Tethered Lipoplex nanoparticle Biochips And Methods Of Use

Abstract

Disclosed are compositions and methods for the use of lipoplex nanoparticle chips and arrays in the detection of/diagnosis of a disease or condition.

What is claimed is: 
     
         1 . A method of making tethered immunolipoplex nanoparticle (iTLN) and cationic nanoparticle (cTLN) biochips comprising:
 (a) surface tethering treatment comprising:
 (1) gold coating of a solid substrate with the substrate being glass, silicon wafer, polymer, ceramics or any solid materials; 
 (2) a thin layer of self-assembly monolayer such as 2-mercaptoethanol (βME), 6-Mercaptohexanol, 16-mercaptohexadecanoic acid (MHA), and other thiol-backfiller molecules; 
 (3) tethering molecules such as WC14, FC16, DC18, and other thiolipids with ethylene oxide units; 
   (b) preparing tethered liposomal nanoparticles comprising:
 (1) lipid mixtures such as 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Cholesterol), and other ionizable lipids, 1,2-di-O-octadecenyl-3-dimethylammonium propane (DODMA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), and other non-ionizable lipids: DODMA), L-α-phosphatidylcholine (EggPC, SoyPC), Cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and other saturated fatty acid, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and other helper lipids and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG), and other PEG phospholipids. 
   
     
     
         2 . The method of  claim 1  wherein said iTLN nanoparticles are functionalized by post-insertion of antibody molecules, pepitide, carbohydrate, DNA/RNA or their mixtures as ligands for detecting specific cell surface receptors. 
     
     
         3 . The method of  claim 1  wherein said iTLN/cTLN nanoparticles are functionalized by avidin-biotin, digoxigenin (Dig)-anti-Dig, fluorescein-anti-FITC or other hapten linkages of antibody molecules, pepitide, carbohydrate, DNA/RNA or their mixtures as ligands for detecting specific cell surface receptors. 
     
     
         4 . The method of  claim 1  wherein said iTLN/cTLN nanoparticles contain reagents or reagent mixtures such as molecular beacons, quantum dots, and/or other sensing molecules and particles as ligands for detecting intra-cellular biomarkers such as messenger RNA, microRNA and proteins. 
     
     
         5 . The method of  claim 1  wherein said iTLN/cTLN nanoparticles contain reagents or reagent mixtures such as drug, DNA/RNA, magnetic particles and/or other therapeutic molecules and particles. 
     
     
         6 . The method of  claim 1  wherein said iTLN/cTLN nanoparticles are placed in the form of microarray. 
     
     
         7 . The method of  claim 1  wherein said iTLN/cTLN nanoparticles are placed in a larger array consisting of many smaller microarray with each small array containing specific cell surface targeting ligands, intra-cellular biomarker ligands and/or therapeutic molecules/particles. 
     
     
         8 . The method of  claim 1  wherein said iTLN/cTLN array is connected to a microfluidic setup so cell mixtures can be brought onto the array and cell washing can be carried out after certain cell incubation time. 
     
     
         9 . The method of  claim 1  where in iTLNs/cTLNs are tethered on nanoscale or microscale particles such as gold or magnetic particles and polymer beads. 
     
     
         10 . The method of  claim 1 , wherein iTLN and cTLN chips, microarrays, arrays and particles capture target cells, microvesicles, exosomes and/or virus with the contained mRNAs, microRNAs and/or proteins being detected by molecular probes such as molecular beacons using a total internal reflective fluorescence (TIRF) microscope, fluorescence microscope, plate reader or portable fluorescence detector. 
     
     
         11 . A method of detecting the presence of a disease or condition in a subject comprising obtaining a tissue or body fluid sample from a subject, contacting a lipoplex nanoparticle chip or array with the tissue or body fluid sample from the subject, wherein the lipoplex comprises a liposome with one or more labeling moieties incorporated into the liposome, wherein the lipoplex further comprises one or more surface targeting moieties on the liposomal surface as receptors for binding to a target cell, cell secreted microvesicles including exosomes, virus, bacteria, or antigen that corresponds to a particular disease or condition, and detecting the presence or absence of a disease or condition, wherein the presence of a disease or condition is indicated by the excitation of a label of the labeling moiety that occurs through the capture and incorporation of a cell, cell secreted microvesicles including exosomes, virus, bacteria, or antigen that corresponds to a particular disease or condition into the lipoplex nanoparticle. 
     
     
         12 . The method of  claim 11 , wherein the lipoplex nanoparticle is immobilized by tethering the lipoplex to a substrate to form a tethered lipoplex nanoparticle (TLN) chip or array. 
     
     
         13 . The method of  claim 11 , wherein the lipoplex nanoparticle is free in solution. 
     
     
         14 . The method of  claim 11 , wherein the lipoplex nanoparticle chip or array is a immunolipoplex nanoparticle chip or array, wherein the surface targeting moiety comprises antibody molecules, pepitides, carbohydrates, aptamer, DNA/RNA or mixtures thereof. 
     
     
         15 . The method of  claim 14 , wherein the lipoplex nanoparticle chip or array comprises antibodies on the surface of liposome, wherein the antibodies on the surface of the liposome are specific for a determinant on an exosome, proteins, RNA, DNA, viral-like particle, virus, bacterial protein, circulating tumor cell, or viral particle. 
     
     
         16 . The method of  claim 11 , wherein the lipoplex nanoparticle chip or array is a cationic lipoplex nanoparticle chip or array, wherein the surface targeting moiety comprises a positive charge on the surface of the liposome, wherein the positively charged liposome binds negatively charged particles in the tissue or body fluid sample. 
     
     
         17 . The method of  claim 11 , wherein the lipoplex nanoparticle array comprises two or more smaller arrays with each small array comprising a specific cell surface target. 
     
     
         18 . The method of  claim 17 , wherein the array comprises one or more immunolipoplex nanoparticle arrays. 
     
     
         19 . The method of  claim 17 , wherein the array comprises one or more cationic nanoparticle arrays. 
     
     
         20 . The method of  claim 17 , wherein the array comprises one or more immunolipoplex nanoparticle arrays and one or more cationic lipoplex nanoparticle arrays. 
     
     
         21 . The method of  claim 11 , wherein in the labeling moiety comprises a molecular beacon. 
     
     
         22 . The method of  claim 21 , wherein the molecular beacon comprises a fluorescence marker or radiomarker. 
     
     
         23 . The method of  claim 11 , wherein in the labeling moiety comprises a quantum dot. 
     
     
         24 . The method of  claim 11 , wherein the lipoplex nanoparticle binds exosomes, proteins, RNA, DNA, viral-like particles, viruses, bacterial proteins, circulating tumor cells, or viral particles. 
     
     
         25 . The method of  claim 11 , wherein the disease or condition is a cancer. 
     
     
         26 . The method of  claim 11 , wherein the disease or condition is a viral infection and the virus or viral antigen incorporated into the lipoplex nanoparticle is or is derived from the viruses selected from the group of viruses consisting of Herpes Simplex virus-1, Herpes Simplex virus-2, Varicella-Zoster virus, Epstein-Barr virus, Cytomegalovirus, Human Herpes virus-6, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus, Influenza virus A (including H1N1 or other Swine H1), Influenza virus B, Measles virus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovirus, Yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Simian Immunodeficiency virus, Human T-cell Leukemia virus type-1, Hantavirus, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, and Human Immunodeficiency virus type-2. 
     
     
         27 . The method of  claim 11 , wherein the disease or condition is a bacterial infection and the bacterium or bacterial antigen captured by the lipoplex nanoparticle is or is derived from the bacteria selected from the group of bacteria consisting of  M. tuberculosis, M. bovis, M. bovis  strain BCG, BCG substrains,  M. avium, M. intracellulare, M. africanum, M. kansasii, M. marinum, M. ulcerans, M. avium  subspecies  paratuberculosis, Nocardia asteroides,  other  Nocardia  species,  Legionella pneumophila,  other  Legionella  species,  Salmonella typhi,  other  Salmonella  species,  Shigella  species,  Yersinia pestis, Pasteurella haemolytica, Pasteurella multocida,  other  Pasteurella  species,  Actinobacillus pleuropneumoniae, Listeria monocytogenes, Listeria ivanovii, Brucella abortus,  other  Brucella  species,  Cowdria ruminantium, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetti,  other  Rickettsial  species,  Ehrlichia  species,  Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Bacillus anthracis, Escherichia coli, Vibrio cholerae, Campylobacter  species,  Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa,  other  Pseudomonas  species,  Haemophilus influenzae, Haemophilus ducreyi,  other  Hemophilus  species,  Clostridium tetani,  other  Clostridium  species,  Yersinia enterolitica,  and other  Yersinia  species. 
     
     
         28 . The method of  claim 11 , wherein the disease or condition is exposure to a toxin and the antigen captured by the lipoplex nanoparticle is a toxin selected from the group of toxins consisting of Abrin, Conotoxins Diacetoxyscirpenol Bovine spongiform encephalopathy agent, Ricin, Saxitoxin, Tetrodotoxin, epsilon toxin,  Botulinum  neurotoxins, Shigatoxin,  Staphylococcal  enterotoxins, T-2 toxin, Diphtheria toxin, Tetanus toxoid, and pertussis toxin or a fragment thereof. 
     
     
         29 . The method of  claim 11 , wherein the disease or condition is a cancer and the antigen or cell captured by the lipoplex nanoparticle is derived from a cancer selected from the group consisting of lymphomas (Hodgkins and non-Hodgkins), B cell lymphoma, T cell lymphoma, myeloid leukemia, leukemias, mycosis fungoides, carcinomas, carcinomas of solid tissues, squamous cell carcinomas, adenocarcinomas, sarcomas, gliomas, blastomas, neuroblastomas, plasmacytomas, histiocytomas, melanomas, adenomas, hypoxic tumors, myelomas, AIDS-related lymphomas or sarcomas, metastatic cancers, bladder cancer, brain cancer, nervous system cancer, squamous cell carcinoma of head and neck, neuroblastoma/glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, colon cancer, cervical cancer, cervical carcinoma, breast cancer, epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, hematopoietic cancers, testicular cancer, colon-rectal cancers, prostatic cancer, pancreatic cancer, or cancer cachexia. 
     
     
         30 . The method of  claim 29 , wherein the antigen is an exosome, protein, peptide, or nucleic acid from the cancer. 
     
     
         31 . The method of  claim 11 , wherein the body fluid sample is blood, serum, urine, sputum, or saliva from the subject.