ANTI-CANCER COMPOSITIONS AND METHODS
20230024584 · 2023-01-26
Inventors
- Chengfeng Yang (Nicholasville, KY, US)
- Zhishan Wang (Nicholasville, KY, US)
- Yunfei Li (Lexington, KY, US)
Cpc classification
A61K9/0053
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K31/4468
HUMAN NECESSITIES
A61K47/6929
HUMAN NECESSITIES
A61K47/62
HUMAN NECESSITIES
International classification
A61K47/69
HUMAN NECESSITIES
A61K31/4468
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K47/62
HUMAN NECESSITIES
Abstract
Treatments for cancer include miriplatin assembled into an ultra-small dot (uPtD) and/or lomitapide or a pharmaceutically-acceptable salt thereof. The uPtD and/or lomitapide can be encapsulated in a nanoparticle for administration to a subject. Some embodiments further include paclitaxel or a pharmaceutically-acceptable salt thereof.
Claims
1. A composition for treating cancer in a subject, comprising: miriplatin assembled into an ultrasmall dot (uPtD).
2. The composition of claim 1, wherein the uPtD is encapsulated in a nanoparticle.
3. The composition of claim 2, wherein the nanoparticle is a lipid-polymer hybrid (LPH) nanoparticle.
4. The composition of claim 2, wherein an exterior layer of the nanoparticle is anchored with a polar species.
5. The composition of claim 4, wherein the polar species is PEG.sub.2000.
6. The composition of claim 2, wherein the nanoparticle is decorated with a targeting agent.
7. The composition of claim 6, wherein the targeting agent is RGD for targeting triple-negative breast cancer (TNBC) cells.
8. A composition for treating cancer in a subject, comprising: lomitapide or a pharmaceutically-acceptable salt thereof in an effective amount for inhibiting the growth of and/or killing a cancer cell.
9. The composition of claim 8, and further comprising paclitaxel or a pharmaceutically-acceptable salt thereof.
10. The composition of claim 8, and further comprising a nanoparticle, wherein the lomitapide or a pharmaceutically-acceptable salt thereof is packaged within the nanoparticle.
11. The composition of claim 10, wherein the nanoparticle is a lipid-polymer hybrid (LPH) nanoparticle.
12. The composition of claim 11, wherein an exterior layer of the nanoparticle is anchored with a polar species.
13. The composition of claim 11, wherein the nanoparticle is decorated with a targeting agent.
14. (canceled)
15. A method of inhibiting the growth of and/or killing a cancer cell, comprising: administering an effective amount of the composition of claim 1, thereby inhibiting the growth of and/or killing the cancer cell.
16. The method of claim 15, wherein the cancer cell is a breast cancer cell, a lung cancer cell, or a colon cancer cell.
17. The method of claim 15, wherein the breast cancer cell is a triple-negative breast cancer (TNBC) cell.
18-19. (canceled)
20. The method of claim 15, wherein the cancer cell is in a subject.
21-27. (canceled)
28. The method of claim 15, comprising administering the composition by injection.
29. The method of claim 28, wherein the composition comprises a platinum (II) genotoxic agents (Pt(II)) packaged within an ultra-small nanoparticle.
30. The method of claim 15, comprising orally-administering the composition.
31-36. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are used, and the accompanying drawings of which:
[0019]
[0020]
[0021]
[0022]
[0023]
[0024]
[0025]
[0026]
[0027]
[0028]
[0029]
[0030]
[0031]
[0032]
[0033]
[0034]
[0035]
[0036]
[0037]
[0038]
[0039]
[0040]
[0041]
[0042]
[0043]
[0044]
[0045]
[0046]
[0047]
[0048]
[0049]
[0050]
[0051]
[0052]
[0053]
[0054]
[0055]
DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0056] The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.
[0057] The presently-disclosed subject matter includes compositions and methods for use in treating cancers. The presently-disclosed subject matter also includes methods for preparing compositions having anti-cancer utility.
[0058] The presently-disclosed subject matter includes platinum (II) genotoxic agents (Pt(II)) assembled as ultrasmall dots. For example, in some embodiments, ultrasmall miriplatin dots (uPtDs) are provided. The dots are “ultra-small,” in that they have a diameter that beneficially allows them to fit into the DNA helix structure of cancer cells, to cause severe and/or lethal DNA damage to the cancer cells, such as double-stranded breaks (DSBs). See
[0059] As disclosed herein, the uPtDs has anti-cancer utility. Accordingly, the presently-disclosed subject matter includes pharmaceutical compositions comprising the uPtDs, and methods for use thereof.
[0060] The presently-disclosed subject matter also relates to the surprising discovery that lomitapide has anti-cancer utility. It was further discovered that a combination of lomitapide and paclitaxel synergistically produce an anti-cancer effect. In this regard, the presently-disclosed subject matter includes pharmaceutical compositions comprising lomitapide or a pharmaceutically-acceptable salt thereof, or a combination of lomitapide and paclitaxel, or pharmaceutically-acceptable salts thereof, and methods for use thereof.
[0061] The compositions disclosed herein can further include, in some embodiments, an additional anti-cancer agent as is known in the art, or can be administered in a combination treatment with, or simultaneously with, an additional anti-cancer agent known in the art.
[0062] The presently-disclosed subject matter also includes compositions wherein the anti-cancer agent(s) is encapsulated in a nanoparticle. For example, in some embodiments, the uPtDs are encapsulated in a nanoparticle. For another example, in some embodiments, the lomitapide is encapsulated in a nanoparticle. For another example, in some embodiments, the lomitapide and paclitaxel are encapsulated in a nanoparticle.
[0063] As will be recognized by the skilled artisan upon studying this document, a variety of nanoparticles can be selected and used. Indeed, an nanoparticle with core-shell structure that is capable of loading the uPtD, lomitapide, and/or paclitaxel can be used. As will be appreciated, once the relevant anti-cancer agent(s) is encapsulated in the nanoparticle for delivery the composition would be stable, as evidenced by the exemplary nanoparticles shown in
[0064] In some embodiments, the nanoparticle can be a lipid-polymer hybrid (LPH) nanoparticle. As will be appreciated, in some embodiments, it can be useful to anchor the exterior layer of the nanoparticle with a polar species. As will also be appreciated, in some embodiments, it can be useful to decorate the exterior of the nanoparticle with a targeting agent. For example, in some embodiments, the exterior layer of the nanoparticle can be anchored with a polar species such as PEG.sub.2000. For another example, in some embodiments, the exterior layer of the nanoparticle can be decorated with a targeting agent to direct the nanoparticle to a target cell of interest. In this regard, for example, the targeting agent could be an RGD peptide for targeting triple-negative breast cancer (TNBC) cells.
[0065] The presently-disclosed subject matter further includes anti-cancer methods, such as methods of inhibiting the growth of and/or killing a cancer cell, which involve administering an effective amount of an agent or composition as disclosed herein, thereby inhibiting the growth of and/or killing the cancer cell.
[0066] The cancer cell can be any type of cancer cell. For example, in some embodiments, the cancer cell is a breast cancer cell, a lung cancer cell, or colon cancer cell. In some embodiments, the cancer cell is a triple-negative breast cancer cell.
[0067] In some embodiments of the method, the cancer cell is in a subject. In this regard, in some embodiments, the cancer cell is in a tumor. Accordingly, methods as disclosed herein include administering the composition to a subject, such as a human or other animal subject. In some embodiments, administering the composition reduces the tumor growth. In some embodiments, administering the composition reduces metastasis.
[0068] In some embodiments, the composition is administered to a subject in need of cancer treatment. In some embodiments, the subject has breast cancer, lung cancer, or colon cancer. In some embodiments, the subject has triple-negative breast cancer.
[0069] In some embodiments of the method, the composition can be administered by injection. In some embodiments, the composition comprises a uPtD, and is administered by injection. In some embodiments, the composition can be administered orally. In some embodiments, the composition comprises lomitapide, and is administered orally. In some embodiments, the lomitapide is administered at a dose of about 10 mg/kg body weight of the subject.
[0070] While the terms used herein are believed to be well understood by those of ordinary skill in the art, certain definitions are set forth to facilitate explanation of the presently-disclosed subject matter.
[0071] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong.
[0072] All patents, patent applications, published applications and publications, GenBank sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety.
[0073] Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.
[0074] As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, Biochem. (1972) 11(9):1726-1732).
[0075] Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are described herein.
[0076] The present application can “comprise” (open ended) or “consist essentially of” the components of the present invention as well as other ingredients or elements described herein. As used herein, “comprising” is open ended and means the elements recited, or their equivalent in structure or function, plus any other element or elements which are not recited. The terms “having” and “including” are also to be construed as open ended unless the context suggests otherwise.
[0077] Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth.
[0078] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.
[0079] As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, in some embodiments ±0.1%, in some embodiments ±0.01%, and in some embodiments ±0.001% from the specified amount, as such variations are appropriate to perform the disclosed method.
[0080] As used herein, ranges can be expressed as from “about” one particular value, and/or to “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
[0081] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally variant portion means that the portion is variant or non-variant.
EXAMPLES
[0082] The presently-disclosed subject matter is further illustrated by the following specific but non-limiting examples. The following examples may include compilations of data that are representative of data gathered at various times during the course of development and experimentation related to the present invention.
Example 1: Cell Lines, Chemicals and Animals
[0083] Human TNBC cell lines MDA-MB-231, LM2 and SUM159 cells were obtained and cultured as previously described[19]. The LM2 cell is a derivative of MDA-MB-231 cells, which was selected for its strong ability to metastasize to lung in vivo [20].
[0084] Miriplatin was purchased from MedChemExpress USA (Monmouth Junction, N.J.). Thia-zolyl Blue Tetrazolium Bromide (MTT), poly(lactic-co-glycolic acid) (PLGA, easter terminated, lactide:glycolide 75:25, M.W. 76,000-115,000) were purchased from Sigma-Aldrich (St Louis, Mo.).
[0085] PEG2000-DSPE was purchased from NOF corporation (Tokyo, Japan). RGD peptide with a terminal cysteine (c(RGDfC), M.W. 578.65, RGD-SH) was purchased from Peptides International, Inc. (Louisville, Ky.). Maleimide(mal)-PEG2000-DSPE was purchased from Jenkem Technology USA Inc. (Allen, Tex.).
[0086] RGD peptide and mal-PEG2000-DSPE were used for the synthesis of RGD-PEG2000-DSPE by the published method [18] and the successful conjugation has been characterized by .sup.1H NMR (
[0087] Nude mice (female, 6-8 weeks old at the beginning of each experiment) were purchased from the Charles River. All mice were housed in groups with unlimited access to food and water.
Example 2: Preparation and Characterization of uPtDs NP
[0088] For the studies described herein, an ultrasmall Pt(II) dot (uPtD) was developed from miriplatin and encapsulated it into integrin α5 (ITGA5) active targeting nanoparticles (uPtDs NPs) and tested their therapeutic efficacy against TNBC metastasis.
##STR00001##
[0089] ITGA5 was previously found to be highly expressed in strongly migratory and invasive TNBC cells as well as their lung metastatic foci [18]. In this study, ITGA5 targeting lipid-polymer hybrid NPs [18] were used to produce uPtDs NPs, which renders lipophilic uPtDs water soluble and confers uPtDs active targeting capability.
[0090] Ultra-small miriplatin particles were prepared, having a diameter of approximate 1 nm, and are referred to as ultra-small miriplatin nanodots (uPtDs). These uPtDs can also be encapsulated in a nanoparticle. Aspects of encapsulation within a nanoparticle (NP) of an agent were previously described [18]. However, uPtD preparation, and subsequent encapsulation, has not been previously described.
[0091] For uPtDs preparation, first, 3 mg of miriplatin was suspended in 300 μL of DMSO and 700 μL of acetone, which was then heated in a water bath under 65° C. to a transparent solution.
[0092] Meanwhile, lecithin (2 mg), .sub.PEG2000-DSPE (16 mg), and RGD-PEG2000-DSPE (2 mg) were dissolved in 30 mL of 4% ethanol aqueous solution and heated to 65° C. (Of course, other lipids could be selected of preparation of encapsulating nanoparticles, as desired, and as selected based on knowledge in the art about lipid nanoparticles).
[0093] Next, 1 mg of PLGA was mixed with miriplatin solutions followed by poured into the preheated lipid solutions, which then stood there still at room temperature to make sure the thorough self-assemble of uPtDs NP as well as the acetone evaporation.
[0094] Finally, the remaining free molecules (ethanol and acetone) were removed by an Amicon Ultra-4 centrifugal filter (Molecular cut-off 100,000, Millipore, Billerica, Mass.) and then resuspended in PBS to obtain a final desired concentration. The size (diameter, nm) of uPtDs NP was obtained using a Malvern Instruments Zetasizer ZS-90 instrument. The morphologies and sizes of uPtDs as well as uPtDs NP were observed utilizing transmission electron microscopy (TEM) under 80 kv and scanning transmission electron microscopy (STEM) under 200 kv (Talos, ThermoFisher Scientific, Hillsboro, Oreg.). NP blank was prepared by a similar method without the addition of miriplatin acetone-DMSO solution.
Example 3: High-Angle Annular Dark-Field Scanning Transmission Election Microscopy (HAADF-STEM) Study of LM2 Cell-Epoxy Ultrathin Films
[0095] LM2 cells were seeded into 6 Well CELLSTAR® Cell Culture Multiwell Plates at a confluence of 50-70%. At the 2nd day, cisplatin, miriplatin and uPtDs NP (6.4 μM) were added into wells. After 6 h incubation, cells were fixed chemically by glutaraldehyde and osmium, dehydrated, embedded in epoxy resins, sliced and immobilized onto grids following common protocols but without the staining of salts of uranium and/or lead. HAADF-STEM under 200 kv (Talos, ThermoFisher Scientific, Hillsboro, Oreg.) was utilized to observe Pt element distributions inside the cells.
Example 4: Determination of Miriplatin Contents and In Vitro Release Profile of uPtDs NP
[0096] A Varian Vista Pro CCD simultaneous ICP-OES was used to determine the concentration of Pt element in samples. Samples were digested with 0.5 mL aqua regia, then diluted with DI water to a total volume of 5 mL. This resulted in a 50× sample dilution prior to analysis. A 50 ppb analytical detection limit and a 2.5 ppm sample detection limit were established with platinum calibrations standards prepared in 5% HCl. Standard curve correlations maintained a corr. Coeff.>0.995. Sample measurements were read in triplicate. Quality control measures include a diluent blank, standard control, and yttrium internal standard measurements with each sample reading. Method conditions: Power (kW): 1.20; Plasma flow (L/min): 15.0; Auxiliary flow (L/min): 1.5; Nebulizer flow (L/min): 0.90; Replicate read time (s): 35.00; Instrument stabilization delay (s): 20. The miriplatin content was converted from Pt content by the following formula:
miriplatin content=(Pt content×Molecular weight of miriplatin)/(Molecular weight of Pt)
[0097] The weight ratio of miriplatin to NPs was defined as drug loadings (DL), while the weight ratio of entrapped miriplatin to added miriplatin was defined as entrapment efficiency (EE). Drug release over time was characterized by dialyzing samples against 1×PBS (pH 5.5 and 7.4) in 10 kDa MWCO Slide-A-Lyzer MINI dialysis units (Thermo Scientific) according to the reported methods [18]. The amount of drug retained in the NP samples was assessed at 0, 4, 12, 24, 48, 72 and 96 h and analyzed by method as described above.
Example 5: DNA Electrophoresis to Study Interaction Between uPtDs NP and Plasmid DNA (pDNA)
[0098] Interactions of cisplatin, miriplatin, uPtDs NP and NP blank with pDNA (250 ng) were investigated by incubating 6 h and 12 h at 37° C. water bath. To make sure uPtDs inside the NPs fully exposed to the exterior environment, uPtDs NP were incubated overnight in PBS at 37° C. water bath while NP blank was used as a control. Samples were loaded into 2% agarose gel to determine the pDNAs shift. Naked pDNA and naked pDNA incubated at 37° C. water bath served as controls. The pDNA bands were visualized by EB staining and photographed using the Gel Doc EZ Imager (Bio-Rad).
Example 6: MTT Assay, Clonogenic Assay, Soft Agar Colony Formation Assay and Suspension Serum-Free Culture Tumorigenic Sphere Formation Assay
[0099] The MTT assay, clonogenic assay, soft agar colony formation experiment and suspension serum-free culture tumorigenic sphere formation assay were carried out as described previously [18].
Example 7: Western Blot Analysis, Immunofluorescence (IF) Staining and Immunohistochemistry (IHC) Staining
[0100] Cells were lysed using Tris-sodium dodecyl sulfate for Western blot analysis as previously described[18]. The following primary antibodies were used: cyclin A, cyclin E (Santa Cruz Biotechnology, Dallas, Tex.); p-CHK1, total CHK1, p-CHK2, total CHK2, CDC25A, c-myc, nanog, klf4 (Cell Signaling Technology, Beverly, Mass.); γ-H2A (Millipore Sigma, Burlington, Mass.); Cyclin D1 (BD Bioscience); and anti-β-actin (Sigma). The IF staining was performed following the previously described protocols [18]. The IHC staining against green fluorescent protein (GFP) in mouse lung tissues was performed following previously-described protocols [21].
Example 8: Cell Cycle Analysis
[0101] LM2 cells were seeded into 6 cm dishes (density: 3×10.sup.5 cells per dish) and allowed to attach and grow overnight. After 24 h synchronization, cells were then exposed to 6.4 M of cisplatin, miriplatin, uPtDs NP or NP blank for 3 h respectively. After treatment withdrawal, LM2 cells were continued to culture 24 h. At end, cells were scrapped, washed, fixed and permeabilized with 70% ice-cold ethanol in 4° C. for 2 h. Cells were then incubated with freshly prepared propidium iodide (PI) staining buffer (0.1% Triton X-100, 200 μg/mL RNase A and 20 μg/mL PI in PBS) for 15 min at 37° C., followed by routine flow cytometry analysis of 20,000 cells in each group.
Example 9: Evaluation on the Therapeutic Effect of Systemic Administration of uPtDs NP on TNBC Using a Nude Mouse Orthotopic Mammary Xenograft Tumor Model and an Experimental Lung Metastasis Model
[0102] Nude mouse orthotopic mammary xenograft tumors were established by directly injecting 1 million of LM2 cells into the 4th mammary gland fat pad as described previously [18]. The treatment started at day 14 post LM2 cell inoculation when mammary tumor volumes reached about 150-250 mm.sup.3 and terminated at day 49 post LM2 cell inoculation. Cisplatin, uPtDs NP and NP blank were administered thrice a week at the dosages of 2, 2, 3 mg cisplatin/kg body weight and 5, 5, 7.5 mg miriplatin/kg body weight [Pt(II) moles from cisplatin≈Pt(II) moles from miriplatin]; NP blank was set as a negative control. Tumor volumes were measured weekly. At day 49 post LM2 cell inoculation, all mice were sacrificed. The lungs were harvested and imaged by IVIS imaging individually using the same settings: exposure time 60s; F/Stop 1; Binning Small, Field of View D; all images captured were piled together and normalized by IVIS software. The mammary tumors were collected, weighted and imaged. For the experimental lung metastasis model experiment, one million of LM2 cells were injected via tail vein to each nude mouse to produce experimental lung metastasis at day 0. When lung metastatic foci were confirmed on day 13 by IVIS imaging, uPtDs NP administration began at the dosage of 5 mg/kg, 5 mg/kg and 7.5 mg/kg per week, and NP blank was set as the control. On day 49, all mice were imaged by IVIS machine to examine the bioluminescence intensity at thoracic area. After euthanasia, the lungs were collected and imaged ex vivo by IVIS machine as well.
Example 10: Capture and Cyanine 5 (Cy5) Identification of Mimic CTCs in Mouse Peripheral Bloodstream
[0103] Nanovehicle-Cy5 were the same as RGD-LPH(Cy5), as previously described [18]. LM2 cells expressing GFP (0.5 million per mouse) were i.v. injected and 0.5 h later 300 μL of free Cy5 or nanovehicle-Cy5 solutions were i.v. injected into each mouse at the dosage of 0.6 mg Cy5/kg mouse body weight. Three hours later 0.5 mL of blood was drawn from abdominal artery by 25G syringe needle and spiked into 10 mL of RBC Lysis Buffer (BioLegend®) for 10 min incubation under room temperature. The pellets got after 10 min of centrifugation (1700 rpm) were resuspended by 0.5 mL of PBS with 2% FBS for flow cytometry examination. GFP expressing LM2 captured by nanovehicle-Cy5 or free Cy5 will present both GFP and Cy5 behavior while LM2 not being captured by Cy5 only illustrate GFP behavior.
Example 11: PCR Method for the Detection of Human LM2 Cells in Xenotransplantation Mice
[0104] Nude mouse orthotopic mammary xenograft were established by the same above-mention method. At day 17 post-inoculation, the tumor sizes reached around 300 mm.sup.3 and treatment was scheduled trice per week at a dosage of 2 mg cisplatin/kg or 5 mg miriplatin/kg. At day 45 post-inoculation, around 0.5 mL mouse blood was collected from each mouse to MiniCollect® Tube (Greiner Bio-one GmbH, Austria) and then poured into 10 mL of 1×RBC lysis buffer (BioLegend®) and incubated for 10 min at room temperature for the removal of red blood cells. The lysis solutions were centrifuged under 1700 rpm for 10 min to collect the pellets, which will be further extracted DNA following the protocol from the manufacturer (DNeasy® Blood & Tissue Kit, QIAGEN GmbH, Germany). Then routine PCR reaction were performed and analyzed by DNA electrophoresis. The PCR reaction mixture (25 μL) contained 1 μL of dNTP (10 mM), 1 μL of each primer (10 μM), 1.5 μL of MgCl2 (50 mM), 2.5 μL of PCR Rxn buffer (10×, Invitrogen®), 1 μL of Taq DNA polymerase and 100 ng of genomic DNA template. Following an initial DNA denaturation and Taq activation at 94° C. for 10 min, 35 1-min cycles of denaturation at 94° C. and annealing/extension at 56° C. were performed followed by a final elongation step at 72° C. for 10 min. Oligonucleotides sequence (5′.fwdarw.3′) used for PCR:
TABLE-US-00001 forward primer(Cr17_1aa): (SEQ ID NO: 1) GGGTAATTTCAGCTGACTAAACAG; reverse primer(Cr17_4bc): (SEQ ID NO: 2) AAACGTCCACTTGCAGATTCTAG; forward primer(Plak_WT): (SEQ ID NO: 3) AACGATGAGGACCCGGTCTGAGAA; reverse primer(Plak_WT): (SEQ ID NO: 4) TGGACAGCTGGTTCACGATCATAG.
Example 12: The Human AP2XCZ6 qPCR Analysis
[0105] DNAs were extracted from the same mouse blood as indicated above. Human AP2XCZ6 primer was designed by Thermo Scientific based on 1-1000 of human alpha satellite from the centromeric region DNA with 16 monomer tandem repeats (Gene Bank Acc. No. 13882). AP2XCZ6 level analysis was carried out in ABI 7500 Fast Real Time PCR system and using mouse actin as the internal control for normalizing relative AP2XCZ6 expression level.
Example 13: Statistical Analysis
[0106] Statistics were computed with GraphPad Prism 8 statistical software. The numerical data are expressed as mean±SD. A p value of <0.05 was considered statistically significant.
Example 14: Results Associated with Physicochemical Characterization of ITGA5-Targeting NPs Loaded with uPtDs
[0107] The fabricating approach of uPtDs NP was adapted from a previously-reported method [18], which is also briefly described in
[0108] When pouring this uPtDs solution into an aqueous solution of lipids together with PLGA, uPtDs NPs self-assembled through nanoprecipitation. Examined by both dynamic light scattering (DLS) and TEM methods, uPtDs NPs were determined having diameters of 10-13 nm (
[0109]
[0110] To explore whether uPtDs could enhance Pt(II) interactions with nucleus DNA in live cells, TNBC LM2 cell-epoxy sectioning was performed after exposure to 6.4 μM of uPtDs NP for 6 h, and then imaged as epoxy ultrathin sections under HAADF-STEM (
[0111] As indicated by
[0112] The bright distinctive spots of uPtDs NPs observed in STEM of
[0113] Single or double-stranded DNA is known to interact with ultrasmall metal NPs such as gold, silver and Pt, to form nano-clusters [24,25]. Some hydrophobic substituents can help ultrasmall metal NPs fit into the non-polar DNA grooves leading to increased NP/DNA interactions [26], and Au NPs grafted with a hydrophobic monolayer have been applied in the gene transfection field [27]. Given the success of ultra-small metal NPs preferably interacting with DNA, spherical uPtDs with exterior hydrophobic myristates layer can potentially enhance DNA affinity (
Example 15: Results Associated with Increased DNA Interactions, Accrued DNA Damages and Cell Cycle Arrest by uPtDs NP Treatment
[0114] In vitro experiments were performed to explore uPtDs NP's abilities to interact with DNA (plasmid DNA used as the model DNA and hereinafter referred as pDNA) and damage DNA in TNBC LM2 cells. Gel electrophoresis in
[0115] The intensities of migrating pDNA bands from cisplatin and miriplatin groups were relatively weaker than those of the other four lanes, which is because ethidium bromide (EB) bound to pDNA could be diminished by cisplatin during the incubation process [30]. Reportedly cisplatin could quench EB within 48 h [30]; and by the same way cisplatin and miriplatin quench EB bound to DNA in present study. Notably, when miriplatin was transformed into dots structure, Pt(II) resided in the dot core and were stable enough to avoid Pt(II) exposure during the incubation process. Due to this, EB quench phenomenon at uPtDs NP group was not observed. Interestingly, a much weaker band close to loading well was also found at NP blank group, suggesting blank NPs had some non-specific interactions with pDNA.
[0116] Next, IF staining of γ-H2A was performed to detect and compare the extent of DNA damage [31]. Due to their reported strong metastatic capability in vivo and inherent resistance to treatment [19,20], TNBC LM2 cells were chosen. In nearly 90% of cells treated with uPtDs NP, typical γ-H2A foci were observed 48 h post treatment (
Example 16: Results Associated with DNA Damage Induced by uPtDs NP Activates the ATR/ATM-CHK1/2-CDC25A-Cyclin A/E Pathway
[0117] The underlying signal pathway was further elucidated. As shown in
[0118] To further validate this DNA damage pathway, rescue experiments were performed utilizing CHK1(CHIR124), ATM (Ku55933) and ATR (AZD6378) inhibitors. By using pharmacological inhibition of CHK1, ATM or ATR activities, .sub.CHK1/2 were identified as the kinases that were activated by DNA damage and involved in uPtDs NP-induced down-regulation of CDC25A and cyclin A/E (
Example 17: Results Associated with uPtDs NP Impairing TNBC Cell CSC-Like Property and Targeting CTCs in Peripheral Bloodstream Reducing TNBC Tumor Lung Metastasis
[0119] The in vivo efficacy of systematic administration of uPtDs NP on TNBC tumor growth and metastasis was then evaluated (
[0120] To investigate the mechanism of uPtDs NP treatment reducing TNBC metastasis, the effect of uPtDs NP treatment on TNBC cell CSC-like property was determined, as CSCs play crucial roles in metastasis. By using SUM159, another typical TNBC cells, a suspension and serum free culture tumorigenic sphere formation assay was performed. This is a well-established and widely-used assay for examining the presence of stem cell/CSC or CSC-like cells [32]. Because LM2 cells could not form spheroid structures under serum-free and suspension culture conditions, LM2 cells were not used for the sphere formation assay. As shown in
[0121] To further investigate the mechanism of uPtDs NP treatment reducing metastasis, the effect of uPtDs NP treatment on CTCs was determined, as CTCs are clinically considered as seeds of metastasis [4, 34-36]. However, the frequency of CTCs is extremely low in the complex peripheral bloodstream, and only up to hundreds of CTCs out of >10.sup.9 hematological cells were reportedly found in 1 mL blood [37]. Hereby, tail vein injection of 0.5 million LM2 cells expressing GFP to immunodeficient mouse was used to mimic CTCs. The studying scheme is briefly shown in
Example 18: Results Associated with uPtDs NP Treatment Reducing the Presence of CTCs in Circulation of TNBC Tumor Bearing Mice
[0122] Furthermore, CTC levels were also determined in mice bearing LM2 xenograft mammary tumor with various treatment. Until now, there have been no in vivo studies using systemic administration of free miriplatin, since miriplatin is poorly soluble in most of the typical medical excipients such as Tween or Poloxamer. That is why the present study only utilized free cisplatin as the control. After orthotopic xenotransplantation of LM2 cells into mice, it is difficult to directly detect the CTCs that are present at a low level in peripheral bloodstream at treatment endpoint.
[0123] A reported sensitive PCR method was used to detect the presence of human cells (CTCs) in tumor bearing mouse blood circulation [38]. This method could detect a human specific 480 bp fragment of α-satellite DNA of the centromere region on human chromosome 17 (Cr17_1aa/4bc) in complex mouse tissue after xenotransplantation of malignant cells. The brief studying scheme is shown in
[0124] To further determine the mechanism of uPtDs NP treatment suppression of TNBC lung metastasis, the effect of uPtDs NP treatment on pre-existed (experimentally-induced) metastatic lung tumors was also investigated. As shown in
Example 19: Discussion Related to Results of Examples 14-18
[0125] The fate of cancer cells following genotoxic insult is greatly impacted by the extent of DNA damage 39. Cisplatin is the most clinically successful DNA covalent binder. However, it usually leads to CSC-resistance due to its moderate DNA damage. Designing more Pt(II) complex molecules is the most popular strategy to enhance the efficacies of Pt(II)-based genotoxic agents but get limited success. There have been few studies on the therapeutic potentials of ultrasmall metal NPs (<5 nm), in one of which ultrasmall transition metal NP could trigger severe DNA damages as a radiosensitizer fitting into DNA helix grooves [40]. As shown in
[0126] In the present study, uPtDs were prepared successfully starting from miriplatin. It is postulated that miriplatin could self-assemble into a sphere structure, the exterior of which were the myristates having good solubility in acetone and the core of which were the relatively lipo-phobic Pt(II) ions. However, the colloidal stability of uPtDs in aqueous environment is poor, due to their hydrophobic myristate exterior. To resolve this issue, additional coatings by NPs are needed. The NP encapsulation could help enhance the colloidal stability of uPtDs in aqueous environment. Because the threshold of renal clearance of NPs is ˜5.5 nm [43], NP encapsulation could help uPtDs (˜1 nm) avoid this problem. As expected, uPtDs were easily incorporated into ITGA5-tar-geting NPs through hydrophobic interactions. Under TEM and STEM, these Pt(II) ions were verified as ultrasmall Pt(II) dots with the diameter of around 1 nm (
[0127] The present study found this ultrasmall dot prepared from mir-iplatin could cause severe DNA damages, possibly through the accumulation at nucleus (
[0128] As for the reasons of uPtDs NP's inability to inhibit primary tumor growth, beside the intrinsic aggressive nature of LM2 cell-derived mammary tumor, the very extended release profile of the uPtDs NP (
[0129] To gain further insights on the mechanism of superior therapeutic effects originating from enhanced DNA damages, the present study also examined the downstream signal pathway of the DNA damage elicited by this novel uPtDs and compared those by free cisplatin and mir-iplatin. Data in support of this conclusion were derived from several experimental approaches. First, as clearly indicated by γ-H2A IF staining shown in
[0130] Collectively, the significant stronger inhibitory effect of uPtDs on tumor metastasis may be attributed to the following three factors. (1) The stability of uPtDs NP. The in vivo stability of nanomedicine is very important for the metastasis therapy, which has been comprehensively discussed by Omid C. Farokhzad et al. using Abraxane and Doxil as examples [47]. Both Doxil and Abraxane have very limited effect in improving patients' overall survival due to the rapid dissociation of payloads and excipients upon intravenous infusion [47]. Cheng Group reported anchoring hydrophobic polylactide (PLA) chains to paclitaxel molecule to avoid burst drug release profile and achieve higher drug loading of polymeric NPs [48]. Similarly, lipophilic corona of uPtDs formed by myristate chains in present study was well compatible with hydrophobic NPs core preventing the premature uPtDs clearance. The 48 h release profile of uPtDs NP was a sustained manner following zero order kinetics, which is indicative of robust NPs (
[0131] (2) By virtue of their superior DNA damaging capabilities (
[0132] (3) The β3 integrin family members (platelet .sub.αIIβ3, tumor αvβ3) can also bind with RGD motifs, which have been anchored at uPtDs NP surface. Notably, the critical role of both .sub.αIIbβ3 and tumor α.sub.vβ3 in CTC adhesion and invasion under blood flow conditions has been disclosed [49]; additionally, CTCs naturally prefer nanostructural surfaces [44] and NPs have large surface-to-volume ratio, both of which will enable efficient cellular binding by NPs.
[0133] The studies described in these Examples highlights the prominent DNA-damaging property of ultrasmall Pt(II) dots with the diameter of 1 nm in comparison to currently clinically-prescribed Pt(II) drugs. This strategy magnifies the DNA damage effects of Pt(II) atoms by leveraging the ultrasmall dot-like design architecture of miriplatin. In the meantime, all the excipients fabricating NP to encapsulate uPtDs are FDA-approved. Hence, uPtDs NP are contemplated to function as a safe and potent cancer chemo-therapeutic agent. While Pt(II) dot treatment and ITGA5-tar-geting NPs were used in the present study as proof-of-concept modalities, given the generalizability of ultrasmall dots design and their NP-encapsulating compatibility, it is envisioned that ultrasmall dot design strategy of Pt(II) atoms can be adapted toward a variety of other active metal complex against cancer metastasis and an alternative strategy for de novo development of metal-based drugs, in view of the information disclosed herein.
Example 20: Lomitapide Treatment Reduces Growth of Breast Cancer Cells
[0134] A library of 1443 FDA-approved drugs was screened (Selleckchem, Cat. No. L1300). It was unexpectedly determined that lomitapide displayed a strong cytotoxic effect on three kinds of triple negative breast cancer cells. In particular, lomitapide treatment at a dose of 10 uM for 48 h reduced the viability of triple negative breast cancer in MDMA-MB-231-LM2, SUM-159 and MDA-MB-231 cells by 86.6%, 87.9%, and 87.4%, respectively.
[0135] It was surprisingly discovered that lomitapide has a cytotoxic effect on other cancer cell lines, including multiple breast cancer cell lines (MCF-7, MDA-MB-453, 4T1 and 4T1-luc), multiple lung cancer cell lines (A549, H460, PC9, PC9GR4, H1975), and multiple colon cancer cell lines (HCT 116, HCT29, DLD1, PT130 and SW480). By using MTT assay, clonogenic assay, soft agar colony formation assay and cell cycle analysis, lomitapide treatment at a dose of 2.5 or 5.0 μM for 72 h displayed strong inhibitory effects on cancer cell growth.
##STR00002##
[0136] With reference to
[0137] Clonogenic assays were also performed to determine the effect of lomitapide treatment on various breast cancer cell lines, with data reflected in
[0138] With reference to
Example 21: Lomitapide Treatment Reduces Cancer Stem Cell-Like Property of Breast Cancer Cells
[0139] Further studies were conducted using a suspension culture sphere formation assay, which is a well-established assay for analyzing cancer stem cell (CSC)-like property. It was discovered that lomitapide treatment displayed a very potent inhibitory effect on cancer stem cell-like property in multiple cancer cell types, including breast cancer and lung cancer cells.
[0140] With reference to
Example 22: Lomitapide Treatment Causes G1 Phase Cell Cycle Arrest in Breast Cancer Cells
[0141] With reference to
Example 23: Lomitapide Treatment Induces Apoptosis in Breast Cancer Cells
[0142] With reference to
Example 24: Lomitapide Treatment Reduces Lung Cancer Cell Growth
[0143]
[0144] Clonogenic assays were also performed to determine the effect of lomitapide treatment on various lung cancer cell lines, with data reflected in
Example 25: Lomitapide Treatment Induces Apoptosis in Lung Cancer Cells
[0145] With reference to
Example 26: Lomitapide Treatment Reduces Cancer Stem Cell-Like Property of Lung Cancer Cells
[0146] With reference to
Example 27: Lomitapide Treatment Reduces Colon Cancer Cell Growth
[0147] With reference to
[0148] With reference to
Example 28: Lomitapide Nanoparticle (NP) Treatment Reduces Tumor Growth and Spontaneous Metastasis
[0149] In vivo animal studies were conducted and demonstrated that systematic administration of nanoparticle-packaged lomitapide reduces mammary tumor growth and/or spontaneous lung metastasis.
[0150] With reference to
[0151]
Example 29: Synergistic Results with Combined Lomitapide (Lomi) and Paclitaxel (PTX) Treatment on Growth of Cancer Cells
[0152]
##STR00003##
[0153] One thousand 4T1 cells were seeded into 96-well plates; at the 2nd day, different concentrations of vehicle control, lomi, PTX or the combination of lomi and PTX were added into the culture media and incubated for 24 hours. The cell growth was assessed by the MTT assay.
[0154]
Example 30: Synergistic Results with Combined Lomitapide (NP-Lomi) and Paclitaxel (PTX) Treatment on Breast Tumor Growth and Metastasis
[0155]
[0156] It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the subject matter disclosed herein. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.
[0157] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference, including the references set forth in the following list:
REFERENCES
[0158] 1. G. Bianchini, J. M. Balko, I. A. Mayer, M. E. Sanders, L. Gianni, Triple-negative breast cancer: challenges and opportunities of a heterogeneous disease, Nat. Rev. Clin. Oncol. 13 (2016) 674-690. [0159] 2. M. Balic, H. Lin, L. Young, D. Hawes, A. Giuliano, G. McNamara, R. H. Datar, R. J. Cote, Most early disseminated cancer cells detected in bone marrow of breast cancer patients have a putative breast cancer stem cell phenotype, Clin. Cancer Res. 12 (2006) 5615-5621. [0160] 3. M. Ksigzkiewicz, A. Markiewicz, A. J. Zaczek, Epithelial-mesenchymal transition: a hallmark in metastasis formation linking circulating tumor cells and cancer stem cells, Pathobiology 79 (2012) 195-208. [0161] 4. U. De Giorgi, V. Valero, E. Rohren, S. Dawood, N. T. Ueno, M. C. Miller, G. V. Doyle, S. Jackson, E. Andreopoulou, B. C. Handy, Circulating tumor cells and [18F] fluorodeoxyglucose positron emission tomography/computed tomography for outcome prediction in metastatic breast cancer, J. Clin. Oncol. 27 (2009) 3303-3311. [0162] 5. B. Rack, C. Schindlbeck, J. Juckstock, U. Andergassen, P. Hepp, T. Zwingers, T. W. P. Friedl, R. Lorenz, H. Tesch, P. A. Fasching, T. Fehm, A. Schneeweiss, W. Lichtenegger, M. W. Beckmann, K. Friese, K. Pantel, W. Janni, T. S. S. Group, Circulating tumor cells predict survival in early average-to-high risk breast cancer patients, JNCI: J. Nat. Cancer Inst. 106 (2014). [0163] 6. J. P. Hong, S. H. Sun, M. Ben-Nakhi, Modified superficial circumflex iliac artery perforator flap and supermicrosurgery technique for lower extremity reconstruction: a new approach for moderate-sized defects, Ann. Plast. Surg. 71 (2013) 380-383. [0164] 7. S. J. Isakoff, Triple negative breast cancer: role of specific chemotherapy agents, Cancer J. (Sudbury, Mass.) 16 (2010) 53. [0165] 8. C. Liedtke, C. Mazouni, K. R. Hess, F. Andre, A. Tordai, J. A. Mejia, W. F. Symmans, A. M. Gonzalez-Angulo, B. Hennessy, M. Green, Response to neoadjuvant therapy and long-term survival in patients with triple-negative breast cancer, J. Clin. Oncol. 26 (2008) 1275-1281. [0166] 9. Y.-P. Liu, C.-J. Yang, M.-S. Huang, C.-T. Yeh, A. T. Wu, Y.-C. Lee, T.-C. Lai, C.-H. Lee, Y.-W. Hsiao, J. Lu, Cisplatin selects for multidrug-resistant CD133+ cells in lung adenocarcinoma by activating notch signaling, Cancer Res. 73 (2013) 406-416. [0167] 10. B. A. Hilton, Z. Li, P. R. Musich, H. Wang, B. M. Cartwright, M. Serrano, X. Z. Zhou, K. P. Lu, Y. Zou, ATR plays a direct antiapoptotic role at mitochondria, which is regulated by prolyl isomerase Pin1, Mol. Cell 60 (2015) 35-46. [0168] 11. V. Ilio, M. Gwenola, M. Ruggero De, K. Guido, G. Lorenzo, DNA damage in stem cells, Mol. Cell 66 (2017) 306-319. [0169] 12. T. M. Phillips, W. H. McBride, F. Pajonk, The response of CD24-/low/CD44+ breast cancer-initiating cells to radiation, J. Natl. Cancer Inst. 98 (2006) 1777-1785. [0170] 13. C. Gong, B. Liu, Y. Yao, S. Qu, W. Luo, W. Tan, Q. Liu, H. Yao, L. Zou, F. Su, Potentiated DNA damage response in circulating breast tumor cells confers resistance to chemotherapy, J. Biol. Chem. 290 (2015) 14811-14825. [0171] 14. S. Bao, Q. Wu, R. E. McLendon, Y. Hao, Q. Shi, A. B. Hjelmeland, M. W. Dewhirst, D. D. Bigner, J. N. Rich, Glioma stem cells promote radioresistance by preferential activation of the DNA damage response, Nature 444 (2006) 756. [0172] 15. L. Kelland, The resurgence of platinum-based cancer chemotherapy, Nat. Rev. Cancer 7 (2007) 573. [0173] 16. J. M. Reichert, A guide to drug discovery: trends in development and approval times for new therapeutics in the United States, Nat. Rev. Drug Discov. 2 (2003) 695. [0174] 17. J. Gilbert, P. Henske, A. Singh, Rebuilding big pharma's business model, Vivo-New York then Norwalk, 21 2003, pp. 73-80. [0175] 18. Y. Li, Y. Xiao, H.-P. Lin, D. Reichel, Y. Bae, E. Y. Lee, Y. Jiang, X. Huang, C. Yang, Z. Wang, In vivo β-catenin attenuation by the integrin α5-targeting nano-delivery strategy suppresses triple negative breast cancer stemness and metastasis, Biomaterials 188 (2019) 160-172. [0176] 19. B. Humphries, Z. Wang, Y. Li, J.-R. Jhan, Y. Jiang, C. Yang, ARHGAP18 down-regulation by miR-200b suppresses metastasis of triple-negative breast cancer by enhancing activation of RhoA, Cancer Res. 77 (15) (2017) 4051-4064. [0177] 20. A. J. Minn, G. P. Gupta, P. M. Siegel, P. D. Bos, W. Shu, D. D. Giri, A. Viale, A. B. Olshen, W. L. Gerald, J. Massague, Genes that mediate breast cancer metastasis to lung, Nature 436 (2005) 518. [0178] 21. B. Humphries, Z. Wang, A. L. Oom, T. Fisher, D. Tan, Y. Cui, Y. Jiang, C. Yang, MicroRNA-200b targets protein kinase Ca and suppresses triple-negative breast cancer metastasis, Carcinogenesis 35 (2014) 2254-2263. [0179] 22. G. Ruan, A. Agrawal, A. I. Marcus, S. Nie, Imaging and tracking of tat peptide-conjugated quantum dots in living cells: new insights into nanoparticle uptake, intracellular transport, and vesicle shedding, J. Am. Chem. Soc. 129 (2007) 14759-14766. [0180] 23. S. Barua, S. Mitragotri, Synergistic targeting of cell membrane, cytoplasm, and nucleus of cancer cells using rod-shaped nanoparticles, ACS Nano 7 (2013) 9558-9570. [0181] 24. W. E. Ford, O. Harnack, A. Yasuda, J. M. Wessels, Platinated DNA as precursors to templated chains of metal nanoparticles, Adv. Mater. 13 (2001) 1793-1797. [0182] 25. A. Kumar, M. Pattarkine, M. Bhadbhade, A. B. Mandale, K. N. Ganesh, S. S. Datar, C. V. Dharmadhikari, M. Sastry, Linear superclusters of colloidal gold particles by electrostatic assembly on DNA templates, Adv. Mater. 13 (2001) 341-344. [0183] 26. C. M. Goodman, N. S. Chari, G. Han, R. Hong, P. Ghosh, V. M. Rotello, DNA-binding by functionalized gold nanoparticles: mechanism and structural requirements, Chem. Biol. Drug Des. 67 (2006) 297-304. [0184] 27. K. K. Sandhu, C. M. McIntosh, J. M. Simard, S. W. Smith, V. M. Rotello, Gold nano-particle-mediated transfection of mammalian cells, Bioconjug. Chem. 13 (2002) 3-6. [0185] 28. D. Zanchet, C. M. Micheel, W. J. Parak, D. Gerion, A. P. Alivisatos, Electrophoretic isolation of discrete Au nanocrystal/DNA conjugates, Nano Lett. 1 (2001) 32-35. [0186] 29. Y. Liu, W. Meyer-Zaika, S. Franzka, G. Schmid, M. Tsoli, H. Kuhn, Gold-cluster degradation by the transition of B-DNA into A-DNA and the formation of nanowires, Angew. Chem. Int. Ed. 42 (2003) 2853-2857. [0187] 30. S. M. Aris, K. M. Knott, X. Yang, D. A. Gewirtz, N. P. Farrell, Modulation of trans-planaramine platinum complex reactivity by systematic modification of carrier and leaving groups, Inorg. Chim. Acta 362 (2009) 929-934. [0188] 31. C. Redon, D. Pilch, E. Rogakou, O. Sedelnikova, K. Newrock, W. Bonner, Histone H2a variants H2AX and H2AZ, Curr. Opin. Genet. Dev. 12 (2002) 162-169. [0189] 32. S. M. Nolte, C. Venugopal, N. McFarlane, O. Morozova, R. M. Hallett, E. O'farrell, B. Manoranjan, N. K. Murty, P. Klurfan, E. Kachur, A cancer stem cell model for studying brain metastases from primary lung cancer, J. Natl. Cancer Inst. 105 (2013) 551-562. [0190] 33. Y. Li, M. Xian, B. Yang, M. Ying, Q. He, Inhibition of KLF4 by statins reverses Adriamycin-induced metastasis and cancer stemness in osteosarcoma cells, Stem Cell Rep. 8 (2017) 1617-1629. [0191] 34. M. G. Krebs, R. Sloane, L. Priest, L. Lancashire, J.-M. Hou, A. Greystoke, T. H. Ward, R. Ferraldeschi, A. Hughes, G. Clack, Evaluation and prognostic significance of circulating tumor cells in patients with non-small-cell lung cancer, J. Clin. Oncol. 29 (2011) 1556-1563. [0192] 35. J. S. De Bono, H. I. Scher, R. B. Montgomery, C. Parker, M. C. Miller, H. Tissing, G. V. Doyle, L. W. Terstappen, K. J. Pienta, D. Raghavan, Circulating tumor cells predict survival benefit from treatment in metastatic castration-resistant prostate cancer, Clin. Cancer Res. 14 (2008) 6302-6309. [0193] 36. B. Rack, C. Schindlbeck, J. Juckstock, U. Andergassen, P. Hepp, T. Zwingers, T. W. Friedl, R. Lorenz, H. Tesch, P. A. Fasching, Circulating tumor cells predict survival in early average-to-high risk breast cancer patients, J. Natl. Cancer Inst. 106 (2014) (dju066). [0194] 37. M. Cristofanilli, G. T. Budd, M. J. Ellis, A. Stopeck, J. Matera, M. C. Miller, J. M. Reuben, G. V. Doyle, W. J. Allard, L. W. Terstappen, Circulating tumor cells, disease progression, and survival in metastatic breast cancer, N. Engl. J. Med. 351 (2004) 781-791. [0195] 38. M. Becker, A. Nitsche, C. Neumann, J. Aumann, I. Junghahn, I. Fichtner, Sensitive PCR method for the detection and real-time quantification of human cells in xe-notransplantation systems, Br. J. Cancer 87 (2002) 1328. [0196] 39. W. P. Roos, A. D. Thomas, B. Kaina, DNA damage and the balance between survival and death in cancer biology, Nat. Rev. Cancer 16 (2016) 20. [0197] 40. E. Porcel, S. Liehn, H. Remita, N. Usami, K. Kobayashi, Y. Furusawa, C. Le Sech, S. Lacombe, Platinum nanoparticles: a promising material for future cancer therapy?Nanotechnology 21 (2010) 085103. [0198] 41. J. Gao, G. Liang, B. Zhang, Y. Kuang, X. Zhang, B. Xu, FePt@ CoS2 yolk-shell nanocrystals as a potent agent to kill HeLa cells, J. Am. Chem. Soc. 129 (2007) 1428-1433. [0199] 42. P. Asharani, N. Xinyi, M. P. Hande, S. Valiyaveettil, DNA damage and p53-mediated growth arrest in human cells treated with platinum nanoparticles, Nanomedicine 5 (2010) 51-64. [0200] 43. H. S. Choi, W. Liu, P. Misra, E. Tanaka, J. P. Zimmer, B. I. Ipe, M. G. Bawendi, J. V. Frangioni, Renal clearance of quantum dots, Nat. Biotechnol. 25 (2007) 1165. [0201] 44. Y. Ming, Y. Li, H. Xing, M. Luo, Z. Li, J. Chen, J. Mo, S. Shi, Circulating tumor cells: from theory to nanotechnology-based detection, Front. Pharmacol. 8 (2017) 35. [0202] 45. P. U. Atukorale, S. P. Raghunathan, V. Raguveer, T. J. Moon, C. Zheng, P. A. Bielecki, M. L. Wiese, A. L. Goldberg, G. Covarrubias, C. J. Hoimes, Nanoparticle encapsulation of synergistic immune agonists enables systemic codelivery to tumor sites and IFNβ-driven antitumor immunity, Cancer Res. 79 (2019) 5394-5406. [0203] 46. H. Du, S. Zhao, Y. Wang, Z. Wang, B. Chen, Y. Yan, Q. Yin, D. Liu, F. Wan, Q. Zhang, Y. Wang, pH/Cathepsin B hierarchical-responsive nanoconjugates for enhanced tumor penetration and chemo-immunotherapy, Adv. Funct. Mater. 30 (2020) 2003757. [0204] 47. J. Shi, P. W. Kantoff, R. Wooster, O. C. Farokhzad, Cancer nanomedicine: progress, challenges and opportunities, Nat. Rev. Cancer 17 (2017) 20. [0205] 48. R. Tong, L. Yala, T. M. Fan, J. Cheng, The formulation of aptamer-coated paclitax-el-polylactide nanoconjugates and their targeting to cancer cells, Biomaterials 31 (2010) 3043-3053. [0206] 49. B. Felding-Habermann, T. E. O'Toole, J. W. Smith, E. Fransvea, Z. M. Ruggeri, M. H. Ginsberg, P. E. Hughes, N. Pampori, S. J. Shattil, A. Saven, Integrin activation controls metastasis in human breast cancer, Proc. Natl. Acad. Sci. 98 (2001) 1853-1858.
[0207] It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the subject matter disclosed herein. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.