Metastasis and Adaptive Resistance Inhibiting Immunotherapy Combined Online Chemotherapy with Radiotherapy's tumor Seeking Extracellular Vesicles with siRNA and Chemotherapeutics
20180356373 ยท 2018-12-13
Inventors
Cpc classification
C12N2521/00
CHEMISTRY; METALLURGY
C12Q1/6876
CHEMISTRY; METALLURGY
C12N2529/00
CHEMISTRY; METALLURGY
International classification
C12N15/63
CHEMISTRY; METALLURGY
C12N15/113
CHEMISTRY; METALLURGY
Abstract
Mutated genome silencing with endogenous RNAi-siRNA and miRNA with near total cellular apheresis with pulse flow apheresis system and EV-exosome-RNA molecular apheresis with sucrose density gradient continuous flow ultracentrifugation combined with array centrifuge for both 50S higher and 50S lower proteomics and genomics apheresis and their fractionated purification with immobilized Tim4-Fc protein Ca2+ magnetic beads affinity chromatography (ACG) and immobilized metal ACG is disclosed. It purifies normal cell derived and tumor cell derived EVs-exosomes, proteomics and subcellular particles. Tumor-specific endogenous siRNA is generated from mutated RNA containing pre-miRNA hairpin through RNA-induced silencing complex (RISC) composed of Dicer, dsRNA binding protein TRBP, and AGO2. Incubating purified RSIC with pre-let-7 hairpin generates siRNA. SiRNA is bonded with T-EVs and T-cells to silence its evasion from tumor immunity. While on radiation therapy or surgery, a patient's blood is continuously processed with above systems. It delivers combined online radiotherapy, and tumor-seeking adoptive extracorporeal chemo-immunotherapy.
Claims
1. A device for circulating cell and subcellular nanoparticle's separation and removal comprising: a. apparatus for pulse flow aphaeresis of red cells, white cells, platelets and tumor cells and plasmapheresis and filtration of said components in blood; b. pulse flow aphaeretic system attached to affinity chromatograms; c. pulse flow aphaeretic system combined with microfilters; d. pulse flow apheresis system attached to immune affinity columns; e. pulse flow apheresis system attached to a continuous flow ultracentrifuge rotor; f. a continuous flow ultracentrifuge for plasma soluble molecule's apheresis; g. a continuous flow ultracentrifuge rotor connected to a series of affinity columns; h. a continuous flow ultracentrifuge rotor connected to size exclusion chromatography columns; i. a continuous flow ultracentrifuge rotor connected to iZON science's modified size exclusion chromatography columns; j. a continuous flow ultracentrifuge rotor connected to immobilized Tim4-Fc protein Ca.sup.2+ magnetic beads affinity columns; k. a continuous flow ultracentrifuge rotor connected to immobilized metal affinity chromatography columns l. a continuous flow ultracentrifuge rotor connected to immuno-affinity chromatography columns; m. a continuous flow ultracentrifuge rotor connected to heparin sulfate pseudo-affinity chromatography columns; n. a continuous flow ultracentrifuge rotor connected to lectin ligand affinity chromatography columns; o. a continuous flow ultracentrifuge rotor connected to lipofectamine 2000 chromatography columns; p. a continuous flow ultracentrifuge rotor and affinity chromatography columns connected to array ultracentrifuge with rotors ranging from 12-96; q. a continuous flow ultracentrifuge rotor and affinity chromatography columns connected to an array ultracentrifuge with rotors ranging from 12-96 and each said rotors spins at adjustable rpm; r. array centrifuge rotors capable of spinning at adjustable g-force ranging from 100,000 to 200,000; s. array centrifuge rotor's spin rate controlling computer; t. a pulse flow apheresis system, a continuous flow ultracentrifuge rotor and affinity chromatography columns connected to array ultracentrifuge rotors and to processed plasma collecting and cooling chambers; u. a pulse flow apheresis system, a continuous flow ultracentrifuge rotor and affinity chromatography columns connected to array ultracentrifuge rotors and to processed plasma cooling and sucrose precipitating chambers; v. a pulse flow apheresis system, a continuous flow ultracentrifuge rotor and affinity chromatography columns connected to processed plasma collecting and cooling chambers; w. a pulse flow apheresis system, a continuous flow ultracentrifuge rotor, affinity chromatography columns and array centrifuge with a series of rotors connected to processed plasma cooling and sucrose precipitating chambers; x. a pulse flow apheresis system, a continuous flow ultracentrifuge rotor, affinity chromatography columns connected to flow cytometer (FCM), atomic force microscope (AFM), nanoparticle tracking analysis (NTA) system, disc centrifuge nanoparticle analysis (DCNA) system and to large-scale targeted proteomics assay resource; y. a pulse flow apheresis system, a continuous flow ultracentrifuge rotor, affinity chromatography columns and array centrifuge with a series of array centrifuge rotors connected to flow cytometer, atomic force microscope, nanoparticle tracking analysis (NTA) system, disc centrifuge nanoparticle analysis (DCNA) system and to large-scale targeted proteomics assay resource; z. a pulse flow apheresis system, a continuous flow ultracentrifuge rotor, affinity chromatography columns connected to sucrose density gradient fraction collector; aa. a pulse flow apheresis system, a continuous flow ultracentrifuge rotor, affinity chromatography columns and an array centrifuge with a series of rotors connected to photometer/flow cell and to sucrose density gradient fraction collector; bb. blood and red blood cells, leucocytes, lymphocytes, platelets and plasma collection bags attachable to pulse flow combined ultracentrifuge apheresis system.
2. Methods of apheresis of circulating cells and plasma soluble subcellular particles, extracellular vesicles, exosomes, proteomics and genomics and therapeutic applications of said endogenous subcellular particles comprising the steps of: a. therapeutic apheresis of circulating mutated cellular and subcellular particles to minimize metastasis and tumor recurrence; b. therapeutic apheresis of large burst of mutated cellular and subcellular particles, extracellular vesicles and exosomes released into circulation in response to cancer treatments to inhibit abscopal metastasis in distant organs; c. therapeutic apheresis of large burst of mutated cellular and subcellular particles, extracellular vesicles and exosomes released into circulation in response to radiation therapy to inhibit abscopal metastasis in distant organs; d. therapeutic apheresis of large burst of mutated cellular and subcellular particles, extracellular vesicles and exosomes released into tumor environment in response to radiation therapy to inhibit metastasis from bystander effect; e. therapeutic apheresis of large burst of mutated cellular and subcellular particles, extracellular vesicles and exosomes released into circulation in response to cancer treatments to inhibit platelet activation and abscopal metastasis in distant organs; f. therapeutic apheresis of large burst of mutated cellular and subcellular particles, extracellular vesicles and exosomes released into circulation in response to cancer treatments to inhibit macrophage activation into tumor promoting M2-like macrophage and abscopal metastasis in distant organs; g. therapeutic apheresis of large burst of mutated cellular and subcellular particles, extracellular vesicles and exosomes released into circulation in response to cancer treatments that inhibit T-lymphocytes, macrophage, platelets and innate immunity against tumor; h. therapeutic apheresis of large burst of mutated cellular and subcellular particles, extracellular vesicles and exosomes released into circulation in response to cancer treatments that cause T-lymphocyte's escape from innate and adaptive immune response to tumor; i. therapeutic apheresis of large burst of mutated cellular and subcellular particles, extracellular vesicles and exosomes released into circulation in response to radiation therapy to overcome therapeutic escape due to proteomic changes in extracellular vesicles and exosomes caused by radiation; j. mutated genome silencing with endogenous RNAi-siRNA generated from DNA damage repair response releasing Ago-2-RISC-Rad-51-diRNA-RNAi-miRNA complexes after radiation therapy and cancer treatments; k. mutated genome silencing with endogenous RNAi-siRNA generated from DNA damage repair response releasing Ago-2-RISC-Rad-51-diRNA-RNAi-miRNA complexes separated by continuous flow ultracentrifugation and siRNA and RNAi precipitation onto array centrifuge rotors and administration such prepared siRNA and RNAi back to patient; l. mutated genome silencing with endogenous RNAi-siRNA generated from DNA damage repair response releasing Ago-2-RISC-Rad-51-diRNA-RNAi-miRNA complexes captured onto affinity columns and RNAi, siRNA elution and administration back to patient; m. mutated genome silencing with endogenous siRNA generated by incubating purified RSIC with pre-let-7 hairpin; n. bonding siRNA with tumor cell derived extracellular vesicles by electroporation for cell silencing; o. bonding siRNA with tumor cell derived extracellular vesicles by photochemical methods for cell silencing; p. bonding siRNA with tumor cell derived extracellular vesicles with lipofectamine 2000 for cell silencing; q. bonding siRNA with T-lymphocytes by electroporation to inhibit T-cell evasion from immunity; r. bonding siRNA with T-lymphocytes by photochemical method to inhibit T-lymphocyte's evasion from immunity; s. bonding siRNA with T-lymphocytes with lipofectamine 2000 to inhibit T-lymphocyte's evasion from immunity; t. inhibition of chronic graft versus host disease with tumor cell derived extracellular vesicles' internalized photosensitive complex-siRNA; u. inhibition of chronic graft versus host disease with T-lymphocytes with internalized photosensitive complex-siRNA; v. internalization of chemotherapeutics into tumor cell's extracellular vesicles and exosomes purified by continuous flow ultracentrifuge and array ultracentrifuge by electroporation for extracorporeal chemotherapy; w. internalization of chemotherapeutics into tumor cell's extracellular vesicles and exosomes purified by continuous flow ultracentrifuge and array ultracentrifuge by photochemical methods for extracorporeal chemotherapy; x. internalization of chemotherapeutics into tumor cell's extracellular vesicles and exosomes purified by continuous flow ultracentrifuge and array ultracentrifuge with lipofectamine 2000 for extracorporeal chemotherapy; y. tumor seeking extracorporeal chemotherapy with tumor cell's extracellular vesicles with internalized chemotherapeutics; z. combined online radiation therapy, surgery, chemotherapy and therapeutic apheresis of large burst of mutated cellular and subcellular particles, extracellular vesicles and exosomes released into circulation in response to such treatments with combined pulse flow apheresis and continuous flow ultracentrifuge and array centrifuge ultracentrifugation apheresis; aa. combined online radiation therapy and chemotherapy and therapeutic apheresis of large burst of mutated cellular and subcellular particles, extracellular vesicles and exosomes released into circulation in response to such treatments with combined pulse flow apheresis and continuous flow ultracentrifuge and array centrifuge ultracentrifugation apheresis; bb. near total apheresis of mutated cellular and subcellular particles, extracellular vesicles and exosomes released into circulation in response to cancer treatments by apheresis of the entire circulating blood and plasma several times during a treatment cycle lasting several hours. cc. therapeutic apheresis of large burst of mutated cellular and subcellular particles, extracellular vesicles and exosomes released into circulation in response to cancer chemotherapy to inhibit abscopal metastasis in distant organs; dd. apheresis of mutated extracellular vesicles carrying apoptotic bodies, microsomes, exosomes, oncosomes, DNA and DNA fragments and microRNAs; ee. apheresis of circulating plasma soluble mutated subcellular particles extracellular vesicles, exosomes, proteosomes to inhibit early niche metastatic process; ff. apheresis of extracellular vesicles carrying vascular endothelial growth factor to inhibit tumor vascular formation; gg. apheresis of early metastatic lymph node seeding of extracellular vesicles and exosomes; hh. apheresis of benign metastatic lymphangioleiomyomatosis (LAM) causing extracellular vesicles; ii. apheresis of extracellular vesicles and exosomes traveling to sentinel lymph nodes and causing metastatic melanomas; jj. apheresis of extracellular vesicles and exosomes causing metastatic melanomas; kk. pulse flow aphaeresis of red cells, white cells, platelets and tumor cells and plasmapheresis and filtration of said components in blood; ll. pulse flow aphaeresis plasma's filtration with microfilters; mm. pulse flow aphaeresis filtered plasma's size exclusion chromatography with chromatographic columns; nn. chromatography of pulse flow aphaeresis' filtered plasma with immune affinity columns; oo. removal of subcellular particle's dissolved in pulse flow apheresis plasma with a continuous flow ultracentrifuge rotor connected to pulse flow apheresis system; pp. a continuous flow plasma ultracentrifugation apheresis, removal and characterization of plasma soluble subcellular particles, extracellular vesicles, exosomes, proteosomes and genomes with higher than 50S sedimentation coefficient in sucrose density gradient; qq. a continuous flow plasma ultracentrifugation aphaeresis for removal and characterization of plasma soluble mutated molecular subcellular particles, extracellular vesicles, exosomes, proteosomes and genomes with higher than 50S sedimentation coefficient in sucrose density gradient; rr. a continuous flow ultracentrifugation rotor and a series of array centrifuge rotors combined plasma ultracentrifugation for removal and characterization of plasma soluble subcellular particles, extracellular vesicles, exosomes, proteosomes and genomes with lower than 50S sedimentation coefficient in sucrose density gradient; ss. a continuous flow ultracentrifugation rotor and a series of array centrifuge rotors combined plasma ultracentrifugation for removal and characterization of mutated plasma soluble molecular subcellular particles, extracellular vesicles, exosomes, proteosomes and genomes with lower than 50S sedimentation coefficient in sucrose density gradient; tt. a continuous flow ultracentrifugation rotor combined with a series of array centrifuge rotors with adjustable rpm and g-force for plasma ultracentrifugation for removal and characterization of plasma soluble subcellular particles, extracellular vesicles, exosomes, proteosomes and genomes with lower than 50S sedimentation coefficient in sucrose density gradient; uu. a continuous flow ultracentrifugation rotor combined with a series of array centrifuge rotors for plasma ultracentrifugation for removal and characterization of mutated plasma soluble molecular subcellular particles, extracellular vesicles, exosomes, proteosomes and genomes with lower than 50S sedimentation coefficient in sucrose density gradient; vv. molecular sieve separation and characterization of plasma soluble molecular subcellular particles with higher than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor connected to a series of affinity columns; ww. molecular sieve separation and characterization of plasma soluble molecular subcellular particles with higher than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor connected to size exclusion chromatography columns; xx. molecular sieve separation and characterization of plasma soluble molecular subcellular particles with higher than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor connected to iZON science's modified size exclusion chromatography columns; yy. molecular sieve separation and characterization of plasma soluble molecular subcellular particles with higher than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor connected to immobilized Tim4-Fc protein Ca.sup.2+ magnetic beads affinity columns; zz. molecular sieve separation and characterization of plasma soluble molecular subcellular particles with higher than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor connected to immobilized metal affinity chromatographic columns; aaa. molecular sieve separation and characterization of plasma soluble molecular subcellular particles with higher than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor connected to immuno-affinity chromatographic columns; bbb. molecular sieve separation and characterization of plasma soluble molecular subcellular particles with higher than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor connected to heparin sulfate pseudo-affinity chromatography columns; ccc. molecular sieve separation and characterization of plasma soluble molecular subcellular particles with higher than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor connected to lectin ligand affinity chromatography columns; ddd. molecular sieve separation and characterization of plasma soluble molecular subcellular particles with higher than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor connected to lipofectamine 2000 chromatography columns; eee. molecular sieve separation and characterization of plasma soluble mutated molecular subcellular particles with higher than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor connected to a series of affinity columns; fff. molecular sieve separation and characterization of plasma soluble mutated molecular subcellular particles with higher than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor connected to size exclusion chromatography columns; ggg. molecular sieve separation and characterization of plasma soluble mutated molecular subcellular particles with higher than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor connected to iZON science's modified size exclusion chromatography columns; hhh. molecular sieve separation and characterization of plasma soluble mutated molecular subcellular particles with higher than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor connected to immobilized Tim4-Fc protein Ca.sup.2+ magnetic beads affinity columns; iii. molecular sieve separation and characterization of plasma soluble mutated molecular subcellular particles with higher than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor connected to immobilized metal affinity chromatographic columns; jjj. molecular sieve separation and characterization of plasma soluble mutated molecular subcellular particles with higher than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor connected to immuno-affinity chromatographic columns; kkk molecular sieve separation and characterization of plasma soluble mutated molecular subcellular particles with higher than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor connected to heparin sulfate pseudo-affinity chromatography columns; lll. molecular sieve separation and characterization of plasma soluble mutated molecular subcellular particles with higher than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor connected to lectin ligand affinity chromatography columns; mmm. molecular sieve separation and characterization of plasma soluble mutated molecular subcellular particles with higher than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor connected to lipofectamine 2000 chromatography columns; nnn. molecular sieve separation and characterization of plasma soluble molecular subcellular particles with lower than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor and affinity chromatography columns connected to a continuous flow array ultracentrifuge having an array of rotors ranging from 12-96 and having rotors with adjustable rpm and g-force ranging from 100,000 to 200,000; ooo. molecular sieve separation and characterization of plasma soluble molecular subcellular particles with lower than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor and size exclusion chromatography columns connected to a continuous flow array ultracentrifuge having an array of rotors ranging from 12-96 and having rotors with adjustable rpm and g-force ranging from 100,000 to 200,000; ppp. molecular sieve separation and characterization of plasma soluble molecular subcellular particles with lower than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor and iZON science's modified size exclusion chromatography columns connected to a continuous flow array ultracentrifuge having an array of rotors ranging from 12-96 and having rotors with adjustable rpm and g-force ranging from 100,000 to 200,000; qqq. molecular sieve separation and characterization of plasma soluble molecular subcellular particles with lower than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor and immobilized Tim4-Fc protein Ca2+ magnetic beads affinity columns connected to a continuous flow array ultracentrifuge having an array of rotors ranging from 12-96 and having rotors with adjustable rpm and g-force ranging from 100,000 to 200,000; rrr. molecular sieve separation and characterization of plasma soluble molecular subcellular particles with lower than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor and immuno-affinity chromatographic columns connected to a continuous flow array ultracentrifuge having an array of rotors ranging from 12-96 and having rotors with adjustable rpm and g-force ranging from 100,000 to 200,000; sss. molecular sieve separation and characterization of plasma soluble molecular subcellular particles with lower than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor and heparin sulfate pseudo-affinity chromatography columns connected to a continuous flow array ultracentrifuge having an array of rotors ranging from 12-96 and having rotors with adjustable rpm and g-force ranging from 100,000 to 200,000; ttt. molecular sieve separation and characterization of plasma soluble molecular subcellular particles with lower than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor and lectin ligand affinity chromatography columns connected to a continuous flow array ultracentrifuge having an array of rotors ranging from 12-96 and having rotors with adjustable rpm and g-force ranging from 100,000 to 200,000; uuu. molecular sieve separation and characterization of plasma soluble molecular subcellular particles with lower than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor and lipofectamine 2000 chromatography columns connected to a continuous flow array ultracentrifuge having an array of rotors ranging from 12-96 and having rotors with adjustable rpm and g-force ranging from 100,000 to 200,000; vvv. molecular sieve separation and characterization of plasma soluble mutated molecular subcellular particles with lower than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor and size exclusion chromatography columns connected to a continuous flow array ultracentrifuge having an array of rotors ranging from 12-96 and having rotors with adjustable rpm and g-force ranging from 100,000 to 200,000; www. molecular sieve separation and characterization of plasma soluble mutated molecular subcellular particles with lower than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor and iZON science's modified size exclusion chromatography columns connected to a continuous flow array ultracentrifuge having an array of rotors ranging from 12-96 and having rotors with adjustable rpm and g-force ranging from 100,000 to 200,000; xxx. molecular sieve separation and characterization of plasma soluble mutated molecular subcellular particles with lower than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor and immobilized Tim4-Fc protein Ca2+ magnetic beads affinity columns connected to a continuous flow array ultracentrifuge having an array of rotors ranging from 12-96 and having rotors with adjustable rpm and g-force ranging from 100,000 to 200,000; yyy. molecular sieve separation and characterization of plasma soluble mutated molecular subcellular particles with lower than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor and immuno-affinity chromatographic columns connected to a continuous flow array ultracentrifuge having an array of rotors ranging from 12-96 and having rotors with adjustable rpm and g-force ranging from 100,000 to 200,000; zzz. molecular sieve separation and characterization of plasma soluble mutated molecular subcellular particles with lower than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor and heparin sulfate pseudo-affinity chromatography columns connected to a continuous flow array ultracentrifuge having an array of rotors ranging from 12-96 and having rotors with adjustable rpm and g-force; ranging from 100,000 to 200,000 aaaa. molecular sieve separation and characterization of plasma soluble mutated molecular subcellular particles with lower than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor and lectin ligand affinity chromatography columns connected to a continuous flow array ultracentrifuge having an array of rotors ranging from 12-96 and having rotors with adjustable rpm and g-force ranging from 100,000 to 200,000; bbbb. molecular sieve separation and characterization of plasma soluble mutated molecular subcellular particles with lower than 50S sedimentation coefficient with a continuous flow ultracentrifuge rotor and lipofectamine 2000 chromatography columns connected to a continuous flow array ultracentrifuge having an array of rotors ranging from 12-96 and having rotors with adjustable rpm and g-force ranging from 100,000 to 200,000; cccc. computer and computer software aided control of array centrifuge rotor's varying spin rate that separates subcellular particles based on their molecular weights and configurations in gradient solutions; dddd. separation of high density sucrose from sucrose density gradient by cold precipitation before administration of the treated aphaeretic plasma back to patient; eeee. aphaeretic processed plasma collection to sterile blood and blood component's collection bags for their return to patients and for such sample's preservation; ffff. monitoring of subcellular particles derived from pulse flow apheresis system, continuous flow ultracentrifuge rotor system with attached affinity chromatography columns with flow cytometer (FCM), atomic force microscope (AFM), nanoparticle tracking analysis (NTA) system, with disc centrifuge nanoparticle analysis (DCNA) system and large-scale targeted proteomics assay resource; gggg. monitoring of subcellular particles derived from pulse flow apheresis system, continuous flow ultracentrifuge rotor system with attached affinity chromatography columns and array centrifuge with a series of array centrifuge rotors with flow cytometer (FCM), atomic force microscope (AFM), nanoparticle tracking analysis (NTA) system, with disc centrifuge nanoparticle analysis (DCNA) system and large-scale targeted proteomics assay resource; hhhh. biochemical and molecular analysis of sucrose density fractions with a photometer and sucrose density gradient fraction collector; iiii. biochemical and molecular analysis of sucrose density gradient fraction's passed through chromatography columns; jjjj. collecting blood, red blood cells, leucocytes, lymphocytes, platelets and plasma into collection bags attachable to pulse flow apheresis system; kkkk collecting continuous flow ultracentrifuge, chromatography columns and array centrifuge processed plasma into sterile collection bags for transfusion back to patient or for preservation and future use; llll. distribution of purified patient specific subcellular particles, proteomics and genomics for research; mmmm. distribution of purified patient specific subcellular particles, proteomics and genomics collected with pulse flow apheresis system, continuous flow ultracentrifugation and array centrifuge centrifugation for inter-laboratory analysis and standardization.
Description
1. BRIEF DESCRIPTION OF THE DRAWINGS
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2. REFERENCE NUMERALS
[0067] 378. Corrugated pipe waveguide [0068] 380. Whole blood reservoir [0069] 382. Densitometer-1 [0070] 384. Pulsed pump [0071] 386. CTC, plasma-platelet and exosomes, microsomes and nanosomes reservoir [0072] 388. Pulsed pump [0073] 390. Densitometer-2 [0074] 392. DNA/RNA/Telomerase, exosomes, nanosomes affinity column-1 with EGCG [0075] 394. Densitometer-3 [0076] 396. Pulsed pump [0077] 398. Purified plasma collecting bag [0078] 400. Densitometer-4 [0079] 402. Reservoir with CTC, platelets, exosomes, microsomes and nanosomes/DNA-Telomerase [0080] 404. Pulsed pump [0081] 406. DNA/RNA/Telomerase, exosomes, nanosomes affinity column-2 with EGCG [0082] 408. Densitometer-5 [0083] 410. Pulsed pump [0084] 412. Purified platelets collecting bag [0085] 414. Densitometer-6 [0086] 416. Pulsed pump [0087] 418. Reservoir for RBC plus WBC and CTC, exosomes, microsomes and nanosomes [0088] 418B. Reservoir with WBC, CTC, exosomes, microsomes and nanosomes/DNA-Telomerase [0089] 420. Densitometer-7 [0090] 422. Pulsed pump [0091] 424. DNA/RNA/Telomerase, exosomes, nanosomes affinity column-3 with EGCG [0092] 426. Densitometer-8 [0093] 428. Pulsed pump [0094] 430. CTC, DNA/RNA/Telomerase, exosome, microsomes and nanosomes free WBC collecting bag [0095] 432. Densitometer-9 [0096] 434. Pulsed pump [0097] 436. Reservoir with concentrated RBC, CTC, exosomes, microsomes and nanosomes/DNA-Telomerase [0098] 438. Densitometer-10 [0099] 440. Pulsed pump [0100] 442. DNA/RNA/Telomerase, exosomes, nanosomes affinity column-4 with EGCG [0101] 444. Densitometer-11 [0102] 446. Pulsed pump [0103] 448. Purified RBC collecting bag [0104] 450. Pulsed pump [0105] 452. Air bubble sensor [0106] 454. Densitometer-12 [0107] 456. Treated return blood in blood flow tubing [0108] 458. Reservoir for DNA/RNA/Telomerase, tumor associated exosome, microsomes, nanosomes and CTC free blood after pulse flow purification [0109] 460. Blood flow inlet channel with clam and sensor [0110] 462. Blood flow return channel with clam and sensor [0111] 464. System clamp with sensors [0112] 466. Diluting normal saline [0113] 468. Anticoagulant reservoir [0114] 470. Blood flow tubing [0115] 472P. Microfilter for CTC removal from plasma [0116] 474P. Microfilter plasma CTC elution collection inlet and outlet [0117] 476P. Purified plasma collection inlet and outlet [0118] 476W Microfilter for removal of CTC bound to WBC [0119] 478R. microfilter for removal of CTC bound to RBC concentrate [0120] 478PL. Microfilter for removal of CTC bound to platelet [0121] 480PL. Microfilter platelet CTC elution collection inlet and outlet [0122] 482PL. Purified platelet collection inlet and outlet [0123] 484W. Microfilter WBC bound CTC elution collection inlet and outlet [0124] 486W. Purified WBC collection inlet and outlet [0125] 488R. Microfilter RBC bound CTC elution collection inlet and outlet [0126] 490R. Purified RBC collection inlet and outlet [0127] 492. Inlet and outlet tube connection [0128] 494. Ultracentrifuge continuous flow rotor [0129] 496. Bottom sample inlet [0130] 498. Mechanical seal [0131] 500. Damper [0132] 502. Bottom hollow driveshaft [0133] 504. Rotation Chamber [0134] 506. Core [0135] 508. High speed rotating cylindrical rotor [0136] 510. Top hollow driveshaft [0137] 512. High frequency motor [0138] 514. Top Mechanical seal [0139] 516. EV-exosome with siRNA effluent plasma flow towards chromatographic columns [0140] 517. EV-exosome with siRNA effluent plasma [0141] 518A. Flow control valve [0142] 518A2. Flow control valve [0143] 518B. SDG fraction collecting air injection [0144] 518C. SDG fraction collecting flow line [0145] 518C2. SDG fraction collecting flow line-2 [0146] 518D. Affinity chromatographic column [0147] 518E. Purified SDG fraction flow to sucrose removing cold reservoir [0148] 518F. Purified SDG fraction collection line [0149] 518G. Purified SDG fraction collection line valve [0150] 518H. SDG fraction outlet and flow control valve [0151] 518-I. SDG fraction sample collection tubes [0152] 519. Processed plasma flow line [0153] 519B. Purifying effluent plasma [0154] 519C. Cooled sucrose free plasma [0155] 520. Sucrose removing cold reservoir [0156] 520B. Sedimented sucrose [0157] 521. Sucrose removed plasma with EV-exosome siRNA or chemotherapeutics [0158] 521B. cooling chamber [0159] 522. Processed plasma flow line [0160] 522A. Affinity chromatography column 1 [0161] 522B. Affinity chromatography column-2 [0162] 522C. Affinity chromatography column-3 [0163] 522D. Affinity chromatography column-4 [0164] 522E. Affinity chromatography column-5 [0165] 522F. Affinity chromatography column-6 [0166] 522G. Affinity chromatography column-7 [0167] 522H. Affinity chromatography column-8 [0168] 522I. Affinity chromatography column-9 [0169] 522J. Affinity chromatography column-10 [0170] 523. Purified, processed plasma with EV-exosome-siRNA-chemotherapeutics reservoir [0171] 524. Purified, processed plasma with EV-exosome-siRNA and or chemotherapeutics [0172] 525A. Purified, processed plasma with EV-exosome-siRNA-chemotherapeutics inlet to Patient [0173] 525B. Plasma inlet valve to patient [0174] 525C. Purified plasma with EV-exosome-proteomics-siRNA collecting bag [0175] 525D. Purified plasma with EV-exosome-proteomics-siRNA to collecting bag inlet valve [0176] 525E. Collecting bag's outflow [0177] 525F. Colleting bag's outflow controlling valve [0178] 526. Control system's LCD [0179] 522A. Affinity chromatography column-1 [0180] 524B. Plasma injector to rotor [0181] 526B. Pulsed flow apheresis plasma into plasma injector [0182] 527A. Cooling chamber filter-1 [0183] 527B. Cooling Chamber filter-2 [0184] 528. Plasma injector [0185] 530A. Cooling Chamber-1 [0186] 530B. Cooling Chamber-2 [0187] 532. Warming coils [0188] 534. Effluent from the chromatographic columns [0189] 536. AFM [0190] 538. NTA [0191] 540. DCNA [0192] 542. FCM [0193] 544. Electronic flow direction control switch [0194] 546. Flow line for lab-testing [0195] 548. Array centrifuge [0196] 550. Titanium rotor for array centrifuge [0197] 552. Upper rotor half [0198] 554. Lower rotor half [0199] 556. Array centrifuge rotor shaft [0200] 558. Electronic inlet and outlet flow directing switch [0201] 560. O-ring [0202] 562. Rotor spin rate controlling computer [0203] 564. Disposable polypropylene rotor inserts [0204] 565 Polyethylene rotor wall [0205] 568. Directional arrows [0206] 570. SDG fraction collecting flow line [0207] 572. Electronic flow control valve [0208] 574. Portable trailer [0209] 576. Continuous flow rotor [0210] 578. Plasma flow control valve [0211] 580. Rotating seal assembly [0212] 582. Central inlet [0213] 584. Upper radial channel [0214] 585. Air block [0215] 586. Inner surface of the bowl wall [0216] 588. Core tapper volume [0217] 590. Core [0218] 592. Connecting channel to central flow [0219] 594. Edgeline outlet [0220] 596. SDG fraction flow to photometer/flow cell [0221] 598. Photometer/Flow cell [0222] 600. SDG Fraction collector [0223] 602. Lid [0224] 604. Bowl [0225] 606. Buffer reservoir [0226] 608. SDG solution reservoir [0227] 610. Buffer line [0228] 612. SDG line [0229] 614. Buffer and SDG flow line valve [0230] 616. Displacement dense fluid reservoir [0231] 618. Displacement dense fluid flow line
3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0232]
[0233] CTC separation by microfiltration is fast and simple. After chemotherapy/radiosurgery large volumes of blood apheresis is processed rapidly to remove CTC, CTC-bound to platelets, exosomes, microsomes and nanosomes and to remove the DNA-DNA fragments, and RNAs. Over 90 percent of CTC can be removed by rapid CTC microfiltration (63). Selected in-vitro and in-vivo methods of EVs-exosome, RNA, DNA cellular interaction described in the literature is used to test patient specific EVs-exosome, RNA and DNA interactions. They are listed below. The blood components are passed thorough siRNA affinity chromatograms Heparin mimics as a DNA binding polyanionic structure nucleic acid (65) Partial purification of DNA binding proteins with HiTrap heparin column is commercially available (66)Cellulose activated charcoal coated with heparin is safely used in hemoperfusion for drug overdose treatment (67). Disposable DNA/RNA/EVs-exosomes, microsomes and nanosomes binding heparin coated cellulose activated charcoal is used to remove the DNA/RNA/EVs-exosomes, microsomes and nanosomes surge caused by chemotherapy-radiosurgery and surgery and or siRNA incorporation into EVs-exosomes. It eliminates and or minimizes the bystander and abscopal effects associated metastasis from mutated genomes.
[0234] Air bubble sensor 452 monitors any air bubbles in the final stretch of the blood flow tubing 470 that connects with the reservoir for DNA/RNA/, tumor associated EVs-exosomes, microsomes, nanosomes and CTC free blood after pulse flow purification 458. If there are air bubbles, they are purged out of the blood flow tubing 470 by opening and closing the system clamps with sensors 464 adjacent to the reservoir for DNA/RNA/, tumor associated EVs-exosomes, microsomes, nanosomes silenced with siRNA 458 The CTCs are filtered out. The densitometer-12 454 monitors the siRNA treated return blood cellular elements diluted with saline in blood flow tubing 456. The purified blood cellular element treated with siRNA and diluted with saline is transfused back to the patients through blood flow return channel with clam and sensor 462. The separated plasma containing DNA/RNAs and exosomes is further treated in DCFUC and array centrifuges combined with size exclusion chromatographic columns (SEC) and immuno-affinity chromatographic columns, (IAC) and affinity chromatography (AC). After apheresis of about 300 ml with the first apheresis system is completed, apheresis with the second set of the pulse flow apheresis system is started by connecting it to the patient at another blood drawing site; say to the left arm if the first pulse flow apheresis system was connected to the right arm. Intermittent apheresis with two such systems facilitates a continuous pulse flow aphaeresis.
[0235] The pulse flow apheresis of cell bound proteomics and genomics combined with siRNA affinity chromatography and molecular apheresis by SDG continuous flow ultracentrifugation improves innate immunity. Tumor-specific endogenous siRNA is generated by incubating purified RSIC with pre-let-7 hairpin. It is internalized into EVs and T-cells by electroporation, by photochemical methods or using lipofectamine 2000. They silence the evasion of immune cells from immunity and inhibit tumor recurrence and metastasis.
[0236]
[0237] The continuous flow ultracentrifuge with continuous flow rotors are generally used to separate micro and nano particles in nanoparticle research and industry. In pharmaceutical industry, they are used to produce vaccines. For the purpose of illustration, such a continuous flow ultracentrifuge rotor made by Hitachi Koki Co. Ltd is described herein in its entirety (26) but with adaptive modifications to suite the removal of remaining nanoparticles after pulse flow apheresis of plasma and its filtration. Any other continuous flow ultracentrifuge and continuous flow rotors could be modified and adapted for molecular apheresis and purification and separation of normal cell derived and tumor cell derived DNA, RNAs, EVs-exosomes nanosomes, and ribosomes in the plasma after pulse flow apheresis. The continuous flow ultracentrifuges and rotors that are suitable for such adapted use include Alpha Wassermann continuous flow ultracentrifuge and rotors, Beckman continuous flow ultracentrifuge and rotors, Sorvall-Thermo Fisher continuous flow ultracentrifuge and rotors or any other similar ones from any other manufacturers. They are also adapted to use with array centrifuge taught in U.S. Pat. No. 6,387,031 which is described herein in its entirety (74) but with adaptive modifications to suite the biochemical testing and removal of still remaining EV-exosome-proteomic nanoparticles after pulse flow apheresis of plasma and its filtration and by continuous flow SDG ultracentrifugation that also separates and removes the subcellular molecules.
[0238] As part of the EV-exosome-proteomic nanoparticle separation and apheresis combined mutated gene silencing with siRNA and extracorporeal chemotherapy, the pulsed flow apheresis plasma is continuously introduced into the high speed rotating cylindrical rotor 508 through its bottom sample inlet 496. High speed rotating cylindrical rotor 508 is connected to the hollow top driveshaft 510 and to the bottom hollow drive shaft 502 for the sample to pass through and are supported by bearings. The driveshaft at the top is connected to a high frequency motor 512. The mechanical seal at the end of the upper driveshaft 502 and the bottom driveshaft 510 seals the sample from any leaks. The rotating cylindrical rotor 508 rotates at any adjustable speeds as desired up to 40,000 rpm/min, 100,000 G and volume. By adjusting the rotor speed and flow rate, plasma subcellular molecules are removed either with the sucrose density gradient in the rotor as SDG fractions or as EV-exosome with siRNA effluent plasma flow towards chromatographic columns 516 (not shown) or as EV-exosome with siRNA and chemotherapeutics containing effluent plasma 517 that flows towards array centrifuges (not shown) that is controlled by the flow control valve 518. After plasma is processed, the purified processed plasma with EV-exosomesiRNA and sucrose flows to sucrose removing cold reservoir 520 through processed plasma flow line 519. Sucrose removed plasma with EV-exosome siRNA or chemotherapeutics 521 flows through processed plasma flow line 522 to purified, processed plasma with EV-exosome-siRNA-chemotherapeutics reservoir 523. Purified, processed plasma with EV-exosome-siRNA and or chemotherapeutics 524 is returned to patient through purified, processed plasma with EV-exosome-siRNA-chemotherapeutics inlet to patient 525A or it is collected into the collecting bag 525C by closing the plasma inlet valve to patient 525B and letting the flow of the purified plasma with EV-exosome and proteomics by opening the purified plasma with EV-exosome-proteomics-siRNA to collecting bag inlet valve 525D. The purified EV-exosome-proteomics-siRNA is collected into purified plasma with EV-exosome-proteomics-siRNA collecting bag 525C. It is transfused back to the patent using the collecting bag's outflow 525E with colleting bag's outflow controlling valve 525F. The operation parameters of the ultracentrifuge with the rotor including electrical, cooling, vacuum and the mechanical seal and status of the motor, are displayed on the control system LCD 526.
[0239] The sucrose gradient solution consisting of 130 ml phosphate buffered saline, 200 ml 17% (W/W) sucrose (density 1.0675 g/cm.sup.2), 130 ml 30% (W/W) sucrose (density 1.1.1268 g/cm.sup.2) and 30 ml 45% (W/W) sucrose (density 1.2028 g/cm.sup.2) (85) is filled into the rotor that can hold about 3 L fluid. Any other small volume rotors and SDG concentration suitable for medical application could also be used. The sucrose gradient solution is filled into the rotor through the bottom hollow driveshaft 502 and the centrifuge is run at 4,000 rpm/min for a few minutes to layer the sucrose gradient solution vertically. It causes the higher concentration sucrose solution to migrate towards the center of the rotor and the lower concentration to move towards the periphery of the rotor forming a density gradient between these layers. After the density gradient is formed, the filtered and cooled pulse flow apheresis plasma without macromolecules from pulse flow plasma cooling chamber 530A and pulse flow plasma cooling chamber 530B is injected into the rotor through the bottom hollow driveshaft 502 at an injection rate of about 5-20 ml/min while the rotor is slowly accelerated to desired speed to separate intended plasma suspended micro or nanomolecules. Before injecting the pulse flow apheresis plasma into the rotor, it is chilled to about 0 C. in the cooling chambers. Cooled-filtered pulse flow apheresis plasma is injected into the rotor through plasma injector 528. The additional cooling is an added precaution to prevent plasma coagulation from the heat generated by the rotation of the rotor. The slow flow rate and high speed rotation of the rotor maintains the sucrose gradient undisturbed (68). Plasma volume for an adult is about 3 L. (69) which is constantly monitored by bioelectrical impedance analysis (BAL) (91X) (70) and maintained at this total body plasma volume with 5% D/0.45 N saline supplemented with electrolytes like potassium, calcium, magnesium if needed to maintain patient's electrolytes and fluid balance. The electrolyte levels are constantly monitored. Continuous plasmapheresis at a rate of about 5-20 ml/min will complete one course of 3 L plasmapheresis within about 10 or 2 hours. In general when continuous flow centrifuges (not the ultracentrifuge) are used for blood component exchanges, the usual flow rate is 40 ml/min (71). Since the pulse flow apheresis system is not based on centrifugation, its flow rate is slower. Safe centrifugal apheresis at rate of 50-150 ml/min is in common practice (72). Because of the intermingling of the plasma with other body fluid compartments, a one or two times whole body plasma aphaeresis is not sufficient to remove and process all the circulating mutated cancer molecules suspended in the whole body plasma. The rate of clearance of the tumor associated mutated nanoparticle in the plasma is monitored with AFM, NTA and DCNA, flow cytometry and other testing listed According to the size and weight of the nanoparticles in the pulsed flow apheresis plasma, they separate towards the inside of the sucrose gradient solution. At the end of the ultracentrifugation, the speed of the rotor is slowly reduced to 4,000 rpm/min and slowly brought to stop. Fractions of the SDG are collected by air injection through the top hollow driveshaft 510.
[0240] The continues-flow ultracentrifuge rotor is run at elective speed and g-force from 50,000 to 100,000 g for about 12 hrs at 4 C. for elective fractionation of plasma soluble and suspended EV-exosomes, exRNAs, tRNAs, DNA fragments, and tumor associated proteins like IDH1 and IDH2 and virus and virus fragments. The rate of clearance of these particles is monitored with AFM, NTA and DCNA and flow cytometry (not shown here). At the end of this ultracentrifugation, the particle that layers in sucrose density gradient contains most of the larger plasma soluble and suspended particles.
[0241] The effluent through the top of the rotor outlet contains plasma soluble and suspended nanoparticles which exit from the top hollow driveshaft 510 of the rotor is directed towards a series of chromatographic columns through EV-exosome with siRNA effluent plasma flow towards chromatographic columns 516 or to EV-exosome with siRNA effluent plasma 517 that flows towards array centrifuges (not shown here) through flow control valve 518A. The flow-line 546 is connected with biochemistry testing devices. The immobilized Tim4-Fc protein Ca2+ magnetic beads affinity column and the series of immobilized metal affinity chromatographic columns and other suitable chromatographic columns selected from the group described above purifies and separates the normal cell derived EVs and the tumor cell derived mutated EVs. The purified EVs-Exosomes are labeled with siRNA and or with chemotherapeutics by electroporation, or by photochemical methods or with lipofectamine 2000. Such labeled EVs containing siRNA and or chemotherapeutics and sucrose gradient plasma is cooled to Oo C in sucrose removing cold reservoir 520 where sedimented sucrose 520B collects at the bottom. The supernatant plasma with EV-exosome-siRNA is warmed to 370 C with a warming coil 532 in purified, processed plasma with EV-exosome-siRNA-chemotherapeutics reservoir 523. Purified, processed plasma with EV-exosome-siRNA and or chemotherapeutics 524 is returned to patient through purified, processed plasma with EV-exosome-siRNA-chemotherapeutics inlet to patient 525A. Alternatively, the purified plasma with EV-exosome-proteomics-siRNA flow valve 525D is opened and the processed plasma with EV-exosome-siRNA and or chemotherapeutics 524 is collected into purified plasma with EV-exosome-proteomics-siRNA collecting bag 525C and preserved for its biochemical analysis and or later administration to the patent.
[0242] The subcellular fractions and the EV-exosome-separated into sucrose gradient cushion are collected from the bottom of the high speed rotating cylindrical rotor 508. The rotational rpm is reduced to 4,000 and the rotor is brought to a halt without disturbing the sucrose density gradient. After rotor comes to a standstill, flow control valves 518A and 518A2 are closed and air is injected into the rotor through the top 0f the rotor to push the SDG layers containing subcellular DNA, RNA, extracellular vesicles-exosomes and nanosomes. Based on molecular weight configuration of the particles and sedimentation coefficients higher than 50S, they sediment into varying layers of the sucrose gradient. Particles with less than 50 S sedimentation coefficients are not suitable for separation by continuous flow ultracentrifugation (75). Subcellular DNA, RNA, extracellular vesicles-exosomes and nanosomes with higher than 50 S sedimentation coefficient separated into glucose density gradient by continuous flow ultracentrifuge with high speed rotating cylindrical rotor 508 flows through SDG fraction collecting flow line 518C to affinity chromatographic column 518D. SDG fractions are also collected per SDG fraction sample outlet and flow control valve 518H into SDG fraction collection tubes 518-I for biochemical testing and to check integrity of EV-exosomes including testing for CD9, CD63, CD73 and CD90, and their size and morphology. SDG fraction collecting flow line 518C leads SDG fraction per fraction to affinity chromatographic column 518D. Only one such affinity chromatographic column 518D is illustrated here but it can be multiple as shown in
[0243]
[0244] For separation of patient specific EV-exosomes and plasma soluble and plasma suspended subcellular micro and nano particle, and proteomics the EV-exosome with siRNA effluent plasma flow towards chromatographic columns 516 exiting from the top hollow driveshaft 510 is directed towards chromatography column 1, 522A and to chromatography column 2, 522B. Alternatively, this EV-exosome with siRNA effluent plasma with EV-exosomes, siRNA and or chemotherapeutics and sucrose gradient is directed towards array centrifuges 517B that is controlled by the flow control valve 518 (array centrifuge is not shown). The flow-line 546 is connected with biochemistry testing devices. The affinity chromatographic columns selected include Tim4-Fc magnetic beads columns, immobilized Tim4-Fc protein Ca2+ magnetic beads affinity column (ITMAC), iZON science's size exclusion chromatographic columns (SEC) qEV, qViro-X, immobilized metal affinity chromatographic columns (IMAC), size exclusion chromatographic columns (SEC), immuno-affinity chromatographic columns, (IAC), heparin sulfate pseudo-affinity chromatographic column (HAC), Lectin ligand affinity chromatographic columns, (LAC) and lipofectamine 2000 column (LF2000) columns. The chromatographic columns are selected on the basis of patient specific subcellular fractions present in the plasma effluents.
[0245] As described under
[0246] The subcellular EVs-exosomes, genomics and proteomics are monitored with AFM 536, NTA 538, and DCNA 540 and to a flow cytometer (FCM) 542 for particle tracking. The effluent supernatant exiting from the chromatographic columns 534 flows back to the high speed rotating cylindrical rotor 508 through its bottom hollow driveshaft 502 and back to the sucrose removing cold reservoir 520 through processed plasma flow line 519 or it re-circulates through the chromatography column1 522A and chromatography column2, 522B through the supernatant outlet 516. Before the effluent supernatant exiting from the chromatographic columns 534 is injected back into the high speed rotating cylindrical rotor 508, it is cooled to 0 C. with the cooling coil 530. The supernatant flow into the rotor, out of the rotor, into the chromatography columns, into AFM, NTA, DCNA and FCM and back to the reservoirs or to the chromatographic columns is controlled by the electronic flow direction control switch 544. Before the effluent supernatant exiting from the high speed rotating cylindrical rotor 508 is returned back to patient, it is warmed to w 37 C. with the warming coil 532. Before the pulsed flow apheresis plasma is injected into the high speed rotating cylindrical rotor 508 through its bottom hollow driveshaft 502 for nanoparticles separation, it is cooled to 0 C. with the cooling coil 530. The chromatography columns are sterilized and kept in a sterile environment. The continuous flow ultracentrifuge is also kept in sterile condition and the rotor is sterilized online as per manufacturer's instructions. It is kept sterile and operated in sterile conditions.
[0247] The extracorporeal continuous flow, or pulse flow apheresis of cell bound proteomics and genomics, combined with molecular apheresis by sucrose density gradient continuous flow ultracentrifugation, and chromatography with siRNA affinity columns, Tim4-Fc magnetic beads columns, immobilized Tim4-Fc protein Ca.sup.2+ magnetic beads affinity column, immobilized metal affinity chromatographic columns, size exclusion chromatographic columns, immuno-affinity chromatographic columns, heparin sulfate pseudo-affinity chromatographic column, Lectin ligand affinity chromatographic columns and lipofectamine 2000 column columns improves innate immunity. Tumor-specific endogenous siRNA is generated from mutated RNA containing pre-miRNA hairpin through RNA induced silencing complex composed of Dicer, dsRNA binding protein TRBP, and Argo2. Endogenous siRNA is generated by incubating purified RSIC with pre-let-7 hairpin. It is internalized into EVs and T-cells by photochemical methods or using lipofectamine 2000. While undergoing radiation therapy, chemotherapy or surgery, a patient's blood is continuously drawn and processed through the above system. The purified and processed plasma containing EV-exosomes-proteomics-siRNA is returned to the patient. Phototherapy with internalized photosensitive complex-siRNA prevents development of chronic graft versus host disease of adaptive immunotherapy.
[0248] As also described under
[0249] Subcellular DNA, RNA, extracellular vesicles-exosomes and nanosomes with higher than 50 S sedimentation coefficient separated into glucose density gradient by continuous flow ultracentrifuge with high speed rotating cylindrical rotor 508 flows through SDG fraction collecting flow line 518C to affinity chromatographic column 518D. SDG fractions are also collected per SDG fraction sample outlet and flow control valve 518H into SDG fraction collection tubes 518-I for biochemical testing and to check integrity of EV-exosomes including testing for CD9, CD63, CD73 and CD90, their size and morphology and for endogenous RNAi, siRNA generation.
[0250] The affinity chromatographic columns selected are the same as used for treatment of effluent existing from the top of the rotor described before. They include immobilized Tim4-Fc protein Ca.sup.2+ magnetic beads affinity column (ITMAC), iZON science's size exclusion chromatographic columns (SEC) qEV, qViro-X or from a series of immobilized metal affinity chromatographic columns (IMAC) or other suitable chromatographic columns selected from the group consisting of other types of SEC, immuno-affinity chromatographic columns, (IAC), heparin sulfate pseudo-affinity chromatographic column (HAC), Lectin ligand affinity chromatographic columns, (LAC) and lipofectamine 2000 column (LF2000) columns. Based on patient specific micro and nano-molecule's separation need, specific chromatographic columns are selected. Such chromatographic columns are routinely used in virus purifications (73).
[0251] Tumor-specific endogenous siRNA is also generated from mutated subcellular fragments sedimented in the sucrose gradient. SDG fractions containing mutated subcellular fractions are combined together and used to generate siRNA. Like with the siRNA generating from the effluent existing from the top of the rotor, the tumor specific endogenous siRNA is generated from sucrose density gradient sediment RNA containing pre-miRNA hairpin through RNA induced silencing complex (RISC) composed of Dicer, dsRNA binding protein TRBP, and Argo2 (76). It is generated by incubating purified RSIC with pre-let-7 hairpin (76). This siRNA is then internalized into EVs and into T-cells by electroporation (77) or by photochemical methods or with lipofectamine 2000. Such T-cell treatments silence its evasion from immunity. Chemotherapeutics are also internalized into SDG sediment EVs by electroporation, photochemical methods or with lipofectamine 2000. Labeled EVs containing siRNA and or chemotherapeutics and sucrose gradient plasma flow to sucrose removing cold reservoir 520. SDG fraction collecting flow line 518C leads SDG fraction per fraction to affinity chromatographic column 518D. Chromatographic purified SDG fraction flows into sucrose removing cold reservoir 520 through SDG fraction collecting flow line-2 518C2 where it mixes with effluent plasma existing from the top of the rotor 514. It is cooled to 0 C. to sediment sucrose and the sedimented sucrose 520B collects at the bottom. As with the effluent plasma existing from the top of the rotor 514 the SDG fraction's purified plasma with EV-exosome-siRNA with or without chemotherapeutics is warmed to 370 C with a warming coil 532 in purified, processed plasma with EV-exosome-siRNA-chemotherapeutics reservoir 523. Purified, processed plasma with EV-exosome-siRNA and or chemotherapeutics 524 is returned to patient through purified, processed plasma with EV-exosome-siRNA-chemotherapeutics inlet to patient 525A. Alternatively, the purified plasma with EV-exosome-proteomics-siRNA flow valve 525D is opened and the processed plasma with EV-exosome-siRNA and or chemotherapeutics 524 is collected into purified plasma with EV-exosome-proteomics-siRNA collecting bag 525C and preserved for its biochemical analysis and or later administration to the patent. The continuous flow ultracentrifuge and all its accessories are kept in sterile conditions in a sterile environment. The rotor is sterilized online as per manufacturer's instructions. It is also kept sterile and operated in sterile conditions.
[0252]
[0253] For simultaneous separation of several tumor derived patient specific plasma soluble micro and nano particles including the EVs-exosomes and proteosomes derived from the normal cells and those mutated ones from the tumor cells, the supernatant exiting from the top hollow driveshaft 510 is directed towards several pairs of affinity chromatography columns. In the example illustrated in this
[0254] The subcellular fractions and the EV-exosome-separated into sucrose gradient cushion are collected from the bottom of the high speed rotating cylindrical rotor 508. In this process, the rotational rpm is reduced to 4,000 and the rotor is brought to a halt without disturbing the sucrose density gradient. After rotor comes to a standstill, flow control valves 518A and 518A2 are closed and air is injected into the rotor through the top of the rotor to push the SDG layers containing subcellular DNA, RNA, extracellular vesicles-exosomes and nanosomes downwards into SDG fraction sample collection tubes 518-I.
[0255] Subcellular DNA, RNA, extracellular vesicles-exosomes and nanosomes with higher than 50 S sedimentation coefficient separated into glucose density gradient by continuous flow ultracentrifuge with high speed rotating cylindrical rotor 508 flows through SDG fraction collecting flow line 518C to affinity chromatographic column 518D. SDG fractions are also collected per SDG fraction sample outlet and flow control valve 518H into SDG fraction collection tubes 518-I for biochemical testing and to check integrity of EV-exosomes including testing for CD9, CD63, CD73 and CD90, their size and morphology and for endogenous RNAi, siRNA generation from pooled mutated and normal cellular RNA containing pre-miRNA hairpin through RNA induced silencing complex (RISC) composed of Dicer, dsRNA binding protein TRBP, and Argo2 (76) in SDG fractions.
[0256] The affinity chromatographic columns selected for treating both effluent supernatant existing from the top of the rotor and in the sucrose gradient fractions are the same. They were described before. The affinity chromatographic columns for treating the effluent existing from the top of the rotor include affinity chromatography column 1, 522A, 522B affinity chromatography column-2 522B, affinity chromatography column-3 522C, affinity chromatography column-4 522D, affinity chromatography column-5 522E, affinity chromatography column-6 522F, affinity chromatography column-7 522G, affinity chromatography column-8 522H, affinity chromatography column-9 522-I and affinity chromatography column-10 522J. Only one of the affinity chromatographic columns, affinity chromatography column 518D is illustrated for treating the SDG fractions existing from the bottom of the rotor. However like for treatment of the effluent existing from the top of the rotor, a series of affinity columns can be used to treat the sucrose gradient fractions existing from the bottom of the rotor. These affinity columns include immobilized Tim4-Fc protein Ca.sup.2+ magnetic beads affinity column (ITMAC), iZON science's size exclusion chromatographic columns (SEC) qEV, qViro-X or from a series of immobilized metal affinity chromatographic columns (IMAC) or other suitable chromatographic columns selected from the group consisting of other types of SEC, immuno-affinity chromatographic columns, (IAC), heparin sulfate pseudo-affinity chromatographic column (HAC), Lectin ligand affinity chromatographic columns, (LAC) and lipofectamine 2000 column (LF2000) columns. Based on patient specific micro and nano-molecule's separation need, specific chromatographic columns are selected.
[0257] Tumor-specific endogenous siRNA is generated from mutated subcellular RNA containing pre-miRNA hairpin through RNA induced silencing complex (RISC) composed of Dicer, dsRNA binding protein TRBP, and Argo2 in effluent existing from the top of the rotor and SDG fractions existing from bottom of the rotor. SDG fractions containing mutated subcellular fractions are pooled together and used to generate siRNA. Like with the siRNA generating from the effluent existing from the top of the rotor, the tumor specific endogenous siRNA is generated from sucrose density gradient sediment RNA containing pre-miRNA hairpin through RISC composed of Dicer, dsRNA binding protein TRBP, and Argo2 (76). It is generated by incubating purified RSIC with pre-let-7 hairpin (76). This siRNA is then internalized into EVs and into T-cells by electroporation (77) or by photochemical methods or with lipofectamine 2000. Such T-cell treatments silence its evasion from immunity. Chemotherapeutics are also internalized into SDG sediment EVs by electroporation, photochemical methods or with lipofectamine 2000. Chemotherapeutics and siRNA labeled and sucrose gradient plasma flow to sucrose removing cold reservoir 520. SDG fraction collecting flow line 518C leads SDG fraction per fraction to affinity chromatographic column 518D. SDG fraction collecting flow line 570 leads SDG fraction per fraction to the series of affinity chromatographic columns that are connected to effluent processing chromatographic columns, 522A-522J. The electronic flow control valve 572 controls the SDG fraction flow direction either to affinity chromatographic column 518D or to effluent processing affinity chromatographic columns 522A-522J. When effluent from the top of the rotor is processed in chromatographic columns 522A-522J, the electronic flow control valve 572 directing the SDG fraction's flow to these columns is closed to prevent mixing of the SDG gradient fraction with effluent existing from the top of the rotor and entering into chromatographic columns 522A-522J. Chromatographic purified SDG fraction flows into sucrose removing cold reservoir 520 through SDG fraction collecting flow line-2 518C2 where it mixes with effluent plasma existing from the top of the rotor 514. It is cooled to 0 C. to sediment sucrose. The sedimented sucrose 520B collects at the bottom. As with the effluent plasma existing from the top of the rotor 514, the SDG fraction's purified plasma with EV-exosome-siRNA with or without chemotherapeutics is warmed to 37 C. with a warming coil 532 in purified, processed plasma with EV-exosome-siRNA-chemotherapeutics reservoir 523. Purified, processed plasma with EV-exosome-siRNA and or chemotherapeutics 524 is returned to patient through purified, processed plasma with EV-exosome-siRNA-chemotherapeutics inlet to patient 525A. Alternatively, the purified plasma with EV-exosome-proteomics-siRNA flow valve 525D is opened and the processed plasma with EV-exosome-siRNA and or chemotherapeutics 524 is collected into purified plasma with EV-exosome-proteomics-siRNA collecting bag 525C and preserved for its biochemical analysis and or later administration to the patent. The series of affinity chromatography columns are placed in a portable trailer 574 that is kept sterilized in a sterile environment. The continuous flow ultracentrifuge is also kept in sterile condition and in sterile environment. The rotor is sterilized online as per manufacturer's instructions. It is kept sterile and operated in sterile conditions.
[0258] The extracorporeal pulse flow apheresis of cell bound proteomics and genomics, combined with molecular apheresis by sucrose density gradient continuous flow ultracentrifugation and affinity chromatography with siRNA affinity columns, Tim4-Fc magnetic beads columns, immobilized Tim4-Fc protein Ca.sup.2+ magnetic beads affinity column, immobilized metal affinity chromatographic columns, size exclusion chromatographic columns, immuno-affinity chromatographic columns, heparin sulfate pseudo-affinity chromatographic column, lectin ligand affinity chromatographic columns and lipofectamine 2000 columns and treatments with tumor specific endogenous siRNA improves innate immunity. It inhibits tumor recurrence and metastasis. Tumor-specific endogenous siRNA is generated from mutated RNA containing pre-miRNA hairpin through RNA induced silencing complex composed of Dicer, dsRNA binding protein TRBP, and Argo2. Endogenous siRNA is generated by incubating purified RSIC with pre-let-7 hairpin which is internalized into EVs and T-cells and returned to the patient. It silences its evasion from immunity against the tumor. Phototherapy with internalized photosensitive complex-siRNA also prevents development of chronic graft versus host disease of adaptive immunotherapy.
[0259]
[0260] The advantages of combined continuous flow ultracentrifugation and ultracentrifugation with array centrifuge include partial separation and purification of less than 50S exosome proteosomes with continuous flow ultracentrifugation at 100,000 G and its additional purification and sedimentation or pelleting with array centrifuge at G-force ranging over 200,000. The rpm of individual rotors in the array centrifuge is adjustable. It is combined with chromatographic additional exosome and proteosomes purification and separation. It is a total analysis of exosomes and proteosomes while the conventional ultracentrifugation based exosomes and proteosomes purification and separation is a partial, incomplete one with discarding valuable subcellular fractions in the supernatants.
[0261] The effluent existing from the top of the rotor and the SDG existing from the bottom of the rotor and their affinity chromatography and further treatments and generation of endogenous siRNA and the EV chemotherapeutics preparation for extracorporeal chemotherapy are the same as described in
[0262]
[0263] The cross section of a zonal continuous flow rotor for example, the Beckman continuous flow rotor model CF 32 Ti and JCF-Z is illustrated as an example. The CF 32 Ti rotor runs at maximum 32,000 rpm. It generates 86,100g at the bottom of the bowl wall and 102,000g at the inner surface of the bowl wall 586. Likewise, The JCF-Z rotor with standard core runs at maximum 20,000 rpm which generates 32,000g at the core bottom and 39,000g at the inner surface of the bowl wall 586. These are low g force for separation of all the subcellular particles and exosomes and proteomics especially those below 50S. Thermo Scientifics' similar rotor, the TCF-32 rotor runs at maximum speed 32,000 rpm that generates maximum 102,000 g which is also insufficient for total separation and pelleting of subcellular particles and exosomes and proteomics especially those below 50S. Even the Thermo-Stovall's alternative CC40 continuous flow ultracentrifuge with cylindrical rotor or other manufacturer's similar continuous flow ultracentrifuges like those from Alpha Wasserman and Hitachi Koki Co., Ltd have only maximum speed of 40,000 rpm which generates maximum 118,000g. They do not separate or pellet EV exosomes and proteosomes with sedimentation below 50S which is quite unsatisfactory for total EV-exosome analysis and apheresis and for cancer treatment that are described in this invention. The continuous flow ultracentrifugation system combined with array centrifuge with series of rotors that can spin at varying G-Force ranging over 200,000 separates and or pellets the total mutated tumor exosomes and proteosomes and removes them from circulation by their apheresis as described in this invention.
[0264] The pulse flow plasma sample flows through plasma injector 528 and enters into continuous flow centrifuge through its central inlet 582. The rotating sealing assembly 580 keeps the fluid lines to remain attached to the rotor during rotation. At low speed rotation, the buffer solution from buffer reservoir 606 or SDG solution reservoir 608 is pumped through buffer line 610 and SDG line 612 by electronically switching on or off the buffer and SDG flow line valve 614. After about a combined volume of about 430 ml buffer and SDG are pumped in at slow rate spinning, the zonal continuous flow rotor 576 is accelerated to operating speed the buffer and SDG flow line valve 614 is switched off and the plasma flow control valve 578 is opened and the pulse flow purified plasma is routed through plasma injector 528 and the central inlet 582 into the bottom of the rotor bowl through the connecting channel to central flow 592. Because of the higher density of the SDG solution, it collects at the periphery of the rotor wall under centrifugal force. At the end of the centrifugation run, rpm is slowly reduced and air is injected through the edge line that makes an airlock at the upper radial channel 584. It prevents disturbance by the dense displacement fluid entering into radial channels 584 during fractionated removal of the SDG layers and disturbing the SDG layers. The dense displacement fluid from the displacement dense fluid reservoir 616 is injected into the rotor through the displacement dense fluid flow line 618 to unload the SDG cushion layers with separated subcellular particles. The rotor contents of SDG fractions flows through the center line to edgeline outlet 594 from where it flows to photometer/flow cell 598 and to SDG fraction collector 600. Based on SDG's density recorded by the photometer/flow cell 598 fractions are collected. It flows to chromatographic affinity columns via electronic flow direction control switch 544. Additional purification of the EV-exosomes and proteosomes and other subcellular contents of the SDG fractions by affinity chromatography and generation of endogenous siRNA and chemotherapeutic-EV for online extracorporeal chemotherapy are the same as those described in
Methods of Operation
[0265] Methods of near total apheresis of tumor cell derived mutated genomics and proteomics, microsomes, EVs-exosomes, DNA, RNAs, apoptotic bodies, nucleosomes, telomerase, (subcellular particles) and genome silencing with endogenous RNAi-siRNA and miRNA with above described devices including those in
[0266] 1. Methods of Pre and Post Treatment Analysis of Circulating CTC and Subcellular Particles.
[0267] Blood is withdrawn from the patient before and after treatments to determine rate of DNA repair and the rate of DNA repair enzymes increase and decrease to normal values after treatments and to determine its relation to abscopal and bystander effects and correlation with its activities in circulating, red cells, granulocytes, macrophages and platelets. Such measurements are repeated daily for 4 days after the treatment and afterwards as needed. In cases of combined radiosurgery and chemotherapy, chemotherapy is administered first and post chemotherapy sample is drawn according to treatment protocols. After radiation therapy a second sample is drawn to assess the combined treatment's effects.
[0268] 2. Methods of Pulse Flow Cellular and Molecular Aphaeresis Combined with Affinity Chromatography Described in
[0269] The CTC, mononuclear white blood cells and platelets carrying tumor specific subcellular particles are removed from circulation first by pulsed flow apheresis combined with affinity chromatography described in
[0270] 3. Methods of Continuous Flow Ultracentrifugation Molecular Aphaeresis Combined with Affinity Chromatography Described in
[0271] Removal of plasma soluble cell debris, larger micro and nano particles, cell membranes, normal cell and tumor cell derived proteins, and subcellular particles including apoptotic bodies, DNA and RNAs, microsomes, exosomes and nanosomes, telomere and telomerase, ATM and ATM kinase after pulse flow apheresis by pulse flow apheresis system 378 is directed into the continuous flow ultracentrifuge systems illustrated in
[0272] 4. After Radiosurgery, Separation of Cancer and Cancer Stem Cell Derived Exosomes from Exosomes Derived from Normal Cells at 24 and or 48 h
[0273] In response to cancer treatments, especially after local radiation therapy, large burst of cellular fragments including micro and nano particles, cell membranes, normal cell and tumor cell derived proteins, and subcellular particles including apoptotic bodies, DNA and RNAs, microsomes, exosomes and nanosomes, telomere and telomerase, ATM and ATM kinase are released into circulation. Its high peak occurs within 24 to 48 hours. Identification of the phenotypic variation among the tumor derived exosomes by various methods during the window of this high peak periods is described below as examples.
[0274] The SDG and SEC fractions are analyzed for patient specific cancer and cancer stem cell phenotype. The generally used genomics and proteomic testing is adapted for such analysis. They include: [0275] 1. Oncosome immunohistochemistry (35), [0276] 2. In-vitro gene silencing, Immunoblotting and siRNA stability assay (63), [0277] 3. size-exclusion chromatography, transmission EM, RNA isolation, qRT-PCR Assays, miRNA profiling, miRNA profiling data analysis, immunoprecipitation and immunoblotting, and data analysis for miRNA in plasma fraction Ago2 immunoprecipitates (22), [0278] 4. Inducible RAD51 assay, indirect immunofluorescence RAD51 staining, confocal microscope image acquisition, gamma H2AX foci counting, Western blotting, subcellular fractionation ssDNA and dsDNA quantification, RNA microarray (64), [0279] 5. Microarray mRNA analysis, microarray miRNA analysis, real-time polymerase chain reaction (PCR), miRNA transfection with Lipofectamine 2000, 3UTR Reporter assay, in vitro translation, flow cytometry 34, [0280] 6. Analysis of exosomal RNA content by sequencing, tracing exosomal uptake in the in vitro system (31). [0281] 7. A large-scale targeted proteomics assay resource based on an in vitro human proteome (81) [0282] 8. EV-Exosomes derived from cancer cells and undifferentiated cancer cells and their phenotyping: [0283] a. AFM measurements of shape, height, width, surface roughness and stiffness combined with fluorescence microscopy of gold nanoparticles coated with antibody against undifferentiated cancer stem cell's antigens and which is bound to normal cells and to exosome and exosome proteins, its antigens, DNAs and RNAs and its comparison with antigen-antibody binding differentiated cancer cell's antigens. [0284] b. AFM measurements of shape, height, width, surface roughness and stiffness of exosomes combined with fluorescence microscopy of exosomes bound to normal cells and to undifferentiated cancer stem cell specific antigens selected from the list of cancer stem cell antigens and they have mutated CTCF with different height and length and DNA looping and its comparison with antigen-antibody binding for the differentiated cancer cell's antigens. [0285] c. AFM measurements of shape, height, width, surface roughness and stiffness of exosomes combined fluorescence microscopy of exosomes bound to normal cells and to undifferentiated cancer stem cell specific antigens and they have cancer treatment resistance like resistance to 5-flurouracil (5-FU) when cytosine deaminase-uracil phosphoribosyl transferase (CD-UPRT) fusion gene is present and its comparison with antigen-antibody binding for differentiated cancer cell's antigens. [0286] d. AFM combined fluorescence microscopy of exosomes bound to normal cells and to undifferentiated cancer stem cell specific antigens showing different height and width, surface roughness and stiffness histograms in the purified exosome DNA and their double strand break and homologues DNA repair deficiency after cancer radiosurgery and chemotherapy and its comparison with antigen-antibody binding for the differentiated cancer cell antigens. [0287] e. AFM combined fluorescence microscopy of exosomes bound to normal cells and to undifferentiated cancer stem cell specific antigens showing different shape, height and width surface roughness and stiffness exosome in response to radiation therapy and chemotherapy and its comparison with antigen-antibody binding for differentiated cancer cell's antigens. [0288] f. AFM combined fluorescence microscopy of exosomes bound to normal cells and to undifferentiated cancer stem cell specific antigens selected from the list of cancer stem cell antigens and the cancer stem cell exosome's poly (ADP) ribose polymerase (PARP) cleavage in response to radiation therapy and chemotherapy and its associated changes in cancer stem cell's shape, height and width surface roughness and stiffness and its comparison with antigen-antibody binding for differentiated cancer cell's antigens. [0289] g. AFM combined fluorescence microscopy of exosomes bound to normal cells and to undifferentiated cancer stem cell specific antigens selected and the presence of Rad50/MRE11/NBS1 (MRN Complex) in ER, PR and HER2 negative breast cancer patient's exosomes with changes in their shape, height and width surface roughness and stiffness and its comparison with antigen-antibody binding to differentiated cancer cell's antigens. [0290] h. AFM combined fluorescence microscopy of exosomes bound to normal cells and to undifferentiated cancer stem cell specific antigens and the presence of Warburg glycolytic glutamate in exosomes with associated changes in their shape, height, width, surface roughness and stiffness and its comparison with antigen-antibody binding to differentiated cancer cell's antigens. [0291] i. AFM combined fluorescence microscopy of exosomes bound to normal cells and to undifferentiated cancer stem cell specific antigens and the cancer stem cell exosomes without greatly diminished caspase activity and its associated changes in exosomes shape, height and width, surface roughness and stiffness in comparison with antigen-antibody binding of differentiated cancer cell's antigens. [0292] 9. Tumor Cell's and Normal Cell's Exosome Analysis by Disc Centrifuge and by Nanoparticle Tracking Analysis (NTA) [0293] A quick analysis of the fraction with peak exosome content and its size and shape ranging from 10 nm to 100 nm is determined with a disc centrifuge and the exosome imaging with AFM. This exosome preparation contains both exosomes from cancer and cancer stem cells and those from the normal cells. [0294] Its aliquot is suspended in the PBS and its size, shape and movements are recorded with NTA software. The video images captured by the NTA automatically and simultaneously record thousands of exosome's locations, their movements and centre of each and every particle and measures the average distance it moves per frame. This information on the characteristics of the exosome like the size, relative intensity and concentration in 2-D and 3-D formats is displayed. [0295] 10. Comparative AFM/NTM/SDNA Phenotypic Analysis of Cancer Stem Cell Exosomes and Normal Tissue Exosomes in SDG Fractions Containing Purified Total Exosomes (PTE) [0296] For the purpose of assessing the exosome in the SDG fractions to determine if they are derived from undifferentiated cancer stem cells, differentiated cancer cells or normal cells, the following guidelines are followed. [0297] First, the exosomes in an aliquot of SDGUF are tested for undifferentiated cancer stem cell antigens and their specific antibody binding. [0298] Second, the remaining exosomes in the same aliquot of SDGUF are tested for known, differentiated cancer cell markers. [0299] If both the first and second group's testing for the antigen antibody binding of exosomes shows their respective antigen-antibody specificity, then those exosomes and subcellular particles and are marked as derived from undifferentiated cancer stem cells. It is one type of undifferentiated cancer stem cell exosome's phenotype. [0300] If the exosomes have only undifferentiated cancer stem cell antigen-antibody binding, and no or poor binding to generally known cancer antigen markers, then such subcellular particles and exosomes are marked as derived from undifferentiated cancer stem cells but of a different phenotype. [0301] If there are no undifferentiated cancer stem cell antigen-antibody binding but has binding only to generally known cancer cell markers, then such subcellular particles and exosomes are marked as derived from more differentiated cancer cells. [0302] The exosomes with poor or no cancer cell antigen-antibody binding are marked as derived from normal tissue. They are the remaining exosome in the same aliquot SDGUF after separation of the exosomes derived from undifferentiated cancer stem cells and those exosomes from differentiated cancer cells. [0303] Thus the characteristics in exosomes derived from the undifferentiated cancer stem cells and differentiated cancer cells and their correlation with presence or absence of generally known cancer cell markers will indicate the predominant phenotype of a tumor from which the tested cancer cell exosome have originated.
[0304] The disclosures of all references cited herein are hereby incorporated as references. Listing of references herein is not intended to be a representation that a complete search of all relevant art has been made, or that no more pertinent art than that listed exists, or that the listed art is material to patentability. Nor should any such representation be inferred.
[0305] While this inventor has described what the prescribed embodiments of the present invention are presently, other and further changes and modifications could be made without departing from the scope of the invention and it is intended by this inventor to claim all such changes and modifications. Accordingly, it should be also understood that the present disclosure has been presented for purposes of example rather than limitation, and does not preclude inclusion of such modifications, variations and/or additions to the present subject matter as would be readily apparent to one of ordinary skill in the art.
TABLE-US-00003 EV-Patent Application: Table of Contents No Title Page 1 CIP-Information 1 2 Background 1 3 EVs and Intercellular communication 4 4 Cellular and organ targeting by EVs 4 5 Circulating RNAs 5 6 Pulse flow cellular apheresis 8 7 Continuous flow cell separator 8 8 Continuous flow ultracentrifugation plasmapheresis of 9 circulating tumor derived EVs 9 Prior art affinity chromatography with lectins 10 10 EVs and exRNA associated diseases 11 12 Lymph node metastasis and circulating EVs and exRNAs 13 13 Large oncosome and metastasis 14 14 Extracellular RNA (exRNA) 15 15 Cancer stem cell EVs and exRNAs and cancer treatment 16 16 MicroRNAs (miRNAs) 18 17 Table 1, Poor prognostic cancer associated miRNAs 18 18 DNA Damage Response (DDR) 24 19 Table 2, Potential metastasis causing mutated DNA 24 Damage Repair gens 20 Extracorporeal Chemotherapy aided by Pulse-Flow- 26 Continuous-Flow Ultracentrifugation and Chromatography 21 Metastasis inhibiting Radiotherapy's AGO2-RISC- 27 RAD51-diRNA-RNAi-miRNA-siRNA enhanced Immunotherapy and Extracorporeal Chemotherapy 22 Brief summary of the invention 28 23 Brief description of the Drawings 24 Reference Numerals 29 25 Detailed Description of the Preferred Embodiments 33 26 FIG. 24 33 27 FIG. 25A 28 FIG. 25B 29 FIG. 25C 30 Selected in-vitro and in-vivo methods of EVs-exosome, 42 RNA, DNA cellular interaction