Polymer composite nanomaterial encapsulation system

Abstract

Generally, polymer nanomaterial encapsulation systems useful in the production of polymer-encapsulated nanoparticles comprised of hydrophobic nanoparticles encapsulated within the hydrophobic regions of a polymer, with the external hydrophilic regions of the polymer ensuring water solubility and providing functional groups that can be utilized for the production of nanoparticle conjugates. Specifically, certain embodiments include polymer nanoparticle structures containing one or more of quantum dots and / or superparamagnetic iron oxide nanoparticles and / or upconversion nanoparticles encapsulated in polystyrene-b-polyethylene glycol amine for the production of antibody conjugates useful in capturing cellular targets.

Description

[Technical Field]

[0001] This International Patent Cooperation Treaty patent application is a continuation of U.S. Non-Provisional Patent Application No. 17 / 943,788, filed September 13, 2022, which is hereby incorporated by reference herein.

[0002] (I.Technical field) Generally, nanomaterial polymer encapsulation systems are useful in the creation of nanocomposites that include hydrophobic nanoparticles encapsulated within the hydrophobic regions of the polymer, where the external hydrophilic regions of the polymer ensure water solubility and provide functional groups that can be utilized to create nanocomposite conjugates.

[0003] In particular, certain embodiments may include nanocomposites comprising one or more nanoparticles (including quantum dots ("QDs") and / or superparamagnetic iron oxide nanoparticles ("SPIONs") and / or upconversion nanoparticles) encapsulated in polystyrene-b-polyethylene glycol amine ("PS-b-PEG-NH2") that provide amine functional groups that can be activated for conjugating antibodies, modified antibodies, or antibody fragments, and in certain embodiments, with methyltetrazine polyethylene glycol-4-N-hydroxysuccinimide ester ("methyltetrazine-PEG4-NHS ester") to enable conjugation of trans-cyclooctyne ("TCO")-modified antibodies or antibody fragments. [Background technology]

[0004] (II. Background) Nanocomposites have a wide variety of potential applications, including, but not limited to, medicine, biomedicine, biotechnology, biomaterials, biomechanics, and energy generation. Current methods for polymer encapsulation of nanoparticles (e.g., QDs or SPIONs) to produce nanocomposites have resulted in aggregation of the nanocomposites, limiting production to small batches, low photochemical stability, lack of uniform size and brightness, lack of specificity of nanocomposite conjugates for cellular targets, and lack of methodologies for rapid purification of nanocomposites bound to cellular targets. Nanocomposites and methods for producing and using nanocomposites that minimize aggregation in large batch production and have high photochemical stability, uniform and narrow size distribution and brightness, high binding specificity for cellular targets, and protocols for rapid purification of nanocomposites bound to cellular targets would be significant advantages. Summary of the Invention [Means for solving the problem]

[0005] III. DISCLOSURE OF THE INVENTION A broad object of embodiments of the present invention may be to provide nanocomposites comprising one or more nanoparticles encapsulated by a polymer having hydrophobic regions associated with the nanoparticles and hydrophilic regions comprising functional groups associated with an aqueous environment, where the nanoparticles may comprise one or more of QDs or SPIONs, or combinations thereof, and the polymer may comprise polystyrene-b-polyethylene glycol comprising functional groups, where the polystyrene may have a molecular weight ranging from about 1.5 kDa to about 40 kDa, and the polyethylene glycol may have a molecular weight ranging from about 10 kDa to about 40 kDa. Thus, combinations and permutations of the selection of the molecular weights of the QDs, SPIONs, polystyrene and / or polyethylene glycol, and the branching structure of the polyethylene glycol, as well as variations in their mass ratios, allow for the production of a wide variety of nanocomposites having substantially uniform hydrodynamic diameters ranging from about 40 nm to about 500 nm, and brightness attributable to QDs having various emission wavelengths ranging from 420 nm to 1000 nm.

[0006] Another broad object of embodiments of the present invention may be to provide nanocomposite antibody conjugates of QDs and / or SPIONs capable of specifically binding to cellular targets, exemplary embodiments of which nanocomposite antibody conjugates include one or more of anti-CD3 or anti-CD4 antibodies capable of specifically binding to CD3 peripheral blood mononuclear cells and / or CD4 peripheral blood mononuclear cells.

[0007] Another broad object of embodiments of the present invention may be to provide nanocomposites comprising polymer-encapsulated QDs or SPIONs or combinations thereof for specific targeting of macrophages, with exemplary embodiments including uptake of nanocomposite embodiments by blood cells.

[0008] Another broad object of embodiments of the present invention may be to provide a method for isolating cell-bound QD and / or SPION nanocomposite antibody conjugates, in which the cell-bound SPION nanocomposite antibody conjugates may be separated and isolated by the influence of a magnetic field, the isolated cell-bound QD or SPION nanocomposite antibody conjugates may be analyzed by flow cytometry, and in certain embodiments, the analyzed cell-bound QD or SPION nanocomposite antibody conjugates may be flow sorted into separate populations based on one or more characteristics of the cells.

[0009] Of course, further objects of the present invention are disclosed throughout other areas of this specification, in the drawings, photographs, and claims. [Brief explanation of the drawings]

[0010] IV. BRIEF DESCRIPTION OF THE DRAWINGS The patent or application file contains at least one drawing executed in color. Copies of this patent or application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0011] [Figure 1] FIG. 1 is a block flow diagram outlining the process for making and using the elements of the nanoparticle-polymer encapsulation system, including: polymer synthesis, preparation of polymer-encapsulated nanoparticles, antibody preparation, generation of antibodies conjugated to polymer-encapsulated nanoparticles, and antibodies conjugated to polymer-encapsulated nanoparticles bound to cellular targets. [Figure 2] FIG. 2 is a block flow diagram including blocks 2A-2I illustrating a process for synthesizing polymers encompassed by the method of block 1A of FIG. 1, including the illustrative example PS-b-PEG-NH2. [Figure 3]FIG. 3 is an NMR spectrum confirming the molecular structure of the illustrative example PS-b-PEG-NH2 obtained by the polymer synthesis process shown in Blocks 2A-2I of FIG. [Figure 4A] FIG. 4A shows an embodiment of the polymer-encapsulated nanoparticles contained by block 1B of FIG. 1, which comprises one or more QDs encapsulated in a polymer obtained by the polymer synthesis process shown in blocks 2A-2I of FIG. 2. [Figure 4B] FIG. 4B shows an embodiment of the polymer-encapsulated nanoparticles contained by block 1B of FIG. 1, which include one or more QDs and one or more SPIONs encapsulated in a polymer obtained by the polymer synthesis process shown in blocks 2A-2I of FIG. 2. [Figure 4C] FIG. 4C shows an embodiment of a nanoparticle encapsulated in a polymer comprised by Block 1B of FIG. 1, comprising one or more QDs and / or one or more SPIONs encapsulated in a polymer obtained by the polymer synthesis process shown in Blocks 2A-2I of FIG. 2, where the polymer may have functional groups that can be activated to bind to one or more agents, illustrative examples of which include one or more linkers, polyethylene glycol, fluorescent probes, aptamers, vitamins, cell surface receptors, cell envelopes, proteins, peptides, radioisotopes, contrast agents, surface charge modifiers, lectins, or antibodies, half-antibodies, antibody fragments, and combinations thereof. [Figure 5] FIG. 5 is a block diagram illustrating a method for assembling the polymer-encapsulated nanoparticles shown in FIGS. 4A and 4B by using electrohydrodynamic mixed-mediated nanoprecipitation. [Figure 6A]FIG. 6A is a micrograph obtained by transmission electron microscopy of polymer-encapsulated nanoparticles comprised by Block 1B of FIG. 1, which contain one or more QDs encapsulated in a polymer obtained by the polymer synthesis process shown in Blocks 2A-2I of FIG. 2, by use of electrohydrodynamic mixed-mediated nanoprecipitation shown in FIG. 5, using 0.5 v / v QDs in the organic phase. [Figure 6B] FIG. 6B is a micrograph obtained by transmission electron microscopy of polymer-encapsulated nanoparticles comprised by Block 1B of FIG. 1, which contain one or more QDs encapsulated in a polymer obtained by the polymer synthesis process shown in Blocks 2A-2I of FIG. 2, by use of electrohydrodynamic mixed-mediated nanoprecipitation shown in FIG. 5, using 0.2 v / v QDs in the organic phase. [Figure 7A] FIG. 7A is a micrograph obtained by transmission electron microscopy of polymer-encapsulated nanoparticles comprised by Block 1B of FIG. 1, which comprise one or more QDs having an emission wavelength of 420 nm, encapsulated in a polymer obtained by the polymer synthesis process shown in Blocks 2A-2I of FIG. 2, by use of electrohydrodynamic mixed-mediated nanoprecipitation as shown in FIG. 5. [Figure 7B] FIG. 7B is a micrograph obtained by transmission electron microscopy of polymer-encapsulated nanoparticles comprised by Block 1B of FIG. 1, which comprise one or more QDs having an emission wavelength of 575 nm, encapsulated in a polymer obtained by the polymer synthesis process shown in Blocks 2A-2I of FIG. 2, by use of electrohydrodynamic mixed-mediated nanoprecipitation as shown in FIG. 5. [Figure 7C]FIG. 7C is a micrograph obtained by transmission electron microscopy of polymer-encapsulated nanoparticles comprised by Block 1B of FIG. 1, which contain one or more QDs having an emission wavelength of 610 nm, encapsulated in a polymer obtained by the polymer synthesis process shown in Blocks 2A-2I of FIG. 2, by use of electrohydrodynamic mixed-mediated nanoprecipitation shown in FIG. 5. [Figure 7D] FIG. 7D is a micrograph obtained by transmission electron microscopy of polymer-encapsulated nanoparticles comprised by Block 1B of FIG. 1, which comprise one or more QDs having an emission wavelength of 655 nm, encapsulated in a polymer obtained by the polymer synthesis process shown in Blocks 2A-2I of FIG. 2, by use of electrohydrodynamic mixed-mediated nanoprecipitation shown in FIG. 5. [Figure 8A] FIG. 8A is a micrograph obtained by transmission electron microscopy of polymer-encapsulated nanoparticles comprised by Block 1B of FIG. 1, which comprise one or more 20 nm SPIONs and one or more QDs having an emission wavelength located at 610 nm, encapsulated in a polymer obtained by the polymer synthesis process shown in Blocks 2A-2I of FIG. 2, by use of electrohydrodynamic mixed-mediated nanoprecipitation as shown in FIG. 5. [Figure 8B] FIG. 8B is a micrograph obtained by transmission electron microscopy of polymer-encapsulated nanoparticles comprised by Block 1B of FIG. 1, which comprise one or more 15 nm SPIONs and one or more QDs having an emission wavelength located at 610 nm, encapsulated in a polymer obtained by the polymer synthesis process shown in Blocks 2A-2I of FIG. 2, by use of electrohydrodynamic mixed-mediated nanoprecipitation as shown in FIG. 5. [Figure 8C]FIG. 8C is a micrograph obtained by transmission electron microscopy of polymer-encapsulated nanoparticles comprised by Block 1B of FIG. 1, which comprise one or more 5 nm SPIONs and one or more QDs having an emission wavelength located at 610 nm, encapsulated in a polymer obtained by the polymer synthesis process shown in Blocks 2A-2I of FIG. 2, by use of electrohydrodynamic mixed-mediated nanoprecipitation as shown in FIG. 5. [Figure 9A] Figure 9A is a micrograph obtained by transmission electron microscopy of polymer-encapsulated nanoparticles comprised by Block 1B of Figure 1, containing a fixed ratio of QD:15 nm SPION:polymer (5:5:20), obtained by using the polymer synthesis process shown in Blocks 2A-2I of Figure 2 and electrohydrodynamic mixed-mediated nanoprecipitation shown in Figure 5. [Figure 9B] Figure 9B is a micrograph obtained by transmission electron microscopy of polymer-encapsulated nanoparticles comprised by Block 1B of Figure 1 containing a fixed ratio of QD:5 nm SPION:polymer (3:5:20) with a hydrodynamic diameter of 210 ± 30 nm, obtained by using the polymer synthesis process shown in Blocks 2A-2I of Figure 2 and electrohydrodynamic mixed-mediated nanoprecipitation shown in Figure 5. [Figure 9C] FIG. 9C is a plot of hydrodynamic diameter versus particle concentration per milliliter for the polymer-encapsulated nanoparticles shown in FIG. 9A, showing a hydrodynamic diameter of approximately 128 nm±22 nm. [Figure 9D] FIG. 9D is a bar graph comparing the iron concentration of nanoparticles encapsulated in polymers containing 15 nm SPIONs and QDs with an emission wavelength located at 610 nm with the iron concentration of nanoparticles encapsulated in polymers containing 5 nm SPIONs and QDs with an emission wavelength located at 610 nm. [Figure 9E] FIG. 9E is a bar graph comparing the fluorescence of nanoparticles encapsulated in polymers containing 15 nm SPIONs with the fluorescence of nanoparticles encapsulated in polymers containing 5 nm SPIONs. [Figure 10A] Figure 10A is a univariate histogram of flow cytometry showing detection of peripheral blood mononuclear cells bound to mouse anti-human CD4 linked to polymer-encapsulated nanoparticles, encompassed by block 1D in Figure 1, containing one or more QDs. [Figure 10B] Figure 10B is a univariate histogram of flow cytometry showing detection of peripheral blood mononuclear cells bound to mouse anti-human CD3 linked to polymer-encapsulated nanoparticles, encompassed by block 1D in Figure 1, containing one or more QDs. [Figure 11A] FIG. 11A is a flow cytometry bivariate dot plot showing the detection of CD3-positive peripheral blood mononuclear cells and CD3-negative cells bound to mouse anti-human CD3 linked to polymer-encapsulated nanoparticles comprising one or more SPIONs and one or more QDs, encompassed by block 1D in FIG. 1, in the reaction solution before magnetic separation. [Figure 11B] FIG. 11B is a flow cytometry bivariate dot plot showing the detection of depleted CD3-positive peripheral blood mononuclear cells in the non-magnetic fraction of the reaction solution after magnetic separation. [Figure 11C] Figure 11C is a flow cytometry bivariate dot plot showing the detection of enriched CD3-positive peripheral blood mononuclear cells bound to mouse anti-human CD3 linked to polymer-encapsulated nanoparticles comprising one or more SPIONs and one or more QDs, encompassed by block 1D in Figure 1, in the magnetic fraction of the reaction solution after magnetic separation. [Figure 12A] FIG. 12A is a bright field image of a host squid hemocyte incubated with a nanocomposite of the present invention comprising one or more SPIONs and one or more QDs having an emission wavelength located at 610 nm. [Figure 12B]Figure 12B is an epifluorescence image of a host squid hemocyte incubated with a nanocomposite of the present invention comprising one or more SPIONs and one or more QDs having an emission wavelength present at 610 nm emission, detected by fluorescence microscopy in the fluorescein isothiocyanate ("FITC") channel. [Figure 12C] Figure 12C is a composite image of bright-field and epifluorescence images of a host squid hemocyte incubated with a nanocomposite of the present invention comprising one or more SPIONs and one or more QDs having an emission wavelength of 610 nm, as detected by bright-field microscopy and fluorescence microscopy measured in the FITC channel. DETAILED DESCRIPTION OF THE INVENTION

[0012] V. MODE FOR CARRYING OUT THE INVENTION The present invention can be understood by reference to the following detailed description of aspects of the invention and the examples and figures contained therein, as well as the above and following descriptions of those figures. It should be understood that compounds, compositions, articles, devices, or methods have been disclosed and described, and are not limited to particular synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, and as such may, of course, vary. It should also be understood that the terminology used herein is intended to describe particular embodiments, even if not explicitly disclosed, in order to enable one of ordinary skill in the art to make and use a wide variety of embodiments of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Any methods and materials similar or equivalent to those described herein may be used in the practice of the present invention.

[0013] All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided herein may be different from the actual publication dates, which may need to be independently confirmed.

[0014] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "component," "polymer," or "particle" includes a mixture of two or more such components, polymers, or particles, etc.

[0015] Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it is understood that the particular value forms another embodiment. It is further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values ​​disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, "about 10" is also disclosed. As appreciated by one of ordinary skill in the art, when a value is disclosed, it is also understood that "less than or equal to," "greater than or equal to," and possible ranges between values ​​are also disclosed. For example, if the value "10" is disclosed, "less than or equal to 10" as well as "greater than or equal to 10" is also disclosed. It is also understood that throughout this application, data are provided in a number of different formats, and that this data sets forth endpoints and starting points, as well as ranges for any combination of those data points. For example, if a specific data point "10" and a specific data point of 15 are disclosed, it is understood that greater than 10, greater than or equal to 10, less than 10, less than or equal to 10, and equal to 10, as well as greater than 15, greater than or equal to 15, less than 15, less than or equal to 15, and equal to 15, and between 10 and 15, are considered to be disclosed. It is also understood that each unit between two specified units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0016] As used herein and in the appended claims, a residue of a chemical species refers to a moiety that is a product of that chemical species resulting from a particular reaction scheme or subsequent formulation or chemical product, regardless of whether that moiety is actually derived from that chemical species. Thus, as an illustrative example, an ethylene glycol residue in a polyester refers to one or more -OCH2CHO- units in the 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 the polyester, regardless of whether that residue is derived by reacting sebacic acid or its ester to obtain the polyester.

[0017] The components to be used for preparing the compositions of the present invention, and the compositions themselves to be used in the methods disclosed herein are disclosed.These and other materials are disclosed herein, and when the combinations, subsets, interactions and groups of these materials are disclosed, it is understood that although specific reference to each of the various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein.For example, when a specific compound is disclosed and discussed, and many modifications that can be made to many molecules including that compound are discussed, all combinations and permutations of that compound and possible modifications are specifically contemplated unless specifically indicated to the contrary.Thus, when classes of molecules A, B and C are disclosed, and classes of molecules D, E and F and an example of a combined molecule AD are disclosed, each is individually and collectively contemplated, even if each is not individually described, which means that the combinations AE, AF, BD, BE, BF, CD, CE and CF are considered to be disclosed.Similarly, any subset or combination of these is also disclosed. Thus, for example, subgroups AE, BF, and CE are considered to be disclosed. This concept applies to all aspects of this application, including but not limited to elements in the methods of making and using the compositions of the present invention. Therefore, if there are various additional elements that can be implemented, it is understood that each of these additional elements can be implemented with any specific embodiment or combination of embodiments of the methods of the present invention. It is understood that the compositions disclosed herein have specific functions. Specific structural requirements for performing the disclosed functions are disclosed herein, and it is understood that there are various structures related to these disclosed structures that can perform the same function, and that these structures usually achieve the same results.

[0018] (Process Overview) Referring now primarily to Figure 1, a block flow diagram provides an overview of a nanomaterial-polymer encapsulation system (1) that includes one or more of the following: polymer synthesis of nanoparticles ("NPs") (2) and encapsulating polymers ("Ps") (3) (Block 1A); formation of polymer-encapsulated nanoparticles ("PENPs") (4) (Block 1B); preparation of antibodies, half-antibodies, and antibody fragments (individually or collectively "Abs") (5) (Block 1C); preparation of PENP-antibody conjugates ("PENP-Abs") (6) (Block 1D); PENP-Ab cell labeling (PENP-Ab-cells") (7), flow cytometry analysis (8), and imaging of PENP-Ab-cells (7) (Block 1E).

[0019] (polymer synthesis) Referring now primarily to FIG. 2, a block flow diagram provides an overview of an exemplary polymer synthesis process of FIG. 1, Block 1A. Referring to Block 2A of that process, the linear formula (C8H8) n Aminopolystyrene ("PS-NH2") having the formula CH5N (wherein the PS molecular weight can range from about 1.5 kDa to about 40 kDa) can be obtained from Polymer Source, Inc., PN P3965-SNH2 having formula I. A particular embodiment can include PS-NH2 having a number average molecular weight of about 9.5 kDa. [ka]

[0020] Now, referring to Block 2B of Figure 2 of the polymer synthesis process, the linear formula C 23 H 18 N2O 5( Dibenzocyclooctyne-N-hydroxysuccinimidyl ester ("DBCO-NHS ester") having CAS number: 1353016-71-3) and a molecular weight of 402.40 g / mol can be obtained from Click Chemistry Tools, PN A133-100, which includes formula II. [ka]

[0021] Now, referring to Block 2C of Figure 2 of the polymer synthesis process, PS-NH2 can be reacted with DBCO-NHS ester to produce polystyrene-dibenzocyclooctyne ("PS-DBCO") comprising Formula III.

[0022] [ka]

[0023] An example of a scalable procedure for the production of PS-DBCO can include melting 20 mg of PS-NH at room temperature ("RT") for 15 minutes and melting 10 mg of DBCO-NHS-ester at room temperature for 15 minutes ("min."). 1 mL of toluene (CHCH) (CAS Number: 108-88-3) is dispensed to the 20 mg of PS-NH and vortexed at 500 RPM for 15 minutes. DBCO is vortexed at 1500 relative centrifugal force ("RCF") (RCF = (RPM) 2 x1.118 x 10 -5 ×r) at approximately 25°C (approximately 77°F) for 30 seconds ("sec."). Dispense 1 mL of toluene into the DBCO. Sonicate the DBCO in room temperature water for 5 minutes. Transfer 1 mL of DBCO in toluene to the PS-NH2 in toluene. Vortex the DBCO in PS-NH2 mixture at 500 RPM at room temperature for 16-24 hours ("hr.").

[0024] Now, referring to Block 2D of Figure 2 of the polymer synthesis process, a multi-arm PEG derivative having an amine group at each end of eight arms attached to a single hexaglycerol core is shown, which can be synthesized according to the linear formula R(O(CHCHO) n CH2CH2NH2)8 and an 8-arm polyethylene glycol-amine ("PEG") having a number average molecular weight of approximately 19.5 kDa. 8アーム—NH2”) is available from Nanosoft Polymers, PN 2443, which contains formula IV and is further shown in formula V. [ka]

[0025] In certain embodiments, 4-arm polyethylene glycol-amine, 6-arm polyethylene glycol-amine, or 8-arm polyethylene glycol-amine, or combinations thereof, may also be utilized, with corresponding PEG molecular weights of 10 kDa, 20 kDa, or 40 kDa.

[0026] Now, referring to Block 2E of Figure 2 of the polymer synthesis process, azido-d-polyethylene glycol 4-N-hydroxysuccinimidyl ester ("Azido-PEG4-NHS ester") (CAS number: 944251-24-5) having a molecular weight of 388.37 g / mol can be obtained from Click Chemistry tools, PN AZ103-100, which contains formula VI. [ka]

[0027] Now, referring to Block 2F of Figure 2 of the polymer synthesis process, PEG 8アーム -NH2 can be reacted with azide-PEG4-NHS ester to produce a branched polyethylene glycol ("PEG-amine-azide") comprising formula VII. [ka]

[0028] An example of a scalable procedure for the production of PEG-amine-azide is 8アームThe method may involve melting 400 μl of methanol (CAS number: 6756-1) with PEG 400 μl of AZIDE-PEG-NHS ester (50 mM) at room temperature for 15 minutes. 8アーム -NH2 and its PEG 8アーム Vortex the AZIDO-PEG4-NH2 at 500 RPM for 15 minutes. Centrifuge the AZIDO-PEG4-NHS at 1500 RCF at 25°C (approximately 77°F) for 30 seconds. Transfer 80 μl of the AZIDO-PEG4-NHS to the 400 μl of PEG8 arm-NH2. Vortex at 500 RPM at room temperature for 16-24 hours.

[0029] Referring now to Block 2G of Figure 2 of the polymer synthesis process, the PS-DBCO obtained in Block 2G of the synthesis process can be purified by the following exemplary scalable procedure, which involves transferring 400 μl of PS-DBCO to each of five centrifuge tubes. Dispense 800 μl of methanol into each of the five centrifuge tubes. Mix the PS-DBCO in the methanol by inverting each of the five centrifuge tubes. Centrifuge the PS-DBCO in the methanol at 20,000 RCF for 5 minutes at 15°C to 25°C (approximately 59°F to approximately 77°F). Decant 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 (approximately 98.6°F) water bath for 2 minutes. Dissolve the PS-DBCO pellet in the 400 μl of toluene by pipetting. 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 minutes at 15°C to 25°C (approximately 59°F to approximately 77°F). Decant 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 (approximately 98.6°F) water bath for 2 minutes. Dissolve the PS-DBCO pellet in the toluene by mixing with a pipette.

[0030] Referring now to block 2H of Figure 2 of the polymer synthesis process, the PS-DBCO obtained in block 2H of the synthesis process can be reacted with the PEG-amine-azide obtained in block 2F of the polymer synthesis process to produce a polystyrene-b-poly(ethylene glycol)amine ("PS-b-PEG-NH") comprising Formula VIII. [ka]

[0031] In this illustrative example of Block 2H, the PS-DBCO in toluene from all five centrifuge tubes from Block 2G and the PEG-amine-azide in methanol from Block 2F can be transferred to a 4 mL glass vial. The PS-DBCO and PEG-amine-azide mixture can be vortexed at 500 RPM at room temperature for 16-24 hours to produce PS-b-PEG-NH2 containing Formula VIII. The PS-b-PEG-NH2 can contain PS having a 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.

[0032] Referring to Block 2I of Figure 2 of the synthetic process, the PS-b-PEG-NH2 obtained in Block 2H can be purified and dried by the following exemplary scalable procedure, which involves chilling hexane (CAS No.: 110-54-3) at -20°C (approximately -4°F) for 15 minutes. Approximately 1.25 mL of PS-b-PEG-NH2 obtained in Block 2I of the synthetic process is dispensed into a 15 mL centrifuge tube. 12 mL of hexane is slowly added to the centrifuge tube containing the PS-b-PEG-NH2. The PS-b-PEG-NH2 is mixed in the toluene / hexane by gently tilting the centrifuge tube 10-15 times. The PS-b-PEG-NH2 in the toluene / hexane is chilled by placing the centrifuge tube at -20°C (approximately -4°F) for 5 minutes. The supernatant is decanted from the PS-b-PEG-NH2 pellet. 1 mL of tetrahydrofuran is dispensed into the centrifuge tube. Place the centrifuge tube in a 37°C (approximately 98.6°F) water bath for 2 minutes. Dissolve the PS-b-PEG-NH2 pellet by mixing with a pipette. Slowly add 6 mL of cold hexane to the centrifuge tube. Gently tilt the centrifuge tube 10-15 times until PS-b-PEG-NH2 precipitates as flakes in the clear supernatant. Place the centrifuge tube at -20°C (approximately -4°F) for 5 minutes. Decant the supernatant from the PS-b-PEG-NH2 pellet. Place the centrifuge tube containing the PS-b-PEG-NH2 pellet at room temperature for 24 hours to remove excess solvent and obtain dry PS-b-PEG-NH2. Store at -20°C (approximately -4°F).

[0033] Referring now primarily to Figure 3, the structure of PS-b-PEG-NH obtained in Block 2I of the synthetic process can be analyzed using nuclear magnetic resonance ("NMR"). The NMR spectrum shown in Figure 3 confirms the molecular structure obtained by the polymer synthesis process shown in Blocks 2A-2I of Figure 2 and the polymer synthesis procedures disclosed above, which is purified PS-b-PEG-NH comprising Formula VIII.

[0034] The illustrative example of 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. The other functional groups may include, by way of example, one or more of the following: acrylate, maleimide, vinyl sulfone, azide, biotin, carboxyl, thiol, alkyne, hydrazide, N-hydroxysuccinimide ester, and nitrophenyl carbonate, as well as combinations thereof, or other similar or equivalent polymers that include one or more functional groups.

[0035] (Formation of polymer-encapsulated nanoparticles) Referring now primarily to Figure 4A, which shows certain embodiments of PENPs (4) comprising one or more QDs (2a) encapsulated in a polymer ("P") (3), the polymer having hydrophobic regions capable of associating with or coordinating the one or more QDs (2a) and hydrophilic regions comprising functional groups (9) capable of associating with an aqueous environment; in certain embodiments, the polymer (P) (3) can comprise PS-b-PEG-NH2, obtained in block 2I, which can generate polymer-encapsulated nanoparticles ("PENP-MultiDot") (4a) comprising one or more QDs (2a) by solution-based assembly.

[0036] Referring now primarily to Figure 4B, which shows certain embodiments of PENP (4) comprising one or more QDs (2a) and one or more SPIONs (2b) encapsulated in a polymer ("P") (3), the polymer having a hydrophobic region capable of associating with or coordinating the one or more SPIONs (2b) and optionally one or more QDs (2a), and a hydrophilic region comprising functional groups (9) capable of associating with an aqueous environment; in certain embodiments, the polymer (P) (3) can comprise PS-b-PEG-NH2 obtained in block 2I, which can undergo solution-based association to produce PENP (4) comprising one or more SPIONs (2b) and one or more QDs (2a) ("PENP-MagDot") (4b).

[0037] Referring now primarily to Figure 4C, functional groups 9 on polymer 3, in certain embodiments amines provided by PS-b-PEG-NH2, can be used to conjugate a wide variety of agents 5 to PENP-MultiDots 4a and / or PENP-MagDots 4b. The example PENP-MagDot 4b shown in Figure 4C illustrates that one or more agents 5 can be conjugated to PENP-MultiDots 4a and / or PENP-MagDots 4b by activating functional groups 9, including, by way of example, one or more linkers 10, 10a, 10b, polyethylene glycol, fluorescent probes, aptamers, vitamins, radioisotopes, contrast agents, surface charge modifiers, lectins, proteins, peptides, cell surface receptors, cell envelopes, and combinations thereof. Specifically, in certain embodiments, the functional group (9) can be used to link an antibody or antibody fragment (Ab) (5') directly or indirectly via one or more linkers (10, 10a, 10b) to produce a PENP-antibody conjugate (6), illustrative examples of which include PENP-MultiDot-Ab (6a) and PENP-MultiDot-Ab (6b).

[0038] Referring now primarily to FIG. 5, scalable solution-based production of PENP (4) encompassed by the present invention (including, but not necessarily limited to, PENP-MagDot (4b) or PENP-MultiDot (4a)) can be achieved by conventional self-assembly, flash nanoprecipitation (FNP), which involves rapid turbulent mixing generated by high-velocity flow as described by Yanjie Zhang and Aaron R. Clapp, RSC Advances, No. 89, 2014, "Preparation of quantum dot-embedded polymeric nanoparticles using flash nanoprecipitation," or rapid mixing induced by electrohydrodynamics ("EHD"): EHD mixing-mediated nanoprecipitation ("EHD-NP"), Kil Ho Lee, Guolingzi Yang, Barbara E. Wyslouzil, and Jessica O. Winter, ACS Appl. Polym. Mater., 2019, 1, 4, 691-700 (each of which is incorporated herein by reference).

[0039] The exemplary EHD mixing system of Figure 5 can include a syringe (11) having a syringe barrel (12) attached to a sliding syringe plunger (13) and a syringe needle (14). A syringe pump (15) is configured to drive the syringe plunger (13) to deliver an organic phase ("OP") of a water-miscible, non-polar, aprotic solvent containing solubilized P (3) and NPs (4) (QDs (2a) and / or SPIONs (2b) (collectively "inorganic")) at a predetermined volumetric flow rate into an aqueous phase ("AP"). The illustrative examples of syringe (11) and syringe driver (15) are not intended to exclude other devices useful in delivering the OP into the AP at a predetermined volumetric flow rate. The concentration of inorganic material per unit volume of the OP can be about 0.1 volume / volume ("v / v") to about 0.5 v / v. The mass concentration of P (3) to QDs (2a) and / or SPIONs (2b) in the OP can be about 1:1 to about 4:1. The amount of P (3) can be adjusted to obtain PENPs (4) with substantially uniform hydrodynamic diameters ("HD") within a size range of about 40 nanometers ("nm") to about 500 nm. In embodiments in which the QDs (2a) and / or SPIONs (2b) are passivated with a ligand (16), the mass of the ligand relative to the total mass of the inorganic material can be about 20% to 40% by mass. A mass percentage of the ligand greater than 40% can interfere with the assembly of the PENPs (4).

[0040] A non-conductive container (17) may hold the AP (typically distilled or deionized water). A positive electrode (18) and a negative electrode (19) may be introduced into the AP held in the non-conductive container (17) at a distance of approximately 1 cm. In certain embodiments, the syringe needle (14), if conductive, may act as the positive electrode (18). A voltage source (20) may supply 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 actuated to introduce the OP into the AP at a consistent flow rate of approximately 8 mL / hour to approximately 15 mL / hour. The voltage (V) may be adjusted between approximately -1 kilovolt ("kV") and approximately -2.5 kV. The electric field can generate a fine dispersion of OPs in the AP to produce substantially uniformly sized PENPs (4), PENP-MagDots (4b), or PENP-MultiDots (4a). The size of the resulting PENPs (4), PENP-MagDots (4b), or PENP-MultiDots (4a) can increase with increasing inorganic concentration per unit volume of V and / or the OPs. The resulting PENP-MagDots (4b) can then be isolated by the influence of a magnetic field (15).

[0041] (PENP-MultiDot) PENP-MultiDot 4a may comprise QD 2a inorganic semiconductor nanocrystals, comprising an inorganic core semiconductor material 2a' (also referred to as "core material") surrounded by a shell semiconductor material 2a'' (also referred to as "shell material") with a different bandgap (annotated as "core material / shell material") (e.g., CdS / ZnS). QD 2a may have a size typically in the range of 1 nm to 10 nm. QD 2a may exhibit size-tunable emission color due to quantum confinement effects, where, for a given composition, smaller QDs 2a emit higher energy (lower wavelength) and larger QDs 2a emit lower energy (higher wavelength). Thus, QD 2a may absorb over a wide range and have a narrow range of photoluminescence emission, which can be tuned depending on the material from which the QD 2a is produced and the size of the QD 2a. Illustrative examples of QD core / shell compositions and combinations thereof suitable for use in PENP-MultiDot (4a) embodiments may include one or more of: CdS / ZnS, CdSSe / ZnS, CdSe / ZnS, CdTe / ZnS, and CdSeTe / ZnS, each of which has an emission photoluminescence in the range of 420 nm to 880 nm; CuInZnS / ZnS, which has an emission photoluminescence in the range of 540 nm to 660 nm; and PbS / CdS, which has an emission photoluminescence in the range of 700 nm to 900 nm. However, these examples are not intended to exclude embodiments having other QD (2a) core / shell compositions.

[0042] The purified QDs (2a) can be bare or capped to control QD particle size and / or prevent QD aggregation. QDs (2a) synthesized by the prototype hot-injection method can be capped with ligands (16) (e.g., oleylamine and oleic acid) after QD purification. 1H NMR spectroscopy has shown that ligand binding can be highly dynamic and that oleylamine selectively binds to the surface as ammonium oleyl bromide in the N-C(X)2 binding motif. Only in the presence of excess oleylamine added after purification does oleic acid bind to the surface in the form of ammonium oleyl oleate. 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). 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) (e.g., trioctylphosphine oxide, L-histidine, chitosan, polyvinyl alcohol, polyvinylpyrrolidone, or combinations thereof).

[0043] Typically, bare or capped QDs 2a reach the required level of water solubility and biocompatibility by surrounding the QDs 2a with P 3 to produce PENP 4. Suitable P 3 for use in embodiments of the present invention may include various embodiments of PS-b-PEG-NH2 obtained in Block 21 of the synthetic process described above. PENP 4 embodiments may be produced through the use of various combinations of one or more of the following: QDs 2a, 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.

[0044] An illustrative example of a scalable method for producing PENP (4) (including, but not necessarily limited to, PENP-MultiDot (4a)) by EHD may include one or more of the following: PS-b-PEG-NH2 obtained in Block 2I of Figure 2 is formulated into a first 1.5 mL tube at 10 mg / mL; 240 μl of the desired QDs are introduced into a second 1.5 mL tube at 5 mg / mL (2a); 480 μl of acetone / methanol (60 / 40) is transferred to the QDs (2a) in the second 1.5 mL tube; the second 1.5 mL tube containing the QDs (2a) is centrifuged at 7000 RCF for 1 minute, after which 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 the QDs and mixed thoroughly with a pipette. In a fresh 1.5 mL centrifuge tube, introduce 240 μl of THF, 240 μl of QDs in THF, and 120 μl of the above solubilized PS-b-PEG-NH2 to generate the OP for EHD.

[0045] In certain embodiments, EHD may be performed by rinsing the EHD mixing system syringe (11) three times with THF. Approximately 0.6 mL of OP is loaded into the syringe barrel (12). The inorganic material is thoroughly mixed into the OP while being loaded into the syringe (11). The syringe (11) is attached to a syringe pump (15), which generates a flow rate of the OP containing the inorganic material from the syringe needle (14) in the range of about 11.00 mL / hour to about 14.00 mL / hour. In certain embodiments, the flow rate may be about 12.5 mL / hour. The syringe needle (14) is primed until a droplet of the OP forms at the end of the syringe needle (14). A 20 mL glass vial (17) is rinsed twice with distilled or deionized water (individually or collectively "DI water"). Approximately 10 mL of DI water is introduced into the 20 mL glass vial. The syringe needle (14) is immersed in the 20 mL glass vial (17). The negative electrode (19) is cleaned by immersion in THF, wiped, and rinsed with DI water. The negative electrode (19) is placed in the AP contained in the 20 mL glass vial. The positive electrode (18) is placed in the AP contained in the 20 mL glass vial (17). In certain embodiments, the syringe needle (14) may act as the positive electrode (18) if it is conductive. It is monitored to ensure that the positive electrode (18), negative electrode (19), and syringe needle (14) do not come into contact. Connect the positive lead (21) from the voltage source (20) to the positive terminal (18) or syringe needle (14), and connect the negative lead (22) from the voltage source (20) to the negative terminal (19). Ensure that the voltage source (20) supplies approximately -1500V.

[0046] The EHD-NP (4) produced by mixing the 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 cutoff centrifugal ultrafiltration column ("CUC") (e.g., Sigma-Aldrich, PN UFC9010D, Amicon® Ultra-15 Centrifugal Filter Unit). The CUC is centrifuged at 3000 RCF for 30 minutes at approximately 25°C (approximately 77°F). 10 mL of 50 mM sodium borate, 100 mM sodium phosphate, pH 7.3-7.5 ("borate buffer") is transferred to the CUC. The CUC is centrifuged at 3000 RCF for 30 minutes at approximately 25°C (approximately 77°F). The EHD-NP filtrate is transferred from the CUC into a 1.5 mL microcentrifuge tube. The volume of the collected EHD-NP filtrate is measured and recorded. Transfer 15 μl of the 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 the EHD-NP filtrate to a spectrophotometer cuvette. Measure the optical density at 450 nm (OD) using a spectrophotometer. 450 ) is measured and recorded.

[0047] The EHD-NP filtrate may contain aggregates of PENP-MultiDot (4a) or aggregates of polymer (P) (3) lacking QDs (2a) (individually and collectively "aggregates"). The aggregates may 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: transferring a 120 μl aliquot of EHD-NP filtrate from the centrifugal ultrafiltration column to a corresponding 1.5 mL tube; centrifuging the 1.5 mL tube containing the EHD-NP filtrate at 3,000 RCF for 10 minutes to pellet aggregates; removing the PENP-MultiDot (4a)-containing supernatant without disturbing the aggregate pellet; measuring and recording the volume of the PENP-MultiDot supernatant. Mix 15 μl of PENP-MultiDot supernatant with 285 μl of borate buffer in a 1.5 mL tube. Transfer 290 μl of the mixture to a spectrophotometer cuvette. Measure the optical density at 450 nm (OD ) using a spectrophotometer. 450 ) is measured and recorded.

[0048] Referring now primarily to Figures 6A and 6B, the morphology of PENP-MultiDots (4a) produced by the EHD method described above at concentrations of 0.5 v / v and 0.2 v / v of P (3) and QDs (2a) in OP can be characterized by the use of transmission electron microscopy ("TEM") to generate photomicrographs of the PENP-MultiDots (4a). PENPs produced at a QD(2a) concentration of 0.5 v / v in the OP as shown in the example of FIG. 6A can load fewer QD(2a) per PENP(4) compared to PENP(4) produced at a QD(2a) concentration of 0.2 v / v in the OP as shown in the example of FIG. 6B (which loads more QD(2a) per PENP(4)), indicating that the concentrations of P(3) and QD(2a) in the OP can be adjusted to affect the size and fluorescence per PENP-MultiDot(4a).

[0049] The PENP-MultiDot (4a) embodiment exhibits a hydrodynamic diameter ("HD") and polydispersity index ("PDI"), as well as size stability, suitable for effective clinical and non-clinical applications. The hydrodynamic diameter and PDI can be obtained by performing dynamic light scattering measurements using a NanoBrook 90 Plus particle size analyzer. A size histogram can be plotted using SigmaPlot (Systat Software Inc., San Jose, CA, USA), and the size distribution can be fitted to a log-normal distribution.

[0050] The HD is defined as the size of a hypothetical hard sphere that diffuses in the same manner as the particle being measured. However, in reality, macromolecules or particles in solution are solvated, dynamic, and non-spherical. Therefore, the diameter calculated from the particle's diffusion properties represents the apparent size of the solvated / dynamically hydrated particle. The HD of PENP-MultiDot (4A) produced by the EHD method described above can vary depending on the parameters used during EHD to produce PENP (4). TEM micrographs shown in Figures 6A and 6B show PENP-MultiDot (4a) with a substantially consistent HD of approximately 40 nm to approximately 50 nm; however, by varying the parameters used during EHD, the HD of PENP-MultiDot (4a) can vary within the range of approximately 40 nm to approximately 100 nm.

[0051] PDI is a representation of the distribution of size populations within a particle sample. PDI values ​​range from 0.0, for a completely homogeneous population within a particle sample, to 1.0, for a highly polydisperse population within a particle sample. PDI values ​​of 0.2 or less are considered acceptable in practice for polymer-based nanoparticle materials. The calculations used to determine PDI are defined in ISO standard documents 13321:1996 E and ISO 22412:2008. PENP-MultiDot (4a) can have a substantially consistent PDI of about 0.1 to about 0.2.

[0052] Referring now primarily to Figures 7A-7E, TEM micrographs show that PENP-MultiDots (4a) can be loaded with QDs (2a) within a range of emission wavelengths in the visible and near-infrared spectrum while maintaining a substantially uniform and narrow size distribution. In certain embodiments, PENP-MultiDots (4a) can be loaded with QDs (2a) or upconversion nanoparticles having emission wavelengths ranging from about 420 nm to about 880 nm. The illustrative examples in Figures 7A-7E show PENP-MultiDots (4a) with emission wavelengths of 420 nm (as shown by the example in Figure 7A), 575 nm (as shown by the example in Figure 7B), 610 nm (as shown by the example in Figure 7C), and 655 nm (as shown by the example in Figure 7D). A long-felt but unresolved concern related to nanocomposites is the lack of uniform and narrow size distribution among nanocomposites containing QDs (2a) with various emission wavelengths. As an illustrative example, QDs (2a) having an emission wavelength of 420 nm may be smaller in size than QDs (2a) having an emission wavelength of 610 nm. Furthermore, for a given composition, the larger the QDs (2a), the brighter they are. Furthermore, under substantially similar preparation, nanocomposites are loaded with a greater number of QDs (2a) having an emission wavelength of 420 nm compared to QDs (2a) having an emission wavelength of 610 nm. These differences between QDs (2a) with varying emission wavelengths in one or more of size, brightness, and loading affinity under similar preparation methods can result in nanocomposites with correspondingly different sizes and brightnesses. Nanocomposite embodiments containing QDs (2a) with various emission wavelengths can be produced with uniform and narrow size distributions by adjusting the ratio of QD inorganic metal mass to polymer mass in the organic phase (OP) introduced into the aqueous phase (AP) during EHD mixing.In the PENP-MultiDot (4a) embodiment, the QD inorganic metal mass to polymer mass ratio can be adjusted between 1:1 and 1:2 to produce PENP-MultiDot (4a) with a uniform and narrow size distribution among nanocomposites containing QD (2a) with various emission wavelengths within the range of 40 nm to 500 nm and substantially similar brightness. In the PENP-MagDot (4b) embodiment, the QD and SPION inorganic metal mass to polymer mass ratio can be adjusted between 1:2 and 1:4 to produce PENP-MagDot (4b) with a uniform and narrow size distribution among nanocomposites containing QD (4b) with various emission wavelengths within the range of 40 nm to 500 nm and substantially similar brightness. These illustrative examples are not intended to exclude other embodiments of PENP-MultiDot (4a) that can be produced as described above, which have other single-wavelength emission, dual-wavelength emission, or emission at more than two wavelengths, depending on the QD (2a) or QDs (2a) encapsulated in the polymer (P) (3).

[0053] (PENP-MagDot) Superparamagnetic iron oxide nanoparticles ("SPIONs") (2b) are small synthetic particles of maghemite (CAS No. 1309-37-1) with the empirical formula Fe2O3, γ-Fe2O3, and a molar mass of 159.69 g / mol, or magnetite (CAS No. 1317-61-9) with the empirical formula Fe3O4 and a molar mass of 231.53 g / mol, or combinations thereof, with cores ranging in diameter from about 5 nm to about 30 nm. Additionally, mixed oxides of iron and transition metal ions (e.g., copper, cobalt, nickel, and manganese) are known to exhibit superparamagnetic properties and also fall into the SPION (2b) category. However, magnetite and maghemite nanoparticles are the most widely used SPIONs (2b) in various biomedical applications. SPIONs (2b) exhibit the phenomenon of "superparamagnetism." Particles that exhibit superparamagnetism are magnetized to their saturation magnetization upon application of an external magnetic field (15) (as shown in the example in Figure 5 ), and no longer exhibit any remanent magnetic interactions upon removal of the field. This property is size-dependent and generally increases when the nanoparticle size reaches approximately 5 nm. At such small sizes, these nanoparticles do not exhibit the multi-domain structure seen in larger magnets; instead, they become single-domain and act as a "single superspin" with high magnetic susceptibility. Therefore, upon application of a magnetic field, these nanoparticles provide a strong and rapid magnetic response compared to bulk magnets, which have negligible residual magnetic flux density (remanent magnetization) and coercivity (the field required to nullify their magnetism). Once the applied magnetic field is removed, the magnetic particles retain no remanent magnetism at room temperature and are easily dispersed. Uncoated iron oxide NPs have very low solubility, which can lead to sedimentation due to gravity and also to high aggregation rates under physiological conditions. Therefore, to be used effectively, SPIONs can be capped with an amphiphilic coating.

[0054] In certain embodiments, SPIONs 2b capped with ligands 16 (e.g., oleic acid) can have particle sizes ranging from about 5 nm to about 30 nm. Various SPIONs 2b are available from Ocean NanoTech, such as PN SOR05 through SOR30. Illustrative examples of embodiments include oleic acid-capped SPIONs 2b with particle sizes of 5 nm, 10 nm, 15 nm, and 20 nm. However, these exemplary SPION 2b particle sizes are not intended to preclude the use of SPIONs 2b with smaller or larger particle sizes, or combinations of SPION 2b particle sizes, which may be used alone or in combination with one or more QDs 2a in PENP 4 embodiments.

[0055] Typically, capped SPIONs (2b) according to the present invention achieve the required level of water solubility and biocompatibility by surrounding the SPIONs (2b) or SPION / QD (2a, 2b) combinations with a polymer (3). Polymer (3) encapsulation layers suitable for use in embodiments of the present invention include PS-b-PEG-NH2, obtained in Block 21 of the synthetic process described above. Embodiments of the present invention can be produced through the use of various combinations of one or more of the following: SPIONs (2b) or SPION / QDs (2a, 2b), capping ligand (16), and PS-b-PEG-NH2, with 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.

[0056] An illustrative example of a scalable method for producing PENP-MagDots (4b) by EHD may include one or more of the following: PS-b-PEG-NH2 obtained in Block 2I of Figure 2 at 10 mg / mL into a first 1.5 mL tube; 13 μl of the desired SPION (2b) particles (e.g., 20 nm) from 25 mg / mL in toluene are introduced into a second 1.5 mL tube; 52 μl of acetone / methanol (60 / 40) is transferred to the SPIONs in the second 1.5 mL tube; Mix by shaking; Centrifuge the second 1.5 mL tube containing the mixed SPIONs (2b) at 7000 RPM for 1 minute at about 25°C (about 77°F); Immediately remove the supernatant with a 200 μl pipette. Immediately, 13 μl of anhydrous tetrahydrofuran ("THF") is transferred to the second 1.5 mL tube containing the SPIONs (2b) and mixed thoroughly with a pipette. 65 μl of QDs (2a) (from 5 mg / mL in toluene) is transferred into a third 1.5 mL microcentrifuge tube. 130 μl of acetone / methanol (60 / 40 v / v) is transferred to the third 1.5 mL microcentrifuge tube containing the QDs. Gently mix the QDs (2a) by tilting. Centrifuge the third 1.5 mL microcentrifuge tube containing the QDs at 7000 RPM at 25°C (approximately 77°F) for 1 minute. Remove the supernatant with a 200 μl pipette. Transfer 65 μl of THF to the third 1.5 mL microcentrifuge tube. Mix thoroughly with an air-displacement pipette. Add 322 μL of THF to a 4 mL vial. Transfer SPION (2b) in 13 μL of THF from a second 1.5 μL microcentrifuge tube to the 4 mL vial. Optionally, transfer QD (2a) in 65 μL of THF from a third 1.5 mL microcentrifuge tube to the 4 mL vial. Transfer 100 μL of the PS-b-PEG-NH2 solution to the 4 mL vial to generate the OP for EHD.

[0057] In certain embodiments, EHD can be performed by rinsing the EHD mixing system syringe (11) three times with THF. Approximately 0.5 mL of OP is loaded into the syringe barrel (12). The inorganic material is thoroughly mixed into the OP while being loaded into the syringe (11). The syringe (11) is attached to a syringe pump (15), which generates a flow rate of the OP containing the inorganic material from the syringe needle (14) in the range of about 11.00 mL / hr to about 14.00 mL / hr. In certain embodiments, the flow rate can be about 12.5 mL / hr. The syringe needle (14) is primed until a droplet of the OP forms at the end of the syringe needle (14). A 20 mL glass vial (17) is rinsed twice with distilled or deionized water (individually or collectively, "DI water"). Approximately 10 mL of DI water is introduced into the 20 mL glass vial (17). The syringe needle (14) is immersed in the 20 mL glass vial (17). The negative electrode (19) is cleaned by immersion in THF, wiped, and rinsed with DI water. The negative electrode (19) is placed in the AP contained in the 20 mL glass vial (17). The positive electrode (18) is placed in the AP contained in the 20 mL glass vial (17). In certain embodiments, the syringe needle (14), if conductive, may act as the positive electrode (18). It is monitored to ensure that the positive electrode (18), negative electrode (19), and syringe needle (14) do not come into contact. Connect the positive lead (21) from the voltage source (20) to the positive terminal (18) or syringe needle (14) and connect the negative lead (22) from the voltage source (20) to the negative terminal (19). Ensure that the voltage source provides approximately -1000V.

[0058] EHD-PENP (4), produced by mixing the organic phase 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 cutoff centrifugal ultrafiltration column (PN UFC9010D, Amicon® Ultra-15 Centrifugal Filter Unit) available from Sigma-Aldrich. Centrifuged at 2000 RCF for 30 minutes at approximately 25°C (approximately 77°F). EHD-PENP (4) is transferred from the centrifugal ultrafiltration column to a 1.5 mL microcentrifuge tube. The volume of the EHD-NP is measured and recorded. 5 μL of the EHD-PENP (4) filtrate is transferred from the 1.5 mL microcentrifuge tube to a 1.5 mL tube, and 295 μL of borate buffer is added. 290 μL of EHD-PENP (4) is transferred to a spectrophotometer cuvette. The optical density (OD) at 450 nm was measured using a spectrophotometer. 450 ) is measured and recorded.

[0059] The EHD-PENP filtrate may contain aggregates of PENP (4) or aggregates of PENP lacking SPION (Sb) or QD (2a) (collectively "aggregates"). The aggregates may be substantially removed from the EHD-NP filtrate to produce substantially pure PENP-MagDots (4b). As an example, a scalable size-exclusion process for purifying PENP-MagDots (4b) may include: placing a magnetic particle separation column ("MPSC") in a magnetic field gradient (e.g., to about 100-200 T / m); transferring 300 μl of borate buffer into the MPSC; allowing the borate buffer to pass through the MPSC; introducing the EHD-NP filtrate into the MPSC in a magnetic field (23); allowing the filtrate to pass through the MPSC; transferring 300 μl of borate buffer into the MPSC; mixing the EHD-NP with the borate buffer in the MPSC. Allow all of the borate buffer to pass through the MPSC. Withdraw the MPSC from the magnetic field gradient. Once the MPSC is withdrawn from the magnetic field gradient (23), it can no longer retain the PENP-MagDots (4b), which can then be eluted. Remove any liquid from the tip of the MPSC. Place the MPSC in a PENP-MagDot (4b) collection tube labeled PENP-MagDot-wavelength. Transfer 200 μl of borate buffer into the MPSC. Pipette the PENP-MagDots (4b) into the borate buffer in the MPSC. Collect the PENP-MagDots (4b) in the PENP-MagDot-wavelength collection tube. Mix the collected PENP-MagDots (4b) thoroughly with an air-displacement pipette. Measure and record the volume of the collected PENP-MagDots (4b). Combine 5 μl of PENP-MagDot (4b) with 295 μl of borate buffer. Transfer 290 μl of the sample to a spectrophotometer cuvette. Measure the optical density at 450 nm (OD ) using a spectrophotometer. 450 ) is measured and recorded.

[0060] Referring now primarily to Figures 8A-8C, which contain TEM micrographs, the TEM micrographs show PENP-MagDots 4b produced by the above method loaded with SPIONs 2b and, optionally, a combination of SPIONs 2b and QDs 2a having emission wavelengths in the visible and near-infrared spectrum. In illustrative examples, PENP-MagDots 4b contain one or more QDs 2a having an emission wavelength of 610 nm in combination with 20 nm (as shown in the example of Figure 8A), 15 nm (as shown in the example of Figure 8B), and 5 nm (as shown in the example of Figure 8C), respectively. The morphology of PENP-MagDots 4b produced by the EHD-EM-NP method can be characterized by the use of TEM images and dynamic light scattering of PENP-MagDots 4b to determine HD and PDI, as described above.

[0061] Referring now primarily to Figures 9A and 9B, which contain TEM micrographs, the TEM micrographs show PENP-MagDots (4b) produced according to the above method. DLS can also be used to determine the size of PENP-MagDots (4b). During DLS measurements, a suspension of PENP-MagDots (4b) can be exposed to a light beam, and when the incident light strikes the PENP-MagDots (4b), the direction and intensity of the light beam can be altered due to scattering. PENP-MagDots (4b) in suspension are in constant random motion due to their kinetic energy; therefore, the change in intensity over time contains information about their random motion and can be used to measure the diffusion coefficient of the particles. The R of the PENP-MagDots H HD is the Stokes-Einstein formula D f =k B T / 6πηR H (In the formula, k B is the Boltzmann constant, T is the temperature of the suspension, and η is the viscosity of the surrounding medium).

[0062] Referring now primarily to Figure 9A, which contains 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), and Figure 9B, which contains TEM micrographs of PENP-MagDot (4b) (“PENP-MagDot5”) prepared using a ratio of QD (2a):5 nm SPION (2b):polymer (3:5:20), they demonstrate that the brightness and magnetic susceptibility of MagDots can be tuned by loading SPIONs of various sizes. PENP-MagDot15 produced using the 5:5:20 ratio can be more magnetic than PENP-MagDot5 using the 3:5:20 ratio. PENP-MagDot 5 produced using the ratio 3:5:20 can be brighter than PENP-MagDot 15 using the ratio 5:5:20.

[0063] Referring now primarily to Figure 9C, the nanoparticle tracking analysis (NTA) for each of the PENP-MagDot15 populations shown in the example of Figure 9A can be plotted as a plot of particle size versus particle concentration, indicating that the HD of each population can have a uniform size with a PDI of about 0.1 to about 0.2. The determined HD of PENP-MagDot15 can be about 120 nm to about 140 nm, and the determined HD of PENP-MagDot5 can be about 180 nm to about 220 nm.

[0064] Based on the above TEM, DLS, or NTA analyses, the HD of PENP-MagDot (4b) produced by the above EHD method may vary depending on the parameters used during EHD and may be within the range of about 40 nm to about 500 nm, indicating that the PENP-MagDot (4b) population exhibits a substantially uniform HD and a PDI of about 0.1 to about 0.2.

[0065] Referring now primarily to Figure 9D, which contains a bar graph comparing the iron concentrations of PENP-MagDot 5 and PENP-MagDot 15, it shows that PENP-MagDot 15 can be produced with a greater iron concentration than PENP-MagDot 5.

[0066] Referring now primarily to Figure 9E, which contains a bar graph comparing the fluorescence of PENP-MagDot 5 and PENP-MagDot 15, it shows that PENP-MagDot 5 can be produced with greater fluorescence than PENP-MagDot 15.

[0067] Thus, by preselecting one or more of the SPION (2b) size, QD (2a), PS-b-PEG-NH2 (3), which may comprise 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, and EHD parameters and QD:SPION:polymer ratio, many different embodiments of PENP-MagDot (4b) can be generated, correspondingly having utility in many clinical and non-clinical applications.

[0068] (Antibody Preparation) Referring again primarily to FIG. 1 , the polymer nanomaterial encapsulation system (1) can further include the preparation of an antibody (Ab) (5). In certain embodiments, PENP (4) generated by FNP, EHD, EM-NPa, self-assembly, or other means can be a target for site-specific conjugation to an antibody (Ab) (5′). As an illustrative example, a half-antibody can be generated by preferential reduction of disulfide bonds in the antibody hinge region to yield a monovalent component with a free thiol group (“SH”) that can be used for site-specific conjugation to PENP, PENP-MultiDot, PENP-MagDot, or a combination thereof. The reduced antibody fragment can be prepared by reacting 2-mercaptoethylamine hydrochloride (2-MEA), dithiothreitol, mercaptoethanol, or tris(2-carboxyethyl)phosphine (TCEP) to generate a reduced half-antibody as shown in Formula IX. [ka]

[0069] However, this example is not intended to exclude embodiments that may include one or more of the following: a whole antibody molecule, or its F(ab')2 fragment, Fab' fragment, Fv fragment, rIgF fragment, Fc fragment obtained after pepsin or other enzymatic digestion, or a combination thereof.

[0070] A specific illustrative example of a scalable process for producing half antibodies useful for conjugation to PENP (4), PENP-MultiDot (4a), and / or PENP-MagDot (4b) can include the following: preparing an antibody stock solution containing immunoglobulin G ("IgG") (0.5 mg / mL) in a pH 7.4 buffer solution of 100 mM phosphate-buffered saline ("PBS") and 10 mM ethylenediaminetetraacetic acid disodium salt dihydrate ("EDTA") ("PBS buffer solution"). An aliquot of 160 μl of the IgG stock solution can be transferred to a first 1.5 mL tube. 2-MEA.HCl (CAS number: 156-57-0), which has the linear formula HSCHCHNHHCl and a molecular weight of 113.60, can be obtained from Fisher Scientific under PN AAA1437714. In a second 1.5 mL tube, 15 mg of 2-MEA.HCl can be dissolved in 264 μl of PBS buffer solution by pipetting. 5.0 μl of the 2-MEA.HCl solution is transferred to 160 μl of the IgG stock solution in the first 1.5 mL tube, respectively, vortexed, and the 1.5 mL tube containing the IgG solution is placed in a 37° C. (approximately 98.6° F.) water bath for 90 minutes.

[0071] 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 while exchanging the half antibody into a reaction buffer for conjugation to PENP (4), PENP-MultiDot (4a), and / or PENP-MagDot (4b). An illustrative example of a scalable procedure for desalting and buffer exchange is ZEBA®, available from ThermoFisher Scientific (PN 89883). (商標)This may involve the use of a spin desalting column, or its equivalent. A pair of desalting columns may be prepared by removing the bottom plugs of each column and placing them in a 1.5 mL collection tube. Centrifuge at 1500 × g for 1 minute to remove the storage solution from the desalting column resin. Add 300 μl of 1× 100 mM sodium phosphate and 1 mM EDTA in water (pH 6.95-7) ("rinse buffer") or an appropriate buffer to the top of the resin bed and centrifuge at 1500 RFC for 1 minute. Discard the flow-through from the collection tube. Repeat the addition of 300 μl of 1× rinse buffer two more times, discarding the buffer from the collection tube each time.

[0072] The desalting column can now be loaded with reduced antibody (5) product by placing the first equilibrated desalting column into a 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 pouring up to 160 μl of reduced antibody product onto the center of the dense resin bed. For sample volumes less than 70 μl, pour 15 μl of buffer (stacker) on top of the resin bed after the sample is fully absorbed to ensure maximum protein recovery. Collect the desalted reduced antibody product by centrifugation at 1500 RCF for 2 minutes at 15°C to 20°C (59°F to 68°F). 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. The collected reduced antibody (5) product is transferred from collection tube R-Ab-1 onto the second desalting column. Centrifuge at 1500 RCF for 2 minutes at 15°C to 20°C (59°F to 68°F) to collect the desalted reduced antibody product into collection tube R-Ab-2. Cap collection tube R-Ab-2 and place at room temperature.

[0073] (Preparation of half antibody-polymer nanocomposite conjugate) Referring now primarily to Figure 1, the polymer nanomaterial encapsulation system (1) can further include surface functionalization of PENP (4), PENP-MultiDot (4a), and / or PENP-MagDot (4b) by addition of a reduced antibody product (Block 1D in Figure 1). Chemical groups (e.g., amines, carboxylates, thiols, or other reactive groups) can be introduced into PENP (4), PENP-MultiDot (4a), and / or PENP-MagDot (4b) during the above synthesis as a basis for modification. These groups can be targeted by heterobifunctional chemical crosslinkers (10) of various lengths and functionality, containing reactive chemical groups linked by spacer arms (e.g., alkanes, polyethylene glycol, cleavable disulfide spacers) that impart flexibility, solubility, or other desirable characteristics to the functionalized PENP (4), PENP-MultiDot (4a), and / or PENP-MagDot (4b). For example, PENP functional group (9) can be reacted with the first reactive group (10') of a heterobifunctional chemical crosslinker (10). As an illustrative example, if functional group (9) of PENP (4), PENP-MultiDot (4a), and / or PENP-MagDot (4b) contains an amine, the amine can be reacted with a succinimidyl ester to form an amide bond; if functional group (9) contains a carboxylate, the carboxylate can be reacted with a carbodiimide to generate an O-acylisourea intermediate, which can be reacted with an amine to form an amide bond; or if functional group (9) contains a thiol, the thiol can be reacted with a maleimide to form a thioether bond. The reduced antibody (5) product can be reacted with the second reactive group (10'') of a heterobifunctional chemical crosslinker (shown in the example of Figure 4C). For example, the second reactive group (10'') of the heterobifunctional chemical cross-linker (10) may comprise a maleimide, which may be reacted with a sulfhydryl of the half antibody (5) to form a thioether bond.

[0074] In certain embodiments, the reactive group associated with PENP (4) can be modified by reaction with a first reactive group (10a') of a first heterobifunctional crosslinker (10a), and the reduced antibody (5) product can be modified by reaction 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 contain second reactive groups (10a", 10b") that remain stable in physiological aqueous buffers and, upon mixing, proceed with fast kinetics and high selectivity to form covalent bonds.

[0075] In certain embodiments, the reduced antibody (5) product in collection tube R-Ab-2 can be modified for subsequent conjugation to PENP (4) by reaction of the free sulfhydryl groups of half antibody (5) with the maleimide-containing first reactive group (10a') of first heterobifunctional crosslinker (10a) to form a thioether bond. In an illustrative example, first heterobifunctional crosslinker (10a) is expressed by the linear formula C 17 H 27 It can include sulfotrans-cyclooctenemaleimide (“Sulfo-TCOmaleimide”), which has N3O8S (CAS number not available) and a molecular weight of 457.50 g / mol, and can be obtained from Click Chemistry Tools, PN 1355, which has formula X. [ka]

[0076] Sulfo-TCO-maleimide constitutes a water-soluble reagent that allows incorporation of the TCO moiety onto thiol-containing half-antibodies. The maleimide group reacts specifically with sulfhydryl groups ("SH") at pH 6.5-7.5 to form stable thioether bonds. The hydrophilic sulfonated spacer arm greatly improves the water solubility of sulfo-TCO antibodies ("sulfo-TCO-Abs") containing Formula XI. [ka]

[0077] An illustrative example of a scalable process for producing sulfo-TCO-Ab useful for conjugation to PENP may include one or more of the following: Remove a tube containing 0.1 mg of sulfo-TCO-maleimide from -20°C (-4°F) and allow it to come to room temperature for 15 minutes. Add 46.4 μl of anhydrous dimethyl sulfoxide ("DMSO") (CAS No. 67-68-5), which has the linear formula CHOS and a molecular weight of 78.13 g / mol, to 0.1 mg of sulfo-TCO-maleimide. Vortex the sulfo-TCO-maleimide in the tube for 10 minutes to ensure dissolution in the DMSO. Mix the sulfo-TCO-maleimide repeatedly by pipetting into the DMSO and 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) described above. Discard the remaining sulfo-TCO-maleimide. Relabel 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 hours.

[0078] The PENP (4) product can be reacted with a first reactive group of a second heterobifunctional crosslinker, and the modified PENP (4) can then be associated with one or more targeting moieties (24) (including one or more of the following: cells, organelles, proteins, peptides, amino acids, oligonucleotides, ligands, or linkers) by selective reaction of the second reactive group with a corresponding functional group within the targeting moiety (24).

[0079] In certain embodiments, the second heterobifunctional crosslinker (10b) has a linear formula C 24 H 31The compound may include methyltetrazine polyethylene glycol-4-N-hydroxysuccinimide ester ("TZ-PEG4-NHS ester") (CAS number 182907-92-1), having Formula XII, having N5O9 and a molecular weight of 533.53 g / mol. [ka]

[0080] As an illustrative example, TZ-PEG4-NHS ester containing formula XIII can be reacted with functional group (9) of multi-arm PEG-NH2 (3) containing multiple amines as shown in formula IV or formula V of PENP (4) to produce PENP-PEG4-TZ containing formula XIII. [ka]

[0081] As an example, a scalable process for producing PENP-PEG4-TZ of Formula XIII using the amine-containing PENP (4) functional group (9) of the multi-arm PEG-NH2 (3) shown in Formula IV or Formula V can include one or more of the following: Bring 1 mg of TZ-PEG-NHS ester to room temperature for approximately 15 minutes; Transfer 93.7 μl or 375 μl of anhydrous DMSO to the TZ-PEG4-NHS ester to produce 20 mM or 5 mM TZ-PEG4-NHS in DMSO; Mix the TZ-PEG4-NHS ester into the DMSO by pipetting to ensure complete dissolution of the TZ-PEG4-NHS ester in DMSO; Label a 1.5 mL tube as PENP-wavelength-TZ; Transfer PENP to the PENP-wavelength-TZ tube (120 μl of PENP-MagDot or 80 μl of PENP-MultiDot). In the PENP-MultiDot (4a) embodiment, approximately 30 nmol of amines can be activated by reaction with approximately 70 nmol of TZ-PEG4-NHS ester, and in the PENP-MagDot (4b) embodiment, approximately 120 nmol of amines can be activated by reaction with 70 nmol of TZ-PEG4-NHS ester. 3.5 μL of 20 mM TZ-PEG4-NHS ester is pipetted into the PENP-MultiDot-wavelength-TZ tube, or 15 μL of 5 mM TZ-PEG4-NHS ester is pipetted into the PENP-MagDot-wavelength-TZ tube. Mix thoroughly with an air-displacement pipette. Allow to react at room temperature for 2 hours.

[0082] In certain embodiments, PENP-PEG4-TZ comprising Formula XIII is reacted with sulfo-TCO-Ab comprising Formula XI in an inverse electron demand Diels-Alder [4+2] cycloaddition reaction of TCO and TZ to form a dihydropyridazine bond to produce a conjugate PENP-antibody (6) comprising Formula XIV ("PENP-Ab") (including, but not limited to, PENP-MultiDot (4a) shown in the example of FIG. 4A, or PENP-MagDot (4b) shown in the example of FIG. 4B), where R1 comprises PENP and R2 comprises Ab. [ka]

[0083] As an example, a scalable process for producing PENP-Ab (6) of Formula XIV using PENP-PEG4-TZ (PENP-MagDot-PEG4-TZ or PENP-MultiDot-PEG4-TZ) and sulfo-TCO-Ab can be performed using a pair of desalting columns (as a non-limiting example, Zeba). (商標) The desalting column pair may comprise a desalting column, each of which is prepared by removing the bottom plug and placing each of the desalting columns in a 1.5 mL collection tube. Centrifuge at 1500 × g for 1 minute to remove the storage solution from the desalting column resin. Add 300 μL of 1× rinse buffer or an appropriate buffer onto the resin bed and centrifuge at 1500 RFC for 1 minute. Discard the flow-through from the individual collection tube. Repeat the addition of 300 μL of 1× rinse buffer (pH 6.0) two more times, discarding the buffer from the individual collection tube each time. Transfer the PENP-PEG4-TZ to the first desalting column of the pair. Transfer the sulfo-TCO-Ab to the second desalting column of the pair. The first and second desalting columns were centrifuged at 1500 RCF for 2 minutes at 15°C–20°C (59°F–68°F) to collect the PENP-PEG4-TZ and sulfo-TCO-Ab products after removing excess unbound linker. The desalted PENP-PEG4-TZ (120 μL of PENP-MagDot or 80 μL of PENP-MultiDot) was mixed in the first collection tube by pipetting. 160 μL (80 μg) of the desalted sulfo-TCO-Ab was transferred from the second collection tube to the PENP-PEG4-TZ in the first collection tube. The mixture was allowed to react for 24 hours at 4°C (39.2°F) to produce PENP-Ab (PENP-MagDot-Ab or PENP-MultiDot-Ab).

[0084] After conjugation, the resulting PENP-Ab (6) product can be purified. As an example, a scalable size-exclusion process for purifying PENP-MultiDot-Ab can include the following: Label a 1.5 mL microcentrifuge tube "PENP-MultiDot-Ab-wavelength." 3 mL of gel filtration resin (e.g., Sephacryl® available from Sigma-Aldrich (PN S400HR)) is introduced into 3 mL of borate-buffered saline (50 mM sodium borate, 100 mM sodium phosphate, pH 7.3-7.5 ("borate buffer")) ("BBS"). The gel filtration resin suspension is transferred into a size-exclusion column ("SEC"). Slowly pipette the PENP-MultiDot-Ab (6a) solution onto the end of the SEC. Attach an air syringe to the SEC. Push 1 mL of air into the SEC via the air syringe. Transfer the PENP-MultiDot-Ab (6a) solution into the gel filtration resin. Disconnect the air syringe from the SEC. Pipette 200 μl of BBS buffer onto the end of the SEC. Reconnect the air syringe to the SEC. Push 1 mL of air into the SEC via the air syringe. Transfer the PENP-MultiDot-Ab (6a) solution into the SEC packing. Disconnect the air syringe from the SEC. Pipette 1000 μl of BBS onto the end of the SEC. Reconnect the air syringe to the SEC. Reduce ambient light incident on the SEC. Direct ultraviolet light ("UV light") onto the SEC. Push 1 mL of air into the SEC via the air syringe. Begin collecting the BBS eluate from the SEC. Upon detecting the first fluorescent droplet from the SEC, collect fluorescent droplets 2 through 5 into a PENP-MultiDot-Ab-wavelength collection tube. The collected fluorescent droplet sample is mixed with a pipette. The air syringe is removed, the gel filtration resin is discarded, and the SEC is cleaned.

[0085] As an example, a scalable size exclusion process for purifying PENP-MagDot-Ab (6b) may include the following: thoroughly mix PENP-MagDot-Ab (6b) into the conjugation buffer using an air-displacement pipette; place an MPSC in a magnetic field gradient (e.g., to about 100-200 T / m); introduce PENP-MagDot-Ab (6b) in the conjugation buffer into the MPSC; allow the conjugation buffer to pass through the MPSC; transfer 300 μL of BBS into the MPSC; pipette PENP-MagDot-Ab to mix with the reaction buffer in the MPSC; allow the reaction buffer to pass through the MPSC; transfer 300 μL of reaction buffer into the MPSC; pipette PENP-MagDot-Ab (6b) to mix with the reaction buffer in the MPSC; allow the reaction buffer to pass through the MPSC; remove any reaction buffer from the tip of the MPSC. The MPSC is withdrawn from the magnetic field gradient. Once the MPSC is withdrawn from the magnetic field gradient, it can no longer retain the PENP-MagDot-Ab (6b), allowing the PENP-MagDot-Ab (6b) to be eluted. The MPSC is placed in a PENP-MagDot-Ab (6b) collection tube labeled PENP-MagDot-Ab-. 200 μl of reaction buffer is transferred into the MPSC. PENP-MagDot-Ab (6b) is mixed into the reaction buffer in the MPSC by pipetting. PENP-MagDot-Ab (6b) is collected in a MagD-antibody-date collection tube. The collected PENP-MagDot-Ab (6b) is thoroughly mixed with an air-displacement pipette.

[0086] (Cell labeling and flow cytometry using PENP-MultiDot-Ab) PENP-MultiDot-Ab (6a) can effectively and specifically label target moieties (24), including cellular targets (24'). Single particle imaging, cell imaging, or flow cytometry using cellular targets (24') labeled with PENP-MultiDot-Ab (6a) demonstrates significantly higher fluorescence intensity than labeling cellular targets (24') with conventional dyes. PENP-MultiDot-Ab (6a) can effectively and specifically label cell surface receptors and intracellular structures without any detectable nonspecific binding in both live and fixed cells. Flow cytometry can be performed to evaluate the performance of PENP-MultiDot-Ab (6a) and demonstrate the high brightness of cell labeling compared to conventional dyes and quantum dot probes.

[0087] As an illustrative example of PENP-MultiDot-Ab (6a) labeling of cellular targets (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: diluting peripheral blood 50:50 with Hank's Balanced Salt Solution ("HBSS") (Sigma Aldrich, PN H6648) as an isotonic solution. The diluted peripheral blood is then passed through a Ficoll-Hypaque gradient (density = 1.077 g / cm). 3) and centrifuged at 1350 RPM for 30 minutes without interruption. Serum can be aspirated and discarded. PBMCs can be removed and transferred to another collection tube and washed with PBS. The PBMC pellet can be resuspended in 10 mL of PBS, and a cell count can be performed using a hemocytometer. One million PBMCs (24') in 200 μl of PBS can be placed in a 12 × 75 mm flow cytometry tube to which 10 μl of PENP-MultiDot-mouse anti-human CD3 (6a) with an emission wavelength of 610 nm has been added. PBMCs (24') and PENP-MultiDot-mouse anti-human CD3 (6a) are incubated for 25 minutes at room temperature. After the incubation period, 500 μl of additional PBS can be added to the PENP-MultiDot-mouse anti-human CD3-bound PBMCs (7) ("PENP-MultiDot-mouse anti-human CD3-PBMCs"). The PENP-MultiDot-mouse anti-human CD3-PBMCs (7) were centrifuged at 1800 RPM for 7 minutes at room temperature. The supernatant was discarded, and the PBMCs / PENP-MultiDot-mouse anti-human CD3-PBMCs (7) were resuspended in 400 μl of PBS for flow cytometry analysis (8) using a Cytek Northern Lights Spectral flow cytometer to detect the PENP-MultiDot-mouse anti-human CD3-PBMC (7) population. The CD3 protein complex can be an important T cell marker for the 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.

[0088] Referring now primarily to Figure 10A, the univariate histogram (fluorescence intensity vs. particle count) shows the detection of a PENP-MultiDot-mouse anti-human CD4-PBMC(7) population ("Peak M1") in a PBMC / PENP-MultiDot-mouse anti-human CD4-PBMC(7) sample obtained by the method described above. The histogram indicates that 38.41% of the cells in the sample can be identified as the PENP-MultiDot-mouse anti-human CD4-PBMC(7) population within the sample. In certain embodiments, the PENP-MultiDot-mouse anti-human CD4-PBMC(7) population can be sorted and isolated for further analysis.

[0089] Referring now primarily to Figure 10B, the univariate histogram (fluorescence intensity vs. particle number) shows the detection of the PENP-MultiDot-mouse anti-human CD3-PBMC(7) population ("Peak M1") prepared according to the method described above. The histogram indicates that 50.09% of the cells in the sample can be identified as the PENP-MultiDot-mouse anti-human CD3-PBMC(7) population within the sample.

[0090] PENP-MagDot-Ab (6b) can also effectively and specifically label cellular targets (24'). Single particle imaging, cell imaging, or flow cytometry analysis (8) using PENP-MagDot-Ab (6b)-labeled cellular targets (24') demonstrates significantly higher fluorescence intensity than labeling cellular targets (24') with conventional dyes or quantum dots. PENP-MagDot-Ab (6b) can effectively and specifically label cell surface receptors and intracellular structures without any detectable nonspecific binding in both live and fixed cells. Flow cytometry (8) can be performed to evaluate the performance of PENP-MagDot-Ab (6b) and demonstrate the high brightness of cell labeling compared to conventional dye and quantum dot probes.

[0091] As an illustrative example of PENP-MagDot-Ab (6b) labeling of cellular targets (24'), peripheral blood mononuclear cells ("PBMCs") (24') may be labeled using PENP-MagDot-Ab (6b). By way of example, a scalable method may include one or more of the following: diluting peripheral blood 50:50 with Hank's Balanced Salt Solution ("HBSS") (Sigma Aldrich, PN H6648) as an isotonic solution. The diluted peripheral blood is then passed through a Ficoll-Hypaque gradient (density = 1.077 g / cm). 3) and centrifuged at 1350 RPM for 30 minutes without interruption. Serum can be aspirated and discarded. PBMCs can be removed and transferred to a PBMC collection tube and washed with PBS. The PBMC pellet can be resuspended in 10 mL of PBS, and a cell count can be performed using a hemocytometer. One million PBMCs (7) in 200 μL of PBS can be introduced into a 12×75 mm flow cytometry tube to which 20 μL of PENP-MagDot-mouse anti-human CD3 (6b) with a 610 nm emission spectrum has been added. The PBMCs and PENP-MagDot-mouse anti-human CD3 (6b) are incubated at room temperature for 25 minutes. After the incubation period, 200 μl of additional PBS can be added to the PENP-MagDot-mouse anti-human CD3-bound PBMCs (7) ("PENP-MagDot-mouse anti-human CD3-PBMCs") in the flow cytometry tube. The flow cytometry tube containing the PENP-MagDot-mouse anti-human CD3-PBMCs (7) in PBS is placed in a magnetic field gradient (23) (e.g., approximately 100-200 T / m). After 15 minutes, the non-magnetic fraction can be aspirated from the flow cytometry tube and placed in a non-magnetic fraction collection tube. The flow cytometry tube containing the magnetically retained PENP-MagDot-mouse anti-human CD3-PBMCs (7) can be removed from the magnetic field gradient, and the PENP-MagDot-mouse anti-human CD3-PBMCs (7) can be resuspended in 400 μl of 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 were each analyzed using a flow cytometer (8).

[0092] Referring now primarily to Figures 11A-11C, they show bivariate dot plots (forward scatter area vs. forward scatter height) obtained by flow cytometry analysis (8) of the PBMC / PENP-MagDot-mouse anti-human CD3 reaction solution before magnetic separation (as shown by the example in Figure 11A), the non-magnetic fraction after magnetic separation (as shown by the example in Figure 11B), and the PENP-MagDot-mouse anti-human CD3-PBMC fraction after magnetic separation (as shown by the example in Figure 11C). These flow cytometry dot plots show the ratio of CD3-negative cells to CD3-positive cells. As shown in Figure 11A, the PBMC / PENP-MagDot-mouse anti-human CD3 reaction solution before magnetic separation contains 51.17% CD3-negative cells and 48.76% CD3-positive cells. Figures 11B and 11C show that after magnetic separation, the non-magnetic fraction contains a large number of CD3-negative cells (97.29%) and very few CD3-positive cells (2.71%), whereas Figure 11C shows that after magnetic separation, the PENP-MagDot-mouse anti-human CD3-PBMC fraction contains very few CD3-negative cells (2.60%) and a large number of CD3-positive cells (97.37%), demonstrating the important advantage of using PENP-MagDot-Ab (6b) to capture and purify cellular targets (24').

[0093] Referring now primarily to Figures 12A-12C, they show the labeling of Hawaiian bobtail squid (Euprymna scolopes) hemocytes with PENP-MagDots. Adult bobtail squid were incubated for 3 hours with 100 μL of PENP-MagDots containing QDs with an emission wavelength of 610 nm. The images show that Hawaiian bobtail squid (Euprymna scolopes) hemocytes passively take up PENP-MagDots 610 nm.

[0094] As can be readily appreciated from the above, the basic concepts of the present invention can be embodied in a variety of ways. The present invention includes many different embodiments of polymer-encapsulated nanoparticles and methods for making and using such polymer-encapsulated nanoparticles.

[0095] Thus, any particular embodiment or element of the invention disclosed herein or shown in any drawing or table accompanying this application is intended to be illustrative, but not limiting, of the many various embodiments encompassed by the invention or equivalents contained in any particular element thereof. Moreover, a specific description of a single embodiment or element of the invention may not explicitly describe all possible embodiments or elements; many alternatives are implicitly disclosed by this specification and drawings.

[0096] It should be understood that each element of an apparatus or each step of a method may be described by apparatus terms or method terms. Such terms may be substituted where desired to make clear the implicitly broad scope to which this invention is entitled. By way of example only, it should be understood that every step of a method may be disclosed as an operation, a means for performing that operation, or an element that causes that operation. Similarly, each element of an apparatus may be disclosed as its physical element or the operation that its physical element facilitates. By way of example only, a disclosure of "encapsulated nanoparticles" should be understood to include a disclosure of the act of "encapsulating nanoparticles," whether or not explicitly discussed, and conversely, if there was effectively a disclosure of the act of "encapsulating nanoparticles," such a disclosure should be understood to also include a disclosure of "encapsulated nanoparticles" and even "means for encapsulating nanoparticles." Such alternative terms for each element or step should be understood to be expressly included herein.

[0097] Furthermore, for each term used, common dictionary definitions, such as those contained in Random House Webster's Unabridged Dictionary, Second Edition (each definition is hereby incorporated by reference), should be understood to be included in the description of each term, unless its usage in this application is inconsistent with such interpretation.

[0098] Accordingly, it should be understood that Applicant claims at least the following: 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 these devices and methods; iv) alternative embodiments that achieve each of the functions shown, disclosed, or described; v) alternative designs and methods that achieve each of the functions shown, as implicitly achieved, as disclosed and described; vi) each feature, component, and step shown as a separate and independent invention; vii) applications that are enhanced by the various systems or components disclosed; viii) the resulting products produced by such systems or components; ix) methods and apparatus substantially as described above with reference to any of the accompanying examples; and x) the various combinations and permutations of each of the above elements that are disclosed.

[0099] The Background section of this patent application provides a description of the field of endeavor to which the present invention pertains, if any. This section may also reference or include paraphrases of particular U.S. patents, patent applications, publications, or claimed subject matter that are useful in describing information, problems, or concerns about the state of the art to which the present invention is directed. It is not intended that any U.S. patent, patent application, publication, statement, or other information cited or incorporated herein be understood, construed, or deemed to be an admission that it is prior art with respect to the present invention.

[0100] The claims set forth in this application, if any, are hereby incorporated by reference as part of this description of the present invention, and Applicant expressly reserves the right to use all or a portion of the so-incorporated content of such claims as additional description to support any or all of such claims or any elements or components thereof, and Applicant further expressly reserves the right to move any part or all of the incorporated content of such claims or any elements or components thereof from the description into the claims, or vice versa, as necessary to define the matter for which protection is sought by this application or any subsequent application or continuation, divisional, or continuation-in-part thereof, or to obtain any benefit of, obtain fee reductions pursuant to, or comply with the patent laws, regulations, or implementing rules of any country or treaty, and such content incorporated by reference will survive the entire pendency of this application, including any subsequent continuation, divisional, or continuation-in-part thereof, or any reissue or extension thereunder. Elements preceded by an open-ended transitional phrase such as "comprising," whether or not expressly stated in the description portion of this specification, may alternatively be claimed using a closed-ended transitional phrase such as "consisting essentially of" or "consisting of."

[0101] Furthermore, the claims set forth in this application are further intended to describe the metes and bounds, if any, of a limited number of preferred embodiments of the present invention, and should not be construed as the broadest embodiment of the invention that may be claimed, nor as an exhaustive recitation of embodiments of the invention. Applicant does not waive any right to claim further rights as part of any continuation, divisional, or continuation-in-part application, or similar application, based on the description set forth above.

[0102] (Item 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 encapsulated by association with the hydrophobic region of said amphiphile; wherein the hydrophilic domains of the amphiphile comprise functional groups. (Item 2) Item 2. The nanocomposite of item 1, wherein the at least one quantum dot has a core comprising cadmium sulfide and a shell of zinc sulfide, the at least one quantum dot has an emission wavelength selected from the group consisting of 420 nm, 440 nm, and 460 nm, and the at least one quantum dot has a diameter in the range of about 5 nm to about 8 nm. (Item 3) Item 10. The nanocomposite of item 1, wherein the at least one quantum dot has a core comprising cadmium sulfide selenide and a shell of zinc sulfide, the at least one quantum dot having an emission wavelength selected from the group consisting of 480 nm, 500 nm, and 520 nm, and the at least one quantum dot has a diameter in the range of about 4 nm to about 6 nm. (Item 4) Item 10. The nanocomposite of item 1, wherein the at least one quantum dot has a core comprising cadmium selenide and a shell of zinc sulfide, the at least one quantum dot 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, and the at least one quantum dot has a diameter in the range of about 4 nm to about 8 nm. (Item 5) Item 2. The nanocomposite of item 1, wherein the at least one quantum dot has a core comprising cadmium telluride and a shell of zinc sulfide, the at least one quantum dot has an emission wavelength selected from the group consisting of 680 nm and 700 nm, and the at least one quantum dot has a diameter in the range of about 6 nm to about 7 nm. (Item 6) Item 10. The nanocomposite of item 1, wherein the at least one quantum dot has a core comprising cadmium selenide telluride and a shell of zinc sulfide, the at least one quantum dot 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, and the at least one quantum dot has a diameter in the range of about 4.5 nm to about 12 nm. (Item 7) Item 2. The nanocomposite of item 1, wherein the at least one quantum dot has a core comprising copper indium zinc sulfide and a shell of zinc sulfide, the at least one quantum dot 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, and the at least one quantum dot has a diameter in the range of about 3.5 nm to about 4 nm. (Item 8) Item 10. The nanocomposite of item 1, wherein the at least one quantum dot has a core comprising lead sulfide and a shell of cadmium sulfide, the at least one quantum dot having an emission wavelength selected from the group consisting of 900 nm, 920 nm, 940 nm, 960 nm, 980 nm, and 1000 nm, and the at least one quantum dot has a diameter in the range of about 3.5 nm to about 5 nm. (Item 9) Item 2. The nanocomposite according to item 1, wherein the at least one magnetic particle has an average particle size within the range of about 5 nm to about 20 nm. (Item 10) 10. The nanocomposite of claim 9, wherein the at least one magnetic particle has an average particle size selected from the group consisting of 5 nm, 10 nm, 15 nm, 20 nm, and combinations thereof. (Item 11) Item 11. The nanocomposite of item 10, wherein the magnetic particles comprise one or more of magnetite and maghemite. (Item 12) Item 11. The nanocomposite of item 10, wherein the magnetic particles comprise superparamagnetic iron oxide nanoparticles. (Item 13) Item 10. The nanocomposite of item 1, wherein the amphiphilic material comprises polystyrene-b-polyethylene glycol containing the functional group. (Item 14) Item 14. The nanocomposite of item 13, wherein the polystyrene-b-polyethylene glycol comprises polystyrene having a molecular weight ranging from about 1.5 kDa to about 40 kDa. (Item 15) Item 14. The nanocomposite of item 13, wherein the polystyrene-b-polyethylene glycol comprises polyethylene glycol having a molecular weight within the range of about 10 kDa to about 40 kDa. (Item 16) 16. The nanocomposite of claim 15, wherein the polyethylene glycol comprises branched polyethylene glycol. (Item 17) Item 14. The nanocomposite of item 13, wherein the amphiphilic material comprises polystyrene-b-polyethylene glycolamine 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 combinations thereof. (Item 18) Item 14. The nanocomposite of item 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 flow into an aqueous phase at a predetermined volumetric flow rate under the influence of an electric field. (Item 19) Item 19. The nanocomposite of item 18, wherein the mass concentration of the at least one quantum dot and the at least one magnetic particle to the mass concentration of the polystyrene-b-polyethylene glycol in the organic phase comprises a ratio that is in the range of about 1:1 to about 1:4. (Item 20) 20. The nanocomposite of item 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 under an external stimulus. (Item 21) 21. The nanocomposite of item 20, wherein the mass concentration of the polystyrene-b-polyethylene glycol is adjusted within the ratio to form the nanocomposite under electrohydrodynamic mixing-mediated nanoprecipitation having a substantially uniform hydrodynamic diameter within a range of about 40 nanometers to about 1000 nanometers. (Item 22) 21. The nanocomposite of claim 20, wherein the mass concentration of the polystyrene-b-polyethylene glycol is adjusted by varying the mass concentration of the polystyrene. (Item 23) 23. The nanocomposite of claim 22, wherein the mass concentration of the polystyrene-b-polyethylene glycol is adjusted by varying the mass concentration of the polyethylene glycol. (Item 24) 23. The nanocomposite of claim 22, wherein the mass concentration of the polystyrene-b-polyethylene glycol is adjusted by varying the branching of the polyethylene glycol, and the branching of the polyethylene glycol is selected from the group consisting of 4-arm polyethylene glycol and 8-arm polyethylene glycol. (Item 25) Item 19. The nanocomposite of item 18, wherein the polystyrene-b-polyethylene glycol, the at least one quantum dot, and the at least one magnetic particle have a concentration in the organic phase of about 0.1 mg / mL to about 5.0 mg / mL. (Item 26) 26. The nanocomposite according to item 25, wherein the organic phase has a concentration in the aqueous phase of about 0.2 v / v to about 1 v / v. (Item 27) Item 10. The nanocomposite of item 1, further comprising a ligand bound to the at least one quantum dot and the at least one magnetic particle, wherein the ligand comprises a mass concentration present in a range of about 10 percent to about 40 percent. (Item 28) 28. The nanocomposite of claim 27, wherein the ligand comprises oleic acid or oleylamine, and combinations thereof. (Item 29) Item 29. The nanocomposite of item 28, wherein the mass concentration of oleic acid bound to the at least one quantum dot is greater than or equal to 10 percent and less than or equal to 40 percent. (Item 30) 2. The nanocomposite of claim 1, further comprising an agent bound to the functional 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 envelope, a protein, a peptide, a radioisotope, an imaging agent, a surface charge modifier, a lectin, and combinations thereof. (Item 31) Item 1, wherein the nanocomposite further comprises a linker that binds to the functional group. (Item 32) 32. The nanocomposite of claim 31, wherein the linker comprises a heterobifunctional linker having a first reactive group that reacts with a functional group of the amphiphile encapsulating the nanoparticle and a second reactive group adapted to react with an agent. (Item 33) 33. The nanocomposite of claim 32, wherein the functional group comprises an amine or a carboxylate. (Item 34) 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. (Item 35) 35. The nanocomposite of claim 34, wherein the linker is selected from the group consisting of acrylate polyethylene glycol succinimidyl carboxymethyl ester, biotin polyethylene glycol succinimidyl carboxymethyl ester, maleimide polyethylene glycol succinimidyl carboxymethyl ester, azido polyethylene glycol succinimidyl carboxymethyl ester, and combinations thereof. (Item 36) 35. The nanocomposite of claim 34, wherein the second reactive group is selected from the group consisting of acrylate, maleimide, vinyl sulfone, and azide, biotin, amine, carboxylic acid, thiol, n-hydroxysuccinimide ester, alkyne, hydrazide, and 4-hydroxy-3-nitrophenylacetyl-ε-aminocaproic acid anion, methyltetrazine polyethylene glycol 4-N-hydroxysuccinimide ester, and combinations thereof. (Item 37) 33. The nanocomposite of claim 32, further comprising an agent that binds 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 envelope, a protein, a peptide, a radioisotope, an imaging agent, a surface charge modifier, a lectin, and combinations thereof. (Item 38) 32. The nanocomposite of claim 31, further comprising an antibody or antibody fragment attached to the linker. (Item 39) 39. The nanocomposite of claim 38, further comprising a cellular target bound by the antibody or antibody fragment. (Item 40) 40. The nanocomposite of claim 39, wherein the antibody or antibody fragment specifically binds to the cellular target. (Item 41) 41. The nanocomposite of claim 40, wherein the antibody or antibody fragment binds to the cellular target substantially without non-specific binding of other molecules. (Item 42) 40. The nanocomposite of claim 39, wherein the nanocomposite linked to the antibody or antibody fragment that binds to the cellular target is analyzed by flow cytometry for detection of the cellular target. (Item 43) 40. The nanocomposite of claim 39, wherein the nanocomposite linked to the antibody or antibody fragment that binds to the cellular target is placed in a magnetic field to isolate the nanocomposite linked to the antibody or antibody fragment that binds to the cellular target. (Item 44) 44. The nanocomposite of claim 43, wherein the nanocomposite linked to the antibody or antibody fragment that binds to the cellular target, isolated by placement in the magnetic field, is analyzed by flow cytometry for detection of the cellular target. (Item 45) 39. The nanocomposite of claim 38, wherein the antibody or antibody fragment comprises a mouse anti-human CD3 and the cellular target comprises a human CD3 peripheral blood mononuclear cell. (Item 46) 39. The nanocomposite of claim 38, wherein the antibody or antibody fragment comprises a mouse anti-human CD4 and the cellular target comprises a human CD4 peripheral blood mononuclear cell. (Item 47) 46. ​​The nanocomposite of claim 45, wherein the nanocomposite linked to mouse anti-human CD3 that binds to human CD3 peripheral blood mononuclear cells is analyzed by flow cytometry for detection of human CD3 peripheral blood mononuclear cells. (Item 48) 48. The nanocomposite of paragraph 47, wherein the mouse anti-human CD3-linked nanocomposite that binds to the human CD3 peripheral blood mononuclear cells, as detected by flow cytometry, is flow sorted into an isolated population of mouse anti-human CD3-linked nanocomposite that binds to the human CD3 peripheral blood mononuclear cells. (Item 49) 47. The nanocomposite of claim 46, wherein the nanocomposite linked to mouse anti-human CD4 that binds to human CD4 peripheral blood mononuclear cells is analyzed by flow cytometry for detection of human CD4 peripheral blood mononuclear cells. (Item 50) 50. The nanocomposite of paragraph 49, wherein the mouse anti-human CD4-linked nanocomposite that binds to the human CD4 peripheral blood mononuclear cells, as detected by flow cytometry, is flow sorted into an isolated population of mouse anti-human CD4-linked nanocomposite that binds to the human CD4 peripheral blood mononuclear cells. (Item 51) 47. The nanocomposite of claim 46, wherein the nanocomposite linked to mouse anti-human CD3 that binds to the human CD3 peripheral blood mononuclear cells is isolated under the influence of a magnetic field. (Item 52) 52. The nanocomposite of claim 51, wherein the nanocomposite linked to the mouse anti-human CD3 that binds to the human CD3 peripheral blood mononuclear cells isolated by the influence of the magnetic field is analyzed by flow cytometry for detection of human CD3 peripheral blood mononuclear cells. (Item 53) 53. The nanocomposite of claim 52, wherein the mouse anti-human CD3-linked nanocomposite that binds to the human CD3 peripheral blood mononuclear cells, as detected by flow cytometry, is flow sorted into an isolated population of mouse anti-human CD3-linked nanocomposite that binds to the human CD3 peripheral blood mononuclear cells. (Item 54) 47. The nanocomposite of claim 46, wherein the nanocomposite linked to mouse anti-human CD4 that binds to human CD4 peripheral blood mononuclear cells is isolated under the influence of a magnetic field. (Item 55) 55. The nanocomposite of claim 54, wherein the nanocomposite linked to mouse anti-human CD4 that binds to the human CD4 peripheral blood mononuclear cells isolated under the influence of a magnetic field is analyzed by flow cytometry for the detection of human CD4 peripheral blood mononuclear cells. (Item 56) A nanocomposite comprising: Polystyrene-b-polyethylene glycol having hydrophobic and hydrophilic regions; and At least one quantum dot or at least one magnetic particle encapsulated by association with the hydrophobic region of said amphiphile. wherein the hydrophilic domains of the amphiphile comprise functional groups. (Item 57) 57. The nanocomposite of item 56, wherein the polystyrene-b-polyethylene glycol comprises polystyrene having a molecular weight ranging from about 1.5 kDa to about 40 kDa. (Item 58) 57. The nanocomposite of item 56, wherein the polystyrene-b-polyethylene glycol comprises polyethylene glycol having a molecular weight ranging from about 10 kDa to about 40 kDa. (Item 59) 59. The nanocomposite of claim 58, wherein the polyethylene glycol comprises branched polyethylene glycol. (Item 60) 57. The nanocomposite of item 56, wherein the polystyrene-b-polyethylene glycol comprises polystyrene-b-polyethylene glycolamine 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 combinations thereof. (Item 61) Quantum dots comprising a cadmium sulfide core and a zinc sulfide shell, the quantum dots having an emission wavelength selected from the group consisting of 420 nm, 440 nm, and 460 nm, and at least one of the quantum dots having a diameter in the range of about 5 nm to about 8 nm. (Item 62) Quantum dots comprising a cadmium sulfide selenide core and a zinc sulfide shell, the quantum dots having an emission wavelength selected from the group consisting of 480 nm, 500 nm, and 520 nm, and the at least one quantum has a diameter in the range of about 4 nm to about 6 nm. (Item 63) Quantum dots comprising a cadmium selenide core and a zinc sulfide shell, said quantum dots 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, said at least one quantum having a diameter in the range of about 4 nm to about 8 nm. (Item 64) Quantum dots comprising a cadmium telluride core and a zinc sulfide shell, the quantum dots having an emission wavelength selected from the group consisting of 680 nm and 700 nm, and the at least one quantum having a diameter in the range of about 6 nm to about 7 nm. (Item 65) Quantum dots comprising a cadmium telluride selenide core and a zinc sulfide shell, said quantum dots 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, said at least one quantum dot having a diameter in the range of about 4.5 nm to about 12 nm. (Item 66) Quantum dots comprising a copper indium zinc sulfide core and a zinc sulfide shell, the quantum dots 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, and the at least one quantum has a diameter in the range of about 3.5 nm to about 4 nm. (Item 67) Quantum dots comprising a lead sulfide core and a cadmium sulfide shell, said quantum dots having an emission wavelength selected from the group consisting of 900 nm, 920 nm, 940 nm, 960 nm, 980 nm, and 1000 nm, said at least one quantum having a diameter in the range of about 3.5 nm to about 5 nm. (Item 68) A quantum dot, Core material; a shell material surrounding the core material; and Ligands bound to the quantum dots wherein the ligand has a mass concentration present in the range of about 10 percent to about 60 percent. (Item 69) Item 69. The quantum dot according to item 68, wherein the ligand has a mass concentration present in the range of about 10 percent to about 40 percent. (Item 70) 70. The quantum dot of item 69, wherein the ligand comprises oleic acid or oleylamine, and combinations thereof. (Item 71) 71. The quantum dot of item 70, wherein the oleic acid bound to the at least one quantum dot has a mass concentration of 10 percent or more and 40 percent or less. (Item 72) A nanocomposite comprising: at least one quantum dot; at least one magnetic particle; and a ligand bound to the quantum dot and the at least one magnetic particle; wherein the ligand has a mass concentration present in the range of about 10 percent to about 40 percent. (Item 73) Amphiphilic substances having hydrophobic and hydrophilic regions 73. The nanocomposite of claim 72, further comprising:

Claims

1. It is a nanocomposite, An amphiphilic substance having a hydrophobic region and a hydrophilic region, wherein the amphiphilic substance comprises polystyrene-b-polyethylene glycol containing a functional group, and the polystyrene-b-polyethylene glycol comprises a polystyrene block and a polyethylene glycol block containing a branched polyethylene glycol selected from the group consisting of 4-arm polyethylene glycol, 6-arm polyethylene glycol and 8-arm polyethylene glycol; and At least one quantum dot and at least one magnetic particle are encapsulated by association of the amphiphilic material with the hydrophobic region. Nanocomposites including

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

3. The nanocomposite according to claim 1, wherein the at least one magnetic particle has an average particle diameter in the range of about 5 nm to about 20 nm.

4. The nanocomposite according to claim 1, further comprising an active substance that binds to the functional group, wherein the active substance is selected from the group consisting of polyethylene glycol, antibodies, half-antibodies, antibody fragments, fluorescent probes, aptamers, vitamins, cell surface receptors, extracellular coverings, proteins, peptides, radioisotopes, contrast agents, surface charge modifiers, lectins, and combinations thereof.

5. The nanocomposite according to claim 1, further comprising a linker bonded to the functional group of the nanocomposite.

6. The nanocomposite according to claim 5, wherein the linker comprises a heterobifunctional linker having a first reactive group adapted to react with the functional group of the amphiphilic material encapsulating the nanoparticles and a second reactive group adapted to react with the active substance.

7. The nanocomposite according to claim 6, wherein the functional group comprises an amine, and the first reactive group of the heterobifunctional linker comprises succinimidyl carboxymethyl ester.

8. The nanocomposite according to claim 6, further comprising an active substance bound to the second reactive group, wherein the active substance is selected from the group consisting of polyethylene glycol, antibodies, half-antibodies, antibody fragments, fluorescent probes, aptamers, vitamins, cell surface receptors, extracellular coverings, proteins, peptides, radioisotopes, contrast agents, surface charge modifiers, lectins, and combinations thereof.

9. The nanocomposite according to claim 5, further comprising an antibody or antibody fragment bound to the linker bound to the functional group of the nanocomposite.

10. The nanocomposite according to claim 9, wherein the antibody or antibody fragment bonded to the linker bonded to the functional group of the nanocomposite is adapted to bind to a cell target.

11. The nanocomposite according to claim 10, wherein the antibody or antibody fragment bound to the linker bound to the functional group of the nanocomposite is adapted to specifically bind to the cell target.

12. The nanocomposite according to claim 11, wherein the antibody or antibody fragment bound to the linker bound to the functional group of the nanocomposite is adapted to bind to the cell target without detectable nonspecific binding of other molecules.

13. The nanocomposite according to claim 12, wherein the antibody or antibody fragment comprises mouse anti-human CD3, and the cell target comprises human CD3 peripheral blood mononuclear cells.

14. The nanocomposite according to claim 13, wherein the antibody or antibody fragment comprises mouse anti-human CD4, and the cell target comprises human CD4 peripheral blood mononuclear cells.

15. The nanocomposite according to claim 13, wherein the nanocomposite linked to the mouse anti-human CD3 that is bound to the human CD3 peripheral blood mononuclear cells is analyzed by flow cytometry for the detection of human CD3 peripheral blood mononuclear cells.

16. The nanocomposite according to claim 13, wherein the nanocomposite linked to the mouse anti-human CD3 that is bound to the human CD3 peripheral blood mononuclear cells is isolated under the influence of a magnetic field.

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