Antibody and sirna nanocarriers and uses thereof

Abstract

The present invention provides a method for suppressing a target gene involved in the Wnt / β-catenin signaling pathway in target cells expressing a receptor capable of binding an extracellular Wnt ligand and an intracellular Wnt effector. The method comprises providing nanocarriers and binding the nanocarriers to the target cells and / or internalizing of the nanocarriers into the target cells, whereby the target gene is suppressed in the target cells. In each nanocarrier, an antibody specific for the receptor, a siRNA specific for the effector, or a combination thereof may be attached to a nanoparticle. The target cells may be in a subject. The method may further comprise reducing disease burden and / or inducing stabilization of a disease in the subject. Also provided are methods for synthesizing the nanocarriers.

Description

[0001] ANTIBODY AND SIRNA NANOCARRIERS AND USES THEREOF

[0002] This application claims priority to United States Provisional Application No.

[0003] 63 / 565,109. filed March 14, 2024, and the contents of which are incorporated herein by reference in their entireties for all purposes.

[0004] REFERENCE TO U.S. GOVERNMENT SUPPORT

[0005] This invention was made with government support under grant number R01CA221925 awarded by the National Cancer Institute. The United States has certain rights in the invention.

[0006] FIELD OF THE INVENTION

[0007] The invention relates to nanocarriers comprising nanoparticles coated with antibodies and / or siRNAs, methods of making the same, and uses thereof.

[0008] BACKGROUND OF THE INVENTION

[0009] Dysregulated Wnt signaling drives the formation and progression of cancer by facilitating cell survival, proliferation, drug resistance, invasion, stem-like behavior, and other characteristics that allow tumors to grow, resist treatment, metastasize, and recur. This signaling pathway is important in many cancers, including triple-negative breast cancer (TNBC), other breast cancers, skin cancers, bladder cancers, brain cancers, cervical cancers, colorectal cancers, hepatocellular cancers, leukemias, lung cancers, lymphomas, oral cancers, ovarian cancers, pancreatic cancers, prostate cancers, and more. Despite the importance of this signaling pathway as a therapeutic target, it has been challenging to inhibit. One approach to manipulate Wnt signaling involves delivering antibodies that bind Frizzled receptors (e.g., Frizzled?) or low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 / 6 co-receptors) that are overexpressed on diseased cells relative to healthy cells, which blocks Wnt ligands from binding the receptors leading to suppression of downstream intracellular Wnt signaling activity. Unfortunately, freely delivered antibodies have low- binding affinities, poor pharmacokinetics, and limited tissue penetration that reduce their efficacy. An alternative strategy to suppress Wnt signaling focuses on the intracellular protein p-catenin. Historically, P-catenin has been considered undruggable due to lack of an effective binding site for small molecule therapeutics. Using small interfering ribonucleic acids (siRNAs) to suppress P-catenin through RNA interference has potential to overcome this problem. Likewise, siRNAs targeting other Wnt effectors (e.g., Axin2, FZD7 (Frizzled?), TCF / LEF (T-cell factor / lymphoid enhancer-binding factor), Wntl, Wnt2, Wnt3, Wnt3a) could be used to suppress the signaling pathway. However, freely delivered nucleic acids are not suitable for in vivo use owing to their nuclease susceptibility, short circulation half-life, and limited cellular uptake.

[0010] There remains a substantial need for carriers that can improve the delivery of antibodies and siRNA molecules to diseased cells to inhibit Wnt signaling and halt disease progression.

[0011] SUMMARY OF THE INVENTION

[0012] The present invention relates to the use of antibody and siRNA nanocarriers and synthesis of the nanocarriers. The inventors have surprisingly discovered that nanoparticles coated with an antibody targeting a Wnt receptor (e.g.. Frizzled?), a siRNA targeting a Wnt effector (e.g., P-catenin), and a passivating agent (e.g., methoxy-poly(ethylene glycol)-thiol (mPEG-SH)) can bind cells that overexpress the Wnt receptor, block Wnt ligand interaction with the overexpressed receptor, leading to downstream phosphory lation and degradation of -catenin proteins, and deliver the siRNA into the cells to induce RNA interference- mediated degradation of the Wnt effector.

[0013] A method for suppressing a target gene involved in the Wnt / p-catenin signaling pathway in target cells is provided. The target cells express a receptor capable of binding an extracellular Wnt ligand and an intracellular Wnt effector. The suppression method comprises (a) providing nanocarriers, and (b) binding the nanocarriers to the target cells and / or internalizing of the nanocarriers into the target cells, whereby the target gene is suppressed in the target cells. The nanocarriers comprise: (i) first nanocarriers, wherein each of the first nanocarriers comprises a first nanoparticle and an antibody specific for the receptor, and the antibody is attached to the first nanoparticle; (ii) second nanocarriers, wherein each of the second nanocarriers comprises a second nanoparticle and a small interfering RNA (siRNA) specific for the Wnt effector, and the siRNA is attached to the second nanoparticle; and / or (iii) third nanocarriers different from the first nanocarriers and the second nanocarriers, wherein each of the third nanocarriers comprises a third nanoparticle, the antibody, and the siRNA, and the antibody and the siRNA are attached to the third nanoparticle. The first nanoparticle, the second nanoparticle, and the third nanoparticle may be the same. The nanocarriers may comprise the first nanocarriers, and the suppression method may further comprise: (c) binding the antibody to the receptor, binding the first nanocarriers to the target cells, and phosphorylating and degrading the Wnt protein P-catenin in the target cells.

[0014] The nanocarriers may comprise the second nanocarriers, and the synthesis method may further comprise: (d) internalizing of the second nanocarriers into the target cells, and cleaving mRNA of the Wnt effector in the target cells.

[0015] The nanocarriers may comprise the third nanocarriers, and the suppression method may further comprise: (c) binding the antibody to the receptor, binding the third nanocarriers to the target cells, phosphorylating and degrading the Wnt protein p-catenin in the target cells, and (d) internalizing of the third nanocarriers into the target cells, and cleaving mRNA of the Wnt effector in the target cells.

[0016] According to the suppression method, the target gene may be selected from the group consisting of P-catenin (encoded by CTNNB1 (catenin beta 1)), Axin2, Cyclin DI (CCND1), C-myc, Nanog, Snail (SNAI1, or Snail family transcriptional repressor 1), Survivin (BIRC5), SOX9 (SRY-box transcription factor 9), RUNX2 (Runt-related transcription factor 2), MDR1 (multi drug resistance 1, which encodes for P-gly coprotein), CTLA4 (cytotoxic T-lymphocyte-associated protein 4), TCF / LEF (T-cell factor / lymphoid enhancer-binding factor), Oct4 (octamer-binding transcription factor 4, also known as POU5F1). KLF4 (Kriippel-like factor 4), MMP-7 (matrix metallopeptidase 7), VEGF (vascular endothelial growth factor), and CD44 (cluster of differentiation 44). The target gene may be selected from the group consisting of P-catenin, CCND1, C-myc, Snail, and Survivin.

[0017] According to the suppression method, the receptor may be a Frizzled (FZD) receptor or a low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 / 6 co-receptors).

[0018] According to the suppression method, the ligand may be selected from the group consisting of Wntl, Wnt2, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt 10a, Wnt 10b. and Wntl l.

[0019] According to the suppression method, the effector may be selected from the group consisting of P-catenin. Axin2, FZD7, TCF / LEF. Wntl, Wnt2, Wnt3, and Wnt3a. According to the suppression method, the nanocarriers may further comprise a passivating agent attached to the first nanoparticles, second nanoparticles, and / or third nanoparticles. The passivating agent may be selected from the group consisting of linear poly(ethylene glycol) (PEG) molecules, branched PEG molecules, Y-shaped PEG molecules, heterobifunctional PEG molecules, polyoxazolines, poly(N-vinylpyrrolidone, poly(glycerols), polyacrylamides, poly (carboxy betaine), poly(sulfobetaine), and phosphobetaine-base polymers.

[0020] The suppression method may further comprise reducing proliferation of the target cells.

[0021] The suppression method may further comprise suppressing migration of the target cells.

[0022] The suppression method may further comprise suppressing invasion of the target cells.

[0023] The suppression method may further comprise reducing self-renewal by the target cells.

[0024] The suppression method may further comprise reducing metabolic activity of the target cells.

[0025] The suppression method may further comprise reducing drug resistance of the target cells.

[0026] The suppression method may further comprise reducing viability of the target cells.

[0027] The suppression method may further comprise blocking interaction between the Wnt ligand and the receptor.

[0028] The suppression method may further comprise RNA interference of the Wnt effector.

[0029] According to the suppression method, the target cells may be cancer cells. The target cells may be in a subject. The subject may suffer from a cancer. The target cells may be in a tumor in the subject. The suppression method may further comprise reducing growth of the tumor. The suppression method may further comprise reducing metastasis of the tumor. The suppression method may further comprise reducing recurrence of the tumor. The suppression method may further comprise inducing stabilization of a disease in the subject. The suppression method may further comprise reducing disease burden in the subject. The subject may be a human. The subject may be a veterinary subject.

[0030] A first synthesis method for synthesizing a first nanocarrier is provided. The first nanocarrier comprises a nanoparticle and an antibody specific for a receptor capable of binding an extracellular Wnt ligand. The first synthesis method comprises attaching the antibody to the nanoparticle, whereby the first nanocarrier is synthesized. The first synthesis method may further comprise attaching a passivating agent to the nanoparticle.

[0031] The first synthesis method may further comprise attaching the antibody to the nanoparticle through a chemical linker. The chemical linker may be a heterobifunctional PEG derivative.

[0032] The first synthesis method may further comprise attaching the antibody to the nanoparticle using free thiols that are exposed by treating the antibody with a reducing agent.

[0033] A second synthesis method for synthesizing a second nanocarrier is provided. The second nanocarrier comprises a nanoparticle and a small interfering RNA (siRNA) specific for a Wnt effector. The second synthesis method comprises attaching the siRNA to the nanoparticle, whereby the second nanocarrier is synthesized. The second synthesis method may further comprise attaching a passivating agent to the nanoparticle.

[0034] The second synthesis method may further comprise attaching the siRNA to the nanoparticle in a buffer. The buffer may comprise NaCl, CaCh, or ethylenediaminetetraacetic acid (EDTA).

[0035] The second synthesis method may further comprise attaching the siRNA to the nanoparticle through a chemical linker. The chemical linker may be a thiol or disulfide. The thiol or disulfide may be positioned at the 3' end of the sense strand of the siRNA.

[0036] A third synthesis method for synthesizing a third nanocarrier is provided. The nanocarrier comprises a nanoparticle, an antibody specific for a receptor capable of binding an extracellular Wnt ligand, and a small interfering RNA (siRNA) specific for a Wnt effector. The third synthesis method comprises: (a) attaching the antibody to the nanoparticle, whereby the nanoparticle carrying the antibody is obtained; and (b) attaching the siRNA to the nanoparticle carry ing the antibody, whereby the third nanocarrier is synthesized. The third synthesis method may further comprise attaching a passivating agent to the nanoparticle.

[0037] The third synthesis method may further comprise attaching the antibody to the nanoparticle through a chemical linker. The chemical linker may be a heterobifunctional PEG derivative.

[0038] The third synthesis method may further comprise attaching the antibody to the nanoparticle using free thiols that are exposed by treating the antibody with a reducing agent.

[0039] The third synthesis method may further comprise attaching the siRNA to the nanoparticle in a buffer. The buffer may comprise NaCl, CaCh, or ethylenediaminetetraacetic acid (EDTA).

[0040] The third synthesis method may further comprise attaching the siRNA to the nanoparticle through a chemical linker. The chemical linker may be a thiol or disulfide. The thiol or disulfide may' be positioned at the 3' end of the sense strand of the siRNA.

[0041] BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIGS. 1 A-1B show overview of the structure of nanoparticles coated with antibodies and siRNAs and their use to inhibit Wnt signaling in target cells. (A) Scheme of a nanoparticle coated with antibodies targeting Frizzled? (FZD7) receptors, siRNAs targeting P-catenin messenger RNA (mRNA), and poly(ethylene glycol) (PEG). (B) Depiction of the nanocarriers’ mechanism of action. FZD7 antibodies on the nanocarriers enable target cell binding and lock FZD7 receptors in a Wnt-ligand unresponsive state, leading to intracellular phosphorylation and degradation of P-catenin proteins. Upon cellular entry, the siRNAs delivered by the nanocarriers trigger cleavage of P-catenin mRNA through RNA interference. The PEG molecules on the nanocarriers provide stability in physiologic environments. Portions of this figure created w ith BioRender.com.

[0043] FIGS. 2A-2D show sy nthesis and characterization of nanoshells (a specific type of nanoparticle) coated with FZD7 antibodies and P-catenin siRNAs. (A) Scheme depicting the steps to functionalize nanoshells (NS) with FZD7 antibodies, P-catenin siRNAs, and mPEG-SH. Nanoshells coated with antibodies, siRNAs, and mPEG-SH are termed “Combo-NS”, nanoshells coated with only antibodies and mPEG-SH are termed "FZD7- NS”, and nanoshells coated with only siRNAs and mPEG-SH are termed “Pcat-NS”. (B) Hydrodynamic diameter and (C) zeta potential of bare NS compared to NS conjugates. (D) Antibody and / or siRNA loading on each type of conjugate. Data are mean ± standard error. Portions of this figure created with BioRender.com.

[0044] FIGS. 3A-3B show demonstration of nanocarrier cellular binding and uptake by microscopy and flow cytometry. (A) Multiphoton microscopy images of Combo-NS, FZD7- NS, and Pcat-NS binding to MDA-MB-231 triple-negative breast cancer cells that overexpress FZD7 receptors or MCF10A mammary epithelial cells with low FZD7 expression. Scales=20 pm. Cell membranes were labeled with CellVue Red and nanoshells (which appear as bright spots) were visualized by two-photon photoluminescence. PEG-NS refers to nanoshells coated with only mPEG-SH. (B) Cellular uptake of nanocarriers after 1 h incubation quantified via flow cytometry. NT=non-treated cells. Data show the mean ± standard error of n=4 biological replicates per nanocarrier in each cell line, **p<0.01 and *p<0.05 by one-way analysis of variance (ANOVA) with post hoc Tukey. Together, the microscopy and flow cytometry data indicate FZD7-NS and Combo-NS have greater binding to and uptake by targeted MDA-MB-231 cells than nanocarriers that lack FZD7 antibodies in their formulation.

[0045] FIG. 4 shows nanocarrier-mediated gene regulation confirmed by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Relative mRNA expression of several Wnt target genes in MDA-MB-231 cells following treatment with FZD7-NS, Pcat-NS, or Combo-NS. Data are normalized to the non-treated (NT) group within each gene and show the mean ± standard error of n=3 biological replicates for all genes except C-myc, which is n=2 biological replicates. **p<0.01, *p<0.05, and #p<0.10 versus NT within each target gene by ANOVA with post hoc Tukey.

[0046] FIG. 5 shows nanocarrier treatment reduces cancer cell proliferation. Normalized count of proliferating MDA-MB-231 cells following treatment for 48, 72, or 96 hours with FZD7-NS, Pcat-NS, or Combo-NS. Data are normalized to the non-treated (NT) group within each timepoint and depict the mean ± standard error of n=3 biological replicates per nanocarrier for each timepoint. #p<0. 10 and *p<0.05 by ANOVA with post hoc Tukey.

[0047] FIGS. 6A-6C show that cancer cell migration decreases in response to nanocarrier treatment. (A) Scheme of nanocarrier treatment and cell reseeding in trans well insert for migration assay. For this study, GFP-expressing MDA-MB-231 triple-negative breast cancer cells were exposed to FZD7-NS, Pcat-NS, or Combo-NS. Non-treated cells served as a control. (B) Count of migrated MDA-MB-231 cells in each treatment group at various imaging timepoints post reseeding. Data show mean ± standard error (n=3 biological replicates per nanocarrier type at each timepoint). NT=non-treated. (C) Representative fluorescence microscopy images of migrated GFP-expressing MDA-MB-231 cells at the 24 h timepoint in each treatment group. Scales = 1 mm. Portions of this figure created with BioRender.com.

[0048] FIGS. 7A-7C show that spheroid formation of cancer cells is altered in response to nanocarrier treatment. (A) Scheme of nanocarrier treatment of MDA-MB-231 cells and cell reseeding to allow for spheroid formation. (B) Metabolic activity' of spheroids measured by Alamar Blue assay after 1 week of formation. Data are normalized to the NT (non-treated) group at each timepoint and depict the mean ± standard error of n=3 biological replicates per nanocarrier. **p<0.01 by ANOVA with post hoc Tukey. (C) Representative brightfield images of spheroids formed after 1 week with 72 h nanocarrier treatment time. Two t pes of spheres were observed to form after nanocarrier treatment, including loose clusters of cells (top row) and less opaque / dense spheres (bottom row). Scale bars=200 pm. Combo-NS had the largest impact on spheroid formation capacity. Portions of this figure created with BioRender.com.

[0049] FIGS. 8A-8D show that antibody and siRNA nanocarriers limit metastasis burden in an experimental triple-negative breast cancer lung metastasis model. (A) Scheme of experimental design. Firefly luciferase-expressing MDA-MB-231 cells were injected intravenously into nude mice to generate a model of triple-negative breast cancer lung metastasis. This model was used to evaluate the ability' of Combo-NS, FZD7-NS, and Pcat- NS to treat pre-existing metastasis. Created with BioRender.com. (B) Schedule of cell injection and nanocarrier treatment. Mice treated with saline served as a control group. (C) Quantified luminescence of lungs in mice imaged with an IVIS Lumina III In Vivo Imaging System at Day 58. Data are mean ± standard error (n=3 mice in the FZD7-NS and Pcat-NS groups and n=4 mice in the saline and Combo-NS groups). *p<0.05 and #p< 0.10 versus saline by ANOVA with post hoc Tukey-Kramer. (D) Representative IVIS images of mice in each treatment group at Day 55. Bright regions indicate metastasis burden in the lungs.

[0050] FIGS. 9A-9B show that Frizzled7 antibodies increase nanocarrier accumulation in lungs bearing triple-negative breast cancer (TNBC) lesions. Nude mice were injected intravenously with MDA-MB-231 cancer cells to form lung metastases and then treated with nanocarriers as shown in FIG. 8. At the study end, tumor-bearing lungs were excised for analysis of nanocarrier accumulation. (A) Quantification of gold in excised lungs determined by inductively coupled plasma-mass spectrometry (ICP-MS). Data are mean ± standard deviation after subtracting background from saline-injected mice. **p<0.01 and *p<0.05 versus Pcat-NS by ANOVA with post hoc Tukey-Kramer. (B) Representative darkfield image of FZD7 antibody -coated NS accumulation in metastatic lung nodules versus adjacent normal lung tissue. NS appear bright against the tissue background, as indicated by arrows. Scale = 50 / rm. Both FZD7-NS and Combo-NS have greater accumulation in tumor-bearing lungs than Pcat-NS.

[0051] FIGS. 10 A- 10C show that antibody and siRNA nanocarriers slow primary tumor growth in a syngeneic orthotopic murine triple-negative breast cancer model. (A) Overview of murine model and nanocarrier treatment schedule. Balb / c mice were inoculated with 4T1 murine triple-negative breast cancer cells in the mammary fat pad to form tumors that can spontaneously metastasize to the lungs and form recurrent tumors after surgical excision of the primary tumors. This model was used to test the ability of Combo-NS. FZD7-NS. and Pcat-NS to treat primary tumors and prevent or limit metastasis and recurrence. Created with BioRender.com. (B) Analysis of primary tumor growth in the mammaiy fat pad betw een the day of cell injection (Day 0) and the day of primary tumor removal (Day 13). (Left) Mean ± standard error of primary tumor volume in each treatment group at distinct timepoints. (Right) Change in primary tumor volume between the first day of treatment (Day 0) and the day of tumor removal (Day 13). Data show mean ± standard error. n=12-14 mice per group. *p<0.05 by ANOVA with post hoc Tukey-Kramer. (C) Analysis of P- catenin protein expression in primary tumors that were surgically removed on Day 13. Sections were counterstained with hematoxylin (scale bars = 100 / zm). The average percent P-catenin positive cells (shown as mean ± standard deviation) in three tumor sections per group was quantified using QuPath software. **p<0.01, *p<0.05, and #p<0.10 by one-way ANOVA with post hoc Tukey.

[0052] FIGS. 11A-11C show that Wnt inhibitory antibody and siRNA nanocarriers delay recurrence and limit spontaneous triple-negative breast cancer metastasis to lungs. Mice with orthotopic 4T1 triple-negative breast cancer tumors w ere treated once per w eek with FZD7-NS, Pcat-NS, Combo-NS or saline as shown in FIG. 10. Primary tumors were surgically removed on Day 13, then grow th of recurrent tumors and lung metastasis was monitored by bioluminescence imaging. n=6-7 mice per group. (A) Analysis of treatment effect on recurrent tumor growth. Mean ± standard error of tumor luminescence in each group at distinct timepoints. #p<0.10 compared to saline control by one-way ANOVA with post hoc Tukey-Kramer. (B) Quantified luminescence of lung metastases in mice treated with nanocarriers or saline. Data are mean ± standard error. (C) Percentage of mice in each treatment group that did not form metastasis by the end of the study.

[0053] FIG12 shows that Combo-NS, FZD7-NS, and Pcat-NS can accumulate in primary tumors, recurrent tumors, and metastatic lungs of mice after intravenous administration. Mice with orthotopic 4T1 triple-negative breast cancer tumors that recur after surgical excision and spontaneously metastasize to the lungs were treated as shown in FIG. 10. After euthanasia, primary tumors, recurrent tumors, and metastatic lungs were collected for analysis of nanocarrier accumulation. The amount of gold in the tissues was measured by ICP-MS (inductively coupled plasma-mass spectrometry). Data show mean ± standard error. Statistical differences denoted as *p<0.05, **p<0.01 by ANOVA with post hoc Tukey-Kramer.

[0054] FIG. 13 shows that nanocarrier treatment does not alter the appearance of major organs. Hematoxylin & eosin-stained sections of tissues obtained from Balb / c mice in the 4T1 spontaneous metastasis model depicted in FIG. 10 show no differences between treatment groups. Scale bars = 50 pm.

[0055] FIGS. 14A-14D show synthesis and characterization of polymeric nanoparticles coated with FZD7 antibodies and P-catenin siRNAs. (A) Scheme depicting the surface modification of poly(lactic-co-gly colic acid)-poly(ethylene glycolj-maleimide (PLGA-PEG- Mal) nanoparticles (NPs) with antibodies (via OPSS-PEG-SVA linkers), thiol-modified siRNA, and methoxy-PEG-SH (mPEG-SH). (B) Mode diameter (nm) and zeta potential (mV) of nanocarriers (n = 3-6). Bare NP=uncoated nanoparticles, siScr-NP=nanoparticles coated with scrambled siRNA and mPEG-SH, sipcat-NP=nanoparticles coated with p- catenin siRNA and mPEG-SH, FZD7-sipcat-NP=nanoparticles coated with Frizzled7 antibodies, P-catenin siRNA, and mPEG-SH. (C) Quantification of siRNA and antibody loading on the nanoparticle surface. Data display the mean ± standard deviation (n = 3-5). (D) siRNA released over time from the PLGA-PEG-Mal nanoparticle surface after placement in either water at 4 °C (to mimic storage conditions) or pH 5.5 phosphate buffered saline (PBS) at 37 °C (to mimic acidic intracellular compartments) (n = 3). Error bars represent standard deviation.

[0056] FIGS. 15A-15B show that polymer nanoparticles coated with FZD7 antibodies and P-catenin siRNAs can enter triple-negative breast cancer cells to suppress Wnt signaling. (A) Confocal microscopy visualization of Cy5-siRNA in MDA-MB-231 cells treated with FZD7-sipcat-NPs. Cell cytoplasm was labeled with CellTracker Green and siRNA was tagged with Cy5. Arrows in the merged image point to representative nanocarriers within cells. Scale bars = 20 pm. (B) RT-qPCR analysis of relative mRNA expression in MDA- MB-231 cells normalized to non-treated (NT) samples after 48 h treatment with NT or 50 nM siRNA of siScr-NPs, sipcat-NPs, or FZD7-sipcat-NPs. Data are displayed as the mean ± standard deviation, with open circles showing individual data points (n=3-5). *p<0.05 by ANOVA with post hoc Tukey-Kramer.

[0057] FIGS. 16A-16B show migration of MDA-MB-231 cells following 48 hour treatment with 50 nM siRNA of siScr-NPs, si cat-NPs, or FZD7-sipcat-NPs. (A) Representative brightfield images of migrated cells at 0 and 20 h post-treatment. NT=non-treated control. Edge of cell migration indicated by dashed lines. Scale bar = 750 pm. (B) Quantified cell density per pm2in the area exposed after creating a scratch through the cells. Data are displayed as the mean ± standard deviation (n=3-4). *p<0.05 by ANOVA with post hoc Tukey-Kramer.

[0058] FIG. 17 shows quantification of MDA-MB-231 cell metabolic activity after 48 h of treatment with nanocarriers. Relative metabolic activity of cells treated with either NT (no treatment) or 50 nM siRNA of siScr-NPs, sipcat-NPs, or FZD7-sipcat-NPs. Data are normalized to NT and show the mean ± standard deviation (n = 4-6). *p < 0.05 by ANOVA with post hoc Tukey-Kramer.

[0059] FIGS. 18A-18C show impact of nanocarrier treatment on MDA-MB-231 spheroids after 72 h exposure to NT or 50 nM siRNA of siScr-NPs, sipcat-NPs, or FZD7-sipcat- NPs.

[0060] (A) Representative images of spheroids labeled with live / dead staining. Live cells are shown in first column, and dead cells are shown in second column. Scale bars = 150 pm.

[0061] (B) Quantification of live reagent fluorescence normalized to NT (n = 3-4). (C) Quantification of dead reagent fluorescence normalized to NT (n = 3-4). Data are displayed as mean ± standard deviation (n = 3-5). **p < 0.01, *p < 0.05 by ANOVA with post hoc Tukey-Kramer. DETAILED DESCRIPTION OF THE INVENTION

[0062] The present invention relates to antibody and siRNA nanocarriers for suppressing the Wnt / p-catenin signaling pathway in target cells. These antibody and siRNA nanocarriers may be used to treat any cancer or disease characterized by hyperactive Wnt / p-catenin signaling. The present invention also relates to the synthesis of the antibody and siRNA nanocarriers.

[0063] The inventors have surprisingly discovered that, when the antibody and siRNA nanocarriers are administered to cells overexpressing the Wnt pathway receptor, the nanocarriers can bind the target cells, enter the target cells, and suppress Wnt signaling in the cells. The nanocarriers may be used to treat any cancer or disease characterized by hyperactive Wnt signaling. The invention is based on the surprising discovery’ by the inventors that nanoparticles coated with an antibody targeting a Wnt receptor (e.g., Frizzled?), a siRNA targeting a Wnt effector (e.g., P-catenin), and optionally a passivating agent (e.g., methoxy-poly(ethylene glycol)-thiol (mPEG-SH)) that minimizes protein adsorption to the nanoparticles, can bind cells that overexpress the Wnt receptor (while exhibiting lesser binding to / uptake by cells that do not overexpress the receptor), block Wnt ligand interactions with the overexpressed receptor leading to downstream phosphorylation and degradation of P-catenin proteins, and also deliver the siRNA into the cell to induce RNA interference-mediated degradation of the Wnt effector (FIG. 1). In cancer cells, this down-regulation of Wnt signaling activity (i.e., suppression of P-catenin and downstream Wnt target genes) can reduce cancer cell proliferation, migration, invasion, metabolic activity, self-renewal (measured as a reduction in spheroid formation), and other oncogenic behaviors. In a subject with cancer, the nanocarriers can accumulate in primary’ tumors, recurrent tumors, and / or metastatic lesions after systemic administration. Administration of the nanocarriers to a subject with cancer in a therapeutically effective amount may reduce Wnt signaling activity' in primary tumors, recurrent tumors, and / or metastatic lesions. Administration of the nanocarriers to a subject with cancer in a therapeutically effective amount may slow the growth of primary tumors and / or metastatic lesions, reduce disease burden, cause stable disease, prevent recurrence and / or metastasis, delay recurrence and / or metastasis, reduce recurrence and / or metastasis, or eliminate the disease. Administration of the nanocarriers to a subject in a therapeutically effective amount may not cause off-target damage to major organs (e.g., kidney, spleen, liver, lungs, heart), indicative of nanoparticle biocompatibility.

[0064] The term “nanocarrier” as used herein refers to a nanomaterial useful for delivering a substance, for example, biological molecules, chemical compounds, or a combination thereof.

[0065] The term “nanoparticle” as used herein refers to a particle having an average diameter of about 1-1000 nm. For systemic administration, an average diameter of about SO- SOO nm may be preferred.

[0066] The term ‘'nanoshell” refers to a specific type of nanoparticle comprising a dielectric core (e.g., a 120-130 nm diameter silica sphere) surrounded by a metallic shell (e.g., 15-25 nm thick gold).

[0067] The terms “Wnt / p-catenin signaling pathway” and “Wnt signaling” are used herein interchangeably and refer to the canonical Wnt (Wingless-type mouse mammary tumor virus integration site family) pathway, which plays a critical role in embryonic development, carcinogenesis, and adult tissue homeostasis. This pathway involves the cytoplasmic accumulation and nuclear translocation of P-catenin and the activation of target genes via TCF / LEF transcription factors. The canonical Wnt / p-catenin signaling pathway is different from the non-canonical Wnt signaling such as the Wnt / planar cell polarity pathway or the Wnt / calcium pathway.

[0068] The terms “suppressing” and “reducing” are used herein interchangeably and refer to decreasing, slowing, or preventing (e.g., reducing the expression of a gene, or slowing or preventing the movement of cells from one location to another).

[0069] The term “reducing” as used herein refers to decreasing.

[0070] The term “migration” as used herein refers to the process by which cells move from one location to another, for example, in response to a stimulus.

[0071] The term “invasion” as used herein refers to the process by which cells move through tissues and change the structure of the extracellular matrix (ECM).

[0072] The term “self-renewal” as used herein refers to the ability of certain cells (such as cancer stem cells) to divide and produce more of the same types of cells.

[0073] The term “metabolic activity” as used herein refers to the totality’ of chemical reactions that sustain life in a cell, also known as metabolism. The term “drug resistance” as used herein refers to the ability of cells to withstand the effects of a drug.

[0074] The term “regression of a tumor” as used herein refers to shrinkage or disappearance of the tumor.

[0075] The term “metastasis of a tumor” as used herein refers to spreading cancer cells from the tumor site to other parts of the body of a subject.

[0076] The term “recurrence of a tumor” as used herein refers to reappearance of cancer after a period of remission or treatment.

[0077] The term “stabilization of a disease” as used herein refers to a state where a tumor neither shrinks significantly nor grows significantly and also does not spread beyond the original location in the body of a subject.

[0078] The term “disease burden” as used herein refers to the impact of a disease on a patient’s well-being, including its severity, effects on daily life, physical impacts, and potential for long-term consequences. To reduce disease burden is to lessen the negative impacts of a disease or decrease its severity.

[0079] The term “treatment” or “treating” as used herein refers to therapeutic application of nanocarriers to a subject with the goal to lessen or cure a disease or condition, for example, a pathologic condition, or prevent its recurrence or progression in the subject. A subject is successfully “treated” if administration of the nanocarriers in a therapeutically effective amount results in observable and / or measurable reduction in or absence of one or more signs and symptoms of the disease or condition.

[0080] The term “therapeutically effective amount” as used herein refers to an amount of nanocarriers sufficient to achieve a desired therapeutic effect in a subject when the nanocarriers are administered to the subject, for example, to cure, prevent, inhibit, or partially prevent or inhibit a disease or condition in the subject. Stabilization of a disease in a subject without progression may be considered successful treatment of the subject. Parameters for assessing treatment success in a subject with a disease or condition may be measurable by routine procedures familiar to a physician or veterinarian who manages care of the subject.

[0081] Nanoparticles coated with antibodies, siRNAs, and / or passivating agents, or any combination of these three, are nanocarriers while nanoparticles are not coated with biomolecules. The antibodies may enable the nanocarriers to bind the targeted receptors (e.g., Frizzled? receptor or LRP5 / 6 co-receptors) that are overexpressed on the cancer cells, which in turn blocks Wnt ligands from binding the receptors; this suppresses downstream intracellular Wnt signaling activity (e.g., by leading to phosphorylation and degradation of P-catenin proteins). Additionally, the nanocarriers may be internalized by the cancer cells, which allows them to deliver the siRNA molecules into the cell to enable RNA interference- mediated inhibition of the Wnt effectors at the messenger RNA (mRNA) level.

[0082] Downregulation of Wnt signaling activity (i.e.. suppression of Wnt target genes) caused by the antibody and siRNA nanocarriers may reduce cell proliferation, migration, invasion, self-renewal, spheroid formation, metabolic activity, chemoresistance, or other oncogenic behaviors. When administered into a subject intravenously, the nanocarriers may enter primary tumors, recurrent tumors, and / or metastatic lesions. Reductions in Wnt signaling in the tumors / lesions caused by the nanocarriers may result in decreased tumor growth, recurrence, and / or metastasis. The nanocarriers may not change the appearance or function of major organs (e.g., kidney, spleen, liver, lungs, heart), indicating biocompatibility.

[0083] The present invention provides a nanocarrier. The nanocarrier comprises a nanoparticle. The nanocarrier also comprises an antibody, siRNA, or a combination thereof. The antibody, siRNA or a combination thereof is attached to the nanoparticle. The nanoparticle may be coated with the antibody, a siRNA, and / or a passivating agent, either directly or through a chemical linker. The antibody may be specific for a receptor capable of binding an extracellular Wnt ligand. The siRNA may be specific for an intracellular Wnt effector. The nanocarrier may further comprise a passivating agent. The passivating agent may be attached to the nanoparticle.

[0084] The nanoparticle may comprise various materials. The nanoparticle may comprise silica core / gold shell “nanoshells” with an average diameter of about 100-350 nm, colloidal gold with an average diameter of about 15-350 nm, poly(lactic-co-gly colic acid) (PLGA) or poly(lactic-co-gly colic acid)-poly(ethylene glycol)-maleimide (PLGA-PEG-MAL) with an average diameter of about 50-500 nm. The nanoparticle may comprise a liposome or a lipid nanoparticle with an average diameter of about 50-300 nm.

[0085] The nanocarrier may have an average diameter of about 1-1000, 1-500, 1-100, 1-50, 10-1000, 10-500, 10-100, 10-50, 50-1000, 50-500, 50-100, 100-1000, 100-500, or 500-1000 nm. The target gene involved in the Wnt / 0-catenin signaling pathway may be selected from the group consisting of P-catenin, axis inhibition protein 2 (Axin2), cell-cycle regulator cyclin Dl(CCNDl), C-myc, Nanog, Snail, Survivin. SOX9, RUNX2, MDR-1, CTLA4, TCF / LEF, Oct4, KLF4, MMP-7, VEGF, and CD44. For example, the target gene may be P-catenin, CCND1, C-myc, Snail, or Survivin.

[0086] The antibody may target a Frizzled receptor (e.g., Frizzled? (FZD7)). The antibody may target the LRP5 / 6 co-receptors.

[0087] The receptor capable of binding an extracellular Wnt ligand may be a Frizzled (FZD) receptor or a low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 / 6 coreceptors).

[0088] The extracellular Wnt ligand may be selected from the group consisting of Wntl, Wnt2, Wnt3, Wnt3a, Wnt4. Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b. Wnt8a, WntlOa, WntlOb, and Wntl 1. For example, the extracellular Wnt ligand may be Wntl. Wnt2, Wnt3, or Wnt3a.

[0089] The intracellular Wnt effector may be selected from the group consisting of P- catenin, axis inhibition protein 2 (Axin2), Frizzled-7 (FZD7), T-cell factor / lymphoid enhancer factor (TCF / LEF), Wntl, Wnt2, Wnt3, and Wnt3a.

[0090] The siRNA may suppress the Wnt effector -catenin. The siRNA may suppress Axin2. The siRNA may suppress FZD7. The siRNA may suppress TCF / LEF. The siRNA may suppress Wntl. The siRNA may suppress Wnt2. The siRNA may suppress Wnt3. The siRNA may suppress Wnt3a.

[0091] The passivating agent may be selected from the group consisting of linear poly(ethylene glycol) (PEG) molecules, branched PEG molecules, Y-shaped PEG molecules, heterobifunctional PEG molecules, polyoxazolines, poly(N-vinylpyrrolidone, poly(glycerols), polyacrylamides, poly(carboxybetaine), poly(sulfobetaine), and phosphobetaine-base polymers. The heterobifunctional PEG molecule may be methoxy- PEG-thiol (mPEG-SH). The molecular weight of the mPEG-SH may be about 1-50 kDa (e.g., 2 kDa, 5 kDa, or 10 kDa).

[0092] The present invention also provides a method for suppressing a target gene involved in the Wnt / p-catenin signaling pathway in target cells. The target cells express a receptor capable of binding an extracellular Wnt ligand and an intracellular Wnt effector. The suppression method comprises providing nanocarriers, and binding the nanocarriers to the target cells and / or internalizing of the nanocarriers into the target cells, whereby the target gene is suppressed in the target cells.

[0093] According to the suppression method, the nanocarriers may comprise first nanocarriers, second nanocarriers, and / or third nanocarriers.

[0094] Each of the first nanocarriers may comprise a first nanoparticle and an antibody specific for a receptor capable of binding an extracellular Wnt ligand. The antibody may be attached to the first nanoparticle. The attachment of the antibody to the first nanoparticle may be direct or through a chemical linker. The first nanocarrier may further comprise a passivating agent. The passivating agent may be attached to the first nanoparticle. The attachment of the passivating agent to the first nanoparticle may be direct or through a chemical linker.

[0095] The nanocarriers may comprise the first nanocarriers, and the suppression method may further comprise binding the antibody to the receptor, binding the first nanocarriers to the target cells, and phosphorylating and degrading the Wnt protein P-catenin in the target cells.

[0096] Each of the second nanocarriers may comprise a second nanoparticle and a small interfering RNA (siRNA) specific for the Wnt effector. The siRNA may be attached to the second nanoparticle. The attachment of the siRNA to the second nanoparticle may be direct or through a chemical linker. The second nanocarrier may further comprise a passivating agent. The passivating agent may be attached to the second nanoparticle. The attachment of the passivating agent to the second nanoparticle may be direct or through a chemical linker.

[0097] The nanocarriers may comprise the second nanocarriers, and the suppression method may further comprise internalizing of the second nanocarriers into the target cells, and cleaving mRNA of the Wnt effector in the target cells.

[0098] The third nanocarriers are different from the first nanocarriers and the second nanocarriers. Each of the third nanocarriers may comprise a third nanoparticle, an antibody specific for a receptor capable of binding an extracellular Wnt ligand, and a small interfering RNA (siRNA) specific for the Wnt effector. The antibody and the siRNA may be attached to the third nanoparticle. The attachment of the antibody to the third nanoparticle may be direct or through a chemical linker. The attachment of the siRNA to the third nanoparticle may be direct or through a chemical linker. The third nanocarrier may further comprise a passivating agent. The passivating agent may be attached to the third nanoparticle. The attachment of the passivating agent to the third nanoparticle may be direct or through a chemical linker.

[0099] The nanocarriers may comprise the third nanocarriers, and the suppression method may further comprise binding the antibody to the receptor, binding the third nanocarriers to the target cells, phosphorylating and degrading the Wnt protein [Tcatcnin in the target cells. The suppression method may further comprise internalizing of the third nanocarriers into the target cells, and cleaving mRNA of the Wnt effector in the target cells.

[0100] According to the suppression method, the first nanoparticle, the second nanoparticle, and the third nanoparticle may be the same or different.

[0101] According to the suppression method, the target gene involved in the Wnt / p-catenin signaling pathway may be selected from the group consisting of P-catenin, axis inhibition protein 2 (Axin2), cell-cycle regulator cyclin Dl(CCNDl), C-myc, Nanog, Snail, Survivin, SOX9, RUNX2, MDR-1, CTLA4, TCF / LEF, Oct4, KLF4, MMP-7, VEGF, and CD44. For example, the target gene may be P-catenin. CCND1, C-myc. Snail, or Survivin.

[0102] According to the suppression method, the antibody may target a Frizzled receptor (e.g., Frizzled? (FZD7)). The antibody may target the Lrp5 / 6 co-receptors.

[0103] According to the suppression method, the receptor capable of binding an extracellular Wnt ligand may be a Frizzled (FZD) receptor (e.g., FZD7) or a low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 / 6 co-receptors).

[0104] According to the suppression method, the extracellular Wnt ligand may be selected from the group consisting of Wnt 1, Wnt2, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, WntlOa, WntlOb, and Wntl 1. For example, the extracellular Wnt ligand may be Wntl, Wnt2, Wnt3, or Wnt3a.

[0105] According to the suppression method, the intracellular Wnt effector may be selected from the group consisting of P-catenin. axis inhibition protein 2 (Axin2), Frizzled-7 (FZD7), T-cell factor / lymphoid enhancer factor (TCF / LEF), Wntl, Wnt2, Wnt3, and Wnt3a.

[0106] According to the suppression method, the siRNA may suppress the Wnt effector P- catenin. The siRNA may suppress Axin2. The siRNA may suppress FZD7. The siRNA may suppress TCF / LEF. The siRNA may suppress Wntl. The siRNA may suppress Wnt2. The siRNA may suppress Wnt3. The siRNA may suppress Wnt3a.

[0107] According to the suppression method, the passivating agent may be selected from the group consisting of linear poly(ethylene glycol) (PEG) molecules, branched PEG molecules, Y-shaped PEG molecules, heterobifunctional PEG molecules, polyoxazolines, poly(N-vinylpyrrolidone, poly(glycerols), polyacrylamides, poly(carboxybetaine), poly(sulfobetaine), and phosphobetaine-base polymers. The heterobifunctional PEG molecule may be methoxy-PEG-thiol (mPEG-SH). The molecular weight of the mPEG-SH may be about 1-50 kDa (e.g., 2 kDa, 5 kDa, or 10 kDa).

[0108] The suppression method may further comprise reducing proliferation of the target cells. The proliferation of the target cells may be reduced by at least about 10%. 20%. 30%. 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 10-80%, 10-90%, 10- 100%, 50-80%, 50-90%, 50-100%, 80-90%, 80-100%, or 90-100%.

[0109] The suppression method may further comprise suppressing migration of the target cells. The migration of the target cells may be suppressed by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 10-80%, 10-90%, 10- 100%. 50-80%, 50-90%, 50-100%, 80-90%, 80-100%. or 90-100%.

[0110] The suppression method may further comprise suppressing invasion of the target cells. The invasion of the target cells may be suppressed by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 10-80%, 10-90%, 10- 100%, 50-80%, 50-90%, 50-100%, 80-90%, 80-100%, or 90-100%.

[0111] The suppression method may further comprise reducing self-renewal of the target cells. The self-renewal of the target cells may be reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 10-80%, 10-90%, 10- 100%. 50-80%, 50-90%, 50-100%. 80-90%, 80-100%. or 90-100%.

[0112] The suppression method may further comprise reducing metabolic activity, or metabolism, of the target cells. The metabolic activity of the target cells may be reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 10-80%, 10-90%, 10-100%, 50-80%, 50-90%, 50-100%, 80-90%, 80-100%, or 90- 100%. The reduction in metabolic activity may be measured through AlamarBlue assay, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, or other similar assays.

[0113] The suppression method may further comprise reducing drug resistance of the target cells. The drug resistance of the target cells may be reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 10-80%, 10-90%, 10- 100%, 50-80%, 50-90%, 50-100%, 80-90% 80-100%, or 90-100%.

[0114] The suppression method may further comprise reducing viability of the target cells. The viability of the target cells may be reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 10-80%, 10-90%, 10-100%, 50- 80%, 50-90%, 50-100%, 80-90%, 80-100%, or 90-100%.

[0115] The suppression method may further comprise blocking interaction between the extracellular Wnt ligand and the receptor capable of binding the extracellular Wnt ligand. The interaction may be blocked by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 10-80%, 10-90%, 10-100%, 50-80%, 50-90%, 50- 100%. 80-90%, 80-100%, or 90-100%.

[0116] The suppression method may further comprise RNA interference of the intracellular Wnt effector. The translation of the intracellular Wnt effector may be reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 10- 80%, 10-90%, 10-100%, 50-80%, 50-90%, 50-100%, 80-90%, 80-100%, or 90- 100%.

[0117] According to the suppression method, the target cells may be cancer cells. The cancer may be selected from the group consisting of breast cancers, skin cancers, bladder cancers, brain cancers, cervical cancers, colorectal cancers, hepatocellular cancers, leukemias, lung cancers, lymphomas, oral cancers, ovarian cancers, pancreatic cancers, and prostate cancers.

[0118] The target cells may be in a subject. The subject may be a human. The subject may be a veterinary subject.

[0119] The suppression method may further comprise administering the nanocarriers to the subject. The subject may not experience a significant or life-threatening adverse effect. The subject may experience minimal / manageable off-target toxicity. The subject may tolerate the nanocarriers well or without the need for additional medical intervention. The nanocarriers may be biocompatible within the subject. The nanocarriers may not cause damage to a major organ in the subject. The nanocarriers may not cause off- target toxicity in the subject in need of a substantial medical intervention. The nanocarriers may not cause a severe or life-threatening adverse effect in the subject.

[0120] According to the suppression method, the subject may suffer from a cancer. The cancer may be selected from the group consisting of breast cancers, skin cancers, bladder cancers, brain cancers, cervical cancers, colorectal cancers, hepatocellular cancers, leukemias, lung cancers, lymphomas, oral cancers, ovarian cancers, pancreatic cancers, and prostate cancers.

[0121] According to the suppression method, the target cells may be in a tumor in the subject.

[0122] The suppression method may further comprise inhibiting Wnt signaling in the target cells in the subject. The inhibition of the Wnt signaling may be detected and quantified by measuring expression of a gene in the Wnt signaling pathway at the mRNA or protein level through reverse transcription-quantitative polymerase chain reaction (RT-qPCR), RNA sequencing (RNA-seq). immunohistochemistry, Western blot, microarrays, transcriptomics, or other molecular biology techniques.

[0123] The suppression method may further comprise reducing growth of the tumor. The growth of the tumor may be reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 10-80%, 10-90%, 10-100%, 50-80%, 50- 90%, 50-100%, 80-90%, 80-100%, or 90-100%.

[0124] The suppression method may further comprise inducing regression of the tumor in the subject. After regression, the size of the tumor may be reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 10-80%, 10- 90%, 10-100%, 50-80%, 50-90%, 50-100%, 80-90% 80-100%, or 90-100%.

[0125] The suppression method may further comprise reducing metastasis of the tumor. The metastasis of the tumor may be reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 10-80%, 10-90%, 10-100%, 50-80%, 50- 90%, 50-100%, 80-90% 80-100%, or 90-100%. The suppression method may further comprise preventing metastasis in the subject. The suppression method may further comprise reducing recurrence of the tumor. The recurrence of the tumor may be reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 10-80%, 10-90%, 10-100%, 50-80%, 50-90%, 50-1 0%, 80-90% 80-100%, or 90-100%. The suppression method may further comprise preventing recurrence of the tumor in the subject. They method may further comprise delaying recurrence of the tumor in the subject.

[0126] The suppression method may further comprise inducing stabilization of a disease in the subject. The disease may be characterized by hyperactive Wnt / p-catenin signaling. The disease may be selected from the group consisting of breast cancers, skin cancers, bladder cancers, brain cancers, cervical cancers, colorectal cancers, gastric cancers, hepatocellular cancers, leukemias, lung cancers, lymphomas, oral cancers, ovarian cancers, pancreatic cancers, prostate cancers, inflammatory' bowel disease, ulcerative colitis, kidney diseases, neurodegenerative diseases, graft-versus-host disease, asthma, cardiovascular diseases, and neurodevelopmental disorders.

[0127] The suppression method may further comprise reducing disease burden in the subject. The disease may be characterized by hyperactive Wnt / p-catenin signaling. The disease burden may be reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 10-80%, 10-90%, 10-100%, 50-80%, 50-90%. 50- 100%. 80-90%, 80-100%, or 90-100%. The disease may be selected from the group consisting of breast cancers, skin cancers, bladder cancers, brain cancers, cervical cancers, colorectal cancers, gastric cancers, hepatocellular cancers, leukemias, lung cancers, lymphomas, oral cancers, ovarian cancers, pancreatic cancers, prostate cancers, inflammatory bowel disease, ulcerative colitis, kidney diseases, neurodegenerative diseases, graft-versus-host disease, asthma, cardiovascular diseases, and neurodevelopmental disorders.

[0128] The present invention further provides a method for treating a disease characterized by hyperactive Wnt / p-catenin signaling in a subject. The treatment method comprises administering to the subject a therapeutically effective amount of the nanocarriers. The nanocarriers may be administered via injection. The nanocarriers may be administered at different doses. The administration of the nanocarriers may have a dosing schedule of, for example, once every 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. The total number of administration (e.g., injections) may vary', for example, from 1 total injection to 20 total injections. The concentration of the nanocarriers may be adjusted. The loading density' of the antibody, siRNA and / or passivating agent on the nanoparticles in the nanocarriers may be adjusted. The therapeutically effective amount of the nanocarriers may vary based on the severity' and type of the disease, the characteristics of the nanocarriers (e.g., size, antibody loading density, siRNA loading density, and other features), and the frequency of injections.

[0129] The present invention further provides a method for synthesizing a nanocarrier. The synthesis method comprises attaching an antibody, siRNA, or a combination thereof to a nanoparticle.

[0130] According to the synthesis method, the nanocarrier comprises a nanoparticle. The nanocarrier also comprises an antibody, siRNA, or a combination thereof. The antibody, siRNA or a combination thereof is attached to the nanoparticle. The antibody may be specific for a receptor capable of binding an extracellular Wnt ligand. The siRNA may be specific for an intracellular Wnt effector. The nanocarrier may further comprise a passivating agent, and the synthesis method further comprises attaching the passivating agent to the nanoparticle.

[0131] The nanoparticle may be synthesized in an RNase-free manner (i.e., using RNase- free materials and reagents) or treated with diethyl pyrocarbonate (DEPC) to render them RNase-free after they are produced.

[0132] The loading density of the antibody, siRNA, and / or passivating agent (e.g., mPEG- SH) on the nanoparticle may be adjusted by altering specific synthesis parameters. The loading density of an antibody may be about 5-2,000 (e.g., 10-350) molecules of the antibody per nanoparticle. The loading density of a siRNA may be about 5-10,000 (e.g., 250-3.500) molecules of the siRNA per nanoparticle. About 1-100% of the nanoparticle surface may be covered with an antibody, siRNA, and / or passivating agent.

[0133] A linker suitable for attaching an antibody to a nanoparticle may vary depending on the nature of the nanoparticle. The nanoparticle may comprise colloidal gold or PLGA- PEG-Mal. and the linker may be a heterobifunctional PEG, for example, orthopyridyl disulfide-poly(ethylene glycol)-succinimidyl valerate (OPSS-PEG-SVA), orthopyridyl disulfide-poly(ethylene glycol)-N-hydroxysuccinimide (OPSS-PEG-NHS), or orthopyridyl disulfide-poly(ethylene glycol)-succinimidyl carboxymethyl ester (OPSS-PEG-SCM). The linker may be of varying molecular weight in the range of about 0. 1-100 kDa (e.g., 2 kDa, 5 kDa, or 10 kDa). The linker may be used as is or modified to expose free thiols (e.g., by pre-treatment with tris(2-carboxy ethyl) phosphine hydrochloride (TCEP)) for attachment to the nanoparticle surface. The antibody may be used as is or modified to expose free thiols (e.g., by pre-treatment with TCEP) for attachment to the linker or direct attachment to the nanoparticle surface. Thiolated antibodies may be attached directly to the nanoparticle if it is of suitable surface chemistry for covalent conjugation or the thiolated antibodies may be attached to heterobifunctional PEG (e.g., OPSS-PEG-SVA or OPSS-PEG-NHS or OPSS- PEG-SCM) prior to nanoparticle conjugation. The antibody may be tethered to lipids for insertion into liposomes or lipid nanoparticles, for example, by conjugating thiolated antibodies with maleimide-functionalized lipids.

[0134] A linker suitable for attaching a siRNA to a nanoparticles may be a thiol (-SH) or disulfide (-SS). The siRNA linker may be attached to the 3' end of the sense strand. When a disulfide-terminated siRNA is used, the siRNA may be used as is or modified to expose free thiols (e.g., by pre-treatment with TCEP). In some embodiments, a spacer (for example, a polymer, an organic moiety, a nucleic acid sequence, or a hexaethylene glycol chain) may be placed between the siRNA and the thiol or disulfide group. The thiol or disulfide may attach to the surface of a nanoparticle comprising colloidal gold via gold-sulfur bonding, or the thiol or disulfide may be attached to the surface of maleimide-terminated PLGA via a thioether bond. Likewise, the thiol of the siRNA molecules may be reacted with maleimide- functionalized lipids for incorporation into liposomes or lipid nanoparticles.

[0135] Synthesis parameters that may be adjusted to tune antibody, siRNA, and / or passivating agent loading on the nanoparticle surface include: the order in which the molecules are attached to the nanoparticle surface; the type of attachment between the entity and the nanoparticle (direct interaction or tethered with a chemical linker); the type, molecular weight, or length of chemical linker used, if any; the amount of each molecule that is attached during the reaction; the concentration of the molecules and the nanoparticles during the reaction; the relative ratio of the molecules to the nanoparticles; the buffer the nanoparticles are placed in during the conjugation reaction (e.g.. NaCl, CaCh. ethylenediaminetetraacetic acid (EDTA)) and its concentration; the presence or absence of Tween-20 during the reaction and its concentration; the molecular weight of the passivating agent; the average diameter of the nanoparticle; the surface chemistry of the nanoparticle. In the embodiment of nanocarriers coated with antibodies, siRNAs, and mPEG-SH, adding the antibodies prior to the siRNAs may yield higher antibody loading on the nanoparticle surface.

[0136] According to the synthesis method, the antibody and siRNA nanocarriers may be purified from the unbound antibody, siRNA and / or passivating agent (e.g., mPEG-SH) after the conjugation reaction. Purification techniques may include centrifugation and resuspension, centrifugal filtration, transflow filtration, diafiltration, density gradient centrifugation, or a combination thereof.

[0137] A first synthesis method for synthesizing a first nanocarrier is provided. The first nanocarrier comprises a nanoparticle and an antibody specific for a receptor capable of binding an extracellular Wnt ligand. The synthesis method comprises attaching the antibody to the nanoparticle such that the first nanocarrier is synthesized. The first synthesis method may further comprise attaching a passivating agent to the nanoparticle.

[0138] The first synthesis method may further comprise attaching the antibody to the nanoparticle through a chemical linker. The chemical linker may be a heterobifunctional PEG derivative.

[0139] The first synthesis method may further comprise attaching the antibody to the nanoparticle using free thiols that are exposed by treating the antibody with a reducing agent.

[0140] A second synthesis method for synthesizing a second nanocarrier is provided. The second nanocarrier comprises a nanoparticle and a small interfering RNA (siRNA) specific for a Wnt effector. The synthesis method comprises attaching the siRNA to the nanoparticle such that the second nanocarrier is synthesized. The second synthesis method may further comprise attaching a passivating agent to the nanoparticle.

[0141] The second synthesis method may further comprise attaching the siRNA to the nanoparticle in a buffer. The buffer may comprise NaCl. CaCh, or ethylenediaminetetraacetic acid (EDTA). The buffer type and concentration may influence final siRNA loading densify on the nanoparticle.

[0142] The second synthesis method may further comprise attaching the siRNA to the nanoparticle through a chemical linker. The chemical linker may be a thiol or disulfide. The thiol or disulfide may be positioned at the 3' end of the sense strand of the siRNA.

[0143] A third synthesis method for synthesizing a third nanocarrier is provided. The third nanocarrier comprises a nanoparticle, an antibody specific for a receptor capable of binding an extracellular Wnt ligand, and a small interfering RNA (siRNA) specific for a Wnt effector. The third synthesis method comprising attaching the antibody to the nanoparticle, whereby the nanoparticle carrying the antibody is obtained; and attaching the siRNA to the nanoparticle carrying the antibody, whereby the third nanocarrier is synthesized. Attaching siRNA to the nanoparticle before attaching the antibody may dramatically reduce the loading of the antibody onto the nanoparticle. The third synthesis method may further comprise attaching a passivating agent to the nanoparticle.

[0144] The third synthesis method may further comprise attaching the antibody to the nanoparticle through a chemical linker. The chemical linker may be a heterobifunctional PEG derivative. The third synthesis method may further comprise attaching the antibody to the nanoparticle using free thiols that are exposed by treating the antibody with a reducing agent.

[0145] The third synthesis method may further comprise attaching the siRNA to the nanoparticle in a buffer. The buffer may comprise NaCl, CaCh, or ethylenediaminetetraacetic acid (EDTA). The buffer type and concentration may influence final siRNA loading density on the nanoparticle.

[0146] The third synthesis method may further comprise attaching the siRNA to the nanoparticle through a chemical linker. The chemical linker may be a thiol or disulfide. The thiol or disulfide may be positioned at the 3 ' end of the sense strand of the siRNA.

[0147] The term "about" as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate.

[0148] Example 1. Frizzled? antibody / p-catenin siRNA nanocarriers based on nanoshell cores suppress Wnt signaling, tumor growth, and lung metastasis in triple-negative breast cancer

[0149] In one embodiment, the inventors synthesized antibody and siRNA nanocarriers using ‘‘nanoshells” as a nanoparticle (FIG. 2), where the nanoshells consisted of -120-130 nm diameter silica spheres surrounded by -15-25 nm thick gold shells (for a total diameter of -150-180 nm). The nanoshells were synthesized by the inventors following the Oldenburg method (Oldenburg et al. Chemical Physics Letters. 1998; 288: 243-247) as described in Dang et al., Advanced Therapeutics, 2024; 7(6): 2300426. Briefly, 3-5 nm diameter gold colloid was made by the Duff method (Duff et al. Langmuir, 1993; 9(9): 2301-2309) from hydrogen tetrachloroaurate (III) hydrate (HauCh), tetrakis(hydroxymethyl)phosphonium chloride, and 1 N sodium hydroxide. The gold colloid was then combined with 120 nm diameter silica spheres functionalized with 3- aminopropyltriethoxysilane (Nanocomposix) and 1 M sodium chloride (NaCl) and rocked for 3-4 days at room temperature (RT) to create “seed” nanoparticles. A typical ratio for the reaction was 180 pL aminated silica spheres + 1 mL 1 M NaCl + 30 mL gold colloid, which was scaled as needed to produce the desired volume of seed. The seed was purified twice via centrifugation (-3000 rpm for 30 min in batches of -20 mL in 50 mL conical tubes) and resuspended in milliQ water to an optical density (OD) of 0. 1 at 530 nm. The diluted seed was mixed with additional H AuCk diluted in potassium chloride followed by addition of a small volume of 37% formaldehyde (VWR). The mixed solution was rapidly agitated to form complete gold shells. Different ratios of the seed, potassium chloride, and 37% formaldehyde were mixed in small volumes to determine the combination that would produce nanoshells (NS) with maximum extinction near 810 nm; this ratio was scaled up to produce the desired volume of NS. Synthesized NS were stored in water at -6x109NS mL’1(OD810 nm= 2) at 4 °C. Additionally, NS were treated with 0.1% diethyl pyrocarbonate (DEPC) for 3 days rocking at 37 °C to render them RNase-free. All materials described were purchased or treated with DEPC to be RNase-free prior to use.

[0150] The DEPC-treated nanoshells were coated with Frizzled7 antibodies and mPEG-SH to form “FZD7-NS”, [3-catenin siRNAs and mPEG-SH to form “Pcat-NS”, or with all three to form “Combo-NS” (FIG. 2). NS coated with only mPEG-SH were termed “PEG-NS.” Details of the synthesis are described below .

[0151] Human and mouse cross-reactive FZD7 antibodies (LSBio; purchased at 1 mg / mL) were diluted in sterile IX PBS at 10X the original volume and purified by centrifugal filtration at 8 °C, 4200rpm for 30 min using 10 kDa centrifugal filtration tubes (Amicon) to remove the 0.02% sodium azide, 50% glycerol, and 0.5% BSA present in the original solution. The purified antibodies were resuspended in IX PBS to the original volume to maintain the concentration of 1 mg mL1. To facilitate conjugation to NS, the FZD7 antibodies were incubated with 5 kDa or 2 kDa orthopyridyl disulfide-PEG-succinimidyl valerate (OPSS-PEG-SVA, Laysan Bio) in 100 mM sodium bicarbonate. Volumetrically nine parts OPSS-PEG-SVA in sodium bicarbonate was reacted with one part antibody at a two PEG: one antibody molar ratio, followed by rocking overnight at 4 °C. The PEGylated antibodies were aliquoted into microcentrifuge tubes and stored at -20 °C until used for NS conjugation.

[0152] P-catenin siRNA oligonucleotides were purchased as single strands from Integrated DNA Technologies. The sense strands had 3' thiol modification to allow functionalization to the gold surface of NS. Complementary sense and antisense strands were mixed in duplex buffer (IDT) in equimolar amounts, heated at 95 °C for 5 min in a thermomixer, and slowly cooled to 37 °C for over 1 h to facilitate duplexing. Duplexed siRNA was aliquoted into small volumes and stored at -80 °C until used for NS attachment. The siRNA sequences used for studies in MDA-MB-231 human triple-negative breast cancer cells were: P-catenin sense— 5'-AGC UGA UAU UGA UGG ACA GTT / iSpl 8 / iSp!8 / / 3ThioMC3-D / -3'; p- catenin antisense — 5'-CUG UCC AUC AAU AUC AGC UTT-3'. The siRNA sequences used for studies in 4T1 murine triple-negative breast cancer cells were: P-catenin sense — 5'- GCU GAU AUU GAC GGG CAG UAU / iSpPC / iSpPC / / 3ThioMC3-D / -3'; p-catenin antisense— 5'- AUA CUG CCC GUC AAU AUC AGC-3'.

[0153] PEGylated FZD7 antibodies and duplexed siRNAs were thawed on ice for ~30 min prior to NS conjugation. To coat NS with cross-reactive FZD7 antibodies and siRNAs targeting human P-catenin (FIG. 2 A), the NS were diluted to an optical density (OD) of 1.5 at 810 nm in RNase-free milliQ water (corresponding to an NS concentration of ~4.5xl09NS / mL), then PEGylated FZD7 antibodies were added at a ratio of 1200 or 1000 antibodies per NS to prepare FZD7-NS or Combo-NS, respectively. The solution was vortexed briefly, rocked at 4 °C for 1 h, then briefly vortexed again and bath sonicated. Next, 0.2% Tween-20 and NaCl (at concentrations of 40 mM and 12 mM for Combo-NS and Pcat-NS, respectively) were added. After 5 min incubation at room temperature (RT). siRNA duplexes were added at 0.25 or 0.1 nmol siRNA per mL of NS for Combo-NS and Pcat-NS, respectively. The solution was vortexed and rocked at 4 °C for 6 h, with brief vortexing and bath sonication performed every 1.5 h. At every 3 h NaCl was added to an end concentration of 100 or 200 mM (for Combo-NS and Pcat-NS. respectively). This salt aging step screens charges between duplexes to maximize siRNA loading on NS. The solution rocked at 4 °C for at least 12-14 h, then the functionalized NS were vortexed and bath sonicated. Lastly, 5 kDa mPEG-SH (Laysan) was added to the NS at a concentration of 10 pM or 20 pM (for Pcat-NS and Combo- / FZD7-NS, respectively). After rocking 4 h at 4 °C, unbound biomolecules were removed via centrifugation (performed thrice at 500 g). After removal of the supernatant, the nanocarriers were diluted in RNase-free lx PBS with 100X less volume than the starting NS volume and stored in LoBIND centrifuge tubes at 4 °C until use. All NS concentrations were calculated based on Beer’s law using the peak extinction (-810 nm) measured on a Can- 60 UV-visible spectrophotometer. When Combo- NS and Pcat-NS were prepared using FZD7 antibodies and siRNAs targeting mouse P- catenin, changes to the protocol included that NaCl was added to a final concentration of 300 mM for Pcat-NS or 1000 mM for Combo-NS and the concentration of mPEG-SH added to both was 20 pM.

[0154] The synthesis protocol described above, which used different concentrations of antibodies, siRNA duplexes, mPEG-SH, Tween-20, and NaCl in the preparation of each nanocarrier type, was developed to yield equivalent antibody loading between Combo-NS and FZD7-NS and equivalent siRNA loading betw een Combo-NS and Pcat-NS (FIG. 2; note that FIG. 2 shows data for nanocarriers prepared using siRNAs targeting human P- catenin). Extensive studies investigating parameters that influence biomolecule loading on NS were performed as detailed in Dang et al., Advanced Therapeutics. 2024; 7(6): 2300426. The inventors initially examined how the order of biomolecule addition (antibody then siRNA versus siRNA then antibody) w ould impact the loading of each molecule on Combo- NS, where the time delay between addition of either molecule was 1 hour. The siRNA and antibody loading on the Combo-NS were measured using a Quant-iT OliGreen ssDNA quantification assay and an enzyme linked immunosorbent assay (ELISA), respectively. This analysis revealed it is important to add antibodies first to yield >100 antibodies per NS, and that adding antibodies first does not hinder the loading of subsequently added siRNA (see FIG. SI in Dang et al., Advanced Therapeutics, 2024; 7(6): 2300426). Then inventors also tested how the salt aging conditions impacted biomolecule loading by adding NaCl to final concentrations ranging from 200 - 1200 mM. This revealed that increasing the concentration of NaCl improved the loading of both antibodies and siRNAs on Combo-NS (see FIG. S2 in Dang et al., Advanced Therapeutics, 2024; 7(6): 2300426). It also revealed that generally -2X the NaCl concentration is required in Combo-NS to yield equivalent siRNA loading as Pcat-NS (i.e., there is similar siRNA loading for Combo-NS made with 800 mM NaCl as Pcat-NS made with 400 mM NaCl). Finally, the inventors also investigated how7mPEG-SH concentration affects biomolecule loading. It was determined that increasing concentrations of mPEG-SH from 5 pM to 20 pM tends to enhance biomolecule loading (see FIG. S3 in Dang et al., Advanced Therapeutics, 2024; 7(6): 2300426). Ultimately, these investigations led to the selection of parameters described in the synthesis protocol above.

[0155] The hydrodynamic diameter and zeta potential of the bare and functionalized NS were measured using an Anton Paar Litesizer with at least three different batches diluted in milliQ water. RNA loading on NS was measured using a Quant-iT OliGreen ssDNA quantification kit. For this, RNA-loaded NPs w ere diluted to OD810 nm= 1, then further diluted with equal volume of 8 M Urea. The solution was heated at 45 °C and vortexed at 400 rpm for 20 min to denature the duplexes such that thiolated sense strands remained bound to NS while antisense strands were released. Following this, the NS were pelleted twice at 1500 g for 5 min, and the supernatant with released antisense strands was collected for quantification. The supernatant w as added to a 96-well plate with black walls and bottom in triplicates and the OliGreen™ dye (diluted per manufacturer instructions) was added in equal volume. The plate was analyzed with 480 nm excitation / 520 nm emission using a Biotek Synergy Hl Microplate Reader. The fluorescence was compared to a standard curve of known antisense strand concentration (ranging from 0 - 40 nM) to reveal the number of antisense strands in the sample. Dividing the number of antisense strands measured by the number of NS present in the solution revealed the siRNA loading per NS.

[0156] Antibody loading on NS was quantified using an enzyme linked immunosorbent assay (ELISA). Briefly, centrifuge tubes were pre-coated with 1 mL of 3% w / v bovine serum albumin in IX PBS (PBSA) and rocked for 30 min at RT. After the blocking step, the PBSA w as removed via aspiration. Meanwhile, antibody-coated NS and control PEG-NS were diluted to OD810 nm= 1. A stock suspension of secondary horseradish peroxidase (HRP)-anti rabbit IgG (HRP-AR. Seracare Life Sciences) was prepared to 100 pg / mL in PBSA. The HRP-AR was then added to the NS suspensions at 10% volumetrically, with an end concentration of 10 pg / mL. The samples were then vortexed, covered, and incubated at RT for 1 h. Following incubation, the samples were centrifuged at 1500 g for 5 min at RT followed by removal of 90% of the supernatant. The pellet was dissociated via bath sonication, and the samples resuspended in the same volume of PBSA as originally removed. These purification steps were repeated two more times. After the last spin step, the samples were resuspended to their original volumes in PBSA. Next, 2.5% of the sample suspension was set aside for antibody quantification (positive samples) and 50% of the sample was set aside for UV-Vis spectrophotometry to quantify the end NS concentration. The remainder of the sample was centrifuged once more at 1500 g for 5 min at RT to use the supernatant for background quantification (negative samples). A standard curve was made from the stock 100 pg mL-1 HRP-AR ranging from 1.9E-4 to IE-1 pg / mL HRP-AR diluted in IxPBS. The positive and negative (pos / neg) samples were plated in clear 96 well plates in technical triplicates at a 10X dilution in IX PBS. The standard curve was plated at the same end volume as the diluted pos / neg samples. A 5X volume of 3, 3', 5,5'- tetramethylbenzidine (TMB Core, BioRad) was added to the samples and standard curve. The plates incubated at RT for 1 min and then the reaction was stopped by adding 2 M sulfuric acid at a one-to-one volume to TMB core. The absorbance at 450 nm was then analyzed using a Biotek Synergy Hl Microplate Reader. The negative sample values were subtracted from the positive samples values to remove background signal and then the data was plotted against the standard curve readings. To calculate the number of antibodies per NS, the number of NS were calculated by measuring the absorbance of the 50% NP suspension that was set aside previously, which was further diluted 2.5X before measuring the extinction spectrum in a Cary 60 UV-vis spectrophotometer. Dividing the concentration of antibodies loaded by the concentration of NS present in the solution revealed the antibody loading per NS.

[0157] Characterization of the Combo-NS. Pcat-NS, and FZD7-NS prepared using human / mouse cross-reactive FZD7 antibodies and siRNAs targeting human P-catenin is shown in FIGS. 2B-D. The conjugated NS were ~20 nm larger than bare NS and were also more neutral in charge, indicating biomolecule attachment (FIGS. 2B, 2C). Combo-NS and FZD7-NS had -130 antibodies per NS and Combo-NS and Pcat-NS had -2400 siRNA duplexes per NS (FIG. 2D).

[0158] The inventors evaluated the binding of each nanocarrier to MDA-MB-231 triplenegative breast cancer (TNBC) cells that have high FZD7 expression versus MCF10A non- cancerous breast epithelial cells that have low FZD7 expression. To visualize nanocarrier binding to each cell t pe, the inventors used two-photon microscopy to detect the photoluminescence emitted by NS in response to their excitation with a femtosecond-pulsed laser tuned to the peak plasmon resonance wavelength. For these studies, MDA-MB-231 and control MCF10A cells were seeded in Nunc Lab-Tek #1 8-well chamber slides at 250,000 and 100,000 cells per well, respectively, and left to adhere for at least 20 h while incubating at 37 °C, 5% CO2. The next day media was removed and 300 pL of PEG-NS. FZD7-NS, ?cat-NS, or Combo-NS was added at OD810 nm= 1 in complete media for both cell lines. The cells were incubated at 37 °C for 1 h, the media was gently removed, and the cells were rinsed thrice with warm Dulbecco’s PBS (DPBS). Cells were then fixed with 4% paraformaldehyde in IxPBS for 15 min at RT and neutralized with lx PBS afterwards. Next, the cell membranes were dyed using CellVue Claret Red Membrane Dye for 8-10 min at RT covered, with the dye prepared per manufacturer’s instructions. The dye was neutralized with 1% w / v BSA in IxPBS. The dye solution and neutralizer were than aspirated and cells covered with lx PBS. Slides were imaged using a Zeiss LSM 880 Multiphoton Microscope with a 20 - / 0.8 NA water objective, which was immersed in a droplet of water to provide the correct contrast and visualization upon connection to the slides. The NPs in the slide samples were excited by the multiphoton laser tuned to Aexcitation = 800 nm with a pinhole of 1.57AU and a detection range Remission = 400-550 nm. The CellVue

[0159] Claret Red-labeled cell membranes were visualized at / Excitation = 655 nm and / Emission of 675 nm. The FZD7 antibody-coated NPs (FZD7-NS and Combo-NS) exhibited greater binding to the MDA-MB-231 cells than the PEG-NS and Pcat-NS controls (FIG. 3 A). There was also minimal attachment of all four NP types to control MCF10A cells.

[0160] To corroborate the imaging data, the inventors used flow cytometry to examine cellular uptake of nanocarriers passivated with Cy5-tagged mPEG-SH. For this, MDA-MB- 231 and MCF10A cells were seeded in 96-well plates at 75,000 or 50,000 cells per well, respectively, allowed to adhere, then treated with nanocarriers for 1, 4, or 8 h at 37 °C . After incubation, the media was removed and the cells rinsed thrice with warm DPBS. Cells were then trypsinzed, neutralized, and transferred to Eppendorf tubes for flow cytometry performed with an Acea Novocyte 2060 Flow Cytometer. The parameters were set to 100 pL / min to a cell cut off at 10,000 cells gated for singlet cells. Cells were then further gated using the APC-Cy7 filter where cells were excited at 640 nm and detected using the 780 / 60 nm filter to assess the shifts in Cy5 signal. In processing the data, the raw median value of Cy5 signal was averaged in triplicate and the NT (non-treated) group values were subtracted from each NS group to evaluate the shift from the baseline cells, which should have minimal Cy5 signal. As with the multiphoton microscopy data, flow cytometry showed the antibody-coated NPs (i.e., FZD7-NS and Combo-NS) were bound or internalized by MDA- MB-231 cells more than the PEG-NS and Pcat-NS (FIG. 3B), as evidenced by greater Cy5 signal (which was taken to correlate with nanocarrier binding / uptake). Additionally, the Cy5 signal was higher in MDA-MB-231 cells than in MCF10A cells, indicating selective binding.

[0161] The inventors next evaluated gene regulation at the mRNA level via RT-qPCR. For this experiment. MDA-MB-231 cells were plated at 40,000 cells per well in 12-well plates and dosed with nanocarriers at a concentration of ~3xl09NS / mL. In addition to analyzing expression of P-catenin, the key mediator of Wnt signaling, the inventors also evaluated several downstream target genes (Axin2, CCND1, C-myc, Nanog, Snail, and Survivin). Details of the PCR protocol including primers are provided in Dang et al., Advanced Therapeutics, 2024: 7(6): 2300426. At 72 h post-addition to cells, all three nanocarriers (Combo-NS, FZD7-NS, and Pcat-NS) decreased mRNA expression of the evaluated genes relative to untreated cells (NT = non-treated), and Combo-NS provided the most robust inhibition of P-catenin, C-myc, and Snail (FIG. 4). PEG-NS did not alter gene expression in cells at any timepoint (see supporting information of Dang et al.. Advanced Therapeutics. 2024; 7(6): 2300426), confirming the gene regulation was due to the delivered FZD7 antibodies and P-catenin siRNAs.

[0162] The inventors proceeded to examine the ability of the antibody and siRNA nanocarriers to cause a reduction in cancer cell proliferation (FIG. 5), migration (FIG. 6), and spheroid formation capacity (FIG. 7). Cellular proliferation is a hallmark of cancer and is associated with genes including C-myc and CCND1. Since the Wnt inhibitory antibody and siRNA nanocarriers reduced expression of these genes, the inventors evaluated their impact on cell proliferation using an EdU assay (FIG. 5). In this experiment, 10,000 cells per well in 24-well plates were treated with nanocarriers at a concentration of ~ 1.5x1010NS / mL. After 48, 72, and 96 h, proliferation was measured with a Click-iT Edu Cell Proliferation Kit. At all three timepoints, Pcat-NS treated cells had comparable proliferation rates as the NT group. In contrast, both FZD7-NS and Combo-NS decreased cell proliferation at 48 h, but only Combo-NS maintained suppression of proliferation through 96 h (FIG. 5).

[0163] Cellular migration and invasion contribute to cancer metastasis and are also regulated by Wnt signaling. To determine whether the nanocarriers could suppress cellular migration, the inventors pretreated GFP-expressing MDA-MB-231 cells with the nanocarriers (1.5 x IO10NS / mL) for 96 h and then reseeded 10,000 cells in transwell porous membranes to evaluate their migratory behavior over the following 6-48 h (FIG. 6A). Images were acquired with an Axioobserver Z1 inverted fluorescence microsope. At 6 h post reseeding, all three Wnt inhibitory nanocarriers reduced cell migration across the membrane compared to the NT group, with the most dramatic reduction observed in Combo-NS treated cells (FIG. 6B). Over time, the cells treated with Combo-NS continued to migrate more slowly than cells in the other treatment groups (FIGS. 6B, 6C). In fact, while the effects of FZD7-NS and [kat-NS were diminished by 16 h, the Combo-NS suppressed migration through 48 h. It should be noted that at this later timepoint the cells may have divided, such that the results indicate effects on both migration and proliferation, but timepoints before 24 h demonstrate the effect on migration alone.

[0164] Wnt signaling is also associated with cancer sternness, which contributes to tumor initiation, treatment resistance, relapse, and metastasis. The inventors evaluated the impact of nanocarrier treatment on stemness / self-renewal through spheroid formation assays (FIG. 7 A). MDA-MB-231 cells plated at 20,000 cells per well in 24-well plates were pretreated with nanocarriers (1.5xl010NS / mL) for 24-72 h and then reseeded at 10,000 cells per well in sphere forming media in low-adhesion U-bottom well plates to facilitate spheroid formation. After 1 w eek of incubation in a 37 °C, 5% CO2 humidified incubator, the spheroids were imaged with a Zeiss Axioobserver Z1 inverted microscope and their metabolic activity assessed via an alamarBlue assay. This revealed that a treatment time of 72 h is needed to impair sphere viability (FIG. 7B). Under these conditions, spheroid metabolic activity was reduced by -34% for Combo-NS, -18% for |3cat-NS, and -13% for FZD7-NS compared to the NT group (FIG. 7B). Upon imaging the spheres, it was observed that all three nanocarriers physically hindered spheroid formation, with some treated samples forming loose cell clusters rather than spheres (FIG. 7C, top row) and others forming less dense spheres as indicated by reduced opacity' (FIG. 7C, bottom row). The appearance of loose cell clusters indicates the nanocarriers have reduced the cells' ability to condense into compact spheroids, while the appearance of less dense spheres indicates the nanocarriers have reduced the proliferation of cells within the spheres (which matches the impact on spheroid metabolic activity). As with the other in vitro assays, Combo-NS had the most dramatic effects on spheroid formation and viability , indicating a potential benefit of suppressing Wnt signaling in TNBC cells at both the receptor and effector level.

[0165] To test the nanocarriers7ability to reduce the growth of established metastases, the inventors used an experimental metastasis model in which 105firefly luciferase-expressing MDA-MB-231 cells are inoculated into the tail vein of female nude mice leading to grow th of metastatic tumor nodules in the lungs. During the study, metastasis burden in the lungs was monitored by bioluminescence imaging in an IVIS Lumina III instrument after injecting mice intraperitoneally with 150 mg / kg of D-luciferin diluted in saline. Once lung metastases formed, mice received saline or nanocarriers intravenously, for six total injections, and bioluminescence imaging was used to monitor tumor growth (FIG. 8A). For these studies, the NS were coated with FZD7 antibodies using 2 kDa orthopyridyl disulfide- PEG-succinimidyl valerate (OPSS-PEG-SVA) linkers rather than 5 kDa OPSS-PEGSVA linkers used in other experiments (note that the 5 kDa linkers increase antibody accessibility, which is why they were used in all other experiments). The injection volume was 100 pL, and the nanocarrier concentration corresponded to -250 x 1012M NS. equal to -300 x | ()9M siRNAs and -15 x 109M antibodies. Using the dose schedule depicted in FIG. 8B, the inventors found that all three Wnt inhibitory nanocarriers decreased metastasis burden, with the lowest luminescent signal observed following treatment with Combo-NS (FIG. 8C, 8D). While saline-treated mice exhibited large increases in mean luminescence versus time indicative of metastatic growth, Combo-NS-treated mice had stable or significantly reduced tumor signal (FIG. 8D). Consequently, there was a large difference in metastatic burden between these groups at the study end. After animal sacrifice, the inventors quantified gold content in excised lungs and other tissues via inductively coupled plasma-mass spectrometry (ICP-MS) and found that the Combo-NS and FZD7-NS exhibited 4-5X higher accumulation in the lungs than the Pcat-NS (FIG. 9A). Darkfield microscopy images of lung sections showed the antibody -modified NS were associated primarily with tumor tissue rather than with adjacent normal lung (FIG. 9B). These data indicate Frizzled7 antibodies increase nanocarrier delivery to the desired site and support the potential of Wnt inhibitory antibody and siRNA nanocarriers to treat pre-existing TNBC metastases.

[0166] Given the promising observations in the experimental lung metastatic model, the inventors proceeded to test whether the antibody and siRNA nanocarriers would also be effective against TNBC in a syngeneic murine model that uses immune competent rather than nude mice. In shifting to this model, they altered the siRNA sequence to target murine P-catenin. and reformulated the nanocarriers using both antibodies and siRNAs designed to target murine FZD7 and P-catenin. These nanocarriers had mean hydrodynamic diameter of -185 nm and zeta potential of — 18 mV. FZD7-NS and Combo-NS had antibody loading of -230 antibodies per NS while Pcat-NS and Combo-NS had siRNA loading of -2000 siRNA molecules per NS. To generate the tumor model, 100,000 4Tl-luc2 murine TNBC cells were injected in the mammary fat pad of Balb / c mice. Treatment began when tumor volume reached -100 mm3(defined as Day 0), and nanocarriers were administered weekly thereafter for 5 total injections (FIG. 10A). The administration volume was 100 pL, and the nanocarrier concentration was 1.9 x io11NS / mL, which corresponds to -73 x | ()9M FZD7 antibodies and -640 x 109M siRNA. Primary tumors were surgically removed from mice on Day 13, after which treatment continued while monitoring for tumor recurrence and metastasis by bioluminescence imaging. All three nanocarriers significantly reduced primary tumor growth compared to saline-injected controls (FIG. 10B). While tumors in saline-treated mice grew by an average of 315 mm3between Day 0 and 13, those in the nanocarrier-treated mice grew by an average of 184-201 mm3, which corresponds to an inhibited growth rate of 36-42% (FIG. 10B, Right). After surgical excision, primary tumors were sectioned and stained for P-catenin with hematoxylin as a counterstain to quantify the average percent positive cells (FIG. 10C). Combo-NS significantly reduced the fraction of P-catenin positive cells to -47%, compared to -71% for saline treated mice, 60% for FZD7- NS treated mice, and 56% for Pcat-NS treated mice. This demonstrates the nanocarriers suppress intratumoral Wnt signaling.

[0167] Following primary' tumor removal, tumor regrowth and lung metastatic spread was monitored via IVIS imaging. All three nanocarriers slowed recurrence compared to saline- treated mice based on average luminescent signal, although the differences were not significant at the 95% confidence level, likely due to the high variability in recurrent tumor burden, particularly in the saline control (FIG. 11A). Evaluation of lung metastasis revealed more differences between the nanocarrier types. Lung metastasis occurred more slowly in mice treated with Wnt inhibitory nanocarriers versus saline (FIG. 1 IB). Mice treated with nanocarriers didn’t have metastatic signal appear until around Day 25 compared to -Day 20 for the saline group. While the metastatic burden (based on luminescent signal) was equivalent between nanocarrier groups for mice that formed distant disease, it is noteworthy that the fraction of mice that formed metastasis differed based on treatment type. Approximately 15% of mice treated with 0cat-NS or FZD7-NS did not form metastasis, compared to 33% of mice treated with Combo-NS (FIG. 1 1 C). This demonstrates a distinct benefit of antibody / siRNA co-delivery.

[0168] To shed light on the therapeutic results, the accumulation of the nanocarriers in primary tumors excised at Day 13, as well as in recurrent tumors and metastatic lungs excised at animal sacrifice, was assessed by measuring gold content in the tissues via ICP- MS. The nanocarriers that incorporated FZD7 antibodies (i.e., FZD7-NS and Combo-NS) exhibited increased delivery to primary tumors and recurrent tumors (FIG. 12). In the lungs, Combo-NS exhibited the most accumulation.

[0169] The inventors also analyzed gold content in major clearance and non-clearance organs, which indicated distribution to the liver and spleen, with lesser delivery to intestines and other tissues. Importantly, microscopic evaluation of H&E-stained liver, spleen, kidney, and heart (FIG. 13) showed that all three nanocarriers were well tolerated in this model. Animal weight versus time was also consistent between treatment groups, suggesting the nanocarriers have satisfactory’ biosafety under the dosing conditions tested.

[0170] Collectively, the results of this study demonstrate Frizzled7 antibodies and 0-catenin siRNAs, along with mPEG-SH, can be loaded on nanoshells to produce antibody / siRNA nanocarriers that can preferentially bind and enter cells that overexpress FZD7, suppress Wnt signaling in these cells, and hinder oncogenic cell behaviors. In vivo, the nanocarriers can accumulate in primary tumors, recurrent tumors, and metastatic lesions to reduce disease burden. Moreover, the nanocarriers are well tolerated, without signs of toxicity under the tested conditions.

[0171] Example 2. Frizzled7 antibody / 0-catenin siRNA nanocarriers based on polymeric cores can suppress oncogenic and stem-like behavior in triple-negative breast cancer cells

[0172] In another embodiment, the inventors synthesized antibody and / or siRNA nanocarriers using poly(lactic-co-glycolic acid)-poly(ethylene glycol)-maleimide (PLGA- PEG-Mal) nanoparticles as the core. In one body of work, the inventors showed how various parameters influence the final loading density of antibodies and siRNAs on PLGA- PEG-Mal nanocarriers (Hoover et al. ACS Omega, 2024; 9: 47637-47646). For this study, the inventors used rabbit anti-human FZD7 antibodies (LS-C383580 from LSBio) and the siRNA sequences were: P-catenin sense — 5'-AGC UGA UAU UGA UGG ACA GTT / 3ThioMC3-D / - 3'; -catemn antisense— 5' - CUG UCC AUC AAU AUC AGC UTT - 3'. It was demonstrated that these antibodies can be treated with TCEP to expose free thiols and conjugated directly to PLGA-PEG-Mal or that antibodies can be attached to TCEP -treated OPSS-PEG-SVA linkers prior to nanoparticle conjugation (Hoover et al. ACS Omega, 2024; 9: 47637-47646). By tuning the number of antibodies added to the reaction (either 1,000 or 3,000 per nanoparticle), the type of conjugation buffer (2 mM EDTA in PBS or 100 mM CaCh in water), and the attachment chemistry (via thiolated antibodies or thiolated PEG linkers), the surface coverage of antibodies on the PLGA-PEG-Mal could be tuned from 17% to 88%. The highest loading was achieved using 3,000 antibodies per nanoparticle in the reaction, direct attachment of thiolated antibodies, and 2 mM EDTA buffer. Similarly, it was shown that adjusting the concentration of CaCh (from 50 mM to 225 mM) and Tween-20 (from 0 to 0.5%) present during attachment of siRNAs could impact the amount of siRNA conjugated to the PLGA-PEG-Mal, with loading ranging from 1.6 pg RNA / mg PLGA to 2.7 pg RNA / mg PLGA. The highest loading of siRNA was achieved using 100 mM CaCh combined with 0.1% Tween-20, so this protocol is detailed later below as it was used in follow-on studies. When both FZD7-PEG linkers and siRNAs were added to PLGA-PEG-Mal, it was observed that increasing the loading of antibodies (from 229 per NP to 358 per NP) decreased the loading of siRNA (from 2.7 pg / mg PLGA to 2.0 pg / mg PLGA). Overall, this work outlined a basis for how conjugation parameters could impact the attachment of two different biomolecules to polymer NPs via maleimide- thiol chemistry (Hoover et al. ACS Omega, 2024; 9: 47637-47646).

[0173] From this work, the following protocol was established for coating PLGA-PEG-Mal nanoparticles (NPs) with Frizzled7 antibodies via direct linkage. PLGA-PEG-Mal NPs were synthesized using a single emulsion oil-in-water method. PLGA-PEG-Mal (20 kDa 50:50 PLGA, 5 kDa PEG, Nanosoft Polymers) was dissolved in di chloromethane (DCM) at 2 mg / mL. 1 rnL of the PLGA-PEG-Mal / DCM solution was added to 3 rnL of either 0.1%, 0.25%, or 0.5% PVA dissolved in PBS. This oil-in-water solution was then probe sonicated on ice with a Fisherbrand model 120 Sonic Dismembrator (Fisher Scientific) at 80% amplitude for 60 s (10 s on, 5 s off). The DCM solvent was allowed to evaporate for 4 h at room temperature under continuous stirring at 800 rpm. Following solvent evaporation, the resulting NPs were purified to remove excess solvent using Millipore 10 kDa molecular weight cut-off (MWCO) filters (4200 g, 30 min, 4 °C) once then transferred to 2 mL Eppendorf tubes, resuspended in 2 mL of MilliQ water and centrifuged for 15 min at 20,000 ref at 4 °C to pellet the NPs. The supernatant was removed, and the NP pellet was resuspended in 2 mL fresh MilliQ water and washed once more before being resuspended in 200 pL MilliQ water. Rabbit anti -human FZD7 antibodies (LS-C383580, LSBio) were used. To modify the antibodies, they were first incubated with 100X molar excess of TCEP (Sigma-Aldrich) in phosphate buffered saline (PBS) containing 2 mM EDTA (ThermoFisher) for 1 h at 4 °C on a rocker. Following incubation with TCEP, the antibodies were washed thrice in 10 kDa Coming Spin-X UF (Sigma- Aldrich) concentrators for 10 min at 12,000 ref, 4 °C with PBS to remove excess TCEP. To conjugate antibodies to the NP surface, thiol-modified antibodies were incubated with the NPs in either PBS containing 2 mM EDTA or MilliQ water containing 100 mM calcium chloride (CaCh, Sigma- Aldrich) overnight on a rocker at 4 °C. The NP concentration in the solution was 1 mg / mL. Next, 10 pg of FZD7-SH was added per mg ofNP for the Low loading condition and 30 pg of FZD7-SH was added per mg of NP for the High loading condition. After overnight incubation while rocking at 4 °C. the NP solution was centrifuged for 15 min at 20,000 ref at 4 °C to pellet the NPs and remove any unconjugated antibody left in the supernatant. The NP pellet was resuspended in MilliQ water and washed twice more.

[0174] In a second body of work, the inventors used specific parameters identified from the above work to produce PLGA-PEG-Mal nanoparticles coated with Frizzled7 antibodies and P-catenin siRNAs or with just P-catemn siRNAs and examined the impact of these nanocarriers on treated triple-negative breast cancer cells (Hoover et al., Journal of Biomedical Materials Research Part A, 2025; 113: e37867). For this study, PLGA-PEG- Mal NPs were synthesized by a single emulsion oil-in-water method. PLGA-PEG-Mal (20 kDa 50:50 PLGA, 5 kDa PEG, Nanosoft Polymers) was dissolved in dichloromethane (DCM) at 2 mg / mL. 1 mL of the PLGA-PEG-Mal / DCM solution was added to 3 mL of 0.25% polyvinyl alcohol (PVA) dissolved in phosphate-buffered saline (PBS). This oil-in- water solution was then probe sonicated on ice with a Fisherbrand model 120 Sonic Dismembrator at 80% amplitude for 60 s (10 s on, 5 s off). The DCM solvent was allowed to evaporate for 4 h at room temperature under continuous stirring at 800 rpm. Following solvent evaporation, the resulting NPs were purified to remove excess solvent using Millipore 10 kDa molecular weight cutoff (MWCO) filters (4200 g, 30 min, 4 °C) once and then transferred to 2 mL Eppendorf tubes, resuspended in 2 mL of MilliQ water, and centrifuged for 15 min at 20,0000 ref at 4 °C to pellet the nanoparticles. The supernatant was removed, and the nanoparticle pellet was resuspended in 2 mL fresh MilliQ water and washed one more before being resuspended in 200 pL MilliQ water.

[0175] P-catenin oligonucleotides (sipcat) or control oligonucleotides using a scrambled P- catenin sequence (siScr) were purchased as separate sense and antisense strands from Integrated DNA Technologies (IDT). The sense strands had a 3' thiol modification to allow for conjugation via maleimide thiol chemistry. The siRNA sequences were: P-catenin sense— 5 -AGC UGA UAU UGA UGG AC A GTT / 3ThioMC3-D / - 3'; P-catenin antisense— 5 -CUG UCC AUC AAU AUC AGC UTT - 3'; Scramble sense 5 -GUC AGA UGU AGG GCG AUU ACU / 3ThioMC3-D / - 3'; Scramble antisense 5 -AGU AAU CGC CCU ACA UCU GAC - 3'. Upon resuspension of the separate oligonucleotides in duplex buffer (IDT), the sense strand was treated with 100X molar excess TCEP for 1 h at 4 °C on a rocker to break the disulfide bond and create a free thiol group. After the disulfide reduction, an equimolar amount of the complementary antisense strand was added, and the solution was heated at 95 °C for 5 min in a thermomixer and then slowly cooled to 37 °C over 1 h to facilitate duplexing. The final siRNA concentration was measured using a Thermo Scientific NanoDrop One Microvolume UV-Vis Spectrophotometer.

[0176] To conjugate only siRNA and mPEG-SH to the PLGA-PEG-Mal surface, thiol- modified P-catenin siRNA (sipcat) or scrambled sequence siRNA (siScr) were incubated with the NPs (at a concentration of 1 mg / mL) in 100 mM calcium chloride (CaCh, Sigma- Aldrich) containing 0.1% Tween-20 (Sigma- Aldrich) overnight stirring at 800 rpm at room temperature. The concentrations of 100 mM calcium chloride and 0.1% Tween-20 were based on the work described above (Hoover et al. ACS Omega, 2024; 9: 47637-47646) that showed these parameters maximize siRNA loading on PLGA-PEG-Mal nanoparticles. The siRNA was added to the nanoparticle solution in a ratio of 1 nmol per mg nanoparticle. Following overnight incubation, mPEG-SH (Laysan Bio) was added to the nanoparticle solution and incubated for 1 h. The solution was then transferred to 1.5 mL Eppendorf tubes and centrifuged for 15 min at 20,000 ref at 4 °C to pellet the nanocarriers and remove any unconjugated siRNA left in the supernatant. The nanocarrier pellet was resuspended in MilliQ water and washed twice more. To conjugate both antibodies and sipcat (as well as mPEG-SH) to the PLGA-PEG- Mal surface (FIG. 14A), the nanoparticles were first suspended in a 0.1% Tween-20 and 100 mM CaCh buffer at a concentration of 1 mg / mL. FZD7-linker antibodies were prepared as follows. Rabbit anti-human FZD7 antibodies were purchased from LSBio. To attach a linker to the anti-FZD7 antibodies, a 5 kDa orthopyridyl disulfide-PEG succinimidyl valerate (OPSS-PEG-SVA) linker was first reconstituted in 100 mM sodium bicarbonate before being incubated with 100* molar excess TCEP for 1 h at 4°C on a rocker to break the disulfide bond and create a free thiol. The thiolated linker was then incubated with the anti-FZD7 antibodies in a 2: 1 PEG: antibody molar ratio overnight on a rocker at 4 °C. Following overnight incubation, the antibodies were washed thrice in 10 kDa Coming Spin- X UF concentrators for 10 min at 12,000 ref and 4 °C with PBS containing 2 mM ethylenediaminetetraacetic (EDTA) to remove unbound linker. After preparation. FZD7- antibody linkers were added to the PLGA-PEG-MAL solution at a ratio of 30 pg antibodies per mg PLGA and allowed to stir at 800 rpm for 1 h at room temperature before adding 1 nmol of the TCEP -treated siRNA per mg PLGA. The solution was left to stir overnight at room temperature. Following overnight incubation, mPEG-SH was added to the solution and incubated for 1 h. The solution was transferred to 1.5 mL Eppendorf tubes and centrifuged for 15 min at 20,000 ref and 4 °C to pellet the nanocarriers and remove any unconjugated FZD7-linker or sipcat left in the supernatant. The nanocarrier pellet was resuspended in MilliQ water and washed twice more.

[0177] Following surface modification, the nanocarriers were characterized for their hydrodynamic diameter, zeta potential, siRNA loading, antibody loading, and RNA release kinetics (FIG. 14B-D). A LiteSizer 500 dynamic light scattering instrument was used to measure zeta potential of samples diluted 1: 100 in MilliQ water. A NanoSight NS300 nanoparticle tracking analysis system was used to measure hydrodynamic diameter and nanoparticle concentration. siRNA loading was measured using Quant-iT PicoGreen dsDNA reagent and antibody loading was quantified using a solution-based ELISA as detailed in Hoover et al., Journal of Biomedical Materials Research Part A, 2025; 113: e37867. The siRNA nanocarriers had a mode hydrodynamic diameter of -176 nm and -166 nm for siScr-NPs and si cat-NPs, respectively. The FZD7-siPcat-NPs had a hydrodynamic diameter of -174 nm while the bare, unmodified NPs had a hydrodynamic diameter of -166 nm (FIG. 14B). The slightly larger diameter of the siScr-NPs as compared to the sipcat- NPs may be related to the higher loading density7of the siScr (FIG. 14C), though differences in diameter and loading density between these nanocarrier types were not statistically significant.

[0178] The zeta potential for the nanocarriers shifted from —31.1 ± 2.2 mV for the bare NPs to -14.4 ± 10, -21.4 ± 8.5, and -12.3 ± 7.8 mV for the siScr-NPs, sipcat-NPs, and FZD7- sipcat-NPs, respectively (FIG. 14B). This slight positive shift in zeta potential is indicative of the nanoparticle surface modifications, including the biomolecules and the mPEG passivation. Quantification of siRNA loading on the NP surface by PicoGreen assay yielded similar loading between the sipcat-NPs and the FZD7-sipcat-NPs while a slightly higher, but not significantly increased, loading was seen with the siScr-NPs (FIG. 14C).

[0179] RNA release was measured by adding 0.5 mg of the nanocarriers to 1 mL of either a storage condition (pH 7.4 MilliQ water, 4 °C), a physiological condition (pH 7.4 PBS containing 10% FBS, 37 °C shaking at 100 rpm), or a lysosomal microenvironment condition (pH 5.5 PBS, 37 °C shaking at 100 rpm). At each time point (1, 2, 4, 24, 48, 72 h), the sample was centrifuged for 15 min at 20,000 ref, and the supernatant was collected. The supernatant was read in triplicate in 100 pL volumes on a plate reader and compared to a known standard curve of siRNA suspended in each condition. Analysis of siRNA released from the sipcat-NPs indicated minimal loss over time when the NPs were stored in water at 4 °C for a 72-h time period (FIG. 14D). After the first 4 h, an insignificant amount (< 1%) of the loaded siRNA was released. Over the 72-h incubation, only 8. 1% ± 0.68% of the total loaded siRNA was lost. When the si0cat-NPs were incubated in a more acidic pH 5.5 PBS solution at 37°C, similar to the intracellular lysosomal environment, faster release of the siRNA was seen over the 72-h period (FIG. 14D). This faster release is attributed to the biodegradability of PLGA in acidic conditions. The nanocarriers suspended in this environment released almost 25% of their cargo during the first 4 h. Thereafter, the release became more gradual, with a final 55% ± 6.4% of the conjugated siRNA being released by the end of the 72-h incubation.

[0180] To assess nanocarrier uptake and confirm internalization of the siRNA cargo, the nanocarriers were synthesized using -catenin siRNA tagged with a Cy5-fluorophore (positioned at the 3' end of the antisense strand). After 24-h incubation with the nanocarriers (wherein 20,000 MD A-MB-231 cells seeded in 4-well chambered cover glass were treated with nanocarriers at a dose of 50 nM siRNA), treated MD A-MB-231 cells were fixed with 4% paraformaldehyde, permeabilized, stained with CellTracker Green chloromethyl derivatives of fluorescein diacetate (CMFDA). and imaged using a Zeiss LSM880 confocal microscope. The microscopy images confirmed that the siRNA cargo delivered by either the sipcat-NPs or the FZD7-sipcat-NPs were internalized by the cells and accumulated in the cytoplasm (FIG. 15 A). Note that while both NPs delivered siRNA into the cells, the mechanism of cellular entry is distinct, as siRNA-only NPs likely employ scavenger receptor-mediated uptake (as has been reported for other nucleic acid-coated nanocarriers) while antibody-modified NPs likely rely on receptor-mediated uptake.

[0181] Gene regulation in MDA-MB-231 cells due to the delivery of P-catenin siRNA was analyzed using RT-qPCR. Details of the PCR protocol may be found in Hoover et al., Journal of Biomedical Materials Research Part A, 2025; 113: e37867. Following 48-h cell treatment with nanocarriers (where IxlO5cells plated in 6-well plates were exposed to nanocarriers at a dose corresponding to 50 nM siRNA), a significant decrease in P-catenin mRNA expression was observed in cells treated with the sipcat-NPs compared to the NT (no treatment) control group (FIG. 15B). A marked decrease in P-catenin mRNA expression was also found when compared to the control siScr-NP treatment (0.69 ± 0.2 compared to 0.98 ± 0.1). FZD7-sipcat-NPs also decreased P-catenin mRNA expression, although to a lesser extent than sipcat-NPs. Other downstream Wnt target genes were also analyzed including Survivin, MDR-1 (multidrug resistance 1), and Axin2. The sipcat-NPs and FZD7- sipcat-NPs both inhibited Axin2 to a greater extent than controls, and reduction in survivin and MDR-1 was also observed.

[0182] One of the driving factors behind cancer metastasis is cell migration and invasion. Accordingly, the impact of nanocarrier treatment on cell migration w as determined using a scratch migration assay. Following treatment of 4xl04cells / well in 24-well plates with nanocarriers for 48 h at a dose corresponding to 50 nM siRNA, a scratch was made through the confluent MDA-MB-231 cells, and fresh media was added. Over the subsequent 20 h, cell infiltration into the open area was microscopically observed using an EVOS M5000 microscope and quantified. Analysis was performed using ImageJ. The area of the scratch was outlined in ImageJ based on the 0 h image, and the number of cells was counted using the ‘'Analyze Particles” feature. The same area outline for each well was then copied to the corresponding 20 h image, and the number of cells w as again counted. All experiments were performed in triplicate. After the 20-h period, cells that had been treated with either the sipcat-NPs or FZD7-sipcat-NPs showed less migration into the open scratch area as seen visually in FIG. 16A. Additionally, the samples treated with either NT (non-treated) or the siScr-NPs saw greater migration of the edges of the scratch and overall greater infiltration into the open area. Cell density within the scratch area was quantified in TmageJ. Cells treated with the sipcat-NPs had a significantly lower cell density at the 20-h time point (0.0005 ± 8.2e-5 cells / pm2) compared to the NT (0.0012 ± 0.0004 cells / pm2) and siScr-NP (0.0013 ± 0.0005 cells / pm2) groups (FIG. 16B). While not statistically significant at the 95% confidence level, the cells treated with FZD7-siPcat-NPs also showed a marked decrease in cell migration and cell density (0.0007 ± 4.9e-9 cells / pm2).

[0183] The inventors further evaluated the impact of the sipcat-NPs and FZD7-sipcat-NPs on cellular metabolic activity (using MTT assay) and spheroid formation capacity (indicative of self-renewal). After 48 h treatment of 5xl03cells per well in 96-well plates at a dose corresponding to 50 nM siRNA, MDA-MB-231 cells exposed to sipcat-NPs and FZD7-sipcat-NPs had statistically significantly lower metabolic activity than cells treated with siScr-NPs or NT (FIG. 17). To evaluate impact on spheroid formation, MDA-MB-231 cells were plated in triplicate in a low-adhesion 96-well U-bottom plate (BrandTech) at 5 x 103cells per well and incubated for 5 days in media containing 4% rat tail collagen type I. Spheroids were then treated with either NT or 50 nM siRNA of siScr-NPs, sipcat-NPs, or FZD7-sipcat-NPs for 72 h. Brightfield images of the spheroids were acquired at 4* magnification on day 0 and after 72 h of treatment using an EVOS M5000 microscope. Cell spheroids formed and treated for 72 h as described above were stained using a live / dead viability / cytotoxicity kit (Thermo Fisher) per the manufacturer's instructions. After the media / treatments were removed, 100 pL of a staining solution containing 1 :500 ethidium homodimer-1 and 1:2000 calcein-AM in PBS was added to each well. Samples were incubated in the dark for 45 min at room temperature. Images were acquired at 10x magnification on an EVOS M5000 microscope using Texas Red (586 nm excitation / 603 nm emission) and GFP (488 nm emission / 510 nm excitation) channels. Fluorescence was measured on a Synergy Hl plate reader using the same channels. Qualitative analysis through both brightfield imaging and live / dead staining indicated treatment with si(3cat-NPs and FZD7-siPcat- NPs led to greater structural changes. These spheroids did not maintain their round morphology throughout the treatment and saw greater levels of cell dissociation from the spheroids (FIG. 18A). Upon quantification of live signal in each spheroid using a plate reader (FIG. 18B), the FZD7-si0cat-NPs demonstrated significantly lower levels of viable, live cells (-28% lower) when compared to the NT control. The live signal from the FZD7-siPcat-NP treatment was also -12% lower than both the siScr-NPs and sipcat-NPs. Congruently, quantification of the dead signal (FIG. 18B) indicated a significantly greater (-50%) dead cell population after treatment with the FZD7-sipcat-NPs when compared to the NT control. The FZD7-sipcat-NP treatment led to -30% greater cell death than the siScr-NP and sipcat-NP treatments. These changes in spheroid morphology and viability after treatment with either sipcat-NPs or FZD7-siPcat-NPs suggest the nanocarriers can reduce the stem-like properties of treated cells.

[0184] Overall, this example demonstrates the synthesis and characterization of antibody and siRNA nanocarriers with a polymeric core and their use to suppress Wnt signaling and oncogenic behaviors in TNBC cells, including inhibition of cell migration, metabolic activity, and spheroid formation / self-renewal. Collectively, the results indicate Wnt-targeted therapeutics have great promise as a treatment for TNBC and other cancers characterized by hyperactive Wnt signaling through interactions at both the receptor (FZD7) and effector (P- catenin) levels.

[0185] All documents, books, manuals, papers, patents, published patent applications, guides, abstracts, and / or other references cited herein are incorporated by reference in their entirety. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

Claims

WHAT IS CLAIMED:

1. A method for suppressing a target gene involved in the Wnt / 0-catenin signaling pathway in target cells expressing a receptor capable of binding an extracellular Wnt ligand and an intracellular Wnt effector, comprising:(a) providing nanocarriers comprise:(i) first nanocarriers, wherein each of the first nanocarriers comprises a first nanoparticle and an antibody specific for the receptor, and the antibody is attached to the first nanoparticle;(ii) second nanocarriers, wherein each of the second nanocarriers comprises a second nanoparticle and a small interfering RNA (siRNA) specific for the Wnt effector, and the siRNA is attached to the second nanoparticle; and / or(iii) third nanocarriers different from the first nanocarriers and the second nanocarriers, wherein each of the third nanocarriers comprises a third nanoparticle, the antibody, and the siRNA, and the antibody and the siRNA are attached to the third nanoparticle; and(b) binding the nanocarriers to the target cells and / or internalizing of the plurality of the nanocarriers into the target cells, whereby the target gene is suppressed in the target cells.

2. The method of claim 1, wherein the first nanoparticle, the second nanoparticle, and the third nanoparticle are the same.

3. The method of claim 1 or 2, wherein the nanocarriers comprise the first nanocarriers, the method further comprising:(c) binding the antibody to the receptor, binding the first nanocarriers to the target cells, and phosphorylating and degrading the Wnt protein P-catenin in the target cells.

4. The method of claim 1 or 2, wherein the nanocarricrs comprise the second nanocarriers, the method further comprising:(d) internalizing of the second nanocarriers into the target cells, and cleaving mRNA of the Wnt effector in the target cells.

5. The method of any one of claims 1-4, wherein the nanocarriers comprise the third nanocarriers, the method further comprising:(c) binding the antibody to the receptor, binding the third nanocarriers to the target cells, phosphorylating and degrading the Wnt protein p-catenin in the target cells; and(d) internalizing of the third nanocarriers into the target cells, and cleaving mRNA of the Wnt effector in the target cells.

6. The method of any one of claims 1-5, wherein the target gene is selected from the group consisting of P-catenin. Axin2, CCND1. C-myc, Nanog, Snail, Survivin, SOX9, RUNX2, MDR-1. CTLA4. TCF / LEF, Oct4. KLF4, MMP-7. VEGF, and CD44.

7. The method of claim 6, wherein the target gene is selected from the group consisting of P-catenin, CCND1, C-myc, Snail, and Survivin.

8. The method of any one of claims 1-7, wherein the receptor is a Frizzled (FZD) receptor or a low -density lipoprotein receptor-related proteins 5 and 6 (LRP5 / 6 co-receptors).

9. The method of any one of claims 1-8, wherein the ligand is selected from the group consisting of Wntl, Wnt2, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, WntlOa, Wntl Ob, and Wntll.

10. The method of any one of claims 1-9, wherein tire effector is selected from the group consisting of P-catenin, Axin2, FZD7, TCF / LEF. Wntl, Wnt2, Wnt3, and Wnt3a.

11. The method of any one of claims 1-10, wherein the nanocarriers further comprise a passivating agent attached to the first nanoparticles, second nanoparticles, and / or third nanoparticles, wherein the passivating agent is selected from the group consisting of linear poly (ethylene glycol) (PEG) molecules, branched PEG molecules, Y -shaped PEG molecules, heterobifunctional PEG molecules, polyoxazolines, poly(N-vinylpyrrolidone. poly(glycerols), polyacrylamides. poly(carboxybetaine), poly(sulfobetaine), and phosphobetaine-base polymers.

12. The method of any one of claims 1-11. further comprising reducing proliferation of the target cells.

13. The method of any one of claims 1-12, further comprising suppressing migration of the target cells.

14. The method of any one of claims 1-13, further comprising suppressing invasion of the target cells.

15. The method of any one of claims 1-14, further comprising reducing self-renewal by the target cells.

16. The method of any one of claims 1-15, further comprising reducing metabolic activity of the target cells.

17. The method of any one of claims 1-16, further comprising reducing drug resistance of the target cells.

18. The method of any one of claims 1-17, further comprising reducing viability of the target cells.

19. The method of any one of claims 1-18, further comprising blocking interaction between the Wnt ligand and the receptor.

20. The method of any one of claims 1-19, further comprising RNA interference of theWnt effector.

21. The method of any one of claims 1-20, wherein the target cells are cancer cells.

22. The method of claim 21, wherein the target cells are in a subject.

23. The method of claim 22. wherein the subject suffers from a cancer.

24. The method of claim 22 or 23, wherein the target cells are in a tumor in the subject.

25. The method of claim 24. further comprising reducing growth of the tumor.

26. The method of claim 24, further comprising reducing metastasis of the tumor.

27. The method of claim 24, further comprising reducing recurrence of the tumor.

28. The method of any one of claims 22-27, further comprising inducing stabilization of a disease in the subject.

29. The method of any one of claims 22-28, further comprising reducing disease burden in the subject.

30. The method of any one of claims 22-29, wherein the subject is a human.

31. The method of any one of claims 22-29, wherein the subject is a veterinary subject.

32. A method for synthesizing a first nanocarrier, wherein the first nanocarrier comprises a nanoparticle and an antibody specific for a receptor capable of binding an extracellular Wnt ligand, the method comprising attaching the antibody to the nanoparticle, whereby the first nanocarrier is synthesized.

33. A method for synthesizing a second nanocarrier, wherein the second nanocarrier comprises a nanoparticle and a small interfering RNA (siRNA) specific for a Wnt effector, the method comprising attaching the siRNA to the nanoparticle, whereby the second nanocarrier is synthesized.

34. A method for synthesizing a third nanocarrier, wherein the nanocarrier comprises a nanoparticle, an antibody specific for a receptor capable of binding an extracellular Wnt ligand, and a small interfering RNA (siRNA) specific for a Wnt effector, the method comprising:(a) attaching the antibody to the nanoparticle, whereby the nanoparticle carrying the antibody is obtained; and(b) attaching the siRNA to the nanoparticle cartying the antibody, whereby the third nanocarrier is synthesized.

35. The method of any one of claims 32-34, further comprising attaching a passivating agent to the nanoparticle.

36. The method of claim 32 or 34, further comprising attaching the antibody to the nanoparticle through a chemical linker.

37. The method of claim 36. wherein the chemical linker is a heterobifunctional PEG derivative.

38. The method of claim 32 or 34, further comprising attaching the antibody to the nanoparticle using free thiols that are exposed by treating the antibody with a reducing agent.

39. The method of claim 33 or 34, further comprising attaching the siRNA to the nanoparticle in a buffer, wherein the buffer comprises NaCl, CaCfi. or ethylenediaminetetraacetic acid (EDTA).

40. The method of claim 33 or 34, further comprising attaching the siRNA to the nanoparticle through a chemical linker.

41. The method of claim 40, wherein the chemical linker is a thiol or disulfide.

42. The method of claim 41, wherein the thiol or disulfide is positioned at the 3 ' end of tire sense strand of the siRNA.

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