Polymer composite nanomaterial encapsulation system

Abstract

In general, a polymeric nanomaterial encapsulation system can be used to manufacture polymer encapsulated nanoparticles, the polymer encapsulated nanoparticles comprising hydrophobic nanoparticles encapsulated in a hydrophobic region of the polymer, the hydrophobic region comprising a hydrophobic region of the polymer, the hydrophobic region comprising a hydrophobic region of the polymer, and a hydrophobic region of the polymer, the hydrophobic region comprising a hydrophobic region of the polymer. Wherein the outer hydrophilic region of the polymer ensures water solubility and provides functional groups, which can be used to make nanoparticle conjugates. In particular, particular examples include a polymeric nanoparticle structure comprising one or more of quantum dots and / or superparamagnetic iron oxide nanoparticles and / or upconverting nanoparticles encapsulated in polystyrene-b-polyethylene glycol amine, and / or one or more polymeric nanoparticles encapsulated in polystyrene-b-polyethylene glycol amine and / or one or more polymeric nanoparticles encapsulated in polystyrene-b-polyethylene glycol amine and / or one or more polymeric nanoparticles encapsulated in polystyrene-b-polyethylene glycol amine, and / or one or more polymeric nanoparticles encapsulated in polystyrene-b-polyethylene glycol amine. The method is used for manufacturing antibody conjugates which can be used for capturing cell targets.

Description

[0001] This International Patent Cooperation Treaty patent application is a continuation of U.S. non-provisional patent application No. 17 / 943,788 filed on September 13, 2022, which is hereby incorporated by reference into this document. Technical Field

[0002] In general, a nanomaterial polymer encapsulation system is useful for making nanocomposites comprising hydrophobic nanoparticles encapsulated in a hydrophobic region of a polymer, wherein the outer hydrophilic region of the polymer ensures water solubility and provides functional groups, which can be used to make nanocomposite conjugates.

[0003] Specifically, a particular embodiment may include a nanocomplex comprising one or more nanoparticles, wherein the one or more nanoparticles include quantum dots ("QDs") and / or superparamagnetic iron oxide nanoparticles ("SPIONs") and / or upconversion nanoparticles, wherein the one or more nanoparticles are encapsulated in polystyrene-b-polyethylene glycol amine ("PS-b-PEG-NH2"), which provides amine functional groups that can be activated to bind antibodies, modified antibodies, or antibody fragments, and in a particular embodiment, activated with methyltetrazine polyethylene glycol-4-N-hydroxysuccinimide ester ("methyltetrazine-PEG4-NHS ester") to allow binding of antibodies or antibody fragments modified with trans-cyclooctyne ("TCO"). Background Art

[0004] Nanocomplexes have a wide variety of potential applications, including, but not limited to, medicine, biomedicine, biotechnology, biomaterials, biomechanics, and energy production. Current methods of polymer encapsulation of nanoparticles such as QDs or SPIONs used to make nanocomplexes have resulted in agglomeration that limits the manufacture of nanocomplexes to small batches, low photochemical stability, lack of consistent particle size and brightness, lack of specificity of nanocomplex conjugates to cellular targets, and lack of methodology for rapid purification of nanocomplexes bound to cellular targets. Nanocomplexes and methods of making and using nanocomplexes such as the following would have substantial advantages: minimized agglomeration in large-scale batch manufacturing, high photochemical stability, consistent and narrow particle size distribution and brightness, high binding specificity to cellular targets, and experimental guidance for rapid purification of nanocomplexes bound to cellular targets. Summary of the invention

[0005] A broad object of particular embodiments of the present invention is to provide a nanocomposite comprising one or more nanoparticles encapsulated by a polymer, the polymer having a hydrophobic region attached to the nanoparticle and a hydrophilic region including a functional group attached to an aqueous environment, wherein the nanoparticle may comprise one or more of QDs or SPIONs, and combinations thereof, and the polymer may comprise polystyrene-b-polyethylene glycol including functional groups, wherein the polystyrene may have a molecular weight ranging from about 1.5 kDa to about 40 kDa, and wherein the polyethylene glycol may have a molecular weight ranging from about 10 kDa to about 40 kDa, whereby the combination and arrangement of variations in the mass ratios of the QDs, SPIONs, the molecular weight of the polystyrene and / or the molecular weight of the polyethylene glycol and the branching structure of the polyethylene glycol, and the like, allows for the manufacture of a large and varied number of nanocomposites having substantially consistent hydrodynamic diameters ranging from about 40 nm to about 500 nm and brightness resulting from QDs having different emission wavelengths ranging from 420 nm and 1000 nm.

[0006] Another broad object of particular embodiments of the present invention is to provide a QD and / or SPION nanocomplex antibody conjugate capable of specifically binding to a cellular target, wherein illustrative embodiments of the nanocomplex antibody conjugate include one or more anti-CD3 or anti-CD4 antibodies capable of specifically binding to CD3 and / or CD4 peripheral blood mononuclear cells.

[0007] Another broad object of particular embodiments of the invention is to provide nanocomplexes comprising polymer-encapsulated QDs or SPIONs or a combination thereof for specific targeting of macrophages, wherein illustrative embodiments comprise uptake of embodiments of the nanocomplexes by blood cells.

[0008] Another broad object of a particular embodiment of the present invention is to provide a method for separating QD and / or SPION nanocomplex antibody conjugates bound to cells, wherein the SPION nanocomplex antibody conjugates bound to the cells can be separated and isolated by the influence of a magnetic field, and wherein the separated QD or SPION nanocomplex antibody conjugates bound to cells can be analyzed by flow cytometry, and in a particular embodiment, the analyzed QD or SPION nanocomplex antibody conjugates bound to the cells can be flow sorted into separate populations based on one or more characteristics of the cells.

[0009] Naturally, further objects of the present invention are disclosed throughout the specification, drawings, paragraphs, and other parts of the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] This patent or application file contains at least one drawing rendered in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0011] Figure 1 is a process block diagram that provides an overview of the process for making and using elements of a nanoparticle polymer encapsulation system including: polymer synthesis, preparation of polymer-encapsulated nanoparticles, antibody preparation, manufacture of antibodies that bind to polymer-encapsulated nanoparticles, and antibodies that bind to polymer-encapsulated nanoparticles that bind to cellular targets.

[0012] Figure 2 is a program block diagram, which includes blocks 2A to 2I, and its description is as follows Figure 1 The method of Block 1A encompasses procedures for synthesizing polymers including the illustrative example of PS-b-PEG-NH2.

[0013] Figure 3 is an NMR spectrum, which is verified by Figure 2 Molecular structure of an illustrative example of PS-b-PEG-NH2 obtained by the polymer synthesis procedure shown in Blocks 2A to 2I.

[0014] Figure 4A Explanation 1 Figure 1 Specific examples of polymer-encapsulated nanoparticles encompassed by Block 1B include nanoparticles encapsulated in Figure 2 The polymer synthesis procedures shown in Blocks 2A to 2I result in one or more QDs in the polymer.

[0015] Figure 4B Explanation 1 Figure 1 Specific examples of polymer-encapsulated nanoparticles encompassed by Block 1B include nanoparticles encapsulated in Figure 2 The polymer synthesis procedures shown in Blocks 2A to 2I result in one or more QDs and one or more SPIONs in the polymer.

[0016] Figure 4C Explanation 1 Figure 1 Specific examples of polymer-encapsulated nanoparticles encompassed by Block 1B include nanoparticles encapsulated in Figure 2 One or more QDs and / or one or more SPIONs in a polymer obtained by the polymer synthesis procedure shown in blocks 2A to 2I, wherein the polymer may have functional groups that can be activated to bind one or more agents including (as illustrative examples): one or more linkers, polyethylene glycol, fluorescent probes, aptamers, vitamins, cell surface receptors, cell coats, proteins, peptides, radioisotopes, contrast media, surface charge modifiers, agglutinating proteins, or antibodies, half antibodies, antibody fragments, and combinations thereof.

[0017] Figure 5 is a block diagram illustrating the assembly of Figure 4A and 4B The method of polymer encapsulation of nanoparticles is shown in FIG.

[0018] Fig. 6A For one Figure 1 A photomicrograph obtained by transmission electron microscopy of a polymer-encapsulated nanoparticle as encompassed by Block 1B, the polymer-encapsulated nanoparticle comprising: Figure 2 One or more QDs in a polymer obtained by the polymer synthesis process shown in Blocks 2A to 2I and by using Figure 5 The electrohydrodynamic mixing-mediated nanoprecipitation described in was obtained using 0.5 v / v of QDs in the organic phase.

[0019] Figure 6B for Figure 1 A micrograph obtained by transmission electron microscopy of a polymer-encapsulated nanoparticle as encompassed by Block 1B, the polymer-encapsulated nanoparticle comprising: Figure 2 One or more QDs in a polymer obtained by the polymer synthesis process shown in Blocks 2A to 2I and by using Figure 5 The electrohydrodynamic mixing-mediated nanoprecipitation described in was obtained using 0.2 v / v of QDs in the organic phase.

[0020] Fig. 7A for Figure 1 A photomicrograph obtained by transmission electron microscopy of a polymer-encapsulated nanoparticle as encompassed by Block 1B, the polymer-encapsulated nanoparticle comprising: Figure 2 The polymer synthesis process shown in Blocks 2A to 2I obtains one or more QDs in the polymer having an emission wavelength of 420 nm and is obtained by using Figure 5 The electrohydrodynamic mixing-mediated nanoprecipitation described in was obtained.

[0021] Figure 7B for Figure 1 A photomicrograph obtained by transmission electron microscopy of a polymer-encapsulated nanoparticle as encompassed by Block 1B, the polymer-encapsulated nanoparticle comprising: Figure 2 The polymer synthesis process shown in Blocks 2A to 2I obtains one or more QDs in the polymer having an emission wavelength of 575 nm and is obtained by using Figure 5 The electrohydrodynamic mixing-mediated nanoprecipitation described in was obtained.

[0022] Figure 7C for Figure 1 A photomicrograph obtained by transmission electron microscopy of a polymer-encapsulated nanoparticle as encompassed by Block 1B, the polymer-encapsulated nanoparticle comprising: Figure 2 The polymer synthesis process shown in Blocks 2A to 2I obtains one or more QDs with an emission wavelength of 610 nm in the polymer and is obtained by using Figure 5 The electrohydrodynamic mixing-mediated nanoprecipitation described in was obtained.

[0023] Fig.7D for Figure 1 A photomicrograph obtained by transmission electron microscopy of a polymer-encapsulated nanoparticle as encompassed by Block 1B, the polymer-encapsulated nanoparticle comprising: Figure 2 The polymer synthesis process shown in Blocks 2A to 2I obtains one or more QDs in the polymer having an emission wavelength of 655 nm and is obtained by using Figure 5 The electrohydrodynamic mixing-mediated nanoprecipitation described in was obtained.

[0024] Fig. 8A for Figure 1 A photomicrograph obtained by transmission electron microscopy of a polymer-encapsulated nanoparticle as encompassed by Block 1B, the polymer-encapsulated nanoparticle comprising: Figure 2 The polymer synthesis process shown in Blocks 2A to 2I obtains one or more 20 nm SPIONs and one or more QDs having an emission wavelength present at 610 nm in the polymer and is obtained by using Figure 5 The electrohydrodynamic mixing-mediated nanoprecipitation described in was obtained.

[0025] Figure 8B for Figure 1 A photomicrograph obtained by transmission electron microscopy of a polymer-encapsulated nanoparticle as encompassed by Block 1B, the polymer-encapsulated nanoparticle comprising: Figure 2 The polymer synthesis process shown in Blocks 2A to 2I obtains one or more 15 nm SPIONs and one or more QDs having an emission wavelength present at 610 nm in the polymer and is obtained by using Figure 5 The electrohydrodynamic mixing-mediated nanoprecipitation described in was obtained.

[0026] Figure 8C for Figure 1 A photomicrograph obtained by transmission electron microscopy of a polymer-encapsulated nanoparticle as encompassed by Block 1B, the polymer-encapsulated nanoparticle comprising: Figure 2The polymer synthesis process shown in Blocks 2A to 2I obtains one or more 5 nm SPIONs and one or more QDs having an emission wavelength present at 610 nm in the polymer and is obtained by using Figure 5 The electrohydrodynamic mixing-mediated nanoprecipitation described in was obtained.

[0027] Fig.9A for Figure 1 Micrograph obtained by transmission electron microscopy of polymer-encapsulated nanoparticles encompassed by Block 1B, the polymer-encapsulated nanoparticles comprising a ratio of QD:15 nm SPION:polymer (5:5:20) and obtained by Figure 2 The polymer synthesis process shown in blocks 2A to 2I and by using Figure 5 The electrohydrodynamic mixing-mediated nanoprecipitation described in was obtained.

[0028] Fig. 9B for Figure 1 Micrograph obtained by transmission electron microscopy of polymer-encapsulated nanoparticles covered by Block 1B, the polymer-encapsulated nanoparticles comprising a ratio of QD:5 nm SPION:polymer (3:5:20) and obtained by Figure 2 The polymer synthesis process shown in blocks 2A to 2I and by using Figure 5 The nanoprecipitation was obtained by electrohydrodynamic mixing mediated nanoprecipitation as described in and had a hydrodynamic diameter of 210±30 nm.

[0029] Fig. 9C for Fig.9A A graph of the hydrodynamic diameter of polymer encapsulated nanoparticles versus the concentration of particles per milliliter is shown in , which demonstrates a hydrodynamic diameter of approximately 128 nm ± 22 nm.

[0030] Fig.9D is a bar graph comparing the iron concentration of polymer-encapsulated nanoparticles comprising 15 nm SPIONs and QDs having an emission wavelength present at 610 nm versus polymer-encapsulated nanoparticles comprising 5 nm SPIONs and QDs having an emission wavelength present at 610 nm.

[0031] Fig.9E is a bar graph comparing the fluorescence of polymer-encapsulated nanoparticles comprising 15 nm SPIONs versus polymer-encapsulated nanoparticles comprising 5 nm SPIONs.

[0032] Fig. 10A is a flow cytometry univariate histogram depicting the detection of peripheral blood mononuclear cells bound to mouse anti-human CD4 Figure 1Block 1D encompasses polymer-encapsulated nanoparticles comprising one or more QDs.

[0033] Fig. 10B is a flow cytometry univariate histogram depicting the detection of peripheral blood mononuclear cells bound to mouse anti-human CD3 linked to Figure 1 Block 1D encompasses polymer-encapsulated nanoparticles comprising one or more QDs.

[0034] Fig.11A is a flow cytometry two-point plot depicting the detection of CD3-positive peripheral blood mononuclear cells and CD3-negative cells in the reaction solution before magnetic separation, wherein the mouse anti-human CD3 is bound to Figure 1 Block 1D encompasses polymer-encapsulated nanoparticle connections comprising one or more SPIONs and one or more QDs.

[0035] Fig. 11B Flow cytometry two-point plot depicting the detection of depleted CD3-positive peripheral blood monocytes in the non-magnetic portion of the reaction solution after magnetic separation.

[0036] Fig. 11C is a flow cytometry two-point plot depicting the detection of enriched CD3-positive peripheral blood mononuclear cells bound to mouse anti-human CD3 in the magnetic portion of the reaction solution after magnetic separation. Figure 1 Block 1D encompasses polymer-encapsulated nanoparticle connections comprising one or more SPIONs and one or more QDs.

[0037] Fig. 12A FIG. 1 is a bright field image of a host squid blood cell cultured with the nanocomplex of the present invention, wherein the nanocomplex includes one or more SPIONs and one or more QDs having an emission wavelength at 610 nm.

[0038] Fig. 12B An epifluorescence image of a host squid blood cell cultured with a nanocomplex of the present invention comprising one or more SPIONs and one or more QDs having an emission wavelength at 610 nm, the emission being detected by fluorescence microscopy in the fluorescein isothiocyanate ("FITC") channel.

[0039] Fig. 12C Composite image of bright field and epi-fluorescence images detected by bright field and fluorescence microscopy measured in FITC channel of host squid blood cells cultured with the nanocomplex of the present invention comprising one or more SPIONs and one or more QDs with emission wavelength at 610 nm. DETAILED DESCRIPTION

[0040] The present invention can be understood by reference to the following detailed description of various aspects of the invention and the examples included therein and the above and below descriptions with reference to the drawings and the like. Compounds, compositions, articles, devices, or methods are disclosed and described, it should be understood that they are not limited to specific synthetic methods (unless otherwise specifically indicated) or to specific reagents (unless otherwise specifically indicated), as these can of course vary. It should also be understood that the terms used herein are used for the purpose of describing specific embodiments for the purpose of enabling those with ordinary knowledge in the technical field to which the invention belongs to make and use a large number of various embodiments of the invention (even if not explicitly disclosed). Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those with ordinary knowledge in the technical field to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used to implement the present invention.

[0041] All publications mentioned herein are incorporated herein by reference to disclose and narrate the method and / or material relevant to the cited publications. The publications discussed herein are provided only for the disclosure content of the application date prior to the present application. Nothing herein should be interpreted as admitting that the present invention has no right to precede such publications due to previous inventions. In addition, the date of the publications provided herein may be different from the actual publication date, and it may need to be independently confirmed.

[0042] As used in this specification and the appended claims, the singular forms "a" and "an" and "the" include plural referents unless the context clearly requires otherwise. Thus, for example, reference to "a component," "a polymer," or "a particle" includes mixtures of two or more such components, polymers, or particles, and so forth.

[0043] Ranges may be expressed herein as from "about" a particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the particular value and / or to the other particular value. Similarly, when a value is expressed as an approximation by using the antecedent "about", it should be understood that the particular value forms another embodiment. It should be further understood that the endpoints of each of the ranges are important relative to and independent of the other endpoints. It should also be understood that there are some values ​​disclosed herein, and each value is also disclosed herein as "about" the particular value in addition to the value itself. For example, if the value "10" is disclosed, "about 10" is also disclosed. It should also be understood that when a value is disclosed, "less than or equal to" the value, "greater than or equal to" the value, and possible ranges between the values ​​are also disclosed, as appropriately understood by those with ordinary knowledge in the art to which the invention belongs. For example, if the value "10" is disclosed, "less than or equal to 10" and "greater than or equal to 10" are also disclosed. It should also be understood that throughout this application, data is provided in a number of different formats and that this data represents endpoints and starting points, and ranges of any combination of data points. For example, if a specific data point "10" and a specific data point 15 are disclosed, it should be understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15, and between 10 and 15 should be considered disclosed. It should also be understood that each unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0044] As used in this specification and in the claims at the end, a residue of a chemical species refers to a portion of the resulting product of that chemical species in a particular reaction scheme or subsequent formulation or chemical product, regardless of whether that portion is actually obtained from that chemical species. Thus, as an illustrative example, an ethylene glycol residue in a polyester refers to one or more -OCH2CH2O- units in that polyester, regardless of whether ethylene glycol was used to prepare the polyester. Similarly, a sebacic acid residue in a polyester refers to one or more -CO(CH2)8CO- moieties in that polyester, regardless of whether that residue is obtained by reacting sebacic acid or an ester thereof to obtain the polyester.

[0045] Disclosed is the component to be used to prepare the composition of the present invention and the composition itself to be used in the method disclosed herein. These and other materials are disclosed herein, and it should be understood that when the combination, subgroup, interaction, and group of these materials are disclosed but the special mention of each individual and common combination and arrangement of these compounds may not be clearly disclosed, each is specifically considered to be possible and described herein. For example, if a specific compound is disclosed and discussed and some modifications that can be made to some molecules including these compounds are discussed, it is specifically considered to be possible that each combination and arrangement of the compound and possible modifications is possible, unless specifically indicated as the opposite. Therefore, if the group of molecules A, B, and C is disclosed and the group of molecules D, E, and F and an example AD of the combined molecule are disclosed, even if each is not independently described in detail, each is independently and jointly considered to be possible, meaning that the combination AE, AF, BD, BE, BF, CD, CE, and CF is considered to be disclosed. Similarly, any subgroup or combination of these is also disclosed. Thus, for example, subgroups of AE, BF, and CE should be considered disclosed. This concept applies to all aspects of this application, including, but not limited to, elements in methods of making and using the compositions of the present invention. Thus, if there are a variety of additional elements that can be performed, it should be understood that each of these additional elements can be performed with any particular embodiment or combination of embodiments of the methods of the present invention. It should be understood that the compositions disclosed herein have certain functions. Disclosed herein are certain structural requirements for performing the disclosed functions, and it should be understood that there are a variety of structures that can perform the same functions associated with the disclosed structures, and these structures typically achieve the same results.

[0046] Program Overview

[0047] Now, the main reference Figure 1 , a process block diagram provides an overview of a nanomaterial polymer encapsulation system (1) that includes one or more of the following: polymer synthesis of nanoparticles ("NP") (2) encapsulating polymers ("P") (3) (Block 1A); formation of polymer-encapsulated nanoparticles ("PENP") (4) (Block 1B); preparation of antibodies, half antibodies, antibody fragments (individually or collectively, "Ab") (5) (Block 1C); preparation of PENP antibody conjugates ("PENP-Ab") (6) (Block 1D); PENP-Ab cell labeling (PENP-Ab-cell") (7), flow cytometric analysis (8), and imaging of PENP-Ab-cell (7) (Block 1E).

[0048] Polymer synthesis

[0049] Now, the main reference Figure 2 , a program block diagram provides Figure 1 An overview of the illustrative polymer synthesis procedure of Block 1A. Referring to Block 2A of the procedure, an amino polystyrene ("PS-NH2") of Formula I (having a linear formula (C8H8) n CH5N, wherein the PS molecular weight may exist in the range of about 1.5 kDa to about 40 kDa) can be obtained from Polymer Source, Inc. as PNP3965-SNH2. A particularly specific example may include PS-NH2 having a number average molecular weight of about 9.5 kDa.

[0050]

[0051] Now, referring to the polymer synthesis procedure Figure 2 Block 2B, comprising dibenzocyclooctyne-N-hydroxysuccinimidyl ester ("DBCO-NHS ester") of Formula II (having a linear formula C 23 H 18 N2O5 (CAS No.: 1353016-71-3) and having a molecular weight of 402.40 g / mol) is available from Click Chemistry Tools, PN A133-100.

[0052]

[0053] Now, referring to the polymer synthesis procedure Figure 2 Block 2C, PS-NH2 can be reacted with DBCO-NHS ester to produce polystyrene-dibenzocyclooctyne ("PS-DBCO") comprising Formula III.

[0054]

[0055] An example of a scalable process for making PS-DBCO may include thawing PS-NH2 20 mg at room temperature ("RT") for 15 minutes and thawing DBCO-NHS-ester 10 mg at RT for 15 minutes ("min."). 1 mL of toluene (C6H5CH3) (CAS No.: 108-88-3) is added to PS-NH2 20 mg and vortexed at 500 RPM for 15 minutes. The mixture is stirred at about 25°C (about 77°F) at 1500 relative centrifugal force ("RCF") (RCF = (RPM) 2 ×1.118×10 -5=Centrifuge DBCO at 400 × 400 °C for 30 seconds ("sec."). Add 1 mL of toluene to DBCO. Sonicate DBCO in RT water for 5 min. Transfer 1 mL of DBCO in toluene to PS-NH2 in toluene. Vortex the DBCO mixture in PS-NH2 at 500 RPM at RT for 16 to 24 hours ("hr.").

[0056] Now, referring to the polymer synthesis procedure Figure 2 Block 2D, comprising an 8-arm polyethylene glycol-amine ("PEG 8臂 -NH2")) (which comprises a multi-arm PEG derivative having an amine group attached to a hexaglycerol core at each end of its eight arms, having a linear formula R(O(CH2CH2O) n CH2CH2NH2)8 and a number average molecular weight of about 19.5 kDa) can be obtained from Nanosoft Polymers, PN 2443.

[0057]

[0058] In particular embodiments, 4-arm polyethylene glycol-amine, 6-arm polyethylene glycol-amine, or 8-arm polyethylene glycol-amine, or combinations thereof, having a corresponding PEG molecular weight of 10 kDa, 20 kDa, or 40 kDa may also be utilized.

[0059] Now, referring to the polymer synthesis procedure Figure 2 Block 2E, comprising azido-d-polyethylene glycol 4-N-hydroxysuccinimidyl ester ("azido-PEG4-NHS ester") of Formula VI, having a molecular weight of 388.37 g / mol (CAS No.: 944251-24-5), is available from Click Chemistry tools, PN AZ103-100.

[0060]

[0061] Now, referring to the polymer synthesis procedure Figure 2 Block 2F, PEG 8臂 -NH2 can react with azido-PEG4-NHS ester to produce a branched polyethylene glycol ("PEG-amine-azide") comprising Formula VII.

[0062]

[0063] An example of a scalable procedure for making PEG-amine-azide may include thawing 40 mg of PEG at RT. 8臂-NH2 for 15 min. and thaw 80 μl of azido-PEG4-NHS ester (50 mM) at RT for 15 min. Add 400 μl of methanol (CAS No.: 6756-1) to the PEG 8臂 -NH2 and vortex PEG at 500RPM 8臂 -NH2 for 15 min. Centrifuge the azido-PEG4-NHS at 1500 RCF for 30 sec. at 25°C (about 77°F). Transfer 80 μl of azido-PEG4-NHS to 400 μl of PEG 8臂 -NH2. Vortex at 500 RPM for 16 to 24 hr. at RT.

[0064] Now, referring to the polymer synthesis procedure Figure 2 Block 2G, the PS-DBCO obtained in Block 2C of the synthetic procedure can be purified by the following illustrative scalable procedure comprising the following: Transfer 400 μl of PS-DBCO to each of five centrifuge tubes. Add 800 μl of methanol to each of the five centrifuge tubes. Mix the PS-DBCO in methanol by inverting each of the five centrifuge tubes. Centrifuge the PS-DBCO in methanol at 20,000 RCF for 5 min. at 15°C to 25°C (about 59°F to about 77°F). Pour the supernatant from each of the five centrifuge tubes. Add 400 μl of toluene to each of the five centrifuge tubes. Place the five centrifuge tubes in a water bath at 37°C (about 98.6°F) for 2 min. Dissolve the PS-DBCO pellet in 400 μl of toluene by mixing with a pipette. Add 800 μl of methanol to each of the five centrifuge tubes. Invert each centrifuge tube to mix the contents. Centrifuge at 20,000 RCF for 5 min. at 15° to 25°C (about 59°F to about 77°F). Pour off the supernatant from each of the five centrifuge tubes. Add 400 μl of toluene to each of the five centrifuge tubes. Place the five centrifuge tubes in a 37°C (about 98.6°F) water bath for 2 min. Dissolve the PS-DBCO pellet in toluene by mixing with a pipette.

[0065] Now, referring to the polymer synthesis procedure Figure 2 Block 2H, the PS-DBCO obtained in Block 2G of the synthesis procedure can be reacted with the PEG-amine-azide obtained in Block 2F of the polymer synthesis procedure to produce a polystyrene-b-poly(ethylene glycol)amine ("PS-b-PEG-NH2") comprising Formula VIII.

[0066]

[0067] In the illustrative example of this block 2H, all five centrifuge tubes of PS-DBCO in toluene and PEG-amine-azide in methanol obtained in block 2F can be transferred to a 4 mL glass vial. The mixture of PS-DBCO and PEG-amine-azide is vortexed at 500 RPM at RT for 16 to 24 hr. to produce PS-b-PEG-NH2 comprising Formula VIII. PS-b-PEG-NH2 can comprise PS having a molecular weight ranging from about 1.5 kDa to about 40 kDa and can comprise PEG having a molecular weight ranging from about 10 kDa to about 40 kDa.

[0068] According to the synthesis procedure Figure 2 The PS-b-PEG-NH2 obtained in Block 2I, Block 2H can be purified and dried by the following illustrative scalable procedure including the following: Cool hexane (CAS No.: 110-54-3) at -20°C (about -4°F) for 15 min. Add approximately 1.25 mL of the PS-b-PEG-NH2 obtained in Block 2I of the synthetic procedure to a 15 mL centrifuge tube. Slowly add 12 mL of hexane to the centrifuge tube containing the PS-b-PEG-NH2. Mix the PS-b-PEG-NH2 in toluene / hexane by gently tilting the centrifuge tube 10 to 15 times. Place the centrifuge tube at -20°C (about -4°F) for 5 min. to cool the PS-b-PEG-NH2 in toluene / hexane. Pour off the supernatant from the PS-b-PEG-NH2 pellet. Add 1 mL of tetrahydrofuran to the centrifuge tube. Place the centrifuge tube in a water bath at 37°C (about 98.6°F) for 2 min. Dissolve the PS-b-PEG-NH2 precipitate pellet by mixing with a pipette. Slowly add 6 mL of cold hexane to the centrifuge tube. Gently tilt the centrifuge tube 10 to 15 times until a thin sheet of PS-b-PEG-NH2 precipitate is seen and a clear supernatant is formed. Place the centrifuge tube at -20°C (about -4°F) for 5 min. Pour off the supernatant from the PS-b-PEG-NH2 precipitate pellet. Place the centrifuge tube containing the PS-b-PEG-NH2 precipitate pellet at RT for 24 hr. to remove excess solvent and obtain dry PS-b-PEG-NH2. Store at -20°C (about -4°F).

[0069] Now, the main reference Figure 3 The structure of PS-b-PEG-NH2 obtained in block 2I of the synthesis procedure can be analyzed using nuclear magnetic resonance ("NMR"). Figure 3 The NMR spectra shown in Figure 2 The molecular structure obtained by the polymer synthesis process shown in Blocks 2A to 2I and the polymer synthesis process disclosed above is the purified PS-b-PEG-NH2 comprising Formula VIII.

[0070] The illustrative example of the polymer PS-b-PEG-NH2 obtained in Block 2I is not intended to exclude embodiments of PS-b-PEG that include functional groups other than NH2. Other functional groups may include, as examples, one or more of acrylates, maleimides, vinyl sulfones, azides, biotin, carboxyls, thiols, alkynes, hydrazides, N-hydroxysuccinimide esters, and nitrophenyl carbonates, and combinations thereof, or embodiments of other similar or equivalent polymers that include one or more functional groups.

[0071] Formation of polymer-encapsulated nanoparticles.

[0072] Now, the main reference Figure 4A , which illustrates a specific specific example of PENP (4) comprising one or more QDs (2a) encapsulated in a polymer ("P") (3), wherein the polymer ("P") (3) has a hydrophobic region that can be connected or coordinated to the one or more QDs (2a) and a hydrophilic region comprising a functional group (9) that can be connected to an aqueous environment, and in a specific specific example, the polymer (P) (3) may include PS-b-PEG-NH2 obtained in block 2I, which can produce polymer-encapsulated nanoparticles ("PENP-MultiDot") (4a) comprising one or more QDs (2a) through solution-based connection.

[0073] Now, the main reference Figure 4B , which illustrates a specific embodiment of a PENP (4) comprising one or more QDs (2a) and one or more SPIONs (2b) encapsulated in a polymer ("P") (3), wherein the polymer ("P") (3) has a hydrophobic region that can be connected or coordinated to the one or more SPIONs (2b) and, if necessary, one or more QDs (2a) and a hydrophilic region comprising a functional group (9) that can be connected to an aqueous environment, and in a specific embodiment, the polymer (P) (3) may include PS-b-PEG-NH2 obtained in block 2I, which can produce a PENP (4) ("PENP-MagDot") (4b) comprising one or more SPIONs (2b) and one or more QDs (2a) by solution-based connection.

[0074] Now, the main reference Figure 4C A large number and variety of agents (5) can be conjugated to PENP-MultiDot (4a) and / or PENP-MagDot (4b) using the functional group (9) of the polymer (3), and in a particular embodiment, the amine is provided by PS-b-PEG-NH2. Figure 4CThe example of PENP-MagDot (4b) in the embodiment illustrates that one or more agents (5) can be bound to PENP-MultiDot (4a) and / or PENP-MagDot (4b) by activating the functional group (9) including, as illustrative examples, one or more linkers (10, 10a, 10b), polyethylene glycol, fluorescent probes, aptamers, vitamins, radioisotopes, contrast media, surface charge modifiers, agglutinins, proteins, peptides, cell surface receptors, cell coats, and combinations thereof. Specifically, in a particular embodiment, the functional group (9) can be used to directly or indirectly link an antibody or antibody fragment (Ab) (5') through one or more linkers (10, 10a, 10b) to produce a PENP-antibody conjugate (6) including, as illustrative examples, PENP-MultiDot-Ab (6a) and PENP-MultiDot-Ab (6b).

[0075] Now, the main reference Figure 5 , scalable, solution-based manufacturing of the PENPs (4) encompassed by the present invention (including (but not necessarily limited to) PENP-MagDot (4b) or PENP-MultiDot (4a)) can be prepared by conventional self-assembly, flash nanoprecipitation (FNP), which includes rapid turbulent mixing generated by high-speed flow, as described by the following: Yanjie Zhang, a Aaron R. Clapp, RSC Advances, 89, 2014 "Preparation of quantum dot-embedded polymeric nanoparticles using flash nanoprecipitation", or by rapid mixing induced by electrohydrodynamic force ("EHD"): EHD mixing mediated-nanoprecipitation ("EHD-NP") Kil Ho Lee, Guolingzi Yang, Barbara E. Wyslouzi and Jessica O. Winter, ACS Appl. Polym. Mater. 2019, 1, 4, 691-700, each of which is incorporated herein by reference.

[0076] Figure 5An illustrative EHD mixing system may include a syringe (11) having a syringe barrel (12) equipped with a sliding syringe plunger (13), and a syringe needle (14). A syringe pump (15) is provided to drive the syringe plunger (13) to deliver an organic phase of a water-miscible non-polar aprotic solvent ("OP") to an aqueous phase ("AP") at a predetermined volume flow rate, wherein the water-miscible non-polar aprotic solvent ("OP") includes a dissolved (P) (3) and NPs (4), whether QDs (2a) and / or SPIONs (2b) (together, "inorganic component"). The illustrative examples of syringes (11) and syringe drivers (15) are not intended to exclude other devices that can be used to deliver the OP to the AP at a predetermined volume flow rate. The concentration of the inorganic component per unit volume of the OP can be about 0.1 volume to volume ("v / v") to about 0.5 v / v. The concentration of P(3) to QD(2a) and / or SPION(2b) in the OP can be about 1:1 to about 4:1 by mass. The amount of P(3) can be adjusted to obtain PENP(4) having a substantially uniform hydrodynamic diameter ("HD") existing in a particle size range of about 40 nanometers ("nm") to about 500 nm. In embodiments where the QD(2a) and / or the SPION(2b) are passivated with a ligand(16), the mass of the ligand relative to the mass of the total inorganic component can be about 20 mass % to 40 mass %. A mass percentage of the ligand greater than 40% can interfere with the assembly of the PENP(4).

[0077] A non-conductive container (17) may be loaded with the AP, which is typically distilled or deionized water. A positive electrode (18) and a negative electrode (19) may be introduced into the AP with the non-conductive container (17) fixed about 1 cm apart. In a particular embodiment, if conductive, the syringe needle (14) may act as the positive electrode (18). A voltage source (20) may provide a voltage ("V") to the positive terminal (18) to generate an electric field between the positive electrode (18) and the negative electrode (19) in the AP. The syringe plunger (13) may be driven at a rate of about 8 mL h -1 to about 15 mL h -1The OP is introduced into the AP at a constant flow rate of about 1.5 V. The voltage (V) can be adjusted between about -1 kilovolts ("kV") and about -2.5 kV. The electric field can produce a fine dispersion of the OP in the AP to produce PENPs (4), PENP-MagDots (4b), or PENP-MultiDots (4a) having substantially uniform particle sizes. The resulting particle size of the PENPs (4), PENP-MagDots (4b), or PENP-MultiDots (4a) can increase with increasing V and / or the concentration of the inorganic component of the OP per unit volume. The resulting PENP-MagDots (4b) can then be separated by the influence of a magnetic field (15).

[0078] PENP-MultiDot.

[0079] PENP-MultiDot (4a) may include QD (2a) inorganic semiconductor nanocrystals, which include an inorganic core semiconductor material (2a') (also called "core material") (noted as "core material / shell material") (e.g., CdS / ZnS) surrounded by a shell semiconductor material (2a") (also called "shell material") having a different energy band gap. The QD (2a) may have a particle size typically existing in the range of 1nm to 10nm. The QD (2a) may exhibit particle size-variable emission color due to quantum confinement effects, wherein smaller QDs (2a) emit at higher energy (lower wavelength) and larger QDs (2a) emit at lower energy (higher wavelength) for a given composition. Accordingly, the QD (2a) may absorb over a wide range and have a photoluminescent emission over a narrow range, which can be adjusted based on the material from which the QD (2a) is manufactured and the particle size of the QD (2a). Illustrative examples of QD core / shell compositions suitable for the embodiment of the PENP-MultiDot (4a), and combinations thereof, may include one or more of the following: CdS / ZnS, CdSSe / ZnS, CdSe / ZnS, CdTe / ZnS, and CdSeTe / ZnS, each having an emission photoluminescence in the range of 420nm to 880nm; CuInZnS / ZnS, 540 to 660nm; and PbS / CdS, 700nm to 900nm. However, these examples are not intended to exclude embodiments having other QD (2a) core / shell compositions.

[0080] Purified QDs (2a) can be unmasked or can be capped to control QD size and / or to prevent QD aggregation. QDs (2a) synthesized by prototyping thermal injection methods can be capped with ligands (16) such as oleylamine and oleic acid after QD purification. 1H NMR spectroscopy analysis demonstrates that ligand binding can be highly dynamic and that oleylamine selectively binds to the surface as oleyl ammonium bromide in the NC(X)2 binding motif. Oleic acid binds to the surface as oleyl ammonium oleate only in the presence of excess oleylamine added after purification. Protesescu L, Yakunin S, Bodnarchuk MI, Krieg F, Caputo R, Hendon CH, Yang RX, Walsh A, Kovalenko MV, Nano Lett. 2015, 15, 3692-3696, hereby incorporated by reference herein. While the use of oleylamine and oleic acid as capping ligands (16) is suitable for embodiments of the present invention, this is not intended to exclude embodiments using other capping ligands (16) (for example, trioctylphosphine oxide, L-histidine, chitosan, polyvinyl alcohol, polyvinyl pyrrolidone, or combinations thereof).

[0081] Typically, unshielded QDs (2a) or capped QDs (2a) are prepared by surrounding the QDs (2a) with P (3) to produce PENPs (4) to achieve the desired level of water solubility and biocompatibility. P (3) suitable for use in embodiments of the present invention may include various embodiments of PS-b-PEG-NH2 obtained in block 2I of the synthetic procedure described above. Embodiments of PENPs (4) can be made by using various combinations of one or more of the following: QDs (2a), capping ligands (16), and PS-b-PEG-NH2 having a PS molecular weight ranging from about 1.5 kDa to about 40 kDa and a PEG having a molecular weight ranging from about 10 kDa to about 40 kDa.

[0082] An illustrative example of a scalable method for manufacturing PENPs (4) (including but not necessarily limited to PENP-MultiDot (4a)) by EHD may include one or more of the following: Figure 2 The PS-b-PEG-NH2 obtained in Block 2I is placed in a first 1.5 mL tube. 240 μl of 5 mg / mL desired QD (2a) is introduced into a second 1.5 mL tube. 480 μl of acetone / methanol (60 / 40) is transferred to the QD (2a) in the second 1.5 mL tube. The second 1.5 mL tube containing QD (2a) is centrifuged at 7000 RCF for 1 min. and then the supernatant is removed with a 200 μL pipette. 480 μl of anhydrous tetrahydrofuran ("THF") is transferred to the second 1.5 mL tube containing QD and mixed thoroughly with a pipette. 240 μl of THF, 240 μl of QD in THF, and 120 μl of dissolved PS-b-PEG-NH2 are introduced into a new 1.5 mL centrifuge tube to produce OP for EHD.

[0083] In a particular embodiment, EHD can be performed by: Cleaning the EHD mixing system syringe (11) three times with THF. Place about 0.6 mL of OP into the syringe barrel (12). Thoroughly mix the inorganic components in the OP while placing the syringe (11). Connect the syringe (11) to a syringe pump (15) and set the syringe pump (15) to produce a flow rate of OP containing inorganic components from the syringe needle (14) in the range of about 11.00 mL / hr. to about 14.00 mL / hr. In a particular embodiment, the flow rate can be about 12.5 mL / hr. In a particular embodiment, the flow rate can be about 12.5 mL / hr. Prepare the syringe needle (14) until a drop of OP is formed at the end of the syringe needle (14). Rinse the 20 mL glass vial (17) twice with distilled or deionized water (individually or collectively, "DI water"). Introduce about 10 mL of DI water into the 20 mL glass vial. Submerge the syringe needle (14) into a 20 mL glass vial (17). Clean the negative electrode (19) by immersing in THF, wiping, and rinsing with DI water. Place the negative electrode (19) into the AP contained in the 20 mL glass vial. Place the positive electrode (18) into the AP contained in the 20 mL glass vial (17). In a particular embodiment, if conductive, the syringe needle (14) can act as the positive electrode (18). Ensure that the positive electrode (18), negative electrode (19), and syringe needle (14) are not touching. Connect the positive lead (21) from the voltage source (20) to the positive electrode (18) or syringe needle (14) and connect the negative lead (22) from the voltage source (20) to the negative electrode (19). Confirm that the voltage source (20) delivers approximately -1500V.

[0084] The EHD-NPs (4) produced by mixing OP and AP under the influence of an electric field can be concentrated using centrifugal filtration. The contents of a 20 mL glass vial (17) can be transferred to a 100 kDa cut-off centrifugal ultrafiltration column ("CUC"), for example, SigmaAldrich PN UFC9010D Ultra-15 centrifugal filter unit. Centrifuge CUC at 3000 RCF for 30 min. at about 25°C (about 77°F). Transfer 10 mL of 50 mM sodium borate, 100 mM sodium phosphate, 7.3-7.5 pH ("borate buffer") to CUC. Centrifuge CUC at 3000 RCF for 30 min. at about 25°C (about 77°F). Transfer EHD-NP filtrate from CUC to a 1.5 mL microcentrifuge tube. Measure and record the volume of EHD-NP filtrate collected. Transfer 15 μl of EHD-NP filtrate from the 1.5 mL microcentrifuge tube to a 1.5 mL tube and add 285 μl of borate buffer. Transfer 290 μl of EHD-NP filtrate to the spectrophotometer cell. Measure and record the optical density (OD) at 450 nm on a spectrophotometer. 450 ).

[0085] The EHD-NP filtrate may contain aggregates of PENP-MultiDot (4a) or aggregates of polymer (P) (3) without QD (2a) (individually and collectively, "aggregates"). The aggregates can be substantially removed from the EHD-NP filtrate to produce substantially pure PENP-MultiDot (4a). A scalable method for purifying PENP-MultiDot (4a) may include one or more of the following: Transfer a 120 μl aliquot of the EHD-NP filtrate from a centrifugal ultrafiltration column to a corresponding 1.5 mL tube. Centrifuge the 1.5 mL tube containing the EHD-NP filtrate at 3,000 RCF for 10 min. to precipitate the aggregates into pellets. Remove the supernatant containing PENP-MultiDot (4a) without disturbing the aggregate precipitate pellet. Measure and record the volume of the PENP-MultiDot supernatant. Mix 15 μl of PENP-MultiDot supernatant and 285 μl of borate buffer in a 1.5 mL tube. Transfer 290 μl of the mixture to the spectrophotometer cell. Measure and record the optical density (OD) at 450 nm using a spectrophotometer. 450 ).

[0086] Now, the main reference Fig. 6A and Figure 6B The morphology of the PENP-MultiDot (4a) produced by the above EHD method at the concentrations of P (3) and QD (2a) in the OP of 0.5 v / v and 0.2 v / v can be characterized by using transmission electron microscopy ("TEM") to produce micrographs of the PENP-MultiDot (4a). Figure 6B The PENPs (4) produced at a concentration of 0.2 v / v of QDs (2a) in the OP are shown in the example of Fig. 6AThe PENPs produced at a concentration of 0.5 v / v of QDs (2a) in the OP shown in the example can load fewer QDs (2a) per PENP (4), which proves that the concentrations of P (3) and QDs (2a) in the OP can be adjusted to affect the particle size and fluorescence of each PENP-MultiDot (4a).

[0087] The embodiment of PENP-MultiDot (4a) demonstrates hydrodynamic diameter ("HD") and polydispersity index ("PDI") and particle size stability suitable for effective clinical and non-clinical applications. The hydrodynamic diameter and PDI can be obtained by dynamic light scattering measurements using a NanoBrook 90Plus particle size analyzer. Particle size histograms can be plotted using SigmaPlot (Systat Software Inc., San Jose, CA, USA), and the particle size distribution can be fitted to a log-normal distribution.

[0088] The HD is defined as the particle size of a hypothetical hard sphere that is dispersed in the same manner as the particles being measured. However, in reality, macromolecules or particles in solution are solvated, dynamic, and non-spherical. Because of this, the diameter calculated from the dispersion characteristics of the particles will imply the apparent size of the solvated / dynamically hydrated particles. The HD of the PENP-MultiDot (4a) produced by the above EHD method can be based on the parameter changes used to produce the PENP (4) during EHD. Fig. 6A and Figure 6B The TEM micrographs shown in demonstrate that PENP-MultiDot (4a) has a substantially constant HD of about 40 nm to about 50 nm; however, by varying the parameters used during EHD, the HD of PENP-MultiDot (4a) can be varied in the range of about 40 nm to about 100 nm.

[0089] PDI is the performance of the distribution of the particle size group in the particle sample. The numerical range of PDI is from 0.0 (the perfect consistent group in the particle sample) to 1.0 (the highly polydisperse group in the particle sample). For nanoparticle materials based on polymers, 0.2 and PDI values ​​lower than 0.2 are considered acceptable in implementation. Calculation for determining PDI is defined in ISO standard file 13321:1996E and ISO 22412:2008. PENP-MultiDot (4a) can have a substantially constant PDI of approximately 0.1 to approximately 0.2.

[0090] Now, the main reference Fig. 7A7E, TEM micrographs demonstrate that PENP-MultiDot (4a) can be loaded with QDs (2a) having a range of emission wavelengths in the visible and near infrared spectra while retaining a substantially uniform and narrow particle size distribution in the range. In a particular embodiment, the PENP-MultiDot (4a) can be loaded with QDs (2a) having an emission wavelength in the range of about 420 nm to about 880 nm or upconversion nanoparticles. Fig. 7A The illustrative examples of 7E demonstrate that PENP-MultiDot (4a) has an emission wavelength of 420 nm (e.g. Fig. 7A The example shows), the emission wavelength of 575nm (such as Figure 7B ), an emission wavelength of 610 nm (as shown by Figure 7C ), and an emission wavelength of 655 nm (as shown by Fig.7D). A long-felt but unresolved problem associated with nanocomposites is the lack of consistent and narrow particle size distributions between nanocomposites including QDs (2a) having different emission wavelengths. As an illustrative example, a QD (2a) having an emission wavelength of 420 nm may have a smaller particle size than a QD (2a) having an emission wavelength of 610 nm. In addition, for a given composition, the larger the QD (2a), the brighter the QD (2a). In addition, under substantially similar preparation methods, the nanocomposites are loaded with a greater number of QDs (2a) having an emission wavelength of 420 nm than with QDs (2a) having an emission wavelength of 610 nm. Under similar preparation methods, such differences between QDs (2a) having different emission wavelengths related to one or more of particle size, brightness, and loading affinity can result in nanocomposites that differ accordingly in particle size and brightness. The embodiment of the nanocomposite including QDs (2a) with different emission wavelengths can be produced with a consistent and narrow particle size distribution by the method of the present invention by adjusting the ratio of the QD inorganic metal mass to the polymer mass in the organic phase (OP) introduced into the aqueous phase (AP) during EHD mixing. In the embodiment of the PENP-MultiDot (4a), the QD inorganic metal mass to the polymer mass can be adjusted between 1:1 and 1:2 to produce PENP-MultiDot (4a) with a consistent and narrow particle size distribution between nanocomposites, the nanocomposite containing QDs (2a) with different emission wavelengths in the range of 40nm to 500nm and having substantially similar brightness. In the embodiment of PENP-MagDot (4b), the QD and SPION inorganic metal mass to polymer mass can be adjusted between 1:2 and 1:4 to produce PENP-MagDot (4b) with a consistent and narrow particle size distribution between nanocomposites, the nanocomposites containing QDs with different emission wavelengths in the range of 40nm to 500nm and with substantially similar brightness. These illustrative examples are not intended to exclude other embodiments of PENP-MultiDot (4a) formed in the form of other single wavelength emission, dual wavelength emission, or more than dual wavelength emission based on the one or more QDs (2a) encapsulated in the polymer (P) (3) as described above.

[0091] PENP-MagDot.

[0092] Superparamagnetic iron oxide nanoparticles ("SPION") (2b) are small synthetic particles of hematite (having an empirical formula of Fe2O3, γ-Fe2O3 and a molar mass of 159.69 g / mol (CAS No. 1309-37-1)) or magnetite (having an empirical formula of Fe3O4 and a molar mass of 231.53 g / mol (CAS No. 1317-61-9)) or a combination thereof, having a core with a diameter ranging from about 5 nm to about 30 nm. In addition, mixed oxides of iron and transition metal ions (such as copper, cobalt, nickel, and manganese) are known to exhibit superparamagnetic properties and also fall into the category of SPION (2b). However, in various biomedical applications, magnetite and hematite nanoparticles are the most widely used SPION (2b). SPION (2b) exhibits the phenomenon of "superparamagnetism". Particles exhibiting superparamagnetism are induced when an external magnetic field (15) is applied (such as Figure 5 ) become magnetized up to their saturation magnetization and no longer exhibit any residual magnetic interaction after the magnetic field is removed. This property is size-dependent and generally occurs when the particle size of the nanoparticles reaches about 5 nm. At such a small particle size, these nanoparticles do not exhibit the multiple domains found in large magnets; instead, they become a single magnetic domain and act as a "single superspin" that exhibits a high magnetic susceptibility. Therefore, upon application of a magnetic field, these nanoparticles provide a stronger and faster magnetic response than large magnets, and the residual magnetism (residual magnetization) and coercivity (the field required to make the magnetism zero) are negligible. Once the applied magnetic field is removed, the magnetic particles retain no residual magnetism at room temperature and are easily dispersed. Uncoated iron oxide NPs have extremely low solubility under physiological conditions that can lead to precipitation due to gravity, as well as a high cohesion rate. Therefore, in order to be effectively used, SPIONs can be capped with an amphiphilic coating.

[0093] In a particular embodiment, the SPION (2b) capped with a ligand (16) such as oleic acid can have a particle size in the range of about 5 nm to about 30 nm. Various SPION (2b) are available from Ocean NanoTech, PN SOR05 to SOR30. Illustrative examples of embodiments include SPION (2b) capped with oleic acid having a particle size of 5 nm, 10 nm, 15 nm, and 20 nm. However, these illustrative SPION (2b) particle sizes are not intended to exclude the use of SPION (2b) having a smaller or larger particle size or a combination of SPION (2b) particle sizes, which can be used alone or in combination with one or more QDs (2a) in the embodiment of the PENP (4).

[0094] Typically, the capped SPION (2b) according to the present invention achieves the desired level of water solubility and biocompatibility by surrounding the SPION (2b) or SPION / QD (2a, 2b) combination with a polymer (3). The polymer (3) encapsulation layer suitable for use in embodiments of the present invention includes the PS-b-PEG-NH2 obtained in block 2I of the synthetic procedure described above. Embodiments of the present invention can be produced by using various combinations of one or more of the following: SPION (2b) or SPION / QD (2a, 2b), a capping ligand (16), and PS-b-PEG-NH2 having a PS molecular weight ranging from about 1.5 kDa to about 40 kDa and a PEG having a molecular weight ranging from about 10 kDa to about 40 kDa.

[0095] An illustrative example of a scalable method for producing PENP-MagDot (4b) by EHD may include one or more of the following: Figure 2 The PS-b-PEG-NH2 obtained in Block 2I is placed in a first 1.5 mL tube. 13 μl of the desired SPION (2b) particles from 25 mg / mL (e.g., 20 nm) in toluene is introduced into a second 1.5 mL tube. 52 μl of acetone / methanol (60 / 40) is transferred to the SPION in the second 1.5 mL tube. Mix by shaking. Centrifuge the second 1.5 mL tube containing the mixed SPION (2b) at 7000 RPM for 1 min. at about 25° C. (about 77° F.). Immediately remove the supernatant with a 200 μl pipette. Immediately transfer 13 μl of anhydrous tetrahydrofuran ("THF") to the second 1.5 mL tube containing SPION (2b) and mix thoroughly with a pipette. 65 μl of QD (2a) (from 5 mg / mL in toluene) is transferred to a third 1.5 mL microcentrifuge tube. Transfer 130 μl of acetone / methanol (60 / 40 v / v) to the third 1.5 mL microcentrifuge tube containing QDs. Tilt to gently mix QDs (2a). Centrifuge the third 1.5 mL microcentrifuge tube containing QDs at 7000 R.PM for 1 minute at 25°C (about 77°F). Remove the supernatant with a 200 μl pipette. Transfer 65 μl of THF to the third 1.5 mL microcentrifuge tube. Mix thoroughly with a gas piston pipette. Add 322 μl of THF to a 4 mL vial. Transfer 13 μl of SPION (2b) in THF contained in the second 1.5 uL microcentrifuge tube to a 4 mL vial. If necessary, transfer 65 μl of QD (2a) in THF contained in the third 1.5 mL microcentrifuge tube to a 4 mL vial. Transfer 100 μl of PS-b-PEG-NH2 to a 4 mL vial to generate OP for EHD.

[0096] In a particular embodiment, EHD can be performed by: Cleaning the EHD mixing system syringe (11) three times with THF. Place about 0.5 mL of OP into the syringe barrel (12). Thoroughly mix the inorganic components in the OP while placing the syringe (11). Connect the syringe (11) to a syringe pump (15) and set the syringe pump (15) to produce a flow rate of OP containing inorganic components from the syringe needle (14) in the range of about 11.00 mL / hr. to about 14.00 mL / hr. In a particular embodiment, the flow rate can be about 12.5 mL / hr.. Prepare the syringe needle (14) until a drop of OP is formed at the end of the syringe needle (14). Rinse the 20 mL glass vial (17) twice with distilled or deionized water (individually or collectively, "DI water"). Introduce about 10 mL of DI water into the 20 mL glass vial (17). Submerge the syringe needle (14) into a 20 mL glass vial (17). Clean the negative electrode (19) by immersing in THF, wiping, and rinsing with DI water. Place the negative electrode (19) into the AP contained in the 20 mL glass vial (17). Place the positive electrode (18) into the AP contained in the 20 mL glass vial (17). In a particular embodiment, if conductive, the syringe needle (14) can act as the positive electrode (18). Ensure that the positive electrode (18), negative electrode (19), and syringe needle (14) are not touching. Connect the positive lead (21) from the voltage source (20) to the positive electrode (18) or syringe needle (14) and connect the negative lead (22) from the voltage source (20) to the negative electrode (19). Confirm that the power source delivers approximately -1000V.

[0097] The EHD-PENPs (4) produced by mixing the organic phase mixed with DI water under the influence of an electric field can be concentrated using centrifugal filtration. The contents of a 20 mL glass vial (17) can be transferred to a 100 kDa cut-off centrifugal ultrafiltration column available from Sigma Aldrich, PN UFC9010D Ultra-15 centrifugal filter unit. Centrifuge at 2000 RCF for 30 min. at about 25°C (about 77°F). Transfer EHD-PENP (4) from the centrifugal ultrafiltration column to a 1.5 mL microcentrifuge tube. Measure and record the volume of EHD-NP. Transfer 5 μl of the EHD-PENP (4) filtrate from the 1.5 mL microcentrifuge tube to a 1.5 mL tube and add 295 μl of borate buffer. Transfer 290 μl of EHD-PENP (4) to the spectrophotometer cell. Measure and record the optical density (OD) at 450 nm using a spectrophotometer. 450 ).

[0098] The EHD-PENP filtrate may contain aggregates of PENP (4) or aggregates of PENP lacking SPION (Sb) or QD (2a) (collectively, "aggregates"). Aggregates may be substantially removed from the EHD-NP filtrate to produce substantially pure PENP-MagDot (4b). As an example, a scalable size exclusion procedure for purifying PENP-MagDot (4b) may include: placing a magnetic particle separation column ("MPSC") in a magnetic field gradient (e.g., up to about 100-200 T / m). Transfer 300 μl of borate buffer to the MPSC. Allow the borate buffer to pass through the MPSC. Introduce the EHD-NP filtrate into the MPSC in a magnetic field (23). Allow the filtrate to pass through the MPSC. Transfer 300 μl of borate buffer to the MPSC. Mix the EHD-NPs in borate buffer in the MPSC. Allow all of the borate buffer to pass through the MPSC. Remove the MPSC from the magnetic field gradient. Once the MPSC is removed from the magnetic field gradient (23), the MPSC can no longer retain PENP-MagDot (4b), which can then be eluted. Remove all liquid from the tip of the MPSC. Place the MPSC in a PENP-MagDot (4b) collection tube (labeled PENP-MagDot-wavelength). Transfer 200μl of borate buffer to the MPSC. Mix the PENP-MagDot (4b) in borate buffer in the MPSC by pipetting. Collect the PENP-MagDot (4b) and place it in the PENP-MagDot-wavelength collection tube. Mix the collected PENP-MagDot (4b) thoroughly with a gas piston pipette. Measure and record the volume of the collected PENP-MagDot (4b). Combine 5μl of PENP-MagDot (4b) with 295ul of borate buffer. Transfer 290μl of sample to the spectrophotometer trough. The optical density (OD) at 450 nm was measured and recorded using a spectrophotometer. 450 ).

[0099] Currently, the main reference includes TEM micrographs. Figures 8A to 8C , which illustrate that the PENP-MagDot (4b) produced by the above method is equipped with SPION (2b), and optionally a combination of SPION (2b) and QD (2a) (having an emission wavelength in the visible light and near infrared light spectrum). In this illustrative example, the PENP-MagDot (4b) includes one or more QDs (2a) with an emission wavelength of 610nm, each combined with 20nm (such as Fig. 8A ), 15nm (as shown in the example of Figure 8B ), and 5nm (as shown in the example Figure 8CThe morphology of PENP-MagDot (4b) produced by the above EHD-EM-NP method can be characterized by using TEM imaging and dynamic light scattering of PENP-MagDot (4b) to determine HD and PDI, as described above.

[0100] Currently, the main reference includes TEM micrographs. Fig.9A and 9B , which illustrate PENP-MagDot (4b) produced according to the above method. DLS can also be used to determine the particle size of PENP-MagDot (4b). During DLS measurement, a suspension of PENP-MagDot (4b) can be exposed to a light beam, and when the incident light hits the PENP-MagDot (4b), the direction and intensity of the light beam can change due to scattering. The PENP-MagDot (4b) in the suspension continues to move randomly due to its kinetic energy, and the change in its intensity over time therefore contains information about the random motion and can be used to measure the diffusion coefficient of the particles. HD R of PENP-MagDot H The diffusion coefficient can be obtained from the Stokes-Einstein equation D f =k B T / 6πηR H To calculate, where k B is the Boltzmann constant, T is the temperature of the suspension, and η is the viscosity of the surrounding medium.

[0101] Now, the main reference Fig.9A and Fig. 9B ( Fig.9A TEM micrographs of PENP-MagDot (4b) ("PENP-MagDot15") prepared using a ratio of QD (2a): 15 nm SPION (2b): polymer (P) (3) (5:5:20), Fig. 9B Including TEM micrographs of PENP-MagDot (4b) ("PENP-MagDot 5") prepared using a ratio of QD (2a): 5nm SPION (2b): polymer (3:5:20), they demonstrated that the brightness and magnetic susceptibility of MagDot can be adjusted by loading SPIONs of different particle sizes. PENP-MagDot 15 produced using a ratio of 5:5:20 is more magnetic than PENP-MagDot 5 using a ratio of 3:5:20. PENP-MagDot 5 produced using a ratio of 3:5:20 is brighter than PENP-MagDot 15 using a ratio of 5:5:20.

[0102] Now, the main reference Fig. 9C , you can Fig.9A Nanoparticle tracking analysis (NTA) of each of the PENP-MagDot15 shown in the examples of is illustrated with a graph of particle size versus particle concentration to demonstrate that the HD of each population can have a consistent particle size and have a PDI of about 0.1 to about 0.2. The HD of PENP-MagDot15 measured can be about 120nm to about 140nm and the HD of PENP-MagDot5 measured can be about 180nm to about 220nm.

[0103] Based on TEM analysis or DLS or NTA analysis, the HD of PENP-MagDot (4b) produced by the above EHD method can vary based on the parameters used during EHD and exist in the range of about 40 nm to about 500 nm, and it is demonstrated that the PENP-MagDot (4b) group demonstrates substantially the same HD and a PDI of about 0.1 to about 0.2.

[0104] Now, the main reference Fig.9D , which contains a bar graph comparing the iron concentrations of PENP-MagDot 5 and PENP-MagDot 15, demonstrating that PENP-MagDot 15 can be produced in a manner that has a greater iron concentration than PENP-MagDot 5.

[0105] Now, the main reference Fig.9E , which contains a bar graph comparing the fluorescence of PENP-MagDot 5 and PENP-MagDot 15, which demonstrates that PENP-MagDot 5 can be produced with greater fluorescence than PENP-MagDot15.

[0106] Thus, by preselecting one or more of the following, a large number and variety of embodiments of the PENP-MagDot (4b) can be produced in a manner useful for a large number of clinical and non-clinical applications: SPION (2b) particle size, QD (2a), PS-b-PEG-NH2 (3) which may include PS molecular weight ranging from about 1.5 kDa to about 40 kDa and PEG having a molecular weight ranging from about 10 kDa to about 40 kDa, and parameters of EHD, and ratio of QD:SPION:polymer.

[0107] Preparation of antibodies.

[0108] Again, the main reference Figure 1, the polymer nanomaterial encapsulation system (1) may further include the preparation of an antibody (Ab) (5). In a particular embodiment, PENP (4) produced by FNP, EHD, EM-NPa, self-assembly, or by other means may be a target for site-specific binding to an antibody (Ab) (5'). As an illustrative example, a half antibody may be produced by selective reduction of disulfide bonds in the hinge region of an antibody, the selective reduction producing a monovalent component having a free thiol group ("SH") that can be used for site-specific binding to PENP, PENP-MultiDot, PENP-MagDot, or a combination thereof. The reduced antibody fragment may be prepared by reacting 2-mercaptoethylamine hydrochloride (2-MEA), dithiothreitol, mercaptoethanol, or tris(2-carboxyethyl)phosphine (TCEP) to produce a reduced half antibody as shown in Formula IX.

[0109]

[0110] However, this example is not intended to exclude embodiments that may include one or more of the following: intact antibody molecules or F(ab')2, Fab', Fv, rIgF, Fc fragments thereof obtained after pepsin or other enzyme digestion, or combinations thereof.

[0111] A particularly illustrative example of a scalable procedure for generating half antibodies useful for binding to PENP (4), PENP-MultiDot (4a), and / or PENP-MagDot (4b) may include: preparing an antibody stock solution comprising immunoglobulin G ("IgG") (0.5 mg / mL) in a buffer solution of 100 mM phosphate buffered saline ("PBS") with 10 mM ethylenediaminetetraacetic acid disodium salt dihydrate ("EDTA") at pH 7.4 ("PBS buffer"). A 160 μl aliquot of the IgG stock solution may be transferred to a first 1.5 mL tube. 2-MEA.HCl (CAS NO: 156-57-0) having a linear formula of HSCH2CH2NH2·HCl and a molecular weight of 113.60 may be obtained from Fisher Scientific, PN AAA1437714. In a second 1.5 mL tube, 15 mg of 2-MEA.HCl was dissolved in 264 μl of PBS buffer solution by pipetting. 5.0 μl of 2-MEA.HCl solution was transferred to 160 μl of IgG stock solution in the first 1.5 mL tube, vortexed, and the 1.5 mL tube containing the IgG solution was placed in a 37°C (about 98.6°F) water bath for 90 min.

[0112] The resulting reduced antibody product (also referred to as "half-antibody product") (5) may require desalting and buffer exchange. Desalting removes unreacted MEA contaminants from the half-antibody product and exchanges the half-antibody into a reaction buffer for binding to the PENP (4), PENP-MultiDot (4a), and / or PENP-MagDot (4b). An illustrative example of a scalable procedure for desalting and buffer exchange may include utilizing ZEBA TM Centrifuge desalting columns, PN89883, or equivalent. A pair of desalting columns can be prepared by removing the bottom cap and placing the columns in 1.5 mL collection tubes. Centrifuge at 1500 x g for 1 min. to remove storage solution from the desalting column resin. Add 300 μl of 1x 100 mM sodium phosphate, 1 mM EDTA ("rinse buffer"), pH: 6.95-7 in water, or an appropriate buffer to the top of the resin bed and centrifuge at 1500 RFC for 1 min. Discard the flow-through from the collection tube. Repeat the addition of 300 μl of 1x rinse buffer two more times, discarding the buffer from the collection tube each time.

[0113] The reduced antibody (5) product can now be loaded onto the desalting column by placing the first equilibrated desalting column into the first 1.5 mL collection tube (labeled Reduced Antibody 1 ("R-Ab-1"), removing the cap from the top of the first desalting column and slowly applying up to 160 μl of the reduced antibody product to the center of the dense resin bed. For sample volumes less than 70 μl, 15 μl of buffer (stacker) is applied to the top of the resin bed after the sample has been completely absorbed to ensure maximum protein recovery. Incubate at 1500F at 15°C to 20°C (59°F to 68°F). Centrifuge at CF for 2 min. to collect the desalted reduced antibody product. Place the second equilibrated desalting column into a second 1.5 mL collection tube (labeled Reduced Antibody 1 ("R-Ab-2")) and remove the cap from the top of the second equilibrated desalting column. Transfer the collected reduced antibody (5) product from collection tube R-Ab-1 to the top of the second desalting column. Centrifuge at 1500 RCF for 2 min. at 15°C to 20°C (59°F to 68°F) to collect the desalted reduced antibody product in collection tube R-Ab-2. Cap collection tube R-Ab-2 and place at RT.

[0114] Preparation of half-antibody-polymer nanocomplex conjugates.

[0115] Now, the main reference Figure 1 The polymer nanomaterial encapsulation system (1) may further include adding a reduced antibody product Figure 1Surface functionalization of PENP (4), PENP-MultiDot (4a), and / or PENP-MagDot (4b) of Block 1D. As a basis for modification, chemical groups such as amines, carboxylates, thiols, or other reactive groups can be introduced into the PENP (4), PENP-MultiDot (4a), and / or PENP-MagDot (4b) during the synthesis as described above. These groups can be targeted by heterobifunctional chemical crosslinkers (10) comprising reactive chemical groups connected by spacer arms (as examples: alkanes, polyethylene glycols, cleavable disulfide spacers) having various lengths and functions to impart flexibility, solubility, or other desired properties to the functionalized PENP (4), PENP-MultiDot (4a), and / or PENP-MagDot (4b). As an example, the PENP functional group (9) can react with the first reactive group (10') of the heterobifunctional chemical crosslinker (10). As an illustrative example, when the functional group (9) of the PENP (4), PENP-MultiDot (4a), and / or PENP-MagDot (4b) comprises an amine, the amine can react with a succinimidyl ester to form an amide bond, or comprises a carboxylate, the carboxylate can react with a carbodiimide to produce an O-acylisourea intermediate, which can react with an amine to form an amide bond, or comprises a thiol, the thiol can react with a maleimide to form a thioether bond. The reduced antibody (5) product can react with the second reactive group (10") of the heterobifunctional chemical crosslinker (e.g. Figure 4C For example, the second reactive group (10") of the heterobifunctional chemical crosslinker (10) may comprise a maleimide, which may react with the sulfhydryl group of the half antibody (5) to form a thioether bond.

[0116] In a particular embodiment, both reactive groups attached to the PENP (4) can be modified by reacting with a first reactive group (10a') of a first heterobifunctional crosslinker (10a) and the reduced antibody (5) product can be modified by reacting with a first reactive group (10b') of a second heterobifunctional chemical crosslinker (10b). The first and second chemical crosslinkers (10a, 10b) can be selected to include a second reactive group (10a", 10b") that remains stable in physiological aqueous buffer and after mixing with rapid kinetics and high selectivity to produce a covalent bond.

[0117] In a particular embodiment, the reduced antibody (5) product in collection tube R-Ab-2 can be modified for subsequent conjugation with PENP (4) by reacting the free sulfhydryl group of the half antibody (5) with a first reactive group (10a') of a maleimide comprising a first heterobifunctional crosslinker (10a) to form a thioether bond. In an illustrative example, the first heterobifunctional crosslinker (10a) can comprise sulfonic acid trans-cyclooctene maleimide ("sulfonic acid-TCO maleimide") comprising Formula X, which has a linear formula C 17 H 27 N3O8S (CAS No. n / a) and a molecular weight of 457.50 g / mol, available from Click Chemistry tools, PN 1355.

[0118]

[0119] Sulfonic acid TCO-maleimide comprises a water-soluble reagent that enables the incorporation of a TCO moiety onto a thiol-containing half-antibody. Its maleimide group reacts specifically with sulfhydryl groups ("SH") at pH 6.5 to 7.5 to form a stable thioether bond. The hydrophilic sulfonated spacer arm greatly improves the water solubility of the sulfonic acid TCO antibody ("sulfonic acid-TCO-Ab") comprising Formula XI.

[0120]

[0121]

[0122] An illustrative example of a scalable procedure for generating a sulfonic acid-TCO-Ab useful for conjugation to PENPs may include one or more of the following: Remove a tube containing 0.1 mg of sulfonic acid-TCO-maleimide from -20°C (-4°F) and place at RT for 15 min. Add 46.4 μl of anhydrous dimethyl sulfoxide ("DMSO") (CAS No. 67-68-5) having a linear formula of C2H6OS and a molecular weight of 78.13 g / mol. to 0.1 mg of sulfonic acid-TCO-maleimide. Vortex the sulfonic acid-TCO-maleimide in the tube for 10 min. to ensure dissolution in DMSO. Repeatedly mix the sulfonic acid-TCO-maleimide in DMSO by pipetting set to 60 μl. Add 5 μl or 10 μl of Sulfo-TCO-Maleimide to the R-Ab-2 tube containing the reduced antibody (5). Discard the remaining Sulfo-TCO-Maleimide. Re-label the tube containing the Sulfo-TCO-Ab. Cap the tube containing the Sulfo-TCO-Ab and vortex. Place the Sulfo-TCO-Ab tube at 4°C (39.2°F) for 20-24 hr.

[0123] The PENP (4) product described above can be reacted with the first reactive group of the second heterobifunctional cross-linker. The modified PENP (4) can then be linked to one or more target moieties (24) including one or more of the following: cells, cell organelles, proteins, peptides, amino acids, oligonucleotides, ligands, or linkers by selectively reacting the second reactive group with a corresponding functional group within the target moiety (24).

[0124] In a particular embodiment, the second heterobifunctional cross-linker (10b) may comprise a methyl tetrazine polyethylene glycol-4-N-hydroxysuccinimide ester ("TZ-PEG4-NHS ester") of Formula XII having a linear formula C 24 H 31 N5O9 and has a molecular weight of 533.53 g / mol (CAS No. 182907-92-1).

[0125]

[0126] As an illustrative example, TZ-PEG4-NHS ester comprising Formula XIII can be reacted with the functional group (9) of a multi-arm PEG-NH2 (3) comprising multiple amines as shown in Formula IV or Formula V of the PENP (4) to produce PENP-PEG4-TZ comprising Formula XIII.

[0127]

[0128] As an example, a scalable procedure using a PENP (4) functional group (9) comprising an amine of a multi-arm PEG-NH2 (3) shown in Formula IV or Formula V to produce a PENP-PEG4-TZ of Formula XIII may include one or more of the following: 1 mg of TZ-PEG-NHS ester is placed at RT for approximately 15 min. 93.7 μl or 375 μl of anhydrous DMSO is transferred to the TZ-PEG4-NHS ester to produce 20 mM or 5 mM TZ-PEG4-NHS in DMSO. The TZ-PEG4-NHS ester in DMSO is mixed by pipette to completely dissolve the TZ-PEG4-NHS ester in DMSO. The 1.5 mL tube is labeled PENP-Wavelength-TZ. The PENP is transferred to a PENP-Wavelength-TZ tube (120 μl of PENP-MagDot or 80 ul of PENP-MultiDot). In the PENP-MultiDot (4a) embodiment, about 30 nmol of amine can be activated by reacting with about 70 nmol of TZ-PEG4-NHS ester, and in the PENP-MagDot (4b) embodiment, about 120 nmol of amine can be activated by reacting with 70 nmol of TZ-PEG4-NHS ester. Pipette 3.5 ul of 20 mM TZ-PEG4-NHS ester to the PENP-MultiDot-Wavelength-TZ tube or 15 μl of 5 mM TZ-PEG4-NHS ester to the PENP-MagDot-Wavelength-TZ tube. Mix thoroughly with a gas piston pipette. React at RT for 2 hr.

[0129] In a particular embodiment, PENP-PEG4-TZ comprising Formula XIII can be reacted with sulfonic acid-TCO-Ab comprising Formula XI in a reverse electron demand Diels-Alder [4+2] cycloaddition reaction of TCO with TZ to form a dihydropyridazine bond to produce a conjugate PENP-antibody (6) ("PENP-Ab") comprising Formula XIV, which includes but is not limited to Figure 4A PENP-MultiDot (4a) or as shown in the example Figure 4B PENP-MagDot (4b) is shown in the example of , wherein R1 comprises PENP and wherein R2 comprises Ab.

[0130]

[0131] As an example, a scalable procedure using PENP-PEG4-TZ (PENP-MagDot-PEG4-TZ or PENP-MultiDot-PEG4-TZ) and sulfonic acid-TCO-Ab to produce PENP-Ab (6) of Formula XIV may include a pair of desalting columns (as a non-limiting example, ZebaTM Desalting columns) are prepared by removing the bottom cap and placing each of the pair of desalting columns in a 1.5 mL collection tube. Centrifuge at 1500 x g for 1 min. to remove the storage solution from the desalting column resin. Add 300 μl of 1x rinse buffer or appropriate buffer to the top of the resin bed and centrifuge at 1500 RFC for 1 minute. Discard the flow-through from the individual collection tubes. Repeat adding 300 μl of 1x rinse buffer (pH 6.0) two more times, discarding the buffer from the individual collection tubes each time. Transfer PENP-PEG4-TZ to the first of the pair of desalting columns. Transfer sulfonic acid-TCO-Ab to the second of the pair of desalting columns. The first and second desalting columns were centrifuged at 1500 RCF for 2 min at 15°C to 20°C (59°F to 68°F) to collect PENP-PEG4-TZ and sulfonic acid-TCO-Ab products after removing excess unbound linkers. The desalted PENP-PEG4-TZ (120uL PENP-MagDot or 80uL PENP-MultiDot) in the first collection tube was mixed by pipette. The desalted 160uL (80ug) sulfonic acid-TCO-Ab was transferred from the second collection tube to the PENP-PEG4-TZ in the first collection tube. The reaction was carried out at 4°C (39.2°F) for 24 hrs to produce PENP-Ab (PENP-MagDot-Ab or PENP-MultiDot-Ab).

[0132] After binding, the resulting PENP-Ab (6) product can be purified. As an example, a scalable size exclusion procedure for purifying PENP-MultiDot-Ab may include: labeling a 1.5 mL microcentrifuge tube "PENP-MultiDot-Ab-wavelength". Place 3 mL of a gel filtration resin (e.g. 3 mL of borate buffered saline (50 mM sodium borate, 100 mM sodium phosphate, 7.3-7.5 pH ("borate buffer")) ("BBS") is introduced into the gel filtration resin. The suspension of the gel filtration resin is transferred to a size exclusion column ("SEC"). The PENP-MultiDot-Ab (6a) solution is slowly pipetted to the side of the SEC. An air syringe is connected to the SEC. 1 mL of air is pushed into the SEC using the air syringe. The PENP-MultiDot-Ab (6a) solution is allowed to move into the gel filtration resin. The air syringe is removed from the SEC. 200 μl of BBS buffer is pipetted to the side of the SEC. The air syringe is connected to the SEC again. 1 mL of air is pushed into the SEC using the air syringe. The PENP-MultiDot-Ab (6a) solution is allowed to move into the SEC packing. The air syringe is removed from the SEC. Pipette 1000 μl BBS to the side of the SEC. Connect the air syringe to the SEC again. Reduce ambient light incident on the SEC. Direct ultraviolet light ("UV light") incident on the SEC. Push 1 mL of air into the SEC via the air syringe. Start collecting BBS eluent from the SEC. After detecting the first fluorescent droplet from the SEC, collect fluorescent drops 2 to 5 in the PENP-MultiDot-Ab-wavelength collection tube. Mix the collected fluorescent droplet samples with a pipette. Remove the air syringe, discard the gel filtration resin, and clean the SEC.

[0133] As an example, a scalable size exclusion procedure for purifying PENP-MagDot-Ab (6b) may include: thoroughly mixing the PENP-MagDot-Ab (6b) in binding buffer with a gas piston pipette. Place the MPSC in a magnetic field gradient (e.g., up to about 100-200 T / m). Introduce the PENP-MagDot-Ab (6b) in binding buffer into the MPSC. Allow the binding buffer to pass through the MPSC. Transfer 300 μl of BBS to the MPSC. Pipette to mix the PENP-MagDot-Ab in reaction buffer in the MPSC. Allow the reaction buffer to pass through the MPSC. Transfer 300 μl of reaction buffer to the MPSC. Pipette to mix the PENP-MagDot-Ab (6b) in reaction buffer in the MPSC. Allow the reaction buffer to pass through the MPSC. Remove any reaction buffer from the tip of the MPSC. Remove the MPSC from the magnetic field gradient. Once the MPSCs are removed from the magnetic field gradient, the MPSCs can no longer retain PENP-MagDot-Ab(6b) and can elute PENP-MagDot-Ab(6b). Place the MPSCs in a PENP-MagDot-Ab(6b) collection tube (labeled PENP-MagDot-Ab-). Transfer 200 μl of reaction buffer to the MPSCs. Pipette to mix the PENP-MagDot-Ab(6b) in the reaction buffer in the MPSCs. Collect the PENP-MagDot-Ab(6b) into a MagD-antibody-date collection tube. Thoroughly mix the collected PENP-MagDot-Ab(6b) with a gas piston pipette.

[0134] Cell labeling and flow cytometry analysis using PENP-MultiDot-Ab.

[0135] PENP-MultiDot-Ab (6a) can effectively and specifically label the target portion (24), including the cell target (24'). Compared with the cell target (24') labeled with conventional dyes, single particle imaging, cell imaging, or flow cytometry analysis of the cell target (24') labeled with PENP-MultiDot-Ab (6a) demonstrates very high fluorescence intensity. The PENP-MultiDot-Ab (6a) can effectively and specifically label cell surface receptors and subcellular structures in both living or fixed cells without any detectable non-specific binding. Flow cytometry analysis can be performed to evaluate the performance of PENP-MultiDot-Ab (6a) and to show high cell labeling brightness compared to conventional dyes and quantum dot probes.

[0136] As an illustrative example of PENP-MultiDot-Ab (6a) labeling of cell markers (24'), peripheral blood mononuclear cells ("PBMCs") (24') can be labeled using PENP-MultiDot-Ab (4a). A scalable method for labeling PBMCs with PENP-MultiDot-Ab (6a) can include one or more of the following: Peripheral blood is diluted 50:50 with Hank's balanced salt solution ("HBSS") (Sigma Aldrich, PN H6648) to an isotonic solution. The diluted peripheral blood can be washed with a Ficoll-Hypaque gradient (density = 1.077 g / cm 3 ) and centrifuged at 1350 RPM for 30 min. without interruption to separate the layers. The serum can be aspirated and discarded. The PBMCs can be removed and transferred to another collection tube and washed with PBS. The PBMC pellet can be resuspended in 10 mL PBS and the cells can be counted on a hemacytometer. One million PBMCs (24') in 200 μl of PBS can be placed in a 12x75 mm flow cytometer tube, to which 10 μl of PENP-MultiDot-mouse anti-human CD3 (6a) with an emission wavelength of 610 nm can be added. The PBMCs (24') and PENP-MultiDot-mouse anti-human CD3 (6a) are incubated at RT for 25 min. At the end of the incubation period, 500 μl of additional PBS can be added to the PBMCs (7) bound to PENP-MultiDot-mouse anti-human CD3 ("PENP-MultiDot-mouse anti-human CD3-PBMC"). PENP-MultiDot-mouse anti-human CD3-PBMC(7) were centrifuged at 1800 RPM for 7 min. at RT. The supernatant can be discarded and the PBMC / PENP-MultiDot-mouse anti-human CD3-PBMC(7) can be resuspended in 400 μl of PBS for flow cytometric analysis(8) for detection of PENP-MultiDot-mouse anti-human CD3-PBMC(7) populations using a Cytek Northern Lights spectroscopic flow cytometer. The CD3 protein complex is an important T cell marker for classification of malignant lymphomas and leukemias (T cell neoplasms). CD3 can also be used to identify T cells in celiac disease, lymphocytic colitis, and collagenous colitis.

[0137] Now, the main reference Fig. 10A, the univariate histogram (fluorescence intensity vs. number of particles) depicts the detection of the PENP-MultiDot-mouse anti-human CD4-PBMC(7) population ("peak M1") in a sample of PBMC / PENP-MultiDot-mouse anti-human CD4-PBMC(7) obtained by the method described above. The histogram demonstrates that 38.41% of the cells in the sample can be identified as the PENP-MultiDot-mouse anti-human CD4-PBMC(7) population in the sample. In a particular embodiment, the PENP-MultiDot-mouse anti-human CD4-PBMC(7) population can be sorted and isolated for further analysis.

[0138] Now, the main reference Fig. 10B , the univariate histogram (fluorescence intensity vs. number of particles) depicts the detection of the PENP-MultiDot-mouse anti-human CD3-PBMC(7) population ("peak M1") prepared according to the method described above. The histogram demonstrates that 50.09% of the cells in the sample can be identified as the PENP-MultiDot-mouse anti-human CD3-PBMC(7) population in the sample.

[0139] PENP-MagDot-Ab (6b) can also effectively and specifically label cell targets (24'). Single particle imaging, cell imaging, or flow cytometric analysis (8) of cell targets (24') labeled with PENP-MagDot-Ab (6b) demonstrated very high fluorescence intensity compared to cell targets labeled with conventional dyes or quantum dots (24'). The PENP-MagDot-Ab (6b) can effectively and specifically label cell surface receptors and subcellular structures in both living and fixed cells without any detectable non-specific binding. Flow cytometric analysis (8) can be performed to evaluate the performance of PENP-MagDot-Ab (6b) and to show high cell labeling brightness compared to conventional dyes and quantum dot probes.

[0140] As an illustrative example of PENP-MagDot-Ab (6b) labeling of cell markers (24'), peripheral blood mononuclear cells ("PBMC") (24') can be labeled with PENP-MagDot-Ab (6b). As an example, a scalable method may include one or more of the following: Peripheral blood is diluted 50:50 to an isotonic solution with Hank's balanced salt solution ("HBSS") (SigmaAldrich, PN H6648). The diluted peripheral blood can be layered on a Ficoll-Hypaque gradient (density = 1.077 g / cm3) and centrifuged at 1350 RPM for 30 min. without interruption. The serum can be aspirated and discarded. The PBMC can be removed and transferred to a PBMC collection tube and washed with PBS. The PBMC pellet can be resuspended in 10 mL PBS and the cells can be counted on a hemacytometer. One million PBMCs (7) in 200 ul of PBS can be introduced into a 12x75 mm flow cytometer tube, to which 20 μL of PENP-MagDot-mouse anti-human CD3 (6b) with a 610 emission spectrum is added. The PBMCs and PENP-MagDot-mouse anti-human CD3 (6b) are incubated at RT for 25 min. After the incubation period, 200 μl of additional PBS can be added to the PBMCs (7) bound to PENP-MagDot-mouse anti-human CD3 in the flow cytometer tube ("PENP-MagDot-mouse anti-human CD3-PBMC"). The flow cytometer tube containing PENP-MagDot-mouse anti-human CD3-PBMCs (7) in PBS is placed in a magnetic field gradient (23) (e.g., up to about 100-200 T / m). After 15 min., the non-magnetic fraction can be aspirated from the flow cytometer tube and placed in a non-magnetic fraction collection tube. The flow cytometer tube containing the magnetically retained PENP-MagDot-mouse anti-human CD3-PBMC (7) can be removed from the magnetic field gradient and the PENP-MagDot-mouse anti-human CD3-PBMC (7) can be resuspended in 400 μl PBS. The PBMC / PENP-MagDot-mouse anti-human CD3 reaction solution before magnetic separation, the non-magnetic fraction after magnetic separation, and the PENP-MagDot-mouse anti-human CD3-PBMC fraction after magnetic separation are each analyzed by flow cytometry (8).

[0141] Now, the main reference Figures 11A to 11C , etc. describe the PBMC / PENP-MagDot-mouse anti-human CD3 reaction solution before magnetic separation (such as by Fig.11A The non-magnetic part after magnetic separation (such as Fig. 11B ), and the PENP-MagDot-mouse anti-human CD3-PBMC fraction after magnetic separation (as shown by Fig. 11C The flow cytometry analysis of each of the samples (shown in the example of FIG. 8 ) obtained a two-point plot (forward scatter area versus forward scatter height). The flow cytometry dot plot demonstrates the ratio of CD3 negative cells to CD3 positive cells. Fig.11A It was demonstrated that the PBMC / PENP-MagDot-mouse anti-human CD3 reaction solution before magnetic separation contained 51.17% CD3-negative cells and 48.76% CD3-positive cells. Fig. 11B and 11C It was proved that the non-magnetic part after magnetic separation contained a large number of CD3 negative cells (97.29%) and very few CD3 positive cells (2.71%), while on the contrary, Fig. 11C The results showed that the PENP-MagDot-mouse anti-human CD3-PBMC fraction after magnetic separation contained very few CD3-negative cells (2.60%) and a large number of CD3-positive cells (97.37%). This demonstrated the substantial advantage of using PENP-MagDot-Ab (6b) to capture and purify cell targets (24').

[0142] Now, the main reference Figures 12A to 12C , etc., described the labeling of blood cells of Hawaiian bobtail squid (Euprymna scolopes) with PENP-MagDot. Adult bobtail squid can be cultured with 100ul of PENP-MagDot containing QDs with an emission wavelength of 610nm for 3hr. Images prove that blood cells of Hawaiian bobtail squid (Euprymna scolopes) passively absorb PENP-MagDot 610nm.

[0143] As can be readily appreciated based on the above, the basic concepts of the invention can be implemented in a variety of ways. The present invention includes numerous and varied embodiments of polymer-encapsulated nanoparticles and methods for making and using such polymer-encapsulated nanoparticles.

[0144] Therefore, the specific embodiments or elements of the present invention disclosed by the present description or shown in the drawings or tables of the present application are not intended to be limiting, but are illustrative of the numerous and varied embodiments generally encompassed by the present invention or the encompassed equivalents of any specific elements thereof. In addition, the specific description of a single embodiment or element of the present invention may not explicitly describe all possible embodiments or elements; many alternatives are implicitly disclosed by the present description and drawings.

[0145] It should be understood that each element of a device or each step of a method can be described by a device term or method term. When desired, such terms can be replaced to make the implicit broad coverage enjoyed by the present invention clear. As just one example, it should be understood that all steps of a method can be disclosed in the form of an action, a means for doing the action, or an element causing the action. Similarly, each element of a device can be disclosed in the form of a physical element or an action promoted by the physical element. As just one example, the disclosure of "encapsulated nanoparticles" should be understood to cover the disclosure of the action of "encapsulated nanoparticles" (whether or not explicitly discussed), and conversely, if there is actually a disclosure of the action of "encapsulated nanoparticles", such disclosure should be understood to cover the disclosure of "encapsulated nanoparticles" and even "means for encapsulating nanoparticles". Such optional terms for each element or step should be understood to be explicitly included in this narration.

[0146] In addition, with respect to each term used, it should be understood that unless its utilization in this application is inconsistent with such interpretation, the general dictionary definition for each term should be understood to be included in this description, as is contained in RandomHouse Webster's Unabridged Dictionary, Second Edition (each definition is hereby incorporated by reference).

[0147] Therefore, the applicant of the present application should be understood to claim at least: i) each of the polymer-encapsulated nanoparticles disclosed and described herein, ii) the related methods disclosed and described, iii) similar, equivalent, and even implicit variations of each of such devices and methods, iv) alternative specific examples of each of the functions shown, disclosed, or described, v) alternative designs and methods of each of the functions shown to implicitly accomplish the functions disclosed and described, vi) each feature, component, and step shown in the form of separate and independent inventions, vii) applications enhanced by the various systems or components disclosed, viii) the resulting products produced by such systems or components, ix) methods and devices substantially as described above and with reference to any of the attached examples, x) various combinations and arrangements of each of the previous elements disclosed.

[0148] The background section of this patent application (if any) provides a description of the technical field to which the invention belongs. This section may also incorporate or contain certain U.S. patents, patent applications, publications, or explanations of the subject matter of the claimed invention that may be used for relevant information, problems, or concerns about the state of the art described in the present invention. The following is not intended: any U.S. patent, patent application, publication, description, or other information cited or incorporated herein is understood, construed, or deemed to be an admission that it is prior art with respect to the present invention.

[0149] The claims (if any) detailed in this specification are hereby incorporated by reference as part of this description of the invention, and the applicant expressly reserves the right to use the owner or part of the so-incorporated content of the claims as additional description to support any person or owner of any element or component of the claims or their equivalents, and the applicant further expressly reserves the right to move the owner or any part of the so-incorporated content of any element or component of the claims or their equivalents from this specification to the application as necessary. or vice versa to define subject matter claimed by the present application or by any subsequent application or continuation, division, or continuation-in-part thereof, or to obtain any benefit under, reduce costs under, or comply with the patent laws, regulations, or rules or treaties of any country, and such content incorporated by reference shall survive the entire pendency of the present application (including any subsequent continuation, division, or continuation-in-part thereof) or any reissue or extension based thereon. Elements following an open transition phrase (such as "comprising") may also be claimed alternatively by a closed transition phrase (such as "consisting essentially of" or "consisting of"), whether or not explicitly indicated in the recitation portion of the specification.

[0150] In addition, the claims (if any) detailed in this specification are further intended to describe the scope of a limited number of preferred embodiments of the invention and should not be interpreted as the broadest embodiment of the invention that can be requested or the complete list of embodiments of the invention. Applicants do not waive any right to develop further claims based on the above detailed description as part of any continuation, division, or part continuation-in-part application, or similar application.

Claims

1. A nanocomposite comprising: an amphiphile having a hydrophobic region and a hydrophilic region; and At least one quantum dot and at least one magnetic particle are encapsulated by being linked to the hydrophobic region of the amphiphile, the hydrophilic region of the amphiphile comprising functional groups.

2. The nanocomposite of claim 1, wherein the at least one quantum dot has a core comprising cadmium sulfide and a shell comprising zinc sulfide and has an emission wavelength selected from the group consisting of 420 nm, 440 nm, and 460 nm, wherein the at least one quantum has a diameter ranging from about 5 nm to about 8 nm.

3. The nanocomposite of claim 1, wherein the at least one quantum dot has a core comprising cadmium sulfide selenide and a shell of zinc sulfide and has an emission wavelength selected from the group consisting of 480 nm, 500 nm, and 520 nm, wherein the at least one quantum has a diameter ranging from about 4 nm to about 6 nm.

4. The nanocomposite of claim 1, wherein the at least one quantum dot has a core comprising cadmium selenide and a shell comprising zinc sulfide and has an emission wavelength selected from the group consisting of 540 nm, 560 nm, 580 nm, and 600 nm, 620 nm, 640 nm, and 660 nm, wherein the at least one quantum has a diameter ranging from about 4 nm to about 8 nm.

5. The nanocomposite of claim 1, wherein the at least one quantum dot has a core comprising cadmium telluride and a shell of zinc sulfide and has an emission wavelength selected from the group consisting of 680 nm and 700 nm, wherein the at least one quantum has a diameter ranging from about 6 nm to about 7 nm.

6. The nanocomposite of claim 1, wherein the at least one quantum dot has a core comprising cadmium selenide telluride and a shell of zinc sulfide and has an emission wavelength selected from the group consisting of 720 nm, 740 nm, 760 nm, 780 nm, 800 nm, 820 nm, 840 nm, 860 nm, and 880 nm, wherein the at least one quantum has a diameter ranging from about 4.5 nm to about 12 nm.

7. The nanocomposite of claim 1, wherein the at least one quantum dot has a core comprising copper indium zinc sulfide and a shell of zinc sulfide and has an emission wavelength selected from the group consisting of 540 nm, 560 nm, 580 nm, 600 nm, 620 nm, 640 nm, 840 nm, and 669 nm, wherein the at least one quantum has a diameter ranging from about 3.5 nm to about 4 nm.

8. The nanocomposite of claim 1, wherein the at least one quantum dot has a core comprising lead sulfide and a shell comprising cadmium sulfide and has an emission wavelength selected from the group consisting of 900 nm, 920 nm, 940 nm, 960 nm, 980 nm, and 1000 nm, wherein the at least one quantum dot has a diameter ranging from about 3.5 nm to about 5 nm.

9. The nanocomposite of claim 1, wherein the at least one magnetic particle has an average particle diameter ranging from about 5 nm to about 20 nm.

10. The nanocomposite of claim 9, wherein the at least one magnetic particle has an average particle diameter selected from the group consisting of: 5 nm, 10 nm, 15 nm, 20 nm, and combinations thereof. The nanocomposite of claim 10 , wherein the magnetic particles comprise one or more of magnetite and hematite.

12. The nanocomposite of claim 10, wherein the magnetic particles comprise superparamagnetic iron oxide nanoparticles.

13. The nanocomposite of claim 1, wherein the amphiphile comprises polystyrene-b-polyethylene glycol including the functional group.

14. The nanocomposite of claim 13, wherein the polystyrene-b-polyethylene glycol comprises polystyrene having a molecular weight ranging from about 1.5 kDa to about 40 kDa.

15. The nanocomposite of claim 13, wherein the polystyrene-b-polyethylene glycol comprises polyethylene glycol having a molecular weight ranging from about 10 kDa to about 40 kDa. The nanocomposite of claim 15 , wherein the polyethylene glycol comprises branched polyethylene glycol.

17. The nanocomplex of claim 13, wherein the amphiphile comprises polystyrene-b-polyethylene glycol amine comprising a combination of polystyrene having a molecular weight ranging from about 1.5 kDa to about 40 kDa and polyethylene glycol having a molecular weight ranging from about 10 kDa to about 40 kDa, and the like.

18. The nanocomposite of claim 13, wherein the nanocomposite of the polystyrene-b-polyethylene glycol, the at least one quantum dot, and the at least one magnetic particle is formed under electrohydrodynamic mixing mediated nanoprecipitation, wherein an organic phase of a water-miscible non-polar aprotic solvent, the polystyrene-b-polyethylene glycol, the at least one quantum dot, and the at least one magnetic particle flows into an aqueous phase at a predetermined volume flow rate under the influence of an electric field.

19. The nanocomposite of claim 18, wherein the mass concentration of the at least one quantum dot and the at least one magnetic particle in the organic phase to the mass concentration of the polystyrene-b-polyethylene glycol comprises a ratio existing in a range of about 1:1 to about 1:

4.

20. The nanocomposite of claim 19, wherein the mass concentrations of the at least one quantum dot and the at least one magnetic particle in the organic phase are adjusted within the ratio to change the brightness of the nanoparticles formed under electrohydrodynamic mixing-mediated nanoprecipitation when exposed to external stimulation.

21. The nanocomposite of claim 20, wherein the mass concentration of the polystyrene-b-polyethylene glycol is adjusted within the ratio to form the nanocomposite having a substantially uniform hydrodynamic diameter ranging from about 40 nm to about 1000 nm under electrohydrodynamic mixing mediated nanoprecipitation. 22 . The nanocomposite of claim 20 , wherein the mass concentration of the polystyrene-b-polyethylene glycol is adjusted by changing the mass concentration of the polystyrene. 23 . The nanocomposite of claim 22 , wherein the mass concentration of the polystyrene-b-polyethylene glycol is adjusted by changing the mass concentration of the polyethylene glycol. 24 . The nanocomposite of claim 22 , wherein the mass concentration of the polystyrene-b-polyethylene glycol is adjusted by changing the branches of the polyethylene glycol, wherein the branches of the polyethylene glycol are selected from the group consisting of 4-arm polyethylene glycol and 8-arm polyethylene glycol.

25. The nanocomposite of claim 18, wherein the polystyrene-b-polyethylene glycol, the at least one quantum dot, and the at least one magnetic particle have a concentration of about 0.1 mg / mL to about 5.0 mg / mL in the organic phase.

26. The nanocomposite of claim 25, wherein the organic phase has a concentration of about 0.2 v / v to about 1 v / v in the aqueous phase.

27. The nanocomposite of claim 1, further comprising a ligand bound to the at least one quantum dot and to the at least one magnetic particle, wherein the ligand comprises a mass concentration present in a range of about 10% to about 40%.

28. The nanocomposite of claim 27, wherein the ligand comprises oleic acid or oleylamine, or a combination thereof.

29. The nanocomposite of claim 28, wherein the mass concentration of the oleic acid combined with the at least one quantum dot comprises no less than 10% and no more than 40%.

30. The nanocomplex of claim 1, further comprising an agent bound to the functional group, wherein the agent is selected from the group consisting of polyethylene glycol, antibodies, half antibodies, antibody fragments, fluorescent probes, aptamers, vitamins, cell surface receptors, cell coats, proteins, peptides, radioisotopes, contrast media, surface charge modifiers, agglutinins, and combinations thereof.

31. The nanocomplex of claim 1, further comprising a linker bound to the functional group.

32. The nanocomposite of claim 31, wherein the linker comprises a heterobifunctional linker having a first reactive group that reacts with the functional group of the amphiphile encapsulating the nanoparticle and having a second reactive group that is suitable for reacting with an agent.

33. The nanocomposite of claim 32, wherein the functional group comprises an amine or a carboxylate.

34. The nanocomposite of claim 33, wherein the functional group comprises an amine and the first reactive group of the heterobifunctional linker comprises succinimidyl carboxymethyl ester.

35. The nanocomposite of claim 34, wherein the linker is selected from the group consisting of polyethylene glycol succinimidyl carboxymethyl acrylate, biotin polyethylene glycol succinimidyl carboxymethyl ester, maleimide polyethylene glycol succinimidyl carboxymethyl ester, azide polyethylene glycol succinimidyl carboxymethyl ester, and combinations thereof.

36. The nanocomposite of claim 34, wherein the second reactive group is selected from the group consisting of acrylates, maleimides, vinyl sulfones, and azides, biotin, amines, carboxylic acids, thiols, n-hydroxysuccinimide esters, alkynes, hydrazides, and 4-hydroxy-3-nitrophenylacetyl-ε-aminocaproic acid anions, methyltetrazine polyethylene glycol 4-N-hydroxysuccinimide esters, and combinations thereof.

37. The nanocomplex of claim 32, further comprising an agent bound to the second reactive group, wherein the agent is selected from the group consisting of polyethylene glycol, an antibody, a half antibody, an antibody fragment, a fluorescent probe, an aptamer, a vitamin, a cell surface receptor, a cell coat, a protein, a peptide, a radioisotope, a contrast medium, a surface charge modifier, an agglutinin, and combinations thereof.

38. The nanocomplex of claim 31, further comprising an antibody or antibody fragment bound to the linker.

39. The nanocomplex of claim 38, further comprising a cellular target bound to the antibody or the antibody fragment.

40. The nanocomplex of claim 39, wherein the antibody or the antibody fragment specifically binds to the cellular target. The nanocomplex of claim 40 , wherein the antibody or antibody fragment binds to the cell target with substantially no non-specific binding of other molecules.

42. The nanocomplex of claim 39, wherein the nanocomplex linked to the antibody or antibody fragment that binds the cellular target is analyzed by flow cytometry for detecting the cellular target.

43. The nanocomplex of claim 39, wherein the nanocomplex linked to the antibody or antibody fragment that binds to the cell target is placed in a magnetic field to separate the nanocomplex linked to the antibody or antibody fragment that binds to the cell target.

44. The nanocomplex of claim 43, wherein the nanocomplex linked to the antibody or antibody fragment that binds the cellular target separated by placement in the magnetic field is analyzed by flow cytometry for detection of the cellular target.

45. The nanocomplex of claim 38, wherein the antibody or antibody fragment comprises mouse anti-human CD3 and the cell target comprises human CD3 peripheral blood monocytes.

46. ​​The nanocomplex of claim 38, wherein the antibody or antibody fragment comprises mouse anti-human CD4 and the cell target comprises human CD4 peripheral blood monocytes.

47. The nanocomplex of claim 45, wherein the nanocomplex linked to the mouse anti-human CD3 that binds the human CD3 peripheral blood monocytes is analyzed by flow cytometry for detecting human CD3 peripheral blood monocytes.

48. The nanocomplex of claim 47, wherein the nanocomplex linked to the mouse anti-human CD3 peripheral blood monocytes bound to the human CD3 detected by flow cytometry is sorted into a population of isolated nanocomplexes linked to the mouse anti-human CD3 peripheral blood monocytes bound to the human CD3.

49. The nanocomplex of claim 46, wherein the nanocomplex linked to the mouse anti-human CD4 that binds to the human CD4 peripheral blood monocytes is analyzed by flow cytometry for detecting human CD4 peripheral blood monocytes.

50. The nanocomplex of claim 49, wherein the nanocomplex linked to the mouse anti-human CD4 bound to the human CD4 peripheral blood monocytes detected by flow cytometry is sorted into a population of isolated nanocomplexes linked to the mouse anti-human CD4 bound to the human CD4 peripheral blood monocytes.

51. The nanocomplex of claim 46, wherein the nanocomplex linked to the mouse anti-human CD3 that binds to the human CD3 peripheral blood mononuclear cells is separated under the influence of a magnetic field.

52. The nanocomplex of claim 51, wherein the nanocomplex linked to the mouse anti-human CD3 binding human CD3 peripheral blood monocytes separated by the influence of the magnetic field is analyzed by flow cytometry for detecting human CD3 peripheral blood monocytes.

53. The nanocomplex of claim 52, wherein the nanocomplex linked to the mouse anti-human CD3 peripheral blood monocytes bound to the human CD3 detected by flow cytometry is sorted into a population of isolated nanocomplexes linked to the mouse anti-human CD3 peripheral blood monocytes bound to the human CD3.

54. The nanocomplex of claim 46, wherein the nanocomplex linked to the mouse anti-human CD4 binding to the human CD4 peripheral blood mononuclear cell is separated under the influence of a magnetic field.

55. The nanocomplex of claim 54, wherein the nanocomplex linked to the mouse anti-human CD4 binding to the human CD4 peripheral blood monocyte separated under the influence of a magnetic field is detected by flow cytometry for detecting the human CD4 peripheral blood monocyte.

56. A nanocomposite comprising: Polystyrene-b-polyethylene glycol having a hydrophobic region and a hydrophilic region; and At least one quantum dot or at least one magnetic particle is encapsulated by being linked to the hydrophobic region of the amphiphile, the hydrophilic region of the amphiphile comprising functional groups.

57. The nanocomposite of claim 56, wherein the polystyrene-b-polyethylene glycol comprises polystyrene having a molecular weight ranging from about 1.5 kDa to about 40 kDa.

58. The nanocomposite of claim 56, wherein the polystyrene-b-polyethylene glycol comprises polyethylene glycol having a molecular weight ranging from about 10 kDa to about 40 kDa.

59. The nanocomposite of claim 58, wherein the polyethylene glycol comprises branched polyethylene glycol.

60. The nanocomposite of claim 56, wherein the polystyrene-b-polyethylene glycol comprises polystyrene-b-polyethylene glycol amine comprising a combination of polystyrene having a molecular weight ranging from about 1.5 kDa to about 40 kDa and polyethylene glycol having a molecular weight ranging from about 10 kDa to about 40 kDa, and the like.

61. A quantum dot comprising a core of cadmium sulfide and a shell of zinc sulfide and having an emission wavelength selected from the group consisting of 420 nm, 440 nm, and 460 nm, wherein the at least one quantum dot has a diameter ranging from about 5 nm to about 8 nm.

62. A quantum dot comprising a core of cadmium sulfoselenide and a shell of zinc sulfide and having an emission wavelength selected from the group consisting of 480 nm, 500 nm, and 520 nm, wherein the at least one quantum has a diameter ranging from about 4 nm to about 6 nm.

63. A quantum dot comprising a core of cadmium selenide and a shell of zinc sulfide and having an emission wavelength selected from the group consisting of 540 nm, 560 nm, 580 nm, and 600 nm, 620 nm, 640 nm, and 660 nm, wherein the at least one quantum has a diameter ranging from about 4 nm to about 8 nm.

64. A quantum dot comprising a core of cadmium telluride and a shell of zinc sulfide and having an emission wavelength selected from the group consisting of 680 nm and 700 nm, wherein the at least one quantum has a diameter ranging from about 6 nm to about 7 nm.

65. A quantum dot comprising a core of cadmium selenide telluride and a shell of zinc sulfide and having an emission wavelength selected from the group consisting of 720 nm, 740 nm, 760 nm, 780 nm, 800 nm, 820 nm, 840 nm, 860 nm, and 880 nm, wherein at least one quantum has a diameter ranging from about 4.5 nm to about 12 nm.

66. A quantum dot comprising a core of copper indium zinc sulfide and a shell of zinc sulfide and having an emission wavelength selected from the group consisting of 540 nm, 560 nm, 580 nm, 600 nm, 620 nm, 640 nm, 840 nm, and 669 nm, wherein the at least one quantum has a diameter ranging from about 3.5 nm to about 4 nm.

67. A quantum dot comprising a core of lead sulfide and a shell of cadmium sulfide and having an emission wavelength selected from the group consisting of 900 nm, 920 nm, 940 nm, 960 nm, 980 nm, and 1000 nm, wherein at least one quantum has a diameter ranging from about 3.5 nm to about 5 nm.

68. A quantum dot comprising: Nuclear materials; a shell material surrounding the core material; and A ligand is bound to the quantum dot, the ligand having a mass concentration in the range of about 10% to about 60%.

69. The quantum dot of claim 68, wherein the ligand has a mass concentration present in the range of about 10% to about 40%.

70. The quantum dot of claim 69, wherein the ligand comprises oleic acid or oleylamine and combinations thereof.

71. The quantum dot of claim 70, wherein the oleic acid bound to the at least one quantum dot has a mass concentration of not less than 10% and not more than 40%.

72. A nanocomposite comprising: at least one quantum dot; at least one magnetic particle; and A ligand is bound to the quantum dot and the at least one magnetic particle, the ligand having a mass concentration in the range of about 10% to about 40%.

73. The nanocomposite of claim 72, further comprising an amphiphile having a hydrophobic region and a hydrophilic region, wherein the hydrophobic region is connected to the at least one quantum dot and the at least one magnetic particle.

Images