DNA origami nancomplexes for selective delivery of imaging and therapeutic agents to KRAS-mutant pancreatic cancer cells

Abstract

Labelled DNA Origami for selective imaging of KRAS-mutant pancreatic cancer cells in a desmoplastic pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) are disclosed, as well as DNA Origami nanocomplexes for targeted delivery of a therapeutic payload to KRAS-mutant pancreatic cancer cells in a subject.

Description

DNA ORIGAMI NANCOMPLEXES FOR SELECTIVE DELIVERY OF IMAGING AND THERAPEUTIC AGENTS TO KRAS-MUTANT PANCREATIC CANCER CELLSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 670,537, filed July 12, 2024, the entire contents of which are hereby incorporated by reference as if fully set forth.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under 2134603 awarded by the National Science Foundation. The government has certain rights in the invention.TECHNICAL FIELD

[0003] The present disclosure relates to labelled DNA Origami for selective imaging of KRAS-mutant pancreatic cancer cells in a desmoplastic pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) and DNA Origami nanocomplexes for targeted delivery of a therapeutic payload to KRAS-mutant pancreatic cancer cells in a subject.BACKGROUND

[0004] Pancreatic ductal adenocarcinoma (PDAC) poses a significant clinical challenge as shown by five-year survival rates of 13%. PDAC tumor microenvironment (TME) are characterized by a desmoplastic stroma that facilitates tumor growth, drives drug resistance, and impedes the drug delivery to pancreatic cancer cells (PCCs), all leading to poor treatment outcomes. The PDAC stroma accounts for up to 90% of the PDAC tumor volume and is predominantly composed of cancer-associated fibroblasts (CAFs) and ECM components.1'4CAFs synthesize and remodel the stromal ECM, which closely contributes to tumorigenesis, impedes drug delivery, and generally correlates with poor prognosis. CAFs also regulate ECM degradation by expressing numerous proteases (e.g., secreted serine proteases and matrix metalloproteinases) that drive tumor growth and metastasis.5'10Various growth factors secreted by CAFs also enhance an epithelial-mesenchymal transition (EMT) and therapeutic resistance ofPCCs.11, 12The combination of these complex biochemical and biophysical mechanisms with heterogeneous genetic mutations makes PDAC extremely difficult to diagnose and treat.

[0005] To overcome the challenges in PDAC, targeted delivery of imaging and therapeutic agents has been extensively explored, leveraging PD AC-specific antibodies or nanoparticle engineering to achieve tumor-specific delivery to cancer cells. Various nanoparticle carriers have been utilized to load cytotoxic drugs and imaging agents for tumor selective delivery, including DNA / RNA based’ polymeric, lipidic, and inorganic vehicles.13'22Among various types of engineered nanomedicines, DNA nanocarriers have advanced rapidly, demonstrating tremendous potential with functionalities, biocompatibility, addressability, and programmability.

[0006] DNA origami is a self-assembly approach that can form arbitrary nanostructures from a long single-stranded (ss) DNA (termed scaffold) with dozens of oligonucleotides (staples).23Various structures of different sizes and shapes have been demonstrated based on the sequence complementarity, including smiley faces, Mona Lisa, and polyhedral architectures.23'27The engineered topological building blocks can serve as the foundation for DNA-based nanocarriers for various payloads, such as therapeutic drugs and imaging agents, to specific cells or tissues.15, 28-30They hold significant opportunities for drug delivery applications with notable features, including addressability with various molecules, versatility in chemical modifications, flexibility in structural and functional design, biocompatibility, and stability.25, 31-38Novel anticancer drug delivery systems with DNA origami thus offer significant promise to enhanced targeting specificity, improved drug delivery efficiency, and reduced toxicity.I ?‘39By precisely targeting cancer cells and minimizing exposure to healthy cells, DNA origami could become a highly effective approach to cancer treatment.29, 40

[0007] Despite significant advances, the translation of DNA-origami nanocarriers into practical utility in targeted delivery has been largely hindered by the lack of sophisticated evaluation of tumor-targeting delivery mechanisms. Specifically, understanding the tumortargeting specificity at the tissue level poses a significant challenge, as highly heterogeneous PDAC includes a substantial population of CAFs. Recent studies have focused on evaluating transport features of DNA-origami carriers with inconsistent observations. For example, Wang et al. studied cellular uptake and trafficking of DNA origami with gold nanoparticle tags,examining various sizes, shapes, and surface chemistry.40The study found that larger origami nanostructures generally demonstrated greater uptake and that, while scavenger receptors were important in mediating cell uptake, no specific endocytosis pathway was identified. Other reports suggested that more compact structures promote the efficiency of cellular uptake.41'43Another study constructed DNA origami of various geometries (e.g., tetrahedron, cube, icosahedron, and buckyball) and observed a clathrin-mediated pathway for cellular entry44Overall, uptake mechanisms are highly dependent on the cell lines and there is a lack of adequate information on its targeting specificity in the complex and heterogeneous PDAC microenvironment. In-depth studies on targeting efficacy and specificity of DNA origami-based delivery are thus critically needed.SUMMARY

[0008] In one aspect, a DNA Origami is provided for selective targeting of KRAS- mutant Pancreatic Cancer Cells via macropinocytosis. By way of example, the DNA origami herein can be constructed of a single stranded DNA scaffold organized into an arbitrary nanostructure via a plurality of oligonucleotide staples. The arbitrary nanostructure can be configured into a variety of geometries. In one embodiment, the arbitrary nanostructure can be configured to have a substantially tubular architecture. The substantially tubular architecture can be defined by a length and a diameter. For instance, the substantially tubular architectures of certain examples can have a length between -6 nm and -150 nm and a diameter between -20 nm and -50 nm. In one such example, the substantially tubular architectures can have a length -70 nm and a diameter of -30 nm. In another example, the substantially tubular architectures can have a length of -140 nm and a diameter of -30 nm. In yet another example, the substantially tubular architectures can have a length of -6 nm and a diameter of -30 nm.

[0009] In another embodiment, the arbitrary nanostructure of the DNA origami can have a substantially planar tile architecture, in which the substantially planar tile architecture can be defined by a length and a width. For instance, the substantially planar tile architectures of certain examples can have a length between -6 nm and -150 nm and a width between -20 and -50 nm. In one such example, the substantially tubular architectures can have a length -70 nm and a width of -30 nm. In another example, the substantially planar tile architectures can have a lengthof -140 nm and a width of -30 nm. In yet another example, the substantially planar tile architectures can have a length of -6 nm and a width of -30 nm.

[0010] Regardless of geometry, the single-stranded DNA scaffold of the DNA origami can be constructed of a single layer DNA film or multi-layer DNA film. In one example, the multi-layer DNA film construct the film can have a thickness of -4 nm.

[0011] In certain embodiments, a labelled DNA Origami is provided for selective imaging of KRAS-mutant pancreatic cancer cells in a desmoplastic pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME). By way of example, the labelled DNA Origami can be constructed of a plurality of oligonucleotide staples in which one or more staples is labelled with a fluorescent species. For instance, the labelled DNA origami can be labelled with a Cyanine5 (Cy5) conjugate. In examples, plurality of oligonucleotide staples comprises at least 20 staples, wherein at least 50% of the staples are labelled with a respective Cy5 conjugate.

[0012] In the same or other embodiments, DNA Origami is provided for targeted delivery of a therapeutic payload to KRAS-mutant pancreatic cancer cells in a subject. In one embodiment, the arbitrary nanostructure of the DNA Origami can be configured to have a substantially tubular architecture, in which an interior space is defined to transport the therapeutic payload. In that regard, example nanocomplexes are provided in which the DNA origami can include a therapeutic payload arranged within the interior space of the substantially tubular architecture. By way of example, the therapeutic payload can include an anti-cancer agent. The anti-cancer agent can be a therapeutic nucleic acid material. The anti-cancer agent can also be a peptide material.

[0013] In another aspect, reagents are provided for selective imaging of KRAS-mutant pancreatic cancer cells in a desmoplastic pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME), where the reagent includes a labelled DNA origami herein. Relatedly, methods of selective imaging of KRAS-mutant pancreatic cancer cells in a desmoplastic pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) are provided. Example methods can includes the steps of (1) obtaining a sample of PDAC tissue; (2) contacting the sample with a reagent described herein; (3) exposing the reagent-contacted sample to an excitation source to generate an image signal; and (4) processing the image signal toidentify a fluorescent signal associated with the fluorescent species, wherein identification of the fluorescent signal is indicative of the presence of KRAS-mutant pancreatic cancer cells in the sample. In one example, the fluorescent species of the labelled DNA origami can be a Cy5 conjugate.

[0014] In yet another aspect, pharmaceutical compositions are provided for targeted delivery of a therapeutic agent to KRAS-mutant pancreatic cancer cells in a subject. According to examples, the composition can include a therapeutically effective amount of DNA origami nanocomplex herein. By way of example, the therapeutic payload can include any anti-cancer agent. The anti-cancer agent can be a therapeutic nucleic acid material. The anti-cancer agent can also be a therapeutic peptide material. Relatedly, methods of treating a desmoplastic pancreatic ductal adenocarcinoma (PDAC) characterized by KRAS-mutant pancreatic cancer cells with composition containing a DNA origami nanocomplex herein are provided. Example methods can include the step of administering a composition containing a DNA origami nanocomplex to a patient in need of such treatment. In one such example the method can include the step of intracellular delivery of the composition, whereby the therapeutic payload is preferentially internalized by the KRAS-mutant pancreatic cells.BRIEF DESCRIPTION OF THE FIGURES

[0015] FIG. 1A is a schematic drawing of an example DNA origami functionalized with Cy5 dyes (DO-Cy5), in which tubular DNA origami is assembled from a long single-stranded scaffold and dozens of oligo staples and Cy5-modified oligonucleotides are used for fluorescence imaging. FIG. IB is an atomic force microscopy (AFM) image of a tubular DNA origami, in which tubular DNA structures were collapse onto mica surface for AFM imaging, measuring a thickness of ~4 nm, length of ~70 nm, and a width of ~50 nm (corresponding to ~30 nm diameter) (Scale bar: 100 nm.) FIG. 1C shows the average hydrodynamic size distribution of DO-Cy5s at approximately 63 nm using dynamic light scattering (DLS).

[0016] FIGS. 2A-C are schematic drawings of DNA origami nanostructures of different shapes and sizes (0.1 X tubular origami, 2X tubular origami, and tile origami) above a corresponding AFM image. The tubular origami (FIG. 2A-B) measured a thickness of ~4 nm, a width of ~ 50 nm (corresponding to ~30 nm diameter), and a variable length of ~6 nm and ~ 140nm, respectively. The tile origami (FIG. 2C) measured a thickness of ~1 nm, a width of - 100 nm, and a length of -70 nm.

[0017] FIGS. 3A-D demonstrate that DO-Cy5 conjugates accumulate in pancreatic tumor cells preferentially more than stroma cells through macropinocytosis. FIG. 3A shows fluorescence micrograph of DO-Cy5 accumulation after 24-hour exposure in 2D monolayer of pancreatic cancer cells (Pancl0.05 and MIA PaCa-2) and CAFs (CAF19 and CAF02). FIG. 3B shows accumulated intensity of Cy5 phosphor normalized by the cell counts. FIG. 3C shows DO-Cy5 accumulation in pancreatic cancer cells with and without EIP A, a macropinocytosis inhibitor. FIG. 3D shows DO-Cy5 accumulation in KRAS mutation-inducible human pancreatic duct epithelial cells (HPDE iKRAS): KRAS mutant cells (KRAS mut, treated with 25 ng / ml doxycycline for 48 hours) and KRAS wild-type cells (KRAS wt, treated with 1 : 100 dilution of DMSO in HPDE media). (Red represents DO-Cy5, and nuclei are blue. Bars indicate Mean ± S.E. (n > 3). P-values of <0.05, <0.01, <0.001, and <0.0001 are represented as *, **, ***, and ****, respectively. Scale bars indicate 100 pm.)

[0018] FIGS. 4A-D demonstrate that DO-Cy5 conjugates preferentially accumulate in pancreatic tumor cells over the CAF cells in co-cultured PCC-CAF. FIG. 4A shows fluorescence micrograph of DO-Cy5 accumulation after 24-hour exposure in co-cultured monolayer pairing pancreatic cancer cells and CAFs (Pancl0.05+CAF19, MIA PaCa-2+CAF19,Panel 0.05+CAF02, and MIA PaCa-2+CAF02). FIG. 4B shows accumulated intensity of Cy5 phosphors conjugated on DNA origami normalized by the cell counts. FIG. 4C shows DO-Cy5 accumulation with and without EIP A, a macropinocytosis inhibitor. FIG. 4D shows relative Cy5 intensity accumulated per cells (%). (Red represents DO-Cy5, nuclei are blue, and GFP- transfected CAFs are green. Bars indicate Mean ± S.E. (n > 3). P-values of <0.05, <0.01, <0.001, and <0.0001 are represented as *, **, ***, and ****, respectively. Scale bars indicate 100 pm.)

[0019] FIGS. 5A-B demonstrate the effect of size and shape of the DNA origami on the tumor-selective accumulation over CAFs. FIG. 5A and 5B show respective fluorescent micrographs and accumulated intensity with three size variants of IX, 0. IX, and 2X. FIGS. 5C and 5D show respective fluorescent micrographs and accumulated intensity of two shape variants of tubule and tile of DNA origami. (Red represents accumulated DO-Cy5, green indicates transfected CAFs, blue indicates nuclei, and magenta indicates Td-tomato labeled PCC(Panel 0.05). Bars indicate Mean ± S.E. (n > 3). P-values of <0.05, <0.01 , <0.001, and <0.0001 are represented as *, **, ***, **** respectively. Scale bars indicate 100 pm.)

[0020] FIG. 6A is a schematic drawing illustrating the production of a T-model using inkjet printing of cancer cells and CAF and allowing it to cure over 24 hours. FIG. 6B shows the time independent selective accumulation of DO-Cy5 in PCCs over CAFs in 3D tumoroid model. FIG. 6C is a magnification of the tumoroid model at 24 hours to illustrate the distinct lack of overlap between the CAF signal and the signal from the origami.

[0021] FIGS. 7A-E demonstrates quantitative evaluation for selective uptake of DO-Cy5 in in vitro PDAC MPS model. FIG. 7A shows schematic configuration of the PDAC MPS model and its’ operation. FIG. 7B shows micrograph of the drug accumulation of DO-Cy5 and doxorubicin (control for comparison59). Red represents accumulated DO-Cy5 or doxorubicin, and green indicates the GFP-transduced CAFs. FIG. 7C shows relative drug accumulation of DO-Cy5 (solid lines) and doxorubicin (dashed lines) measured for cell type-specific areas, compared with the mean intensity in capillary channels, respectively. Dots indicate Mean intensity ± S.E. (n > 3). FIG. 7D shows quantified influx (Kinflux) and efflux (Kefflux) affinity defined by cellular capability modeled by mass conservation. (Mean ± S.E. (n > 3). P-values of <0.05, <0.01, <0.001, and <0.0001 are represented as *, **, ***, and ****, respectively.) FIG. 7E shows DO-Cy5 accumulation in activated THP-1. Scale bars indicate 100 pm.

[0022] FIGS. 8A-E shows evaluation DO-Cy5 in vivo distribution in xenograft mouse model. FIG. 8A is a schematic illustration representing the PDAC stromal models inoculating PCC with CAFs with a ratio of 1 :5. FIG. 8B provides a histological stain of hematoxylin plus eosin (H&E) and immunofluorescence image of the whole tumor tissue, in which significant CAF population, indicated by GFP staining, is observed and the CAF population is also populated at the tumor core, consistent with observations in the tumoroid model. FIG. 8C shows a histological stain of hematoxylin plus eosin (H&E, left) and anti-FITC (Green) immunohistochemistry with nuclei staining (Blue). FIG. 8D provides immunohistochemistry to visualize the distribution of Cy5 phosphor within the tumors of anti-Cy5 (red) and anti-FITC (green). FIG. 8E illustrates that most of the Do-Cy signals are detected in non-CAF cancer cell regions, indicating selective uptake of Do-Cy by the PCCs. (Mean ± S.E. (n = 4). P-values of <0.05, <0.01, and <0.001 are represented as *, **, and ***, respectively.)DETAILED DESCRIPTION

[0023] Precise delivery of imaging and therapeutic agents to tumor cells in the tumor microenvironment (TME) is a major technical challenge to effective diagnosis and treatments. Especially, strong desmoplasia of pancreatic ductal adenocarcinoma (PDAC) creates complex TME posing multiple barriers of effective delivery of imaging and therapeutic agents. To address this challenge, Labelled DNA Origami are described herein for selective imaging of KRAS- mutant pancreatic cancer cells in a desmoplastic pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) and as well as DNA Origami nanocomplexes for targeted delivery of a therapeutic payload to KRAS-mutant pancreatic cancer cells in a subject.Terms and Concepts

[0024] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0025] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of Tess than x’, less than y’, and Tess than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater thanz’ . In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0026] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0027] As used herein, the term “effective amount” refers to an amount that is sufficient to achieve the desired modification of a physical property of the composition or material and / or achieving the desired level of reduction of withdrawal symptoms. The specific level in terms of wt % in a composition required as an effective amount will depend upon a variety of factors including the amount and type of formulation materials used in the disclosed compositions, amount and type of pharmaceutically acceptable excipients, and disorder being treated using the disclosed compositions.

[0028] As used herein, “treatment / treating” is an approach for obtaining beneficial or desired results including clinical results. For purposes of the present disclosure, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing or improving the quality of life, increasing weight gain, and / or prolonging survival. Also encompassed “treatment / treating” is a reduction of pathological consequence of the disease. The methods provided herein contemplate any one or more of these aspects of treatment.

[0029] As used herein, “patient” or “subject” includes human or non-human (i.e., animal) patients or subjects. In a particular embodiment, the invention encompasses both human and nonhuman. In another embodiment, the invention encompasses nonhuman. In another embodiment, the terms encompass human.

[0030] As used herein, “therapeutically effective amount” refers to an amount effective, when administered to a human or non-human patient, to provide a therapeutic benefit such as amelioration of symptoms, slowing of disease progression, or prevention of disease. The specific dose of substance administered to obtain a therapeutic benefit will, of course, be determined by the particular circumstances surrounding the case, including, for example, the specific substance administered, the route of administration, the condition being treated, and the individual being treated.

[0031] While it is possible for an active agent to be administered alone, it may be preferable to present them as pharmaceutical formulations or pharmaceutical compositions as described above. The formulations, both for veterinary / animal and for human use, of the disclosure comprise at least one of the active agents, together with one or more acceptable carriers therefor and optionally other therapeutic ingredients. The carriers must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and physiologically innocuous to the recipient thereof.

[0032] Each of the active agents can be formulated with conventional biologically active and / or inactive carriers and excipients with or without a biodegradable material, which will be selected in accord with ordinary practice. As used herein, the phrase “biologically active” refers to a characteristic of any substance that has activity in a biological system (e.g., cell culture, organism, etc.). For instance, a substance that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active and may be referred to herein as an “active agent,” or, for the treatment of pancreatic cancer, e.g., an “anti-cancer agent.”

[0033] Carriers, excipients and other additives commonly used for pharmaceutical compositions can be used to prepare pharmaceutical compositions comprising the separate active agents (or pharmaceutically acceptable salts thereof) or combination of active agents (or pharmaceutically acceptable salts thereof) for use in the method of treating a patient forpancreatic cancer. Examples of inert excipients include, but are not limited to, lactose, mannitol, glucose, hydroxypropylcellulose, microcrystalline cellulose, starch, polyvinylpyrrolidone, magnesium aluminum silicate, and the like. Other inert ingredients include, but are not limited to, lubricants, such as magnesium stearate, disintegrating agents, such as sodium carboxymethyl starch, and dissolution aids. “Carrier” is used generically herein to refer to pharmaceutically acceptable carriers, diluents, adjuvants, and excipients.

[0034] Commercially available compositions, or modifications thereof, including combinations thereof, can be used in the method. The formulation will depend, in part, on the route of administration.

[0035] Forms of administration include those suitable for oral administration, and may include forms such as tablets, pills, capsules, granules, powders, emulsions, syrups, solutions, aqueous or oily suspensions, elixirs, and other liquid preparations. Such liquid preparations can include diluents, such as water or alcohol (which can be contra-indicated in the very young), solubilizing agents, wetting agents, suspending agents, sweeteners, flavoring agents, and preservatives. If necessary or desired, tablets and pills can be coated with sugar, a gastric / enteric coating agent, and the like.

[0036] Other forms of administration include those suitable for non-oral administration of active agents, e.g., anti-cancer agents, and may include forms suitable for administration by intravenous injection or infusion, subcutaneous or intramuscular injection, suppository, transdermal implant, or inhalation. Injections for parenteral administration can include sterile aqueous or non-aqueous liquid preparations, suspensions, and emulsions. Diluent aqueous solutions can include distilled water and physiological saline. Non-aqueous diluent solutions can include propylene glycol, polyethylene glycol, vegetable oils, alcohols (which can be contraindicated in the very young), and polysorbate 80. Such compositions can further contain isotonic agents, such as preservatives, wetting agents, emulsifying agents, dispersing agents, stabilizing agents, dissolving aids, and the like. The compositions can be sterilized by filtration, addition of anti-bacterial agents, or irradiation, for example. In addition, these compositions can be made as sterile, solid compositions and dissolved or suspended in sterile water / solvent for injection prior to use. Compositions for transmucosal administration, such as inhalation and nasal absorption, can be solid, liquid, or semi-solid, and can be made in accordance with conventional methods.For example, excipients such as lactose, starch, pH adjusting agents, preservatives, surfactants, lubricants, stabilizing agents, thickening agents, and the like can be added. A suitable inhalation or insufflation device can be used. Examples of such devices include metered dose inhalers and pressurized aerosol spray canisters, which contain a suitable propellant, such as chlorofluoroalkane, hydrofluoroalkane, carbon dioxide, or the like.

[0037] The pharmaceutical composition for the active agent or anti-cancer agent can include those suitable for the foregoing administration routes. The formulations can conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations generally are found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa ). Such methods include the step of bringing into association the active agent or anti-cancer agent with the carrier which constitutes one or more accessory ingredients. In general, the formulations may be prepared by uniformly and intimately bringing into association the active agent or anti-cancer agent with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0038] Appropriate dosages can be determined in accordance with dosage range-finding techniques known in the art. FDA-approved dosages for active agents or anti-cancer agents can be used when appropriate for the patient being treated. In children, clearance is reportedly most associated with body size parameters (body surface area and weight). Age reportedly also affects clearance in very young children; it is hypothesized that this may be due to changes in drug absorption and metabolism as children mature. See, e.g., Scott et al., Ther. Drug Monitor 35(3): 332-337 (2013).

[0039] A daily dose of an active agent administered orally to adult patients can range from about 0.001 mg / kg to 100 mg / kg, whereas a daily dose of an active agent or anti-cancer agent administered intravenously can range from about 0.001 mg / kg to 10 mg / kg. The daily dose can be given in a single dose or divided into 2-4 doses. Fewer doses, such as a single dose, are possible with extended-release formulations. A daily dose of an active agent or anti-cancer agent administered to a child, a toddler, an infant, a newborn, or a pre-term baby will be substantially less than a daily dose of an active agent or anti-cancer agent administered to an adult.

[0040] The combination of pharmaceutical compositions can be formulated to be administered by the same or different routes. Any suitable route or routes (e.g., the active agents of the combination can be administered by the same or different routes) of administration can be used. In this regard, the route(s) of administration can depend, in part, on the age of the patient, such that certain routes may be preferred for pre-term babies and newborns over the routes used for administration to infants, toddlers, and young children. For example, intravenous administration may be preferred for pre-term babies and newborns, whereas injections or liquid formulations suitable for oral administration may be preferred for infants, toddlers, and young children. The combination of pharmaceutical compositions can be formulated to be administered at the same or different times. The disclosed pharmaceutical compositions can further comprise other therapeutically active compounds, which are usually applied in the treatment of the above mentioned pathological or clinical conditions.

[0041] In some embodiments, compositions described herein include one or more active agents. Exemplary agents include, but are not limited to, small molecules (e.g. cytotoxic agents), nucleic acid (e.g., siRNA, RNAi, and microRNA agents), proteins (e.g. antibodies), peptides, lipids, carbohydrates, hormones, metals, radioactive elements and compounds, drugs, vaccines, immunological agents, etc., and / or combinations thereof.

[0042] As used herein, an “optic signal,” “output signal,” “emission signal,” or “signal” refers to a detectable event such as an emission, such as light emission, for example, in an image. Thus, in some implementations, a signal may represent any detectable light emission that is captured in an image (z.c., a “spot”) from a fluorescing analyte (as a “point source”). Thus, as used herein, “signal” may refer to both an actual emission from an analyte of the specimen and may refer to a spurious emission that does not correlate to an actual analyte. Thus, a signal could arise from noise and could be later discarded as not representative of an actual analyte of a specimen. A signal consistent with the disclosure includes, for example, fluorescent, luminescent, scatter, or absorption signals. Signals can be detected in the ultraviolet (UV) range (about 200 to 390 nm), visible (VIS) range (about 391 to 770 nm), infrared (IR) range (about 0.771 to 25 microns), or other ranges of the electromagnetic spectrum. Signals may be detected in a way that excludes all or part of one or more of these ranges.

[0043] A signal may include light emissions from conjugate fluorescent species of a biological material or chemical reagent. A signal may also include transmitted light refracted and / or reflected by optical substrates. Optical signals, including excitation radiation that is incident upon the sample and light emissions that are provided by the sample, may have one or more spectral patterns. For example, more than one type of fluorescent species may be excited in an imaging session. In such cases, the different types of fluorescent species may be excited by a common excitation light source or may be excited by different excitation light sources that simultaneously provide incident light. Each type of fluorescent species may emit optical signals having a spectral pattern that is different from the spectral pattern of other labels. For example, the spectral patterns may have different emission spectra. The light emissions may be filtered to separately detect the optical signals from other emission spectra. As used herein, when the term “different” is used with respect to emission spectra, the emission spectra may have wavelength ranges that at least partially overlap so long as at least a portion of one emission spectrum does not completely overlap the other emission spectrum. Different emission spectra may have other characteristics that do not overlap, such as emission anisotropy or fluorescence lifetime. When the light emissions are filtered, the wavelength ranges of the emission spectra may be narrowed.

[0044] Various types of fluorescence microscopy may be used with assays described herein. Fluorescence microscopy may be performed using an optical detection system that includes a light source (e.g., lasers, light emitting diodes (LEDs)) tuned to wavelengths of light that induce excitation in the fluorescent species used for labelling a sample biological material or probe; one or more optical instruments, such as cameras, lenses, sensors, to capture signals emitted through induced excitation, and one or more processors for developing composite images from captured signals emitted from labelled targets within the optical elements’ field of view (tile) in a given sequencing assay. For example, embodiments may be configured to perform at least one of conventional fluorescent imaging, confocal imaging, epifluorescence imaging, total-intemal-reflectance-fluorescence (TIRF) imaging, near-infrared fluorescence (NIRF) imagin, time-delay integration (TDI) imaging (CCD-TDI or CMOS-TDI), or Super Resolution imaging, e g., Structured Illumination Microscopy (SIM). Furthermore, the imaging sessions may include line scanning one or more samples such that a linear focal region of light is scanned across the sample(s). Imaging sessions may also include moving a point focal region oflight in a raster pattern across the sample(s). Alternatively, one or more regions of the sample(s) may be illuminated at one time in a step and shoot manner.

[0045] In some embodiments, an optical detection system may include high-resolution optical components. Generally, an optical detection system may be limited by the optical resolution of the data capable of being detected by the optical components of the system. In microscopy, optical resolution is the shortest distance between two separate points in a microscope’s field of view that can still be distinguished as distinct entities, i.e., the Rayleigh limit. For example, the optical resolution of such objects may be expressed as a function of a wavelength ( ) of light in the optical sequencing system, in which shorter wavelengths yield higher resolution, and an objective, or optical element (e.g., lens or lenses) used to gather the light from the target objects, which may be measured by a numerical aperture (NA). NA of an objective lens may be given by the formula nsine 9. where n is the index of refraction of the medium in which the lens is working (nair » 1), and 0 is the half-angle of the maximum cone of light that can enter or exit the lens.

[0046] In one example, high-resolution images may be obtained through an optical detection system employing a high NA objective lens. An optical sequencing system using an objective lens having a relatively high NA is capable of resolving more closely adjacent point sources compared to a system characterized by a relatively lower NA. NA thus may determine the resolving power of an objective lens of an optical sequencing system. The higher the NA of the total system, the better the resolution. Higher quality detection lenses and other detection optical elements thus may be used to improve the optical resolution of the optical sequencing systems.

[0047] In another example, high-resolution images may be obtained through implementation of a subpixel imaging system, e.g., TDI using CCD- or CMOS-based sensors. Subpixel imaging is based on increasing a sample rate to raise the Nyquist frequency, which limits the highest frequency the optical sequencing system can reliably measure (e.g., translate digitally) to one half the sample rate at which the equipment operates. Subpixel imaging may be performed by staggering TDI sensors by a subpixel offset and subsampling a given collection area, which effectively doubles the Nyquist frequency along the offset-axis.

[0048] In yet another example, high-resolution images may be obtained through SIM or other SR techniques in connection, e.g., with optimized diffraction-limited imaging. SIM may be implemented by an optical sequencing system to take multiple images of a target object, with varying angles and phase displacements of structured illumination to generate a computational transform (Fourier transform) that is then used to reconstruct closely spaced, otherwise unresolvably high spatial frequency features, into lower frequency signals that may be sensed by an optical system without violating the Abbe diffraction limit. In that manner, captured raw images (e.g., six or nine images) of a same point source or point sources within a same tile may be assembled into a single image having an extended spatial frequency bandwidth, which may be transformed into real space to generate an image having a higher resolution than one captured by other imaging systems. Other apt SR microscopy systems include, e.g., direct stochastic optical reconstruction microscopy (dSTORM)); photo-activated localization microscopy (PALM)) and stimulated emission depletion microscopy (STED).

[0049] In certain embodiments, optical detection systems may also include in vivo imaging systems or surgical -based imaging devices, including, e.g, laparoscopic or endoscopic imaging devices.

[0050] In certain other embodiments, optical detection systems may process signals into spectral data through spectroscopy, using various techniques known in the art, including, e.g., Raman spectroscopy, including surface-enhanced Raman spectroscopy (SERS), and up- conversion spectroscopy. SERS is a spectroscopic technique that can enhance the otherwise feeble Raman scattering effect with the help of plasmonic or dielectric nanoantennae. The enhancement effect originates due to an increase in the local electric field magnitude. SERS allows for the structural fingerprinting of low-concentration analytes through the plasmon- mediated amplification of electrical fields.

[0051] Nucleic acid materials may be referred to herein as “nucleic acids,” “nucleic acid molecules,” “nucleic acid materials,” “nucleic acid sequences,” “polynucleotides,” or “oligonucleotides,” and can comprise a polymeric form of nucleotides of any length, can comprise DNA and / or RNA, and can be single-stranded, double- stranded, or multiple stranded. One strand of a nucleic acid also refers to its complement. Nucleic acid materials, whether as origami scaffolding or therapeutic nucleic acid materials, may be gDNA, including DNAvariants (e.g., alleles, polymorphs, missense), mtDNA, mRNA, cDNA transcribed from mRNA, non-coding RNA, and small RNA. Nucleic acid materials herein may also include polynucleotide analogues, amplicons, conjugates, and substitutions, crosslinked polynucleotides, polynucleotide complexes, and non-natural polynucleotides, including, but not limited to, dideoxynucleotides, or biotinylated, aminated, deaminated, alkylated, benzylated, flourophor- labeled polynucleotides.

[0052] Nucleic acids in certain implementations may include, for instance, linear polymers of deoxyribonucleotides in 3 '-5' phosphodiester or other linkages, such as DNA, for example, single- and double-stranded DNA, genomic DNA, copy DNA or complementary DNA (cDNA), recombinant DNA, or any form of synthetic or modified DNA. In other implementations, nucleic acids include for instance, linear polymers of ribonucleotides in 3'-5' phosphodiester or other linkages such as ribonucleic acids (RNA), for example, single- and double-stranded RNA, messenger (mRNA), copy RNA or complementary RNA (cRNA), alternatively spliced mRNA, ribosomal RNA, small nucleolar RNA (snoRNA), microRNAs (miRNA), small interfering RNAs (sRNA), piwi RNAs (piRNA), or any form of synthetic or modified RNA. Nucleic acids used in the compositions and methods of the present invention may vary in length and may be intact or full-length molecules or fragments or smaller parts of larger nucleic acid molecules. In particular implementations, a nucleic acid may have one or more detectable labels, as described elsewhere herein.

[0053] In one aspect, a DNA Origami is provided for selective targeting of KRAS- mutant Pancreatic Cancer Cells via micropinocytosis. By way of example, the DNA origami herein may be constructed of a single stranded DNA scaffold organized into an arbitrary nanostructure via a plurality of oligonucleotide staples. The arbitrary nanostructure can be configured into a variety of two-dimensional or three-dimensional geometries. Geometries consistent with the present disclosure include, e.g., tubules, rings, tiles, squares, triangles, spheres, and the like.

[0054] In one embodiment, the arbitrary nanostructure can be configured to have a substantially tubular architecture. The substantially tubular architecture can be defined by a length and a diameter. For instance, the substantially tubular architectures of certain examples can have a length between -6 nm and -150 nm, -10 nm and -140 nm, -15 nm and -145 nm,~20 nm and —150 nm, -25 nm and —145 nm, -30 nm and -140 nm, ~35 nm and —135 nm, -40 nm and —130 nm, ~45 nm and —125 nm, ~50 nm and ~120 nm, -55 nm and —115 nm, ~60 nm and -110 nm, -65 nm and -105 nm, -70 nm and -100 nm, -75 nm and -95 nm, or -80 nm and -90 nm. The substantially tubular architectures of the same or certain other examples can have a diameter between -10 nm and -50 nm, -15 nm and -45 nm, -20 nm and -40 nm, or -25 nm and -35 nm. In one such example, the substantially tubular architectures can have a length -70 nm and diameter of -30. In another example, the substantially tubular architectures can have a length of -140 nm and a diameter of -30 nm. In yet another example, the substantially tubular architectures can have a length of -6 nm and a diameter of -30 nm.

[0055] In another embodiment, the arbitrary nanostructure of the DNA origami can have a substantially planar tile architecture, in which the substantially planar tile architecture can be defined by a length and a width. For instance, the substantially tubular architectures of certain examples can have a length between -6 nm and -150 nm, -10 nm and -140 nm, -15 nm and -145 nm, -20 nm and -150 nm, -25 nm and -145 nm, -30 nm and -140 nm, -35 nm and -135 nm, -40 nm and -130 nm, -45 nm and -125 nm, -50 nm and -120 nm, -55 nm and -115 nm, -60 nm and -110 nm, -65 nm and -105 nm, -70 nm and -100 nm, -75 nm and -95 nm, or -80 nm and -90 nm. The substantially tubular architectures of the same or certain other examples may have a width between -10 nm and -50 nm, -15 nm and -45 nm, -20 nm and -40 nm, or -25 nm and -35 nm. In one such example, the substantially tubular architectures can have a length -70 nm and diameter of -30. In another example, the substantially tubular architectures may have a length of -140 nm and a diameter of -30 nm. In yet another example, the substantially tubular architectures can have a length of -6 nm and a diameter of -30 nm.

[0056] Regardless of geometry, the single-stranded DNA scaffold of the DNA origami may be constructed of a single layer DNA fdm or multi-layer DNA fdm. The multi-layer DNA film may have 2 or more layers, between 2 and 20 layers, between 3 and 15 layers, between 4 and 12 layers, between 5 and 10 layers, or between 6 and 8 layers. The multi-layer DNA film may be defined by thickness. In examples, the thickness may be between -3 nm and -12 nm, -4 nm and -10 nm, -5 nm and -8 nm, -6 nm, or -7 nm. In one example of a multi-layer DNA film construct the film can have a thickness of -4 nm.

[0057] In certain embodiments, a labelled DNA Origami is provided for selective imaging of KRAS-mutant pancreatic cancer cells in a desmoplastic pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME). By way of example, the labelled DNA Origami can be constructed of a plurality of oligonucleotide staples in which one or more staples is labelled with a fluorescent species A fluorescent species consistent with the present disclosure may beany target analyte, analyte conjugate, or other moiety that can be detected based on an optical field response to excitation. Fluorescent species may include a target analyte with inherent fluorescence (e.g., peptide tracers) or detectible label moieties. Exemplary labels for use consistent with various embodiments, for example, include chromophores; luminophores; fluorophores; optically encoded nanoparticles; particles encoded with a diffraction-grating; electrochemiluminescent labels such as Ru(bpy)32+ / thioacetamide; or other species capable of detection based on an optical characteristic. In general, fluorophores exist that fluoresce green (emission wavelength -500 nm), orange (emission at 550-570 nm), red (emission at 570-620 nm), and far-red (emission at over 620 nm). Fluorophores that may be useful include, for example, fluorescent lanthanide complexes, including those of Europium and Terbium, fluorescein, rhodamine, tetramethylrhoda- mine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, Cy3, Cy5, stilbene, Lucifer Yellow, Cascade Blue™, methylene blue, Texas Red, alexa dyes, phycoerythin, bodipy, indocyanine green, and others known in the art. In the Examples set forth herein, the labelled NDA origami may be labelled with a Cyanine5 (Cy5) conjugate, or one or more of Cy2, Cy3, Cy3.5, Cy5.5, or Cy7 conjugates. The fluorescent species may be conjugated to the DNA scaffold, the oligonucleotide staples, or both.

[0058] In examples, the plurality of oligonucleotide staples may comprise at least 10 staples, wherein at least 50% of the staples are labelled with a respective Cy5 conjugate or other fluorescent species, at least 20 staples, wherein at least 50% of the staples are labelled with respective Cy5 conjugate or other fluorescent species, at least 30 staples, wherein at least 50% of the staples are labelled with respective Cy5 conjugate or other fluorescent species, at least 40 staples, wherein at least 50% of the staples are labelled with respective Cy5 conjugate or other fluorescent species, at least 50 staples, wherein at least 30% of the staples are labelled with respective Cy5 conjugate or other fluorescent species, at least 50 staples. In one example, the plurality of oligonucleotide staples may comprise 30 staples, each of which is labelled with respective Cy5 conjugate or other fluorescent species. According to examples in which the fluorescentspecies is conjugated to the NDA scaffold, labelled DMA origami may have between 10 and 100 Cy5 conjugates or other fluorescent species, 20 and 90 Cy5 conjugates or other fluorescent species, 30 and 80 Cy5 conjugates or other fluorescent species, or 40 and 60 Cy5 conjugates or other fluorescent species.

[0059] In the same or other embodiments, DNA Origami is provided for targeted delivery of a therapeutic payload to KRAS-mutant pancreatic cancer cells in a subject. In one embodiment, the arbitrary nanostructure of the DNA Origami can be configured to have a substantially tubular architecture, in which an interior space is defined to transport the therapeutic payload. In that regard, example nanocomplexes are provided in which the DNA origami can include a therapeutic payload arranged within the interior space of the substantially tubular architecture. By way of example, the therapeutic payload can include an anti-cancer agent. Anti-cancer agents consistent for use with the invention may include, e.g., cell death inducers, antitumor immunotherapeutic agents, cancer vaccines. The anti-cancer agent can be a therapeutic nucleic acid material. The therapeutic nucleic acid material according to examples may be a therapeutic DNA, which can include one or more of pDNA, mcDNA, linear DNA amplicons, ASOs, CpG oligodeoxynucleotides, DNA enzymes, and DNA methyltransferase inhibitors. The therapeutic nucleic acid material can also be a therapeutic RNA, which can include one or more of mRNA, siRNA, shRNA, miRNA, saRNA, piRNA, eRNA, IncRNA, circRNA, a ribozyme, an antagonist RNA aptamer, and a cell type-specific RNA aptamer. The therapeutic nucleic acid material can also be a CRSPR-Cas9 vector.

[0060] In examples, the DNA Origami for targeted delivery of a therapeutic cargo further includes a fluorescent species, e.g., a Cy5 conjugate, for tracking delivery of the therapeutic cargo to target cancer cells.

[0061] The anti-cancer agent can also be a peptide material and / or protein. Therapeutic peptide materials and / or proteins may include, e.g., peptide-targeted cytotoxic peptides, peptide- drug conjugates, peptide receptor radionuclides, peptide inhibitors, e.g., immune checkpoint inhibitors, and cell surface protein antagonists, e.g., antagonists of hormone receptors, tyrosine kinases, kinase-associated receptors, epidermal growth factor, toll-like receptors.

[0062] In another aspect, reagents are provided for selective imaging of KRAS-mutant pancreatic cancer cells in a desmoplastic pancreatic ductal adenocarcinoma (PDAC) tumormicroenvironment (TME), where the reagent includes a labelled DNA origami herein. Relatedly, methods of selective imaging of KRAS-mutant pancreatic cancer cells in a desmoplastic pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) are provided. Example methods can includes the steps of (1) obtaining a sample of PDAC tissue; (2) contacting the sample with a reagent herein; (3) exposing the reagent-contacted sample to an excitation source to generate an image signal; and (4) processing the image signal to identify a fluorescent signal associated with the fluorescent species, wherein identification of the fluorescent signal is indicative of the presence of KRAS-mutant pancreatic cancer cells in the sample. In one example, the fluorescent species of the labelled DNA origami can be a Cy5 conjugate. In other examples, imaging may be performed on a subject in vivo, e.g., flow-based tumor identification.

[0063] In yet another aspect, pharmaceutical compositions are provided for targeted delivery of a therapeutic agent to KRAS-mutant pancreatic cancer cells in a subject. According to examples, the composition can include a therapeutically effective amount of DNA origami nanocomplex herein. By way of example, the therapeutic payload can include any anti-cancer agent. The anti-cancer agent can be a therapeutic nucleic acid material. The anti-cancer agent can also be a therapeutic peptide material. Relatedly, methods of treating a desmoplastic pancreatic ductal adenocarcinoma (PDAC) characterized by KRAS-mutant pancreatic cancer cells with composition containing a DNA origami nanocomplex herein are provided. Example methods can include the step of administering a composition containing a DNA origami nanocomplex to a patient in need of such treatment. In one such example the method can include the step of intracellular delivery of the composition, whereby the therapeutic payload is preferentially internalized by the KRAS-mutant pancreatic cells.

[0064] In the following Examples, DNA origami-Cy5 (DO-Cy5) nanocomplexes were developed to selectively imaging of KRAS-mutant PCCs in the desmoplastic PDAC TME. This DO-Cy5 nanocomplex was designed based on a rationale that KRAS-mutant PCCs show elevated macropinocytosis than other stromal cells in the TME. Since DNA nanostructures are internalized by macropinocytosis, DO-Cy5 nanocomplex is believed to be internalized preferentially to KRAS-mutant tumor cells over other stroma cells. To that end, the tumor- selective uptake of DNA origami in the PDAC TME was investigated by using both in vitro and in vivo PDAC tumor models developed for mimicking the PDAC stromal microenvironment.The data and results presented herein establish a new understanding of the transport mechanisms of DNA origami having tumor specificity within this complex PDAC TME, characterized by a high degree of heterogeneity and the presence of large populations of CAFs. The results provide insights into precision delivery of therapeutic and imaging agents to KRAS-mutant cancers.EXAMPLESNucleic Acid Constructs

[0065] Nucleic acid constructs synthesized for use in connection with the following examples included Cy5 modified sequences of Table 1; 70 nm Tube Origami Staples of Table 2; Ring Origami of Table 3; Tube 2 for 140 nm tube origami of Table 4; and linkers between tube 1 and 2 of Table 5.Cells and Reagents

[0066] Human pancreatic cancer cells (Pane 10.05, MIA PaCa-2, Panel) and cancer- associated fibroblasts (CAF19 and CAF02) were maintained in Advanced Dulbecco’s Modified Eagle’s Medium / Ham’s F-12 (DMEM / F12, Invitrogen, NY, USA) with 2.05mM L-glutamine (GE Healthcare Bio-Sciences Corp., MA, USA) supplemented by 5% v / v fetal bovine serum (FBS) and 100 pg ml-1 penicillin / streptomycin (P / S). The cells were regularly harvested by 0.05% trypsin and 0.53mM EDTA (Life Technologies, CA, USA) when grown to ~80% confluency in 75 cm2 T-flasks and incubated at 37°C with 5% CO2. Harvested cells were used for experiments by culturing on 2D and iT-MOC, or sub-cultured while maintaining them below 15th passage.67

[0067] The KRAS-inducible human pancreatic ductal epithelial cells (HPDE iKRAS, provided by Dr. Brittney Allen-Peterson at Purdue University) cell line was genetically modified to allow for the KRAS G12D mutation to be induced by the presence of doxycycline.68The modification and characterization of the cell line are described in Tsang et al .48HPDE iKRAS cells were maintained in Keratinocyte-Serum Free medium (Invitrogen, MA, USA) supplemented by Bovine Pituitary Extract (0.05mg / ml), recombinant human epidermal growth factor (5ng / ml) and L-Glutamine.

[0068] For KRAS induction, HPDE iKRAS cells were treated with 25ng / ml doxycycline for 48 hours (HPDE KRASmut), whereas the HPDE iKRAS cells were treated with acorresponding DMSO control media for HPDE KRASwtin the normal culture conditions. The KRAS induction was processed after the cells were harvested and seeded in the experimental platforms.Cellular Uptake Assay

[0069] The cells were seeded on 96 well plates at the density of 2000 cells / well and then pre-cultured in normal media for at least 48-hours before performing the cellular uptake assay. For the inhibitor treatment experiment, cells were pre-incubated with 5-(N-Ethyl-N- isopropyl)amiloride (EIP A, 50 pM) for 1.5 hours before exposing DO-Cy5. The control groups were pre-incubated with DMSO. Afterward, the cells were exposed to DO-Cy5 for 24 hours. At the 24-hour time point, the nuclei were stained with Hoechst 33342 (Thermo Fisher, Waltham, MA). Fluorescence images were obtained at various time points between 0 and 24 hours using an Olympus 1X71 inverted microscope.THP-1 Activation

[0070] THP-1 cells were kept in culture in complete RPMI media supplemented with 10% FBS, 1% MEM non-essential amino acids (Thermo Fisher Scientific, MA, USA), 1% sodium pyruvate, 1% HEPES (Life Technologies, CA, USA), and 1% P / S. Cells were seeded in 96 wells at 5000 cells / well density and treated with 10 ng / mL phorbol 12-myristate 13-acetate (PMA, Sigma Aldrich, MO, USA) for 24 hours. Subsequently, wells were washed with complete RPMI media and cellular uptake assay was performed as previously explained.3D Tumoroids Model

[0071] The tumoroid model comprised of Pane 10.05 and CAF19 was developed through inkjet-printed, cell-laden bioinks.51Briefly, prepared cancer cell ink and CAF ink are printed and cured using the inkjet printing setup. Initially, five drops of cancer cell-laden ink were deposited onto a glass well-plate, creating a line array, and then cured for 1 minute at 37°C for IPI gelation. Subsequently, five drops of CAF-laden ink were deposited adjacent to the first line and similarly cured for 1 minute at 37°C to gel the entire structure. Following this, cell medium was added, and the polymer matrix underwent compaction due to contractile forces generated by the cells. Ultimately, a 3D tumor-stroma model, known as a tumoroid, was formed as the matrix compacted.3D MPS Model

[0072] The uptake of the DO-Cy5 was assessed in PCC-CAF co-cultured T-MOC model to investigate differential response in drug accumulation between PCC and CAF cells. The cocultured T-MOC model was a microfluidic platform to demonstrate dynamic transport.54Briefly, the T-MOC model is composed of capillary, interstitial, and lymphatic channels. The DO-Cy5 is accumulated in the cell body through cellular-uptake while the compound transports from capillary to interstitial, then lymphatic channels. The drug transport is governed by the perfusion flow condition of the T-MOC which is controlled by differences in hydrostatic pressure of the capillary, interstitial, and lymphatic channels.52, 54In the present study, a hydrostatic pressure difference of 20mmH2O was used to mimic the average interstitial flow rates typically observed in TME.69The accumulation was measured using live-cell imaging technique with time-lapse microscopy for fluorescent DO-Cy5 conjugates / Doxorubicin. An inverted microscope (Olympus 1X71, Japan) was equipped with a stage top incubator, which allowed maintaining the microfluidic platform at 37°C with 5% CO2 environment during imaging.70Drug medium (30nM of DO-Cy5) was introduced into the capillary channel, Cy5 fluorescence in the T-MOC platform was captured every 2 hours for 24 hour-duration.

[0073] Temporal drug accumulation in the cells was measured by fluorescence intensity at each cell type where the intensity was calibrated with fluorescence of 30nM of the corresponding compound. The accumulation was measured for each cell type separately. Since the CAF cell lines are transduced with GFP, CAF cell area was defined with the FITC- fluorescence. The control experiment with doxorubicin also followed the above procedure to compare the differential drug accumulation. All experiments were repeated at least 3 times for each treatment group. The data was reported in the form of mean ± standard deviation.Xenograft Model

[0074] MIA PaCa-2 and CAF 19 cells were cultured for subcutaneous model development of xenograft mouse. For subcutaneous injections, cells were prepared in 1 :5 PCC:CAF ratio (1 x 106: 5 x 106cells per mice) were prepared in 100 pL sterile PBS and mixed with 100 pL Matrigel for implantation into NRG mice. After 3 weeks, DNA origami was delivered via tail vein injection 200 pl / mice. After 24 hours of origami injection, tumor, liver,and spleen were harvested and formalin fixed at the end of the experiment for further histology analysis.Histology

[0075] For paraffin sections, tissues were fixed in 10% formalin. Subsequently, they were subjected to dehydration using graded ethanols, followed by clearing in xylene and infiltration with Leica Paraplast Plus paraffin using a Sakura Tissue-Tek VIP6 tissue processor. Following processing, the tissues were embedded in Leica Paraplast Plus paraffin. H&E staining was performed using the Leica Autostainer XL, the slides are stained using Gill’s II hematoxylin, then blued and counterstained with a mixture of eosin and phloxine B. Subsequently, they undergo dehydration, clearing in xylene, and are finally cover-slipped using a toluene-based mounting medium (Leica MM24).

[0076] For immunofluorescence, slides were incubated in 2.5% normal horse serum for 20 minutes. Then, primary antibodies applied accordingly. Anti-GFP was applied at 1 : 100 and anti-Cy5 at 1: 100 for 1 hour. The negative control slide was stained with Rabbit IgG (Vector Labs, 1-1000) at a concentration of lug / mL for 1 hour. Slides were rinsed twice in TBST and anti-goat Alexa 555 secondary (Invitrogen, A21432) applied at 4ug / mL for 30 minutes. An antirabbit dylight 488 (Vector Labs, DI-1488) was then applied at 6ug / mL for 30 minutes. Slides were rinsed in TBST and counterstained with DAPI (Invitrogen, EN62248) at lug / mL for 10 minutes before rinsing and coverslipping with Prolong Gold (Invitrogen, P36934). All images were taken using a Leica Versa8 whole-slide scanner.Statistical Analysis

[0077] All experiments were repeated at least 3 times for each group. The data was reported in the form of mean ± standard estimated error (S .E.) or mean ± standard deviation (S.D.), which is indicated respectively. Data points were statistically analyzed by using a oneway analysis of variance (ANOVA) or student t-test. The statistical significance was represented by p-value (p<0.05).Example 1 - Synthesis and Characterization of DNA origami-CY5 dye (DO-Cy5) Conjugates

[0078] With reference to Fig. 1A, Tube-shaped DNA origami were designed as a nanocarrier for cellular uptake. The template DNA scaffold and ss-staples assembled into in-silico-designed structures via self-assembly under thermal annealing. Thirty staples were modified with an extension for binding with oligos with Cy5 fluorophores, thus ensuring 30 dyes per origami. DNA assemblies were purified with filtration to remove excessive ssDNA. The structural details and assembly conditions can also be found elsewhere (Chen et al., Nanotechnology. 24: 43 (2013)). The designed origami has a length of approximately 70 nm and a diameter of -30 nm. The assembled DNA tubes were characterized by atomic force microscopy (AFM) and dynamic light scattering (DLS), as shown in Figs. 1B-C. AFM measures a length of -70 nm, a width of -50 nm, and a thickness of -4 nm. These dimensions are expected given that the origami collapses onto mica surface, with the width corresponding to ~30 nm diameter. Hydrodynamic size was measured as ~63 nm, consistent with the present design and AFM. With reference to Fig. 2, two other types of 30-nm-diameter DNA tubes were also constructed, with variable lengths: twice long (-140 nm; termed 2X) and about one tens of the length (-6 nm; termed 0.1X). A tile-shaped DNA origami was also designed with a length of -70 nm, a width of -100 nm, and a thickness of -4 nm.Example 2 - The DO-Cy5 Conjugate is Accumulated in Pancreatic Cancer Cells Significantly More than Cancer Associated Fibroblasts through Macropinocytosis

[0079] To evaluate the accumulation pattern of DO-Cy5 in pancreatic tumor tissue, the accumulation patterns of the DO-Cy5 conjugate in vitro was investigated using various cell lines, including pancreatic cancer cells and patient-driven cancer-associated fibroblasts (CAFs). The pancreatic cancer cell panel includes Pancl0.05, MIA PaCa-2 (and supp. Panel), and CAF cells include CAF 19 and CAF02. With reference to Figs. 3A and 3B, the uptake of the DO-Cy5 was captured with the fluorescence intensity of Cy5 accumulated on mono-cultured cells after 24 hours. In the results, it was observed that the DNA origami-Cy5 conjugate was accumulated in the cancer cells significantly more than in CAF cells.

[0080] The majority of pancreatic cancer patients hold oncogenic KRAS mutations which lead to activation of macropinocytosis in cancer.46, 47To test the effect of KRAS mutation on DO-Cy5 uptake for cancer cells, an accumulation assay was performed on the genetically engineered cell lines, KRAS mutation-inducible human pancreatic duct epithelial cells (HPDE iKRAS). HPDE iKRAS cells were developed to induce the KRAS mutation (KRAS G12D) in accordance with doxycycline treatment.48KRAS mutant HPDE cells (KRAS mut) were derivedfrom 25ng / ml doxycycline treatment whereas the control group with normal media is considered as KRAS wild type (KRAS wt). Consequently, as shown in Fig. 3C, it was observed that the DO-Cy5 accumulated in KRAS mut cells significantly more than KRAS wt cells, which supports that the KRAS mutation contributes to the uptake of the DO-Cy5 activating macropinocy tosi s .

[0081] To elucidate the mechanism of tumor-preferential accumulation of DNA origami- Cy5, it has been hypothesized that macropinocytosis derived by oncogenic KRAS transformation in pancreatic cancer plays a key role in tumor-selective uptake of DNA origami- Cy5. That hypothesis is evaluated by inhibiting the macropinocytosis of the cancer cells, Pane 10.05 and MIA PaCa-2 using pharmaceutical macropinocytosis inhibition of 5-[N-ethyl-N- isopropyl] amiloride (EIP A).49As shown in Fig. 3D, DO-Cy5 accumulation in cancer cells is highly suppressed in EIPA-treated groups in both cancer cells, supporting the role of macropinocytosis in the selective uptake of the DO-Cy5 conjugate by cancer cells.

[0082] Furthermore, the tumor-preferential accumulation of the DO-Cy5 was evaluated using the 2D co-culture models of the PCCs and CAFs. With reference to Figs. 4A-D, consistent with findings from the monoculture assays, a significantly higher accumulation of DO-Cy5 in the cancer cells compared to the CAF cells. Specifically, Cy5 signals exhibited notably elevated expression in Pancl0.05 cells within both Pancl0.05+CAF19 and Pancl0.05+CAF02 pairs. This trend was consistently observed across other cancer cell types, providing further evidence of the tumor-selective accumulation patterns of the DO-Cy5 conjugate.

[0083] The macropinocytosis-induced tumor-selective accumulation of DO-Cy5 is additionally investigated in a co-culture pair of Pancl0.05 and CAF 19, as particularly demonstrated in Fig. 4C. The intensity of Cy5 is significantly reduced in the EIPA treated group. To confirm the reduction from the inhibition of EIPA, not from the metabolic activity due to the viability changes, the cell viability of Pancl0.05 and CAF19 was investigated, showing that no significant changes in viability after EIPA treatment. The results indicate that the reduction of DO-Cy5 accumulation is less associated with metabolic activities, supporting the hypothesis that macropinocytosis is a contributor to tumor-selective uptake for DO-Cy5.Example 3 - The Effect of the Size and Shape of DNA Origami in Turn or- Selective Delivery Over the CAFs.

[0084] DNA origami presents promising opportunities for engineering the size and shape of particles to enhance targeted delivery efficiency. Numerous studies have highlighted the crucial role of particle size in triggering endocytic processes, emphasizing the importance of optimizing size parameters for effective delivery systems.14To evaluate the impact of DO-Cy5 size on activating macropinocytosis, an optimal size range was sought. Thus the length of tubular DO-Cy5 was varied, while keeping the diameter constant. The tubular origami tested were (1) approximately 70 nm in length and 30 nm in diameter (IX size), (2) -140 nm in length and 30 nm in diameter (2X size), and (3) -6 nm in length and 30 nm in diameter (0.1X size). These nanostructures were exposed to a co-cultured PCC and CAFs of Pancl0.05-CAF19 pair, and accumulation patterns were transiently monitored.

[0085] With reference to Figs. 5A-D, the results demonstrate that all sizes of DO-Cy5 (IX, 0.1X, and 2X) exhibit significantly higher accumulation in cancer cells compared to CAFs. Notably, size-dependency in the accumulation of DO-Cy5 in pancreatic cancer cells was observed. DO-Cy5 at IX and the smaller variant at 0. IX exhibit similar accumulation patterns. In contrast, the larger 2X variant shows significantly lower accumulation, indicating a sizedependent variation in the cellular uptake efficiency of the cancer cells. This observation implies the importance of optimizing DNA origami size to maximize targeted delivery efficacy.

[0086] Furthermore, the impact of altering the shape of DNA origami structures in tumor-specificity was explored. Various geometries, such as tubules, tiles, and triangular DNA origami structures, have been developed as potential candidates for enhancing targeted delivery efficiency and specificity.28, 29,° In this investigation, a specific focus was put on comparing two representative shapes with similar sizes - tubules (IX) and tiles (100 nm x 70 nm), to recognize potential differences in their transport patterns towards tumor-specific accumulation in the tumor tissue. The results reveal similar trends in the delivery of DO-Cy5 regardless of the shape change. These findings suggest that the tumor specificity of DO-Cy5 delivery is more closely associated with the size of DNA origami structures rather than their shape, particularly in the context of comparing cancer cells and CAFs.Example 4 - D0-Cy5 Conjugate is Preferentially Accumulated in Tumor Cell Populations in 3D Tumoroid Models.

[0087] The tumor preferential accumulation of DO-Cy5 was examined using 3D cell tumoroids models.51The 3D tumoroids were developed to comprise of pancreatic tumor cells and stroma components including CAFs. Specifically, the tumoroids model featured a mechanically dynamic tumor-stroma interface at elevated cell density, achieved through the remodeling of a cell-laden polymer matrix mediated by cellular contractile forces. As was observed from the 2D in vitro models as referenced in Figs. 6A-C, DO-Cy5 was selectively accumulated within the pancreatic cancers than CAFs. However, the tumoroids may not fully capture the dynamic transport schemes of DO-Cy5 due to the absence of the fluidic culture conditions.Example 5 - Quantitative Assessment of the Tumo-Targeting Efficacy of the DO-Cy5 Conjugate Utilizing a Microfluidic-based Tumor Microenvironment-on-chip Model

[0088] With reference to Fig. 7A, a microfluidic tumor microenvironment-on-a-chip model (T-MOC) was developed expand the investigation into the quantitative approaches for the transport and accumulation of DO-Cy5 in targeting cancer cells.52The biomimetic model of PDAC stroma comprises of pancreatic cancer cells and CAFs embedded in dense type I collagen matrix. Moreover, as illustrated in Fig. 7B, the T-MOC system recapitulates pharmacokinetic processes generating fluid flow perfused across an endothelium mimicking membrane interfaced with a capillary channel, mimicking extravasation from capillary vessels, interstitial diffusion and convection, cellular uptake, and lymphatic drainage. In the T-MOC model, DO-Cy5 contained solutions along the capillary channel were perfused, monitoring transient drug accumulation in the cells using time-lapse microscopy over a 24-hour period.52'54Consequently, the fluorescence images displayed in Fig. 7C show significant accumulation of DO-Cy5 (in red) within cancer cells (Pancl0.05), with minimal fluorescence intensity detected within the regions occupied by green fluorescent CAFs (CAF19). In contrast, doxorubicin drugs, characterized by autofluorescence in red, were observed to accumulate regardless of the cell types. The drug accumulation was further quantified with respect to the cell types, as shown in Fig. 7D. The transient drug accumulation of DO-Cy5 notably enhanced within Pancl0.05 compared with CAF19, allowing us to quantitatively measure the tumor-targeting performance of DO-Cy5.

[0089] Furthermore, an additional analysis was conducted to estimate intracellular transport parameters specific to each cell type, employing a transient drug accumulation model based on mass conservation principles.55In this model, the cell surface's drug influx affinity (kinfiux) reflects the cell's capability to uptake the drug, while the drug efflux affinity (kefflux) indicates the cell's capacity for drug outflow, kinfiux and kefflux were assumed to be constant values representing the cellular capability for intracellular transport of the respective drugs. The results reproduced in Fig. 7E demonstrate a significantly higher konfor PCC cells compared to CAF cells for DO-Cy5. Additionally, although the konfor doxorubicin in Pancl0.05 cells surpassed that of CAF 19 cells, the difference was less distinct compared to DO-Cy5, suggesting a potential link between the selective uptake of these conjugates by cancer cells and cancer cellspecific endocytic activity for DO-Cy5, as hypothesized, such as macropinocytosis.

[0090] Given the inherent limitations of both in vitro and in vivo xenograft models, characterized by a deficient immune system, the accumulation patterns of DO-Cy5 using THP-1 immune cell model was further elucidated. THP-1 monocytes and their phorbol ester (PMA) activated macrophages are reported to demonstrate elevated phagocytosis activity of various pathogens and nanoparticles and possess plasticity to be further differentiated to diverse tumor associated macrophage phenotypes.56’58This approach allowed for an estimate of the accumulation in macrophages, as the immune-deficient xenograft model does not accurately depict immune cell uptake of DO-Cy5. These observations indicate that DO-Cy5 is not significantly captured by macrophages.Example 6 - Evaluation of DO-Cy5 Distribution Under Xenograft Mouse Model

[0091] To validate the in vitro findings and explore the distribution of DO-Cy5 in more physiologically relevant conditions, an in vivo experiment using a xenograft mouse model was conducted. The mouse model was developed to feature human pancreatic tumor tissue, incorporating both pancreatic cancer cells and CAF cells to mimic the complex tumor microenvironment. To differentiate between these cell types, transfected CAF cells expressing GFP were utilized within the cytoplasm. Prior to administering DO-Cy5 into the mouse model, tumor growth post-inoculation was carefully studied to establish the optimal proportion of cancer cells and CAF cells. Subsequently, the optimal time point for tail vein injection of DO-Cy5 was determined, which was conducted on the 28th day of the experiment, coinciding with the presence of sufficient populations of CAFs as confirmed by GFP antibody staining.

[0092] Following injection, DO-Cy5 was allowed to circulate for 24 hours before harvesting tumor tissues to capture the accumulation of DO-Cy5 within the tumors. The 24-hour time point was chosen to minimize potential interference from high concentrations of DO-Cy5 in the bloodstream, thus ensuring that fluorescence detected was primarily due to uptake by cells. Then, the harvested tumor tissues underwent further analysis via histological sections to elucidate the distribution of DO-Cy5 within the tumor microenvironment. With reference to Figs. 8A-E, as consistently observed in in vitro models, it was found that DO-Cy5 accumulated in PCCs rather than CAFs. This trend mirrors previous findings and reinforces the notion that DO-Cy5 exhibits a selective affinity for cancer cells over stromal cells within the tumor microenvironment.

[0093] In the present disclosure, the cancer-targeting drug delivery of DO-Cy5 was systematically evaluated using various in vitro and in vivo tumor-stroma models with the inclusion of CAFs to evaluate tumor-selective accumulation of DO-Cy5. Despite their dominance in tumor tissues and significant interaction with tumors, prior studies targeting tumors have largely overlooked the role of CAFs. The systematic analysis of DNA origami transport patterns, considering the interactions between cancer cells and CAFs, was conducted across conventional in vitro, MPS, and in vivo models. This comprehensive approach sheds light on the intricate dynamics of tumor-selective accumulation and offers valuable insights for future research and therapeutic strategies.

[0094] The affinity of cancer cells for DO-Cy5 can be quantitatively assessed using microphysiological systems. The findings from MPS-based quantitative analysis support the hypothesis of macropinocytosis. Specifically, a significantly higher uptake affinity compared to efflux affinity has been observed, indicating an upregulation in intake consistent with macropinocytosis. This disclosure highlights the significant potential of leveraging humanmimicking in vitro systems, especially considering recent regulatory shifts. This potential is validated by the findings in in vivo conditions.

[0095] Importantly, the investigation of the uptake mechanism revealed the involvement of macropinocytosis of tumor selectivity.44 60This is particularly significant given the highcorrelation between KRAS mutation in pancreatic cancer cells and macropinocytosis, presenting new avenues for exploration.61'65Macropinocytosis, known for its non-selective uptake of adjacent materials, holds considerable promise for targeting cancer.47’62> 65The KRAS mutation causes various pathological activities of the cancer cells such as remodeling the metabolism.66In adjusting to the harsh condition to survive in tumor microenvironment, macropinocytosis is upregulated through the KRAS mutation. Interestingly, recent studies have shown the macropinocytosis-involved tumor selective uptake in targeting cancer. In this context, the systematic analysis elucidating tumor selectivity within PDAC tumor microenvironment enhances the potential of DNA origami for therapeutic applications.Table 1Cy5-Modified SequencesTable 270 nm Tube Origami Staples (Sequences with underline are used to bind Cy5 fluorophores)Table 3 Ring Origami (Sequences with red color are used to bind Cy5 fluorophores)Table 4 Tube 2 for 140 nm tube origamiTable 5Linkers between tube 1 and 2REFERENCES

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Claims

WHAT IS CLAIMED IS:

1. A DNA Origami for selective targeting of KRAS-mutant Pancreatic Cancer Cells via macropinocytosi s .

2. The DNA origami of claim 1, wherein the DNA origami comprises a single stranded DNA scaffold organized into an arbitrary nanostructure via a plurality of oligonucleotide staples.

3. The DNA origami of claim 2, wherein the arbitrary nanostructure has a substantially tubular architecture.

4. The DNA origami of claim 3, wherein the substantially tubular nanostructure is defined by a length and a diameter.

5. The DNA origami of claim 4, wherein the length is between -6 nm and —150 nm and the diameter is between -20 and ~50 nm.

6. The DNA origami of claim 5, wherein the length is ~70 nm and the diameter is ~30.

7. The DNA origami of claim 5, wherein the length is -140 nm and the diameter is -30.

8. The DNA origami of claim 5, wherein the length is -6 nm and the diameter is -30.

9. The DNA origami of any of claims 1-8, wherein the single-stranded DNA scaffold comprises a single layer DNA film.

10. The DNA origami of any of claims 1-8, wherein the single-stranded DNA scaffold comprises a multi-layer DNA film, wherein the multi-layer DNA film has a thickness of -4 nm.

11. The DNA origami of claim 2, wherein the arbitrary nanostructure has a substantially planar tile architecture.

12. The DNA origami of claim 11 , wherein the substantially tubular nanostructure is defined by a length and a width.

13. The DNA origami of claim 12, wherein the length is between ~6 nm and —150 nm and the width is between ~20 and ~50 nm.

14. The DNA origami of claim 12, wherein the length is ~70 nm and the width is ~30.

15. The DNA origami of claim 12, wherein the length is —140 nm and the width is ~30.

16. The DNA origami of claim 12, wherein the length is ~6 nm and the width is ~30.

17. The DNA origami of any of claims 1-16, wherein the single-stranded DNA scaffold comprises a single layer DNA film.

18. The DNA origami of any of claims 1-16, wherein the single-stranded DNA scaffold comprises a multi-layer DNA film, wherein the multi-layer DNA film has a thickness of ~4 nm.

19. The DNA origami of any of claims 1-18, wherein one or more of the plurality of oligonucleotide staples is labelled with a fluorescent species.

20. The labelled DNA origami of claim 19, wherein the fluorescent species is a Cyanine5 (Cy5) conjugate.

21. The DNA origami of any of claims 1-10, wherein the substantially tubular architecture defines an interior space configured to transport a therapeutic payload.

22. The DNA origami of claim 21, wherein the DNA origami forms a nanocomplex with a therapeutic cargo arranged within the inter space of the substantially tubular architecture.

23. The DNA origami nanocomplex of claim 22, wherein the therapeutic payload comprises an anti-cancer agent.

24. The DNA origami nanocomplex of claim 23, wherein the anti-cancer agent is a therapeutic nucleic acid material.

25. The DNA origami nanocomplex of claim 23, wherein the anticancer agent is a therapeutic peptide material.

26. The labelled DNA origami of claim 19, wherein the plurality of oligonucleotide staples comprises at least 20 staples, wherein at least 50% of the staples are labelled with a respective Cy5 conjugate.

27. A reagent for selective imaging of KRAS-mutant pancreatic cancer cells in a desmoplastic pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) comprising a labelled DNA origami of claim 19.

28. A method of selective imaging of KRAS-mutant pancreatic cancer cells in a desmoplastic pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME), the method comprising: obtaining a sample of PDAC tissue; contacting the sample with a reagent of claim 27; exposing the reagent-contacted sample to an excitation source to generate an image signal; and processing the image signal to identify a fluorescent signal associated with the fluorescent species, wherein identification of the fluorescent signal is indicative of the presence of KRAS-mutant pancreatic cancer cells in the sample.

29. The method of claim 28, wherein the fluorescent species is a Cy5 conjugate.

30. A pharmaceutical composition for targeted delivery of a therapeutic agent to KRAS-mutant pancreatic cancer cells in a subject comprising a therapeutically effective amount of DNA origami nanocomplex of claim 22 .

31. The pharmaceutical composition of claim 30, wherein the therapeutic payload comprises an anti-cancer agent.

32. The pharmaceutical composition of claim 31, wherein the anti-cancer agent is a therapeutic nucleic acid material.

33. The pharmaceutical composition of claim 31, wherein the anticancer agent is a therapeutic peptide material.

34. A method of treating a desmoplastic pancreatic ductal adenocarcinoma (PDAC) characterized by KRAS-mutant pancreatic cancer cells, the method comprising administering a composition of any of claims 30-33 to a patient in need of such treatment.

35. The method of claim 34, wherein the administering comprises intracellular delivery of the composition, whereby the therapeutic payload is preferentially internalized by the KRAS-mutant pancreatic cells.

Images