solution composition comprising:an organic acid;at least one surfactant;at least one dehydrating agent; andat least one solvent,wherein the pretreatment solution facilitates the cleansing of mucosal tissue inAtty. Docket No.: 1249-11470-PCT- 58 - vivo prior to the application of molecular probes.

29. The composition of claim 28, wherein the organic acid comprises citric acid, the at least one surfactant comprises pol oxamer 188, triton x-100, and Masurf FS-2825, the at least one dehydrating agent comprises ethanol, and the at least one solvent comprises water.

30. The composition of claim 29, wherein the organic acid comprises citric acid at a concentration of 10%, the at least one surfactant comprises pol oxamer 188 at a concentration of 2%, triton x-100 at a concentration of 1%, and Masurf FS-2825 at a concentration of 0.05%, the at least one dehydrating agent comprises ethanol at a concentration of 25%, and the at least one solvent comprises water at a concentration of 61.95%.

31. The composition of claim 28, wherein the pretreatment solution facilitates the removal of mucosal debris and mucus, while dehydrating the mucosa, and increasing the permeability of the mucosal epithelium.

32. The composition of claim 28, further comprising at least one mucolytic agent.

33. The composition of claim 28, further comprising at least one lubricant.

34. The composition of claim 33, further comprising at least one lubricant at a concentration between 5% - 35%.

35. The composition of claim 33, wherein the at least one lubricant comprises glycerin.

36. The composition of claim 28, further comprising one or more of the following: at least one lubricant, at least one flavorant, at least one sweetener.

37. The composition of claim 36 further comprising at least one flavorant at a concentration between 0.1% - 20%.

38. The composition of claim 28, wherein the pH of the pretreatment solution is between 3 - 5.5.

39. The composition of claim 28, wherein the surface tension of the pretreatment solution is less than 30 dynes / cm.

40. The composition of claim 28, wherein the pretreatment solution composition removes at least 20% of the surface mucosal epithelium.Atty. Docket No.: 1249-11470-PCT- 59 - 41. The composition of claim 40, wherein the pretreatment solution composition removes at least 20% of the surface mucosal epithelium within five minutes of in vivo application.

42. A nuclear stain-based detection solution composition comprising:at least one nuclear stain;at least one organic acid;at least one salt of the conjugate base of said at least one organic acid;at least one dehydrating agent;at least one preservative; andat least one solvent,wherein the nuclear stain-based detection solution is applied in vivo, wherein the nuclear stain molecules are excited such that they fluoresce, wherein this fluorescence is perceivable by the human eye or a camera, and wherein the nuclear stain molecules detect mucosal-based tissue disease through visible staining differentiation of diseased tissue.

43. The composition of claim 42, wherein the at least one nuclear stain comprises DAPI, the at least one organic acid comprises acetic acid, the at least one salt of the conjugate base of said organic acid comprises sodium acetate, the at least one dehydrating agent comprises ethanol, the at least one preservative comprises thymol, and the at least one solvent comprises water.

44. The composition of claim 43, wherein the at least one nuclear stain comprises DAPI at a concentration between lOuM and ImM.

45. The composition of claim 44, wherein the at least one nuclear stain comprises DAPI at a concentration of about ImM.

46. The composition of claim 45, wherein the at least one organic acid comprises acetic acid at a concentration of about 4.61%, the at least one salt of the conjugate base of said organic acid comprises sodium acetate at a concentration of 2.45%, the at least one dehydrating agent comprises ethanol at a concentration of 7.5%, the at least one preservative comprises thymol at a concentration of 0.06%, and the at least one solvent comprises water at a concentration of 85.35%.Atty. Docket No.: 1249-11470-PCT-60 - 47. The composition of claim 42, further comprising one or more of the following: at least one lubricant, at least one surfactant, at least one flavorant, at least one sweetener, and at least one chelating agent.

48. The composition of claim 47, further comprising at least one lubricant at a concentration between 5% - 35%.

49. The composition of claim 47, further comprising at least one flavorant at a concentration between 0.1% - 20%.

50. The composition of claim 42, wherein the pH of the nuclear stain-based detection solution composition is between 3.5 - 5.5.

51. The composition of claim 42, wherein the surface tension of the nuclear stain-based detection solution is less than 30 dynes / cm.

52. A method of fragmenting a native lectin into at least one biologically active lectin fragment comprising:exposing proteins of the native lectin to at least one reducing agent; isolating at least one lectin fragment; andpurifying the at least one lectin fragment to produce the at least one biologically active lectin fragment,wherein, the at least one biologically active lectin fragment is less than 80% the molecular weight of the native lectin, andwherein the at least one biologically active lectin fragment retains at least 50% glycan affinity of the native lectin.

53. The method of claim 52, wherein the at least one reducing agent is selected from either dithiothreitrol (DTT) or beta-mercaptoethanol (BME).

54. The method of claim 52, further comprising the step of exposing the native lectin to at least one additional fragmentation step selected from the following list: heat and / or pressure, acids, bases, or proteases.

55. A method of fragmenting a native lectin into at least one biologically active lectin fragment comprising:exposing proteins of the native lectin to at least one protease;isolating at least one specific lectin fragment; andAtty. Docket No.: 1249-11470-PCT- 61 - purifying the at least one lectin fragment to produce at least one biologically active lectin fragment,wherein the at least one biologically active lectin fragment is less than 80% the molecular weight of the native lectin, andwherein the at least one biologically active lectin fragment retains at least 50% glycan affinity of the native lectin.

56. The method of claim 55, wherein the at least one protease is selected from a group consisting of: trypsin, chymotrypsin, subtilisin, elastase, thermolysin, or proteinase K.

57. The method of claim 55, further comprising the step of exposing the native lectin to at least one additional fragmentation step selected from a group consisting of: reducing agents, heat and / or pressure, acids, or bases.

58. The method of claim 55, wherein the fragmentation process exposes previously hidden or hindered glycan binding sites that are now capable of binding to cell surface glycans in vivo.

59. The method of claim 55, wherein the at least one biologically active lectin fragment agglutinates a 2% suspension of human erythrocytes at a concentration less than 50 ug / mL.

60. The method of claim 55, further comprising the step of conjugating the at least one biologically active lectin fragment to a fluorophore.

61. The method of claim 55, wherein the biologically active lectin comprises WGA and the at least one lectin fragment comprises at least one WGA fragment between 3kDa and 15kDa that maintains its affinity for n-acetylglucosamine.

62. The method of claim 61, wherein the lectin comprises WGA and the at least one lectin fragment comprises at least one WGA fragment between 5kDa and 13kDa that maintains its affinity for n-acetylglucosamine.

63. The method of claim 62, wherein the native lectin comprises WGA and the at least one biologically active lectin fragment comprises a WGA fragment about 13kDa that maintains its affinity for n-acetylglucosamine.Atty. Docket No.: 1249-11470-PCT- 62 - 64. The method of claim 55, wherein the lectin comprises WGA and the at least one lectin fragment comprises at least one WGA fragment between 3kDa and 15kDa that maintains its affinity for sialic acid.

65. A lectin-based detection solution composition comprising:at least one fluorescently-labeled lectin;at least one dehydrating agent;at least one permeation enhancer; andat least one solvent,wherein the lectin-based detection solution is applied in vivo,wherein the fluorescently-labeled lectin molecules are excited such that they fluoresce,wherein this fluorescence is perceivable by the human eye or a camera, and wherein the fluorescently-labeled lectin molecules detect mucosal -based tissue cancers through visible staining differentiation of diseased tissue.

66. The composition of claim 65, wherein the at least one fluorescently-labeled lectin comprises WGA-IR800CW, the at least one dehydrating agent comprises ethanol, the at least one non-ionic surfactant comprises pol oxamer 188, triton x-100, and Masurf FS-2825, and the at least one solvent comprises IX phosphate buffered saline (PBS).

67. The composition of claim 66, wherein the at least one fluorescently-labeled lectin comprises WGA-IR800CW at a concentration of 0.01%, the at least one dehydrating agent comprises ethanol at a concentration of 25%, the at least one permeation enhancer comprises pol oxamer 188 at a concentration of 2%, triton x-100 at a concentration of 1%, and Masurf FS-2825 at a concentration of 0.25%, and the at least one solvent comprises phosphate buffered saline at a concentration of at least 71.74%.

68. The composition of claim 65, wherein the at least one solvent comprises phosphate buffered saline at a concentration of at least 91.74%.

69. The composition of claim 65, wherein the at least one fluorescently-labeled lectin targets at least one glycan selected from a group consisting of: mannose, glucose, sialic acid, a-L-fucose, blood group H / O, T antigen, Tn antigen, N-acetyl galactosamine, or N-acetyl glucosamine.Atty. Docket No.: 1249-11470-PCT- 63 - 70. The composition of claim 65, wherein the at least one fluorescently-labeled lectin is at a concentration between luM and 50uM.

71. The composition of claim 65, wherein the at least one fluorescently-labeled lectin is at a weight / weight (w / w%) concentration of preferably 0.001% - 0.5%.

72. The composition of claim 65, wherein the at least one permeation enhancer yields an epithelial penetration of the fluorescently-labeled lectin of at least 250 microns.

73. The composition of claim 65, further comprising one or more of the following: at least one lubricant, at least one at least one flavorant, at least one sweetener, at least one preservative, and at least one mucolytic agent.

74. The composition of claim 73, further comprising at least one lubricant at a concentration between 5% - 35%.

75. The composition of claim 73, further comprising at least one flavorant at a concentration between 0.1% - 20%.

76. The composition of claim 65, wherein the pH of the lectin-based detection solution composition is between 6.5 - 7.5.

77. The composition of claim 65, wherein the surface tension of the lectin-based detection solution is less than 50 dynes / cm.

78. The composition of claim 65, wherein the osmolarity of the lectin-based detection solution is preferably between 290 mOsm / L and 310 mOsm / L.

79. The composition of claim 65, wherein the at least one fluorescently-labeled lectin is comprised of a lectin selected from the following list: ConA, LCA, LcH, GNA, PNA, AIL, VVL, WGA, MAL, MAH, SB A, UEA, AAL, BP A, ECA, Jacalin, LEA, MAA, PHA-E, PHA-L, PHA-M, PHA-P, PSA, TJA, VEA, SNA, UDA, NPA, or NPL.

80. The composition of claim 65, wherein the at least one fluorescently labeled lectin is comprised of a fluorophore selected from a group consisting of: fluoresceine, fluoresceine derivatives, rhodamine, cyanine dyes, Alexa Fluor dyes, BODIPY dyes, coumarin, IRDye 800 and conjugates, or quantum dots.

81. The composition of claim 65, wherein the solvent compreises a water-based buffer selected from a group consisting of: MES, TRIS, HEPES, or TEA.Atty. Docket No.: 1249-11470-PCT- 64 - 82. The composition of claim 65, wherein the at least one fluorescently-labeled lectin has a molecular weight less than 50kDa.

83. The composition of claim 65, wherein the at least one fluorescently-labeled lectin is comprised of only lectin monomers.

84. The composition of claim 65 wherein the at least one fluorescently-labeled lectin is an isolectin or lectin isoform.

85. The composition of claim 65, wherein the at least one fluorescently-labeled lectin agglutinates a 2% suspension of human erythrocytes at a concentration less than 50 ug / mL.

86. The composition of claim 65, wherein the at least one fluorescently-labeled lectin is comprised of a lectin and fluorophore conjugate, wherein the molecular weight of the lectin is preferably at least 10 times greater than the molecular weight of the fluorophore.

87. The composition of claim 86, wherein the labeling ratio (fluorophores to protein) is preferably between 1 : 1 and 2:1.

88. The composition of claim 87, wherein the labeling ratio (fluorophores to protein) is preferably about 1.5:1.

89. The composition of claim 65, wherein the at least one fluorescently-labeled lectin is replaced by a fluorescently labeled lectin fragment.

90. The composition of claim 65, wherein the at least one fluorescently-labeled lectin is replaced by a fluorescently labeled WGA fragment of about 13kDa.

91. The composition of claim 65, wherein the at least one fluorescently-labeled lectin consists of WGA-IR800CW and wherein the WGA component of WGA-IR800CW is a 13kDa fragment of the native WGA lectin.

92. The composition of claim 65, wherein the at least one permeation enhancer comprises one or more of the following: Zonyl FS-300, ethanol at a preferable concentration of <25%, Pluronic F-68, Triton X-100 at 1%, Propylene glycol between 5-10%, taurocholic acid (0.5-1%), bile salts (0.5-1%), and methyl sulfonylmethane <12.5%.

93. The composition of claim 65, wherein the at least one permeation enhancer does not decrease lectin glycan binding affinity by more than 30%.Atty. Docket No.: 1249-11470-PCT- 65 - 94. The composition of claim 65, wherein the at least one permeation enhancer increases epithelial penetration of the lectin-fluorophore conjugate by at least lOOum.

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