Gold particles and methods of making and use thereof

Abstract

The disclosed subject matter relates to gold particles, and methods of making and use thereof. For example, disclosed herein are one-step ligand exchange methods. The methods comprise contacting a gold particle capped with a toxic ligand with a less-toxic ligand in a solution, wherein the concentration of the less-toxic ligand and / or the pH is sufficient to cause the toxic ligand to be replaced by the less-toxic ligand. In some examples, the methods comprise contacting a gold particle capped with a toxic ligand with a gold salt and a less-toxic ligand in a solution, wherein the less-toxic ligand is a reducing agent for the gold salt, such that the gold salt is reduced and deposited as a thin layer of gold onto the gold particle, wherein the concentration of the less-toxic ligand is sufficient to cause the toxic ligand to be replaced by the less-toxic

Description

[0001] GOLD PARTICLES AND METHODS OF MAKING AND USE THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 571,056 filed March 28, 2024, which is hereby incorporated herein by reference in its entirety.

[0004] BACKGROUND

[0005] Noble-metal nanocrystals / nanoparticles with diverse shapes have been synthesized by manipulating the experimental parameters, including temperature, surface ligand, and reducing agent, among others, typically involved in a colloidal synthesis. The nanocrystals / nanoparticles have found widespread use in a variety of applications, including plasmonics, catalysis, sensing, biomedicine, and self-assembly. The surface ligand, in particular, can serves as a colloidal stabilizer to prevent the resultant nanocrystals / nanoparticles from aggregation during synthesis or storage in addition to its role in dictating the evolution of shape. Despite its role in a shape- controlled synthesis, however, the surface ligand may compromise or ruin the targeted application like biomedicine due to its intrinsic toxicity. As such, ligand exchange is often necessary to introduce the ligand of interest for the proposed application rather than the synthesis step. However, to date, the ligand exchange methods available suffer from a variety of drawback and / or limitations. Improved methods and compositions are needed. The compositions, methods, and systems discussed herein address these and other needs.

[0006] SUMMARY

[0007] In accordance with the purposes of the disclosed compositions and methods as embodied and broadly described herein, the disclosed subject matter relates to gold particles and methods of making and use thereof.

[0008] For example, disclosed herein are one-step ligand exchange methods, the methods comprising: contacting a gold particle capped with a toxic ligand with a solution, the solution comprising a less-toxic ligand, or contacting a solution comprising a gold particle capped with a toxic ligand with a less-toxic ligand, wherein the concentration of the less-toxic ligand in the solution and / or the pH of the solution is sufficient to cause the toxic ligand to be replaced by the less-toxic ligand at the surface of the gold particle. In some examples, the pH of the solution is selected to control the protonation of the less-toxic ligand, thereby controlling the binding strength of the less-toxic ligand to the gold particle. In some examples, the pH is 7 or less, 4 or less, or 2 or less. In some examples, the pH is from 2 to 4. In some examples, the solution further comprises a gold salt, and the less-toxic ligand further serves as a reducing agent for the gold salt, such that the gold salt is reduced and deposited as a thin layer of gold onto the gold particle.

[0009] Also disclosed herein are one-step ligand exchange methods, the methods comprising: contacting a gold particle capped with a toxic ligand with a solution, the solution comprising a gold salt and a less-toxic ligand, or contacting a solution comprising a gold particle capped with a toxic ligand with a gold salt and a less-toxic ligand, wherein the less-toxic ligand further serves as a reducing agent for the gold salt, such that the gold salt is reduced and deposited as a thin layer of gold onto the gold particle, wherein the concentration of the less-toxic ligand in the solution is sufficient to cause the toxic ligand to be replaced by the less-toxic ligand at the surface of the gold particle. In some examples, wherein the method further comprises controlling the pH of the solution, wherein the pH of the solution is selected to control the protonation of the less-toxic ligand, thereby controlling the binding strength of the less-toxic ligand to the gold particle. In some examples, the pH is 7 or less, 4 or less, or 2 or less. In some examples, the pH is from 2 to 4.

[0010] In some examples, the thin layer of gold is an atomic monolayer.

[0011] In some examples, the gold salt comprises HAuCU.

[0012] In some examples, the concentration of gold salt is selected to control the thickness of the deposited layer. In some examples, the concentration of the gold salt is selected such that the thin layer of gold has a thickness of 3 nm or less. In some examples, the concentration of the gold salt is selected such that the thin layer of gold is an atomic monolayer.

[0013] In some examples, the solution is added dropwise.

[0014] In some examples, the solution is added in one shot.

[0015] In some examples, the toxic ligand comprises cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTAC), dodecyltrimethylammonium bromide, dodecyltrimethylammonium chloride, octadecyltrimethylammonium bromide, octadecyltrimethylammonium chloride, or a combination thereof. In some examples, the toxic ligand comprises cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTAC), or a combination thereof.

[0016] In some examples, the less-toxic-ligand comprises citric acid or a citrate (e.g., a monocitrate, bi-citrate, tri-citrate, or a combination thereof), such as a sodium citrate (e.g., sodium mono-citrate, sodium bi-citrate, sodium tri-citrate, or a combination thereof). In some examples, the less-toxic ligand comprises citrate, such as sodium citrate.

[0017] In some examples, the toxic ligand is more strongly bound to the gold particle relative to the less-toxic ligand.

[0018] In some examples, the gold particle has an average particle size of from 1 nm to 1000 nm. In some examples, the gold particle has an average particle size of from 5 nm to 1000 nm. In some examples, the gold particle has an average particle size of 5 nm to 150 nm. In some examples, the gold particle has an average particle size of from 10 nm to 40 nm.

[0019] In some examples, the gold particle before and / or after the contacting step has a substantially spherical shape.

[0020] In some examples, the shape of the gold particle is preserved after the contacting step.

[0021] In some examples, the gold particle comprises a radioisotope, such as Au- 198 and / or Au- 199.

[0022] In some examples, the concentration of the less-toxic ligand is 200 mM or more.

[0023] In some examples, the method is a one pot method.

[0024] In some examples, the method is performed at room temperature.

[0025] In some examples, the method is completed in an amount of time of from 1 minute to 1 hour. In some examples, the method is completed in an amount of time of from 1 minute to 30 minutes.

[0026] In some examples, the method does not result in agglomeration of the gold particles.

[0027] Also disclosed herein are gold particles made by any of the methods disclosed herein. In some examples, the gold particle after ligand exchange has decreased cell toxicity relative to the gold particle before ligand exchange. In some examples, the gold particle after ligand exchange is colloidally stable for an amount of time of 24 hours or more.

[0028] Also disclosed herein are compositions comprising any of the gold particles disclosed herein.

[0029] Also disclosed herein are pharmaceutical compositions comprising any of the gold particles disclosed herein and a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient, or a combination thereof.

[0030] Also disclosed herein are assays comprising any of the gold particles or compositions disclosed herein. In some examples, the gold particle is further conjugated to a targeting ligand, such as an antibody, a nucleic acid probe, a molecular recognition element, or a combination thereof.

[0031] Also disclosed herein are point of care tests comprising any of the gold particles or compositions disclosed herein.

[0032] In some examples, the point of care test comprises a pregnancy test.

[0033] In some examples, the point of care test comprises a test for a pathogenic microorganism.

[0034] In some examples, the point of care test comprises a coronavirus test, such as a Covid- 19 test. In some examples, the point of care test comprises a cancer screening test.

[0035] Also disclosed herein are methods of use of any of the gold particles or compositions disclosed herein.

[0036] In some examples, the method comprises using the gold particle or composition in biomedical imaging (e.g., as an imaging agent).

[0037] In some examples, the method comprises using the gold particle or composition in photodynamic therapy, photothermal therapy, or a combination thereof.

[0038] In some examples, the method comprises using the gold particle or composition in radiotherapy, preferably wherein the gold particle includes a radioisotope, such as Au- 198 and / or Au- 199.

[0039] In some examples, the method comprises using the gold particle or composition as a sensor.

[0040] In some examples, the method comprises using the gold particle or composition for detection of biomarkers, infectious disease-related antigens, antibodies, or a combination thereof.

[0041] In some examples, the method comprises using the gold particle or composition for drug delivery. In some examples, the gold particle is further conjugated with a drug-carrying ligand. In some examples, the gold particle or composition further comprises a drug, the drug being conjugated to the drug-carrying ligand.

[0042] In some examples, the method comprises using the gold particle or composition as a catalyst.

[0043] In some examples, the method comprises using the gold particle or composition as a photosensitizer.

[0044] Also disclosed herein are methods of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any of the gold particles or compositions disclosed herein. In some examples, the disease comprises cancer.

[0045] Also disclosed herein are methods of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any of the gold particles or compositions disclosed herein.

[0046] In some examples, the method further comprises co-administering an anticancer agent to the subject.

[0047] Also disclosed herein are methods of suppressing tumor growth in a subject in need thereof, the method comprising contacting at least a portion of the tumor with a therapeutically effective amount of any of the gold particles or compositions disclosed herein. In some examples, the methods further comprise irradiating the gold particle with electromagnetic radiation that overlaps with at least a portion of one or more of an absorption, emission, or excitation band of the gold particle.

[0048] Also disclosed herein are methods of imaging a cell or a population of cells within or about a subject, the method comprising administering to the subject an amount of any of the gold particles or compositions disclosed herein, and detecting the gold particle or composition. In some examples, the cell or population of cells is indicative of cancer.

[0049] Additional advantages of the disclosed compositions and methods will be set forth in part in the description which follows, and in part will be obvious from the description. The advantages of the disclosed compositions and methods will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosed compositions and methods, as claimed.

[0050] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0051] BRIEF DESCRIPTION OF THE FIGURES

[0052] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects of the disclosure, and together with the description, serve to explain the principles of the disclosure.

[0053] Figure 1. Schematic showing the exchange of CTAB / CTAC with tri-citrate during the deposition of an ultrathin shell of fresh Au in the presence of tri-citrate at an adequate concentration.

[0054] Figure 2. Schematic showing the exchange of CTAB / CTAC with tri-citrate during the deposition of an ultrathin shell of fresh Au in the presence of tri-citrate at an adequate concentration. The CTAB / CTAC is gradually replaced by citrate and the surface is eventually covered by citrate.

[0055] Figure 3A-Figure 3E. TEM images of the samples obtained by depositing Au shell of (Figure 3A) one atomic layer, (Figure 3B) three atomic layers, (Figure 3C) 1 nm, and (Figure 3D) 3 nm in thickness onto surface of 10-nm Au spheres, with the use of 200 mM aqueous tricitrate for AuClF reduction. The thickness was calculated using the molar ratio between the number of Au nanospheres and the amount of AuCLf added. The particles circled in (Figure 3B) involved symmetry reduction during Au deposition. (Figure 3E) Schematic showing the shape evolution of a spherical particle under three different kinetic conditions.

[0056] Figure 4. FTIR spectra recorded from CTAB / CTAC, citrate, and the Au nanospheres obtained through the deposition of one monolayer of Au with the use of 10, 100, and 200 mM aqueous tri-citrate for Au deposition.

[0057] Figure 5. UV-Vis spectra recorded from the samples of 10-nm Au spheres dispersed in different media.

[0058] Figure 6A-Figure 6B . The activity of ADMSCs after culture with 0, 20, 200, and 400 pg / mL of (Figure 6A) CTAB / CTAC- and (Figure 6B) tri-citrate-capped 10-nm Au spheres for 12 h. Data are presented as the mean ± standard deviation (SD), n = 3. One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test < 0.0001; ns, no significant).

[0059] Figure 7. UV-vis spectra recorded from the samples of CTAB / CTAC-capped 10-nm Au spheres in 20 mM aqueous CT AC solution (black) and samples after ligand exchange with citric acid or mono- / bi-citrate at different pH for 3 h.

[0060] Figure 8. UV-vis spectra recorded from the samples of CTAB / CTAC-capped 10-nm Au spheres in 20 mM aqueous CT AC solution (black) and mono-citrate-capped 10-nm Au spheres in 50 mM aqueous mono-citrate solution (pH = 4) after 24 h (red).

[0061] Figure 9. FTIR spectra recorded from CTAC, citric acid, sodium citrate and the Au nanospheres obtained through the ligand replacement.

[0062] Figure 10. FTIR spectra recorded from CTAB, CTAC, citric acid, sodium citrate, and the Au nanospheres obtained through the ligand replacement at different pH when exchanging time = 30 min.

[0063] Figure HA-Figure 11B. TEM images of CTAB / CTAC-capped Au nanospheres with a diameter of (Figure 11 A) 14 nm, and (Figure 11B) 19.7 nm obtained through two seed-mediated growth steps involving one-shot addition only.

[0064] Figure 12A-Figure 12E. TEM images of CTAB / CTAC-capped Au nanospheres with a diameter of 20.5 nm with the use of CTAC at concentration of (Figure 12A) 200 mM, (Figure 12B) 130 mM, (Figure 12C) 100 mM, (Figure 12D) 70 mM, and ( Figure 12E) 0 mM, obtained through a seed-mediated one-shot injection method.

[0065] Figure 13. Schematic diagram of methods for directly exchanging the toxic cetyltrimethylammonium bromide / chloride (CTAB / C) on Au nanocrystals with citrate species.

[0066] Figure 14A-Figure 14B. (Figure 14A) FTIR spectra recorded from free citric acid, sodium tri-citrate, CTAB / C, and the Au nanospheres obtained at different stages of ligand exchange. (Figure 14B) A segment of the FTIR spectra in the region indicated by the dashed box in (Figure 14A).

[0067] Figure 15. UV-vis spectra recorded from an aqueous suspension of the CTAB / C-capped Au nanospheres in 20 mM aqueous CTAC (black) and aqueous suspensions of the citric acidcapped Au nanospheres in 50 mM aqueous citric acid at pH = 2 before and after storage for 24 h, respectively.

[0068] Figure 16A-Figure 16D. (Figure 16A, Figure 16B) TEM and (Figure 16C, Figure 16D) HAADF-STEM images of the Au nanospheres before and after ligand exchange: (Figure 16A, Figure 16C) CTAB / C-capped and (Figure 16B, Figure 16D) the corresponding citric acidcapped Au nanospheres, respectively.

[0069] Figure 17. FTIR spectra recorded from the references (free sodium tri-citrate, sodium bicitrate, sodium mono-citrate, and citric acid) and Au nanospheres obtained through ligand exchange at different pH for 30 min.

[0070] Figure 18A-Figure 18B. UV-vis spectra recorded from the aqueous suspension of Au nanospheres capped by (Figure 18A) citric acid and (Figure 18B) tri-citrate in aqueous citric acid (pH = 2) and sodium tri-citrate solution (pH = 7) at a concentration of 100 mM, respectively (red trace). UV-vis spectra recorded from the same samples upon dilution by different factors (blue: diluted by 2-fold, green: diluted by 10-fold, and orange: diluted by 100-fold). All spectra were normalized to the peak intensity for easy comparison.

[0071] Figure 19A-Figure 19B. (Figure 19 A) FTIR spectra recorded from the cubic and octahedral Au nanocrystals before and after the ligand exchange for 30 min. (Figure 19B) A segment of the FTIR spectra in the region indicated by the dashed box in (Figure 19A).

[0072] Figure 20. Ratios of Ac=o to Acm in the FTIR spectra recorded from Au nanocrystals with different shapes at different stages of ligand exchange.

[0073] Figure 21. The most stable binding configurations of citric acid, all sodium citrates, and CTAB on the { 100}, { 111 }, {211 }, and {331 } facets at appropriate coverages. Color coding: golden, pink, green, blue, brown, white, and red represent Au, Na, Br, N, C, H, and O, respectively.

[0074] Figure 22. Calculated binding energies for the most stable adsorption states of each adsorbate on different Au facets. Negative values indicate exothermic adsorption of the gasphase species on the Au surface. Lines connecting data for each adsorbed species only serve as guides to the eye.

[0075] Figure 23. The speciation diagram of citric acid as a function of pH, where an increase in pH leads to the progressive deprotonation of citric acid, generating mono-, bi-, and tri-citrate. Figure 24A-Figure 24B. Schematic showing the binding of citrate species to a Au atom in the presence (Figure 24A) H+and (Figure 24B) Na+counterions, respectively, together with the corresponding wavenumbers of the vibrational peaks.

[0076] Figure 25. Calculated wavenumbers of the C=O bond in different citrate species when binding to different types of Au planes.

[0077] Figure 26. Schematic showing the ligand exchange between surface-bound CTAB / C and citric acid on the surface of a Au nanosphere.

[0078] Figure 27. UV-vis spectra recorded from aqueous suspensions of the 20-nm Au spheres, cubes, and octahedra capped by CTAB / C, respectively. For fair comparison, 20-nm instead of 10-nm Au spheres for the recorded UV-vis spectrum. Unless notified, all the Au nanospheres used in this work had an average diameter of 10 nm.

[0079] Figure 28. Langmuir curves proposed for describing the adsorption of citric acid and tricitrate on the surface of Au nanospheres.

[0080] Figure 29. UV-vis spectra recorded from aqueous suspensions of the CTAB / C-capped Au nanospheres in 20 mM aqueous CTAC (black) before and after direct ligand exchange with citric acid, mono-, bi-, and tri-citrate, respectively, by adjusting the pH to different values.

[0081] Figure 30A-Figure 30D. TEM images of cubic and octahedral Au nanocrystals before and after the attempted ligand exchange for 30 min: (Figure 30A) CTAB / C-capped cubic nanocrystals before ligand exchange, (Figure 30B) CTAB / C-capped octahedral nanocrystals before ligand exchange, (Figure 30C) cubic nanocrystals after ligand exchange, and (Figure 30D) octahedral nanocrystals after ligand exchange.

[0082] Figure 31. FTIR spectra recorded from a solid mixture of citric acid and CTAC at a molar ratio of 1 : 1.

[0083] Figure 32A-Figure 32C. (Figure 32A) HAADF-STEM image of a Au nanosphere at atomic resolution, together with the assignments of high-index facets on the surface: pink = {211}; yellow = {311}; and blue = {331 }. (Figure 32B) FFT pattern of the Au nanosphere along [Oil] direction used for the assignment of facets exposed on the surface. (Figure 32C) Theoretical FFT pattern of a face-centered cubic metal along the [Oi l] direction.

[0084] DETAILED DESCRIPTION

[0085] The compositions and methods described herein may be understood more readily by reference to the following detailed description of specific aspects of the disclosed subject matter and the Examples included therein.

[0086] Before the present compositions and methods are disclosed and described, it is to be understood that the aspects described below are not limited to specific synthetic methods or specific reagents, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0087] Also, throughout this specification, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the disclosed matter pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

[0088] General Definitions

[0089] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings.

[0090] Throughout the description and claims of this specification the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps. As used in the specification and in the claims, the term “comprising” can include the aspects “consisting of’ and “consisting essentially of.”

[0091] As used in the description 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 composition” includes mixtures of two or more such compositions, reference to “an agent” includes mixtures of two or more such agents, reference to “the component” includes mixtures of two or more such components, and the like.

[0092] “Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0093] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. By “about” is meant within 5% of the value, e.g., within 4, 3, 2, or 1% of the value. When such a range is expressed, another aspect 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 will be understood that the particular value forms another aspect. It will be 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.

[0094] Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, a description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, 6 and any whole and partial increments therebetween. This applies regardless of the breadth of the range.

[0095] When the specific values are disclosed between two end values, it is understood that these end values can also be included.

[0096] For the terms “for example” and “such as,” and grammatical equivalences thereof, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. It is further understood that these phrases are not used in a restrictive sense, but for explanatory purposes. “Exemplary” means “an example of’ and is not intended to convey an indication of a preferred or ideal embodiment.

[0097] It is understood that throughout this specification the identifiers “first” and “second” are used solely to aid in distinguishing the various components and steps of the disclosed subject matter. The identifiers “first” and “second” are not intended to imply any particular order, amount, preference, or importance to the components or steps modified by these terms.

[0098] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0099] As used herein, the term "substantially" means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance generally, typically, or approximately occurs.

[0100] Still further, the term “substantially” can, in some aspects, refer to at least about 80 %, at least about 85 %, at least about 90 %, at least about 91 %, at least about 92 %, at least about 93 %, at least about 94 %, at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, at least about 99 %, or about 100 % of the stated property, component, composition, or other condition for which substantially is used to characterize or otherwise quantify an amount.

[0101] In other aspects, as used herein, the term “substantially free,” when used in the context of a composition or component of a composition that is substantially absent, is intended to refer to an amount that is then about 1 % by weight, e.g., less than about 0.5 % by weight, less than about 0.1 % by weight, less than about 0.05 % by weight, or less than about 0.01 % by weight of the stated material, based on the total weight of the composition. The expressions “ambient temperature” and “room temperature” as used herein are understood in the art and refer generally to a temperature from about 20°C to about 35 °C.

[0102] References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a mixture containing 2 parts by weight of component X and 5 parts by weight of component Y, components X and Y are present at a weight ratio of 2:5 and are present in such a ratio regardless of whether additional components are contained in the mixture.

[0103] A weight percent (wt.%) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.

[0104] It is understood that the term “salt,” as used herein, refers to a chemical compound that can be formed form a reaction between an acid and a base. It is understood that the term “salt,” as used herein, encompasses both inorganic and organic salts capable of providing the desired properties to the composition. In still further aspects, a cation of the disclosed herein salts is a metal cation.

[0105] While aspects of the present invention can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of ordinary skill in the art will understand that each aspect of the present invention can be described and claimed in any statutory class. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to the arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0106] 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 this invention belongs.

[0107] “Phase,” as used herein, generally refers to a region of a material having a substantially uniform composition which is a distinct and physically separate portion of a heterogeneous system. The term “phase” does not imply that the material making up a phase is a chemically pure substance, but merely that the chemical and / or physical properties of the material making up the phase are essentially uniform throughout the material, and that these chemical and / or physical properties differ significantly from the chemical and / or physical properties of another phase within the material. Examples of physical properties include density, thickness, aspect ratio, specific surface area, porosity, and dimensionality. Examples of chemical properties include chemical composition.

[0108] By “continuous” it is meant a phase such that all points within the phase are directly connected, so that for any two points within a continuous phase there exists a path which connects the two points and does not leave the phase.

[0109] As used herein, by a “subject” is meant an individual. Thus, the “subject” can include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e. ., mouse, rabbit, rat, guinea pig, etc.), and birds. “Subject” can also include a mammal, such as a primate or a human. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0110] The term “inhibit” refers to a decrease in an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This can also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0111] By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control.

[0112] By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed. For example, the terms “prevent” or “suppress” can refer to a treatment that forestalls or slows the onset of a disease or condition or reduced the severity of the disease or condition. Thus, if a treatment can treat a disease in a subject having symptoms of the disease, it can also prevent or suppress that disease in a subject who has yet to suffer some or all of the symptoms.

[0113] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0114] The term “therapeutically effective amount” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0115] The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0116] The term “anticancer” refers to the ability to treat or control cellular proliferation and / or tumor growth at any concentration.

[0117] As used herein, “molecular weight” refers to weight average molecular weight as measured by mass spectrometry, unless indicated otherwise.

[0118] As used herein, the term “delivery” encompasses both local and systemic delivery. For example, delivery of mRNA encompasses situations in which an mRNA is delivered to a target tissue and the encoded protein or peptide is expressed and retained within the target tissue (also referred to as “local distribution” or “local delivery”), and situations in which an mRNA is delivered to a target tissue and the encoded protein or peptide is expressed and secreted into patient's circulation system (e.g., serum) and systematically distributed and taken up by other tissues (also referred to as “systemic distribution” or “systemic delivery).

[0119] As used herein, the term “encapsulation,” or grammatical equivalent, refers to the process of confining an individual nucleic acid molecule within a nanoparticle.

[0120] As used herein, “expression” of a mRNA refers to translation of an mRNA into a peptide (e.g., an antigen), polypeptide, or protein (e.g., an enzyme) and also can include, as indicated by context, the post-translational modification of the peptide, polypeptide or fully assembled protein (e.g., enzyme). In this application, the terms “expression” and “production,” and grammatical equivalent, are used inter-changeably.

[0121] As used herein, the term “messenger RNA (mRNA)” refers to a polynucleotide that encodes at least one peptide, polypeptide or protein. mRNA as used herein encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl- cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8- oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, pseudouridine, and 5- methylcytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphoro thioates and 5'-N- phosphoramidite linkages).

[0122] As used herein, the term “nucleic acid,” in its broadest sense, refers to any compound and / or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, “nucleic acid” refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, “nucleic acid” encompasses RNA as well as single and / or double-stranded DNA and / or cDNA. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and / or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone.

[0123] The organic moieties mentioned when defining variable positions within the general formulae described herein (e.g., the term “halogen”) are collective terms for the individual substituents encompassed by the organic moiety. The prefix Cn-Cmpreceding a group or moiety indicates, in each case, the possible number of carbon atoms in the group or moiety that follows.

[0124] The term “ion,” as used herein, refers to any molecule, portion of a molecule, cluster of molecules, molecular complex, moiety, or atom that contains a charge (positive, negative, or both at the same time within one molecule, cluster of molecules, molecular complex, or moiety (e.g., zwitterions)) or that can be made to contain a charge. Methods for producing a charge in a molecule, portion of a molecule, cluster of molecules, molecular complex, moiety, or atom are disclosed herein and can be accomplished by methods known in the art, e.g., protonation, deprotonation, oxidation, reduction, alkylation, acetylation, esterification, de-esterification, hydrolysis, etc.

[0125] The term “anion” is a type of ion and is included within the meaning of the term “ion.” An “anion” is any molecule, portion of a molecule (e.g., zwitterion), cluster of molecules, molecular complex, moiety, or atom that contains a net negative charge or that can be made to contain a net negative charge. The term “anion precursor” is used herein to specifically refer to a molecule that can be converted to an anion via a chemical reaction (e.g., deprotonation).

[0126] The term “cation” is a type of ion and is included within the meaning of the term “ion.” A “cation” is any molecule, portion of a molecule (e.g., zwitterion), cluster of molecules, molecular complex, moiety, or atom, that contains a net positive charge or that can be made to contain a net positive charge. The term “cation precursor” is used herein to specifically refer to a molecule that can be converted to a cation via a chemical reaction (e.g., protonation or alkylation).

[0127] Gold Particles and Methods of Making Thereof

[0128] Disclosed herein are gold particles and methods of making and use thereof.

[0129] As used herein, “a particle” and “the particle” are meant to include any number of particles. Thus, for example “a particle” includes one or more particles. In some embodiments, the particle can comprise a plurality of particles.

[0130] For example, disclosed herein are one-step ligand exchange methods.

[0131] In some examples, the one-step ligand exchange method comprises contacting a gold particle capped with a toxic ligand with a solution, the solution comprising a less-toxic ligand, wherein the concentration of the less-toxic ligand in the solution and / or the pH of the solution is sufficient to cause the toxic ligand to be replaced by the less-toxic ligand at the surface of the gold particle. In some examples, the one-step ligand exchange method comprises contacting a solution comprising a gold particle capped with a toxic ligand with a less-toxic ligand, wherein the concentration of the less-toxic ligand in the solution and / or the pH of the solution is sufficient to cause the toxic ligand to be replaced by the less-toxic ligand at the surface of the gold particle. In some examples, the one-step ligand exchange method comprises contacting a gold particle capped with a toxic ligand with a less-toxic ligand in a solution, wherein the concentration of the less-toxic ligand in the solution and / or the pH of the solution is sufficient to cause the toxic ligand to be replaced by the less-toxic ligand at the surface of the gold particle

[0132] In some examples, the pH of the solution is selected to control the protonation of the less- toxic ligand, thereby controlling the binding strength of the less-toxic ligand to the gold particle. For example, selecting the pH of the solution can comprise controlling the pH via addition of an appropriate amount of an acid or base.

[0133] In some examples, the pH is 7 or less (e.g., 6.5 or less, 6 or less, 5 or less, 4.5 or less, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less, or 1.5 or less). In some examples, the pH is 1 or more (e.g., 1.5 or more, 2 or more, 2.5 or more, 3 or more, 3.5 or more, 4 or more, 4.5 or more, 5 or more, 5.5 or more, or 6 or more). The pH of the solution can range from any of the minimum values described above to any of the maximum values described above. For example, the pH can be from 1 to 7 (e.g., from 1 to 4, from 4 to 7, from 1 to 3, from 3 to 5, from 5 to 7, from 1 to 6, from 1 to 5, from 2 to 7, from 3 to 7, from 2 to 6, or from 2 to 4). In some examples, the pH is from 2 to 4.

[0134] In some examples, the solution further comprises a gold salt, and the less-toxic ligand further serves as a reducing agent for the gold salt, such that the gold salt is reduced and deposited as a thin layer of gold onto the gold particle. For example, gold salt is reduced to gold atoms, and the resulting gold atoms are deposited as a thin layer on the gold particle.

[0135] In some examples, the one-step ligand exchange methods comprise: contacting a gold particle capped with a toxic ligand with a solution, the solution comprising a gold salt and a less- toxic ligand, wherein the less-toxic ligand further serves as a reducing agent for the gold salt, such that the gold salt is reduced and deposited as a thin layer of gold onto the gold particle, wherein the concentration of the less-toxic ligand in the solution is sufficient to cause the toxic ligand to be replaced by the less-toxic ligand at the surface of the gold particle. In some examples, the one-step ligand exchange methods comprise: contacting a solution comprising a gold particle capped with a toxic ligand with a gold salt and a less-toxic ligand, wherein the less- toxic ligand further serves as a reducing agent for the gold salt, such that the gold salt is reduced and deposited as a thin layer of gold onto the gold particle, wherein the concentration of the less- toxic ligand in the solution is sufficient to cause the toxic ligand to be replaced by the less-toxic ligand at the surface of the gold particle. In some examples, the one-step ligand exchange methods comprise: contacting a gold particle capped with a toxic ligand with a gold salt and a less-toxic ligand in a solution, wherein the less-toxic ligand further serves as a reducing agent for the gold salt, such that the gold salt is reduced and deposited as a thin layer of gold onto the gold particle, wherein the concentration of the less-toxic ligand in the solution is sufficient to cause the toxic ligand to be replaced by the less-toxic ligand at the surface of the gold particle.

[0136] In some examples, the thin layer of gold is a monolayer (e.g., an atomic monolayer).

[0137] In some examples, the gold salt comprises HAuCU.

[0138] In some examples, the concentration of gold salt is selected to control the thickness of the deposited layer. In some examples, the concentration of the gold salt is selected such that the thin layer of gold has a thickness of 3 nanometer (nm) or less (e.g., 2 nm or less, or 1 nm or less). In some examples, the concentration of the gold salt is selected such that the thin layer of gold is a monolayer (e.g., an atomic monolayer).

[0139] In some examples, the method further comprises controlling the pH of the solution, wherein the pH of the solution is selected to control the protonation of the less-toxic ligand, thereby controlling the binding strength of the less-toxic ligand to the gold particle. For example, selecting the pH of the solution can comprise controlling the pH via addition of an appropriate amount of an acid or base. In some examples, the pH is 7 or less (e.g., 6.5 or less, 6 or less, 5 or less, 4.5 or less, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less, or 1.5 or less). In some examples, the pH is 1 or more (e.g., 1.5 or more, 2 or more, 2.5 or more, 3 or more, 3.5 or more, 4 or more, 4.5 or more, 5 or more, 5.5 or more, or 6 or more). The pH of the solution can range from any of the minimum values described above to any of the maximum values described above. For example, the pH can be from 1 to 7 (e.g., from 1 to 4, from 4 to 7, from 1 to 3, from 3 to 5, from 5 to 7, from 1 to 6, from 1 to 5, from 2 to 7, from 3 to 7, from 2 to 6, or from 2 to 4). In some examples, the pH is from 2 to 4.

[0140] In some examples, the method comprises contacting the gold particle with the solution by adding the solution drop wise.

[0141] In some examples, the method comprises contacting the gold particle with the solution adding the solution in one shot.

[0142] Suitable capping ligands for gold particles are known in the art. "Capping" refers to the formation of an ionic or covalent bond of molecules to the surface atoms of a particle, this molecule is referred to as a capping ligand. "Capping ligand" refers to a molecule or ion possessing a functional group capable of binding to the surface atoms of a particle by ionic or covalent bond. In some examples, the capping ligands comprise a group that can form a bond with gold atoms, such as a group comprising S, Se, N, P, C, or a combination thereof. In some examples, capping ligands contain functional groups such as thiol (-SH), carboxyl (-COOH), or amine (-NH2) groups. The capping ligands can be used to stabilize the gold particles. The capping ligands can, for example, prevent the oxidation on the exterior surface of the gold particles and / or increase the dispersibility of the gold particles.

[0143] In some examples, the toxic ligand comprises cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTAC), dodecyltrimethylammonium bromide, dodecyltrimethylammonium chloride, octadecyltrimethylammonium bromide, octadecyltrimethylammonium chloride, or a combination thereof. In some examples, the toxic ligand comprises cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTAC), or a combination thereof.

[0144] In some examples, the less-toxic ligand comprises citric acid or a citrate (e.g., monocitrate, bi-citrate, tri-citrate, or a combination thereof), such as a sodium citrate (e.g., sodium mono-citrate, sodium bi-citrate, sodium tri-citrate, or a combination thereof). In some examples, the less-toxic ligand comprises citrate, such as sodium citrate.

[0145] In some examples, the toxic ligand is more strongly bound to the gold particle relative to the less-toxic ligand.

[0146] The gold particle can have an average particle size. “Average particle size” and “mean particle size” are used interchangeably herein, and generally refer to the statistical mean particle size of the particles in a population of particles. For example, the average particle size for a plurality of particles with a substantially spherical shape can comprise the average diameter of the plurality of particles. For a particle with a substantially spherical shape, the diameter of a particle can refer, for example, to the diameter measured using electron microscopy (e.g., transmission electron microscopy). As used herein, the diameter of a particle can refer to the largest linear distance between two points on the surface of the particle. For an anisotropic particle, the average particle size can refer to, for example, the average maximum dimension of the particle (e.g., the length of a rod shaped particle, the diagonal of a cube shape particle, the bisector of a triangular shaped particle, etc.) For an anisotropic particle, the average particle size can refer to, for example, the hydrodynamic size of the particle. Mean particle size can be measured using methods known in the art, such as evaluation by scanning electron microscopy, transmission electron microscopy, and / or dynamic light scattering. As used herein, the mean particle size is measured by electron microscopy (e.g., transmission electron microscopy).

[0147] In some examples, the gold particle can have an average particle size of 1 nanometer or more (e.g., 5 nm or more, 10 nm or more, 15 nm or more, 20 nm or more, 25 nm or more, 30 nm or more, 35 nm or more, 40 nm or more, 45 nm or more, 50 nm or more, 55 nm or more, 60 nm or more, 65 nm or more, 70 nm or more, 75 nm or more, 80 nm or more, 85 nm or more, 90 nm or more, 95 nm or more, 100 nm or more, 125 nm or more, 150 nm or more, 175 nm or more, 200 nm or more, 225 nm or more, 250 nm or more, 300 nm or more, 350 nm or more, 400 nm or more, 450 nm or more, 500 nm or more, 600 nm or more, 700 nm or more, 800 nm or more, or 900 nm or more). In some examples, the gold particle can have an average particle size of 5 nanometers (nm) or more. In some examples, the gold particle can have an average particle size of 1000 nm or less (e.g., 900 nm or less, 800 nm or less, 700 nm or less, 600 nm or less, 500 nm or less, 450 nm or less, 400 nm or less, 350 nm or less, 300 nm or less, 250 nm or less, 225 nm or less, 200 nm or less, 175 nm or less, 150 nm or less, 125 nm or less, 100 nm or less, 95 nm or less, 90 nm or less, 85 nm or less, 80 nm or less, 75 nm or less, 70 nm or less, 65 nm or less, 60 nm or less, 55 nm or less, 50 nm or less, 45 nm or less, 40 nm or less, 35 nm or less, 30 nm or less, 25 nm or less, 20 nm or less, 15 nm or less, 10 nm or less, or 5 nm or less). The average particle size of the gold particle can range from any of the minimum values described above to any of the maximum values described above. For example, the gold particle can have an average particle size of from 1 nm to 1000 nm (e.g., from 1 to 500 nm, from 500 to 1000 nm, from 1 to 200 nm, from 200 to 400 nm, from 400 to 600 nm, from 600 to 800 nm, from 800 to 1000 nm, from 1 nm to 800 nm, from 1 nm to 600 nm, from 1 nm to 400 nm, from 1 nm to 100 nm, from 1 nm to 50 nm, from 10 nm to 1000 nm, from 50 nm to 1000 nm, from 100 to 1000 nm, from 200 to 1000 nm, from 400 to 1000 nm, from 600 to 1000 nm, from 5 nm to 900 nm, from 10 nm to 500 nm, or from 10 nm to 40 nm). In some examples, the gold particle can have an average particle size of from 5 nm to 1000 nm (e.g., from 5 to 500 nm, from 500 to 1000 nm, from 5 to 200 nm, from 200 to 400 nm, from 400 to 600 nm, from 600 to 800 nm, from 800 to 1000 nm, from 5 nm to 800 nm, from 5 nm to 600 nm, from 5 nm to 400 nm, from 5 nm to 100 nm, from 5 nm to 50 nm, from 10 nm to 1000 nm, from 50 nm to 1000 nm, from 100 to 1000 nm, from 200 to 1000 nm, from 400 to 1000 nm, from 600 to 1000 nm, from 5 nm to 900 nm, from 10 nm to 500 nm, or from 10 nm to 40 nm). In some examples, the gold particle has an average particle size of from 5 nm to 150 nm. In some examples, the gold particle can have an average particle size of from 10 nm to 40 nm. In some examples, the gold particle has an average particle size of 10 nm, 20 nm, or 40 nm.

[0148] In some examples, the gold particle can be substantially monodisperse. “Monodisperse” and “homogeneous size distribution,” as used herein, and generally describe a population of particles where all of the particles are the same or nearly the same size. As used herein, a monodisperse distribution refers to particle distributions in which 80% of the distribution (e.g., 85% of the distribution, 90% of the distribution, or 95% of the distribution) lies within 25% of the median particle size (e.g., within 20% of the median particle size, within 15% of the median particle size, within 10% of the median particle size, or within 5% of the median particle size).

[0149] The gold particle can comprise any shape (e.g., a sphere, a rod, a quadrilateral, an ellipse, a triangle, a polygon, etc.). In some examples, the gold particle can have an isotropic shape. In some examples, the gold particle can have an anisotropic shape. In some examples, the gold particle can have a shape that is substantially spherical, cuboidal, octahedral, or a combination thereof. In some examples, the gold particle is substantially spherical in shape. In some examples, the gold particle before and / or after the contacting step has a substantially spherical shape. In some examples, the shape of the gold particle is preserved after the contacting step.

[0150] In some examples, the gold particle can comprise a plurality of gold particles and the plurality of gold particles can comprise: a first population of particles having a first average particle size and a first particle shape; and a second population of particles having a second average particle size and a second particle shape; wherein the first average particle size and the second average particle size are different, the first particle shape and the second particle shape are different, or a combination thereof. In some examples, the plurality of gold particles can comprise a mixture of a plurality of populations of particles, wherein each population of particles within the mixture has a different size, shape, or combination thereof.

[0151] In some examples, the gold particle can comprise a radioisotope, such as Au- 198 and / or Au- 199.

[0152] In some examples, the concentration of the less-toxic ligand is 200 mM or more. In some examples, the concentration of the less-toxic ligand is at or below the solubility limit of the less- toxic ligand.

[0153] In some examples, the method is a one pot method.

[0154] In some examples, the method is performed at room temperature.

[0155] In some examples, the method is completed in an amount of time of 1 minute or more (e.g., 2 minutes or more, 3 minutes or more, 4 minutes or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, 25 minutes or more, 30 minutes or more, 35 minutes or more, 40 minutes or more, 45 minutes or more, 50 minutes or more, or 55 minutes or more). In some examples, the method is completed in an amount of time of 1 hour or less (e.g., 55 minutes or less, 50 minutes or less, 45 minutes or less, 40 minutes or less, 35 minutes or less, 30 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less). The amount of time in which the methods is completed can range from any of the minimum values described above to any of the maximum values described above. For example, the method can be completed in an amount of time of from 1 minute to 1 hour (e.g., from 1 minute to 30 minutes, from 30 minutes to 1 hour, from 1 minute to 20 minutes, from 20 minutes to 40 minutes, from 40 minutes to 1 hour, from 1 minute to 50 minutes, from 1 minute to 40 minutes, or from 1 minute to 10 minutes). . In some examples, the method is completed in an amount of time of from 1 minute to 30 minutes.

[0156] In some examples, the method does not result in agglomeration of the gold particles.

[0157] Also disclosed herein are gold particles made by any of the methods described herein. In some examples, the gold particle after ligand exchange has decreased cell toxicity relative to the gold particle before ligand exchange. In some examples, the gold particle after ligand exchange is colloidally stable for an amount of time of 24 hours or more (e.g., 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 1 week or more, 1.5 weeks or more, 2 weeks or more, 2.5 weeks or more, 3 weeks or more, 3.5 weeks or more, or 1 month or more).

[0158] In some examples, the gold particle is further conjugated to a targeting ligand, such as an antibody, a nucleic acid probe, a molecular recognition element, or a combination thereof.

[0159] In some examples, the gold particle is further conjugated to a drug-carrying ligand.

[0160] Compositions

[0161] Also disclosed herein are compositions comprising any of the gold particles disclosed herein. For example, the composition can comprise the gold particle at least partially dispersed in a solvent (e.g., a colloidal dispersion). The solvent can, for example, comprise water, ethylene glycol, polyethylene glycol, glycerol, alkane diol, ethanol, methanol, propanol, isopropanol, dimethyl sulfoxide (DMSO), acetonitrile, methylene chloride, or combinations thereof. In some examples, the solvent comprises water (e.g., an aqueous colloidal dispersion).

[0162] In some examples, the colloidal dispersion of the gold particle is stable for an amount of time of 24 hours or more.

[0163] In some examples, the gold particle is further conjugated to a drug-carrying ligand. In some examples the gold particle or composition further comprises a drug, the drug being conjugated to the drug-carrying ligand.

[0164] Also disclosed herein are pharmaceutical compositions comprising any of the gold particles disclosed herein and a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient, or a combination thereof.

[0165] In some examples, the pharmaceutical composition is administered to a subject. In some examples, the subject is a mammal. In some examples, the mammal is a primate. In some examples, the mammal is a human. In some examples, the human is a patient.

[0166] In some examples, the disclosed compositions comprise the disclosed gold particles as an active ingredient, a pharmaceutically acceptable carrier, and, optionally, other therapeutic ingredients or adjuvants. The instant compositions include those suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

[0167] Methods of Use

[0168] Also disclosed herein are methods of use of any of the gold particles or compositions described herein. For example, the gold particles or compositions disclosed herein have potential applications in a number of fields such as imaging (e.g., biological imaging, biomedical imaging), sensing (e.g., chemical sensing, biological sensing), medical applications (e.g., imaging, therapy, diagnostics, photothermal therapy, combinations thereof).

[0169] In some examples, the method comprises using the gold particle or composition in biomedical imaging (e.g., as an imaging agent).

[0170] In some examples, the method comprises using the gold particle or composition in photodynamic therapy, photothermal therapy, or a combination thereof.

[0171] In some examples, the method comprises using the gold particle or composition as a sensor.

[0172] In some examples, the method comprises using the gold particle or composition for detection of biomarkers, infection disease-related antigens, antibodies, or a combination thereof.

[0173] In some examples, the method comprises using the gold particle or composition for drug delivery. In some examples, the gold particle is further conjugated with a drug-carrying ligand. In some examples, the gold particle or composition further comprises a drug, the drug being conjugated to the drug-carrying ligand.

[0174] In some examples, the method comprises using the gold particle or composition as a catalyst.

[0175] In some examples, the method comprises using the gold particle or composition as a photosensitizer.

[0176] Also disclosed herein are assays comprising any of the gold particles or compositions herein. Also disclosed herein are point of care tests comprising any of the gold particles or compositions herein. For example, the point of care test can comprise a pregnancy test. In some examples, the point of care test can comprise a test for a pathogenic microorganism. In some examples, the point of care test can comprise a coronavirus test, such as a Covid- 19 test. In some examples, the point of care test can comprise a cancer screening test. Also disclosed herein are methods of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any of the gold particles or compositions disclosed herein. In some examples, the disease comprises cancer.

[0177] Also disclosed herein are methods of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any of the gold particles or compositions disclosed herein.

[0178] For example, the gold particles and compositions described herein are useful for treating cancer in humans, e.g., pediatric and geriatric populations, and in animals, e.g., veterinary applications. The disclosed methods can optionally include identifying a patient who is or may be in need of treatment of a cancer. Examples of cancer types include bladder cancer, brain cancer, breast cancer, colorectal cancer, cervical cancer, gastrointestinal cancer, genitourinary cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer, and testicular cancer. Further examples include cancer and / or tumors of the anus, bile duct, bone, bone marrow, bowel (including colon and rectum), eye, gall bladder, kidney, mouth, larynx, esophagus, stomach, testis, cervix, mesothelioma, neuroendocrine, penis, skin, spinal cord, thyroid, vagina, vulva, uterus, liver, muscle, blood cells (including lymphocytes and other immune system cells). Further examples of cancers treatable by the gold particles and compositions described herein include carcinomas, Karposi’s sarcoma, melanoma, mesothelioma, soft tissue sarcoma, pancreatic cancer, lung cancer, leukemia (acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myeloid, and other), and lymphoma (Hodgkin’s and non-Hodgkin’s), and multiple myeloma.

[0179] The methods of treatment or prevention of cancer described herein can, in some examples, further include treatment with one or more additional agents e.g., an anti-cancer agent or ionizing radiation). For example, the gold particles or compositions as described herein can be combined into a pharmaceutical composition with an anticancer agent, such as a chemotherapeutic agent, an immunotherapeutic agent, or a combination thereof.

[0180] In some examples, the anticancer agent can comprise a chemotherapeutic agent. Chemotherapy is the treatment of cancer with one or more cytotoxic anti-neoplastic drugs (e.g., chemotherapeutic agents) as part of a standardized regimen. Chemotherapy may be given with a curative intent or it may aim to prolong life or to palliate symptoms. In some cases, it can be used in conjunction with other cancer treatments, such as radiation therapy, surgery, hyperthermia therapy, or a combination thereof.

[0181] The anti-cancer agent can also include biopharmaceuticals such as, for example, antibodies. Many tumors and cancers have viral genome present in the tumor or cancer cells. For example, Epstein-Barr Virus (EBV) is associated with a number of mammalian malignancies. The gold particles disclosed herein can also be used alone or in combination with anticancer or antiviral agents, such as ganciclovir, azidothymidine (AZT), lamivudine (3TC), etc., to treat patients infected with a virus that can cause cellular transformation and / or to treat patients having a tumor or cancer that is associated with the presence of viral genome in the cells. The gold particles disclosed herein can also be used in combination with viral based treatments of oncologic disease.

[0182] Also described herein are methods of suppressing tumor growth in a subject. The method includes contacting at least a portion of the tumor with a therapeutically effective amount of any of the gold particles or compositions as described herein.

[0183] In some examples, it is desirable to target the gold particle using a targeting moiety that is specific to a cell type and / or tissue type. In some embodiments, gold particle may be targeted to a particular cell, tissue, and / or organ using a targeting moiety. Exemplary non-limiting targeting moieties include ligands, cell surface receptors, glycoproteins, vitamins (e.g., riboflavin) and antibodies (e.g., full-length antibodies, antibody fragments (e.g., Fv fragments, single chain Fv (scFv) fragments, Fab' fragments, or F(ab')2 fragments), single domain antibodies, camelid antibodies and fragments thereof, human antibodies and fragments thereof, monoclonal antibodies, and multispecific antibodies (e.g.,. bispecific antibodies)). In some embodiments, the targeting moiety may be a polypeptide. The targeting moiety may include the entire polypeptide (e.g., peptide or protein) or fragments thereof. A targeting moiety is typically positioned on the outer surface of the particle in such a manner that the targeting moiety is available for interaction with the target, for example, a cell surface receptor. A variety of different targeting moieties and methods are known and available in the art, including those described, e.g., in Sapra et al., Prog. Lipid Res. 42(5):439-62, 2003 and Abra et al., J. Liposome Res. 12:1-3, 2002.

[0184] The targeting moiety can target any known cell type, including, but not limited to, hepatocytes, colon cells, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells (including primary tumor cells and metastatic tumor cells).

[0185] In some examples, the methods further include the step of irradiating at least a portion of the tumor with a therapeutically effective amount of ionizing radiation. Additionally, methods of radiotherapy of tumors are provided herein. The methods include contacting the tumor cell with an effective amount of a compound or composition as described herein, and irradiating the tumor with an effective amount of ionizing radiation. As used herein, the term ionizing radiation refers to radiation comprising particles or photons that have sufficient energy or can produce sufficient energy via nuclear interactions to produce ionization. An example of ionizing radiation is X- radiation. A therapeutically effective amount of ionizing radiation refers to a dose of ionizing radiation that produces an increase in cell damage or death when administered in combination with the gold particles described herein. The ionizing radiation can be delivered according to methods as known in the art, including administering radiolabeled antibodies and radioisotopes. In some examples, the gold particle can comprise a radioisotope, such as Au-198 and / or Au-199.

[0186] In some examples, the methods further comprise irradiating the gold particle with electromagnetic radiation that overlaps with at least a portion of one or more of an absorption, emission, or excitation band of the gold particle.

[0187] The methods of treatment of the disease or disorder described herein can further include treatment with one or more additional agents. The one or more additional agents and the gold particles and compositions as described herein can be administered in any order, including simultaneous administration, as well as temporally spaced order of up to several days apart. The methods can also include more than a single administration of the one or more additional agents and / or the gold particles and compositions as described herein. The administration of the one or more additional agents and the gold particles and compositions as described herein can be by the same or different routes. When treating with one or more additional agents, the gold particles and compositions as described herein can be combined into a pharmaceutical composition that includes the one or more additional agents.

[0188] Also disclosed herein are methods of imaging a cell or a population of cells within or about a subject, the method comprising administering to the subject an amount of any of the gold particles or compositions disclosed herein and detecting the gold particle or composition. The detecting can involve methods known in the art. In some examples, the gold particles or compositions can further comprise a detectable label, such as a radiolabel, fluorescent label, enzymatic label, and the like. In some examples, the detectable label can comprise a radiolabel, such as Au-198 and / or Au-199. Such imaging methods can be used, for example, for assessing the extent of a disease and / or the target of a therapeutic agent. In some examples, the cell or population of cells is indicative of cancer.

[0189] The methods, gold particles, and compositions as described herein are useful for both prophylactic and therapeutic treatment. As used herein the term treating or treatment includes prevention; delay in onset; diminution, eradication, or delay in exacerbation of signs or symptoms after onset; and prevention of relapse. For prophylactic use, a therapeutically effective amount of the gold particles and compositions as described herein are administered to a subject prior to onset (e.g., before obvious signs of the disease or disorder), during early onset (e.g., upon initial signs and symptoms of the disease or disorder), or after an established development of the disease or disorder. Prophylactic administration can occur for several days to years prior to the manifestation of symptoms of a disease or disorder. Therapeutic treatment involves administering to a subject a therapeutically effective amount of the gold particles or compositions as described herein after the disease or disorder is diagnosed.

[0190] Compositions, Formulations, and Methods of Administration

[0191] In vivo application of the disclosed gold particles, and compositions containing them, can be accomplished by any suitable method and technique presently or prospectively known to those skilled in the art. For example, the disclosed gold particles can be formulated in a physiologically- or pharmaceutically-acceptable form and administered by any suitable route known in the art including, for example, oral, nasal, rectal, topical, and parenteral routes of administration. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, and intrasternal administration, such as by injection. Administration of the disclosed gold particles or compositions can be a single administration, or at continuous or distinct intervals as can be readily determined by a person skilled in the art.

[0192] The gold particles disclosed herein, and compositions comprising them, can also be administered utilizing liposome technology, slow release capsules, implantable pumps, and biodegradable containers. These delivery methods can, advantageously, provide a uniform dosage over an extended period of time.

[0193] The gold particles disclosed herein can be formulated according to known methods for preparing pharmaceutically acceptable compositions. Formulations are described in detail in a number of sources which are well known and readily available to those skilled in the art. For example, Remington ’ Pharmaceutical Science by E.W. Martin (1995) describes formulations that can be used in connection with the disclosed methods. In general, the gold particles disclosed herein can be formulated such that an effective amount of the gold particles is combined with a suitable excipient in order to facilitate effective administration of the gold particles. The compositions used can also be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspension, suppositories, injectable and infusible solutions, and sprays. The preferred form depends on the intended mode of administration and application. The compositions can also include conventional pharmaceutically-acceptable carriers and diluents which are known to those skilled in the art.

[0194] Examples of carriers or diluents for use with the gold particles include ethanol, dimethyl sulfoxide, glycerol, alumina, starch, saline, and equivalent carriers and diluents. To provide for the administration of such dosages for the desired application, compositions disclosed herein can comprise between about 0.1% and 100% by weight of the total of one or more of the subject gold particles based on the weight of the total composition including carrier or diluent.

[0195] The pharmaceutical carrier employed can be, for example, a solid, liquid, or gas. Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen.

[0196] Formulations suitable for administration include, for example, aqueous sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions, which can include suspending agents and thickening agents. The formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powder, granules, tablets, etc. It should be understood that in addition to the excipients particularly mentioned above, the compositions disclosed herein can include other agents conventional in the art having regard to the type of formulation in question.

[0197] The gold particles disclosed herein, and compositions comprising them, can be delivered to a cell either through direct contact with the cell or via a carrier means. Carrier means for delivering gold particles and compositions to cells are known in the art.

[0198] For the treatment of oncological disorders, the gold particles or compositions disclosed herein can be administered to a patient in need of treatment in combination with other antitumor or anticancer substances and / or with radiation and / or photodynamic therapy and / or with surgical treatment to remove a tumor. These other substances or treatments can be given at the same as or at different times from the gold particles or compositions disclosed herein. For example, the gold particles or compositions disclosed herein can be used in combination with mitotic inhibitors such as taxol or vinblastine, alkylating agents such as cyclophosamide or ifosfamide, antimetabolites such as 5-fluorouracil or hydroxyurea, DNA intercalators such as adriamycin or bleomycin, topoisomerase inhibitors such as etoposide or camptothecin, antiangiogenic agents such as angiostatin, antiestrogens such as tamoxifen, and / or other anti-cancer drugs or antibodies, such as, for example, GLEEVEC (Novartis Pharmaceuticals Corporation) and HERCEPTIN (Genentech, Inc.), respectively, or an immunotherapeutic such as ipilimumab and bortezomib.

[0199] In certain examples, the gold particles and compositions disclosed herein can be locally administered at one or more anatomical sites, such as sites of unwanted cell growth (such as a tumor site or benign skin growth, e.g., injected or topically applied to the tumor or skin growth), optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent. The gold particles and compositions disclosed herein can be systemically administered, such as intravenously or orally, optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent, or an assimilable edible carrier for oral delivery. They can be enclosed in hard or soft shell gelatin capsules, can be compressed into tablets, or can be incorporated directly with the food of the patient’s diet. For oral therapeutic administration, the gold particles can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, aerosol sprays, and the like.

[0200] The tablets, troches, pills, capsules, and the like can also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; diluents such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring can be added. When the unit dosage form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials can be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules can be coated with gelatin, wax, shellac, or sugar and the like. A syrup or elixir can contain the gold particles, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the gold particles can be incorporated into sustained-release preparations and devices.

[0201] The gold particles and compositions disclosed herein can be administered intravenously, intramuscularly, or intraperitoneally by infusion or injection. Solutions of the gold particles can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.

[0202] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the gold particles, which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. Optionally, the prevention of the action of microorganisms can be brought about by various other antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents that delay absorption, for example, aluminum monostearate and gelatin.

[0203] Pharmaceutical compositions disclosed herein suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In some examples, the final injectable form can be sterile and can be effectively fluid for easy syringability. In some examples, the pharmaceutical compositions can be stable under the conditions of manufacture and storage; thus, they can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

[0204] Sterile injectable solutions are prepared by incorporating the gold particles and / or compositions disclosed herein in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

[0205] Pharmaceutical compositions disclosed herein can be in a form suitable for topical use such as, for example, an aerosol, cream, ointment, lotion, dusting powder, mouth washes, gargles, solution, tincture, and the like. In some examples, the compositions can be in a form suitable for use in transdermal devices. In some examples, it will be desirable to administer them topically to the skin as compositions, in combination with a dermatologically acceptable carrier, which can be a solid or a liquid. The gold particles and compositions disclosed herein can be applied topically to a subject’s skin. These formulations can be prepared, utilizing any of the gold particles disclosed herein, via conventional processing methods.

[0206] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol / glycol blends, in which the gold particles can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers, for example.

[0207] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

[0208] Pharmaceutical compositions disclosed herein can be in a form suitable for rectal administration wherein the carrier is a solid. In some examples, the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories can be conveniently formed by first admixing the composition with the softened or melted carriers) followed by chilling and shaping in molds.

[0209] In addition to the aforementioned carrier ingredients, the pharmaceutical formulations described above can include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like. Furthermore, other adjuvants can be included to render the formulation isotonic with the blood of the intended recipient. Compositions containing any of the gold particles disclosed herein can also be prepared in powder or liquid concentrate form.

[0210] Useful dosages of the gold particles and compositions disclosed herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms or disorder are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.

[0211] Also disclosed are kits that comprise a gold particle disclosed herein in one or more containers. The disclosed kits can optionally include pharmaceutically acceptable carriers and / or diluents. In one embodiment, a kit includes one or more other components, adjuncts, or adjuvants as described herein. In one embodiment, a kit includes instructions or packaging materials that describe how to administer a gold particle or composition of the kit. Containers of the kit can be of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration. In one embodiment, a gold particle disclosed herein is provided in the kit as a solid, such as a tablet, pill, or powder form. In another embodiment, a gold particle disclosed herein is provided in the kit as a liquid or solution. In one embodiment, the kit comprises an ampoule or syringe containing a gold particle disclosed herein in liquid or solution form.

[0212] In some examples, the kit further comprises at least one agent, wherein the gold particle and the agent are co-formulated.

[0213] In some examples, the gold particle and the agent are co-packaged.

[0214] The kits can also comprise gold particles and / or products co-packaged, co-formulated, and / or co-delivered with other components. For example, a drug manufacturer, a drug reseller, a physician, a compounding shop, or a pharmacist can provide a kit comprising a disclosed gold particles and / or product and another component for delivery to a patient.

[0215] It is contemplated that the disclosed kits can be used in connection with the disclosed methods of making, the disclosed methods of using, and / or the disclosed compositions.

[0216] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0217] The examples below are intended to further illustrate certain aspects of the systems and methods described herein, and are not intended to limit the scope of the claims. EXAMPLES

[0218] The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.

[0219] Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of measurement conditions, e.g., component concentrations, temperatures, pressures and other measurement ranges and conditions that can be used to optimize the described process.

[0220] Example 1 - Tri-Citrate-Capped Gold Nanospheres with Uniform and Well-Controlled Sizes

[0221] Described herein is a method for preparing tri-citrate-capped Au nanospheres with uniform and well-controlled sizes and the potential application of this class of nanomaterials in healthcare and biomedicine. Gold (Au) nanoparticles exhibit favorable optical properties for a range of applications, including sensing, imaging, and therapy. However, the low biocompatibility and high toxicity of the organic surfactants typically used as surface ligands for their synthesis, including cetyltrimethylammonium bromide (CTAB) and cetyltrimethylammonium chloride (CTAC), greatly limit their use in bio-related applications. To address these issues, a simple and versatile method to effectively replace the toxic CTAB / CTAC ligands on the surface of Au nanospheres with tri-citrate, an organic species known for its high biocompatibility and low toxicity, has been developed.

[0222] Specifically, a method had been developed to replace the CTAB / CTAC pre-adsorbed on the surface of Au nanospheres with tri-citrate by depositing an ultrathin shell of fresh Au in the presence of excess sodium tri-citrate. The tri-citrate serves as a reducing agent for the AuCh precursor and the ligand of interest for bio-related applications. As reported in a prior study, the CTAB / CTAC-capped Au nanospheres could still grow into larger sizes, indicating that the binding between CTAB / CTAC and Au surfaces was not very strong and could be replaced by other ligands. When a fresh surface is formed during the Au deposition process, it will be preferentially covered by tri-citrate, which is more abundant in the reaction solution. When the synthesis is conducted at an adequate concentration of sodium tri-citrate, the initially-adsorbed CTAB / CTAC will be readily desorbed into the reaction solution and replaced by tri-citrate. Multiple techniques are then used to confirm that the surface of the resultant Au nanospheres are indeed covered by tri-citrate and free of CTAB / CTAC. The tri-citrate-capped Au nanospheres show the same optical properties as the original CT AB / CT AC-capped sample, indicating no aggregation during the ligand exchange process. Besides, an in vitro study is performed to demonstrate the effectiveness of this method in mitigating the cytotoxicity associated with the ligand on the surface of Au nanospheres.

[0223] Since Au nanospheres have strong optical absorption in the visible region, the tri-citrate- capped Au nanospheres can be directly used as a color maker for sensitive detection. For instance, the strong red / pink color of the tri-citrate-capped Au nanospheres can be used for rapid and accurate detection of biomarkers, infectious disease-related antigens, or antibodies. The tri- citrate-capped Au nanospheres can also serve as signal amplifiers or carriers, facilitating high- sensitivity signal enhancement and detection. Due to the presence of Au surface, the tri-citrate- capped nanospheres can be robustly conjugated with antibodies to interact with biological targets or molecular recognition elements, enabling efficient detection of specific bioactive substances through molecular-specific binding. Additionally, due to their high stability, the tri-citrate- capped Au nanospheres can be employed as signal amplifiers or labels in portable biosensors such as lateral flow biosensors for point-of-care test (POCT) or molecular diagnosis, such as pregnancy test and Covid- 19 screening. By conjugating these tri-citrate-capped Au nanospheres with specific molecules like antibodies or nucleic acid probes, highly sensitive, rapid, and reliable detection of specific disease markers or pathogenic microorganisms can be achieved. This technology presents a nanomaterial and preparation method for immediate use in biorelated applications.

[0224] The uniform and tri-citrate-capped Au nanospheres can find immediate use in replacing the non-uniform Au nanoparticles currently used in the commercial kits for point-of-care test (POCT) such as pregnancy test and Covid- 19 screening.

[0225] The Au nanospheres can also serve as a contrast agent for cancer diagnosis through computed tomography (CT). They can also serve as an electron- scattering probe in transmission electron microscopy (TEM)-assisted imaging of pancreatic cancer cells. The low cytotoxicity of the tri-citrate-capped Au nanospheres can ensure a low level of adverse impacts on the patients. Additionally, by heating these tri-citrate-capped Au nanospheres via optical irradiation at their absorption peak, cancer cells can be destructed through localized hyperthermia.

[0226] The tri-citrate-capped Au nanospheres can also serve as a carrier for drug delivery. By adsorbing antitumor agents onto the surface of the Au nanospheres, it is possible to enhance the anchoring and penetration in the target tumors to maximize the efficacy of the antitumor drugs. The tri-citrate -capped Au nanospheres can also play a major role in theranostics due to their unique optical properties and low cytotoxicity. They allow for real-time monitoring of treatment by physicians while increasing the treatment efficacy. The different sizes of the nanospheres described herein can be used in future theranostic experiments to gauge the efficacy of treatment as a function of particle size.

[0227] Gold nanoparticles have found widespread use in an array of applications, including those related to point-of-care test (POCT). Although many protocols have been reported for the colloidal synthesis of Au nanoparticles, the products were typically plagued by a number of drawbacks, including a polycrystalline structure, a quasi-spherical shape, and a polydispersed size. In addressing these issues, a method was developed involving seed-mediated growth for the synthesis of Au nanospheres with a single-crystal structure and controlled diameters in the range of 5-150 nm. However, the surface of these nanospheres were capped by CT AB and CTAC, surfactants known for their cytotoxicity. The Au nanospheres described herein are capped by tricitrate, a chemical species known for its negligible cytotoxicity.

[0228] Different from a prior study involving silver (Ag) deposition and then etching for the ligand exchange, this work offers a method free of contamination from Ag, which is known to be toxic to various biological systems.

[0229] The uniform, tri-citrate-capped Au nanospheres open the door to a broad range of applications in healthcare and biomedicine. The Au nanospheres have unique optical properties such as surface plasmon resonance, making them useful for optical imaging contrast enhancement, drug delivery, photothermal therapy, and biosensing, among others. The ability to exchange the toxic CTAB / CTAC with tri-citrate further enhances the applicability of this class of nanomaterials.

[0230] The market is potentially huge as the nanomaterial described in this disclosure can find immediate use in replacing the non-uniform Au nanoparticles currently used in the commercial kits for pregnancy test and Covid- 19 screening.

[0231] Example 2 - Facile Synthesis and Characterization of Uniform Au Nanospheres Capped by Tri-Citrate for Biomedical Applications

[0232] Abstract. Described herein is a simple and versatile method for effectively replacing the toxic ligands, such as cetyltrimethylammonium bromide (CTAB) and cetyltrimethylammonium chloride (CTAC), on the surface of Au nanospheres with different sizes by tri-citrate (Figure 1). The method involves the deposition of an ultrathin shell of fresh Au in the presence of sodium tri-citrate at an adequate concentration. After the ligand exchange process, multiple techniques are used to confirm that the surface of the resultant Au nanospheres is covered by tri-citrate while there is no sign of aggregation. The mitigation of cell toxicity after exchanging the surface-bound CTAB / CTAC with tri-citrate is also demonstrated, opening the door to a range of biomedical applications.

[0233] Introduction. Noble-metal nanocrystals with diverse shapes have been synthesized by manipulating the experimental parameters, including temperature, surface ligand, and reducing agent, among others, typically involved in a colloidal synthesis.[1’7]The nanocrystals have found widespread use in a variety of applications, including plasmonics, catalysis, sensing, biomedicine, and self-assembly.[8‘13]The surface ligand, in particular, can serve as a colloidal stabilizer to prevent the resultant nanocrystals from aggregation during synthesis or storage in addition to its role in dictating the evolution of shape.11 1- 16Despite its essential role in a shape- controlled synthesis, however, the surface ligand may compromise or ruin the targeted application like biomedicine due to its intrinsic toxicity.[11,17]As such, ligand exchange is often necessary to introduce the ligand of interest for the proposed application rather than the synthesis step.[18,19]

[0234] In the case of direct ligand exchange, the as-synthesized nanocrystals are collected by centrifugation, washed with a proper solvent, and then redispersed in a solvent containing the desired ligand. The suspension is typically incubated for a relatively long period of time under shaking / stirring to facilitate the ligand exchange process. This approach works the best when the initial ligand has a weaker binding to the surface than the ligand of interest. Notable examples include the exchange of poly(vinylpyrrolidone) (PVP), cetyltrimethylammonium bromide (CTAB), or cetyltrimethylammonium chloride (CTAC) on Au nanoparticles with a thiol compound.[18’20’21]In general, it is challenging to achieve complete exchange if the ligand of interest shares a similar or even lower affinity to the surface than the initial ligand. Besides, aggregation tends to occur if the involved ligands bear opposite charges, making it very difficult or impossible to conduct direct ligand exchange.

[0016]

[0235] Alternatively, ligand exchange can also be conducted in an indirect manner by introducing additional step(s) to facilitate the use of a ligand weaker in binding strength or with an opposite charge.

[0022] In one study, the original ligand was removed using acid treatment under sonication, followed by the deposition of the ligand of interest and then thermal treatment.

[0019] This approach tended to trigger aggregation if the pH of the solution is not optimized. In another study, an ultrathin shell of Ag was deposited onto the Au nanospheres to help remove the surface-bound CTAB / CTAC. The Ag shell was subsequently etched away in the presence of sodium tri-citrate to cover the surface with tri-citrate.

[0023] While this approach solved the aggregation issue, it required the removal of Ag and the resultant tri-citrate-capped Au nanospheres were often contaminated by the residual Ag. The multiple steps involved in the deposition and removal of Ag may have also caused potential loss of some nanospheres.

[0236] Herein, a facile, one-step method to effectively replace the initial CTAB / CTAC on Au nanospheres with tri-citrate is described. It involves the deposition of an ultrathin shell of fresh Au on the surface of Au nanospheres in the presence of tri-citrate, which serves as the ligand of interest and a reducing agent for the AuClT precursor (Figure 2). By depositing Au instead of Ag, this method prevents the resultant Au nanospheres from being contaminated by the residual Ag from incomplete etching, which might be detrimental to some biomedical applications. As reported in a prior study, the CTAB / CT AC-capped Au nanospheres could still grow into larger sizes,

[0024] indicating that the binding between CTAB / CTAC and Au surface was not as terminal as a thiol compound. In other words, the Au surface has the potential to uptake tri-citrate, which is more abundant in the reaction solution during the Au deposition process. As a result, the surfacebound CTAB / CTAC can be gradually exchanged with tri-citrate over a period of about 10 min. The results indicate that there is no aggregation during the ligand exchange process and the surface of the resultant Au nanospheres is covered by tri-citrate. Using an in vitro assay, the effectiveness of this method in mitigating the toxicity associated with the ligand on the surface of Au nanospheres was also demonstrated.

[0237] Results and Discussion. First, CTAB / CTAC-capped Au spheres of 10 nm in diameter were prepared by following a previously reported method.

[0024] As documented in literature, CTAB and CTAC can bind to the surface of Au nanoparticles in Au-(Br / Cl)-N or Au-N configuration.[23,25]In either configuration, the hydrophobic tails are pointed away from the Au surface. Since the Au nanospheres were formed and stably suspended in an aqueous solution, they were supposed to be surrounded by a bilayer comprised of CTAB and CTAC,

[0023] giving a highly-positive surface charge (zeta potential: +37.8 mV). Meanwhile, the aqueous sample showed a sharp localized surface plasmon resonance (LSPR) peak at 521 nm in the ultraviolet- visible (UV-vis) spectrum, indicating good uniformity and dispersibility of the nanospheres.

[0238] The surface-bound CTAB / CTAC was exchanged with tri-citrate by depositing an ultrathin (one monolayer in thickness) shell of fresh Au on the nanospheres to facilitate the ligand exchange process. In this case, tri-citrate could serve as both a reducing agent for AuCLf and a ligand for the freshly formed Au surface. In a standard protocol, 200 mM aqueous sodium tri-citrate was used to reduce ALICU" to Au atoms at room temperature. By controlling the amount of AuCLf added, only one atomic layer of fresh Au on each nanosphere was deposited. During the deposition of fresh Au, the surface-bound CTAB / CTAC molecules were quickly desorbed and substituted by tri-citrate, which had a much higher concentration than CTAB / CTAC in the solution. As shown by the TEM image in Figure 3A, the spherical shape of the particles was well preserved due to the deposition of only one atomic layer of fresh Au. For the same reason, the average diameter of the Au nanospheres was essentially maintained at 10 nm.

[0239] The involvement of an ultrathin shell was important to preserving the spherical shape taken by the particles. Using the standard protocol, the concentration of AuCU' used was also increased and thus the amount of fresh Au deposited on each nanosphere increased. Specifically, the theoretical thickness of the freshly formed shell was gradually increased from one to three atomic layers, 1 nm, and 3 nm. As expected, the shape started to deviate from spherical when more AuCU" was added. In the case of three atomic layers, nanoparticles featuring a comic tail were also formed (Figure 3B, marked by red circle). Further increasing the amount of AuCU' resulted in nanoparticles with longer tails or even branched structures (Figure 3C). Only a few spherical particles could be identified in this sample. Interestingly, when the theoretical thickness of the deposited shell was further increased to 3 nm, octahedra instead of particles with longer tails or more branched arms were observed (Figure 3D).

[0240] The results could be attributed to the different kinetics involved in the growth process (Figure 3E). Specifically, when the concentration of the precursor was only adequate for the deposition of one atomic layer, the rates corresponding to atom deposition (Vdeposition) and surface diffusion (Vdiffusion) were comparable to each other (^deposition ~ Vdiffusion), leading to the formation of a uniform shell of Au on the surface of each nanosphere. When more precursor was introduced, Vdeposition would increase. However, Vdiffusion should remain the same since the temperature was kept at 27 °C, hence, Vdeposition > Vdiffusion. At a low flux of Au atoms, only some of the equivalent sites would be able to receive the atoms, resulting in an asymmetric growth pattern. This phenomenon was also observed in the synthesis of nanorods, which were the products of asymmetric growth from cuboctahedral seeds.

[0026] Further increasing the amount of AuCU' led to a higher flux of Au atoms so that all equivalent sites would be able to receive Au atoms for growth. As a result, the growth pattern was switched back to a symmetric mode, with { 111} facets exposed as the most stable atomic plane. It is worth noting that Vdiffusion should be more or less the same among the three different scenarios as they were all carried out at the identical temperature of 27 °C.

[0241] The Au nanospheres were further characterized with spectroscopy methods to confirm the exchange of the surface-bound CTAB / CTAC with tri-citrate. The structural difference between tri-citrate and CTAB / CTAC allowed the use Fourier-transform infrared spectroscopy (FTIR) for this purpose (Figure 4). Firstly, the reference spectra of free CTAB / CTAC and tri- citrate were obtained. Due to the stretching mode of CH2 group in a long hydrocarbon chain, CTAB / CTAC had strong doublet peaks around 2900 cm In contrast, tri-citrate had characteristic doublet peaks in the region of 1400-1600 cm corresponding to the stretching mode of the carboxylate group. FTIR spectra from the Au nanospheres obtained through Au deposition with the use of tri-citrate at different concentrations were then recorded. For the sample prepared using the standard protocol (Figure 4, red trace), the tri-citrate-capped Au nanospheres showed strong doublet peaks in the region of 1400-1600 cm confirming the presence of tri-citrate. Meanwhile, no peak was observed around 2900 cm for CTAB / CTAC. This result suggested that tri-citrate ions could indeed replace the CTAB / CTAC on the surface if they were used at a sufficiently high concentration of 200 mM. The tri-citrate peak showed a slight shift in position, likely due to the binding between the tri-citrate and Au nanospheres.

[0023]

[0242] Decreasing the concentration of tri-citrate used for Au deposition would result in incomplete exchange of the surface-bound CTAB / CTAC. For example, when 100 mM instead of 200 mM aqueous sodium tri-citrate was used for Au deposition, the FTIR spectrum contained characteristic peaks of both CTAB / CTAC and tri-citrate, as shown by the blue trace in Figure 4. This result indicated that some CTAB / CTAC still bound to the Au surface and were not replaced by the tri-citrate. When the concentration of sodium tri-citrate used for Au deposition was further reduced to 10 mM (Figure 4, green trace), the tri-citrate peak in the region of 1400-1600 cm1decreased substantially in intensity when compared with that of the 100-mM or 200-mM sample. In contrast, the CTAB / CTAC doublet peaks were clearly observed around 2900 cm In this case, ligand exchange barely occurred due to the use of tri-citrate at a low concentration of 10 mM. Some tri-citrate could be trapped on the surface due to the possible electrostatic attraction between CTA+cation and tri-citrate anion.

[0243] Zeta potential measurements were also applied to analyze the extent of ligand exchange. The measured zeta potentials shifted from positive to negative since the surface of the CTAB / CTAC-capped and tri-citrate-capped Au nanospheres carried positive and negative charges, respectively. Specifically, the CTAB / CTAC-capped 10-nm Au spheres had a zeta potential of +37.8 mV. The value was reduced to +12.2 mV when 10 mM sodium tri-citrate was used for Au deposition. The decrease in zeta potential could be ascribed to partial exchange of CTAB / CTAC with tri-citrate. The zeta potentials further dropped to +2.07 and -33.42 mV when the concentration of sodium tri-citrate used for Au deposition was increased to 100 and 200 mM, respectively. The value of -33.42 mV was comparable to some of the values reported in the literature for tri-citrate-capped Au nanoparticles.[2,27]The highly -negative zeta potential indicated a surface fully covered by tri-citrate, suggesting that the surface-bound CTAB / CTAC had been mostly replaced by tri-citrate. The zeta potentials and FTIR results were consistent with each other, confirming that the ligand exchange could be achieved by simply increasing the concentration of sodium tri-citrate used for Au deposition to 200 mM.

[0244] After proving the exchange of ligands on the surface, the ability to preserve the colloidal stability of the tri-citrate-capped Au nanospheres against aggregation in a proper medium was further demonstrated by monitoring changes to their LSPR peak. The Au nanospheres prepared with the standard protocol were dispersed in pure water and 200 mM aqueous tri-citrate, respectively, and their UV-vis spectra were recorded. When dispersed in pure water, the tri- citrate-capped Au nanospheres showed a red-shift and broadening in LSPR peak (Figure 5), indicating poor stability against aggregation. In contrast, the position and full width at half maximum (FWHM) of the peak were retained when the nanospheres were dispersed in 200 mM aqueous sodium tri-citrate. The difference could be attributed to the desorption of tri-citrate from the surface of Au nanospheres in pure water. According to the Langmuir model,[10,28]if the concentration of free ligand in the solution drops, the adsorbed ligand will desorb from the surface, reducing the fractional surface coverage. As a result, the nanospheres would lose protection from the ligand and start to aggregate. Additionally, the CTAB / CTAC- and tri-citrate- capped Au nanospheres had zeta potentials of +37.8 and -33.42 mV, respectively. The similarity in absolute value indicated that their suspensions should share comparable stability against aggregation since a higher absolute value of zeta potential corresponded to a stronger electrostatic repulsion.

[0245] To demonstrate that this strategy is applicable to Au nanospheres of different sizes, the standard protocol was extended to CTAB / CTAC-capped Au spheres of 20 and 40 nm in diameter, which were prepared by following the previously reported method.

[0024] It was found that the tri-citrate solution used for Au deposition should have a concentration higher than 200 mM in order to fully replace the surface-bound CTAB / CTAC with tri-citrate. This could be attributed to the larger surface areas of 20- and 40-nm spheres when they were used at the same particle concentration as the 10-nm spheres. As shown by TEM images and UV-vis spectra, the spherical shape was well preserved during ligand exchange for both the 20- and 40-nm Au spheres, together with good dispersibility in 1 M aqueous sodium tri-citrate. For the FTIR spectra obtained from the 20- and 40-nm tri-citrate-capped Au spheres, the characteristic peaks in the region of 1400-1600 cm for tri-citrate was also observed while there was no CTAB / CTAC peaks observed around 2900 cm1. The negative zeta potentials also supported the FTIR data. Altogether, these results confirmed the successful replacement of the surface-bound CTAB / CTAC by tri-citrate when the particle size was increased from 10 to 20 and 40 nm. The biocompatibility of a nanomaterial is one of the most important factors in determining its ultimate use in an in vivo application such as cancer therapy and tissue repair. To this end, the biocompatibility of the CTAB / CTAC- and tri-citrated-capped 10-nm Au spheres were assessed using Cell Counting Kit 8 (CCK8) assay after they had been cultured with adipose-derived mesenchymal stem cells (ADMSCs) at 0, 20, 200, and 400 g / mL concentrations for 12 h (Figure 6A-Figure 6B). In this assay, the cell activity and survival state were analyzed by measuring the concentration of dehydrogenase in mitochondria with 2-(2- methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium sodium salt (WST-8). For the CTAB / CTAC-capped sample, the cell activity gradually decreased to 0.58 (20 g / mL). 0.11 (200 pg / mL), and 0.11(400 ug / mL) relative to the control group involving no Au nanospheres (Figure 6A), indicating the high toxicity of CTAB / CTAC-capped Au nanospheres. This could be attributed to the disruption of the cell membrane integrity by the CTAB / CTAC, which had been leveraged to induce cell destruction during cancer therapy.

[0029] For the tri-citrate- capped Au nanospheres (Figure 6B), the cell activity increased by 1.7 (20 pg / mL), 2.6 (200 pg / mL), and 2.8 (400 pg / mL) folds when compared with the CTAB / CTAC-capped sample at the same particle concentration. Essentially, there was no statistical difference between the groups of 0 and 20 pg / mL tri-citrated-capped Au nanospheres, indicating their excellent biocompatibility when used at a concentration below 20 pg / mL. This result indicated that the tri-citrate-capped Au nanospheres can be potentially used for stem cell tracking or fate regulation during tissue repair.

[0030] In principle, a proper surface ligand and concentration of Au nanospheres can be selected to fit the specific biomedical application.

[0246] Conclusion. In summary, an effective method for ligand exchange to replace the CTAB / CTAC on Au nanospheres of different sizes with tri-citrate was demonstrated. The method involves the deposition of an ultrathin shell of fresh Au on the surface of Au nanospheres in the presence of tri-citrate at an adequate concentration. During Au deposition, the CTAB / CTAC desorb from the surface, while tri-citrate adsorbs and gradually occupies the entire surface. The exchange of ligands was confirmed using multiple techniques. This method offers a facile way to replace a strongly-binding ligand with a weaker one or a ligand with an opposite charge, where the conventional direct ligand exchange methods tend to fail. In addition, this approach outperforms other ligand exchange methods by preventing the resultant nanoparticles from aggregation, contamination, and loss from etching. Most importantly, the tri-citrate-capped Au nanospheres show lower cell toxicity than the original CTAB / CTAC-capped particles, opening the door to a set of bio-related applications such as drug delivery, imaging contrast enhancement, stem cell tracking, and biosensing. Experimental

[0247] Chemicals and Materials. Gold(III) chloride trihydrate (HAuCU-SthO, >99.9%), sodium borohydride (NaBFL, 98%), ascorbic acid (AA, >99.0%), sodium tri-citrate dihydrate (NaaCA- EkO, 99.0%), cetyltrimethylammonium bromide (CTAB, >99.0%), and cetyltrimethylammonium chloride (CTAC, 25% in water) were all obtained from Sigma-Aldrich and used as received. Deionized water with a resistivity of 18.2 MQ cm at room temperature was used throughout the experiments.

[0248] Synthesis of CT AB / CT AC-capped Au nanospheres with different sizes. A published protocol was followed to synthesize CTAB / CTAC-capped Au spheres of 10, 20, and 40 nm in diameter.

[0024] For the 10-nm spheres, Au clusters were firstly synthesized by mixing 5 mL of aqueous CTAB (200 mM) and 5 mL of aqueous H AuCL (0.5 mM) in a 20-mL glass vial, followed by the introduction of freshly-prepared 0.6 mL aqueous NaBFL (10 mM) in one shot at room temperature. The mixture was placed on an orbital shaker at a speed of 270 rpm for 2 min and then kept undisturbed for at least 3 h at 27 °C to allow the NaBFL to completely decompose. Meanwhile, 2 mL of aqueous CTAC (200 mM) and 2 mL of aqueous HAuCL (0.5 mM) were mixed in a separate 20-mL glass vial, followed by one-shot injection of 1.5 mL of aqueous AA (100 mM) under magnetic stirring at a speed of 600 rpm and at 27 °C. Then, 50 pL of the as- obtained suspension of Au clusters was introduced in one shot at the same stirring speed and temperature. The reaction was allowed to proceed for 15 min at 27 °C. The solid products were collected by centrifugation at 14500 rpm for 30 min and washed once with water. The particles were re-dispersed in 1 mL of aqueous CTAC (20 mM) after washing with deionized water and extraction of supernatant for further growth to obtain larger Au nanospheres.

[0249] For the synthesis of 20- and 40-nm Au spheres, 2 mL of aqueous CTAC (100 mM), 130 pL of aqueous AA (10 mM), and a specific amount (see the table below for details) of the 10- or 20-nm Au spheres were mixed in a 20-mL glass vial under magnetic stirring at a speed of 600 rpm and at 27 °C. Then, 2 mL of aqueous HAuCL (0.5 mM) was introduced dropwise using a syringe pump at an injection rate of 2 mL / h at the same stirring speed and temperature. After completion of HAuCL addition, the reaction was allowed to proceed for another 10 min at 27 °C and the solid products were collected by centrifugation at 14500 rpm for 30 min and washed once with water. The particles were re-dispersed in 0.82 mL of water or 1 mL of aqueous CTAC (20 mM) after washing with deionized water and extraction of supernatant for further growth to obtain larger Au nanospheres or ligand exchange with tri-citrate, respectively.

[0250] Exchange of CT AB / CT AC with tri-citrate on the surface of 10-nm Au spheres. In a standard protocol, 200 pL of the seed suspension (7.58 x IO1010-nm spheres per mL in 20 mM aqueous CT AC) was mixed with 2 mL of water and 130 pL of aqueous sodium tri-citrate (200 mM) in a 20-mL glass vial under magnetic stirring at a speed of 600 rpm and at 27 °C. Then, 100 p L of aqueous HAuCL solution (0.25 mM) was added dropwise into the reaction mixture using a syringe pump at an injection rate of 4 mL / h at the same stirring speed and temperature. After completion of injection, the reaction was allowed to proceed for another 10 min at 27 °C. The solid products were collected by centrifugation at 14500 rpm for 30 min and then redispersed in 200 pL of 200 mM aqueous sodium citrate after washing and extraction of supernatant. The mixture was kept undisturbed overnight. The tri-citrate-capped Au nanospheres were collected again by centrifugation at 14500 rpm for 30 min and then re-dispersed in water after washing and extraction of supernatant for characterization to exclude the effect brought by citrate solution. The concentration of tri-citrate and the volume of H AuCL solution were varied while keeping all other parameters the same as those in the standard protocol.

[0251] For the ligand exchange on 20-nm Au spheres, the CTAB / CTAC-capped Au spheres had a concentration of 1.01 X 1010particles / mL in 20 mM aqueous CTAC; the concentration of AuCLf used for Au deposition was 0.1 mM; and the final concentration of sodium tri-citrate in the reaction solution was 0.5 M. The sample was collected at 11000 rpm for 10 min and the final product was re-dispersed in 0.2 mL of 1 M aqueous sodium citrate. Everything else was kept the same as that for the 10-nm sample.

[0252] For the ligand exchange on 40-nm Au spheres, the CTAB / CTAC-capped Au spheres had a concentration of 1.55 X 109particles / mL in 10 mM aqueous CTAC; the concentration of AuClC used for Au deposition was 0.1 mM and the final concentration of aqueous sodium tricitrate was 1 M. The sample was collected at 5500 rpm for 5 min and the final product was redispersed in 0.2 mL of 1 M aqueous sodium tri-citrate. Everything else was kept the same as that for the 10-nm sample. Cell culture and CCK8 assay. ADMSCs were cultured with Au nanospheres in Alpha minimum essential medium (a-MEM, Gibco, USA) with 10% fetal bovine serum (Gibco, USA) and 1% penicillin / streptomycin (Gibco, USA). After culture for 12 h, the cells were performed CCK8 assay. Typically, the cell culture medium was replaced by serum-free a- MEM containing 10% CCK8 (Dojindo, USA). After that, the cells were cultured at 37 °C for 1 h. Finally, the collected medium was analyzed using a microplate reader.

[0253] Characterization methods. Transmission electron microscope (TEM) images were taken on a Hitachi HT7700 microscope operated at 120 kV. Zeta potential measurements were determined using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). The Au content of a sample was determined using an inductively-coupled plasma mass spectrometer (ICP-MS, NexION 300Q, PerkinElmer, Waltham, MA). The UV-vis spectra were recorded on a Cary 60 spectrometer (Agilent Technologies, Santa Clara, CA). All IR spectra were recorded on a Varian 640 IR spectrometer (Agilent Technologies, Santa Clara, CA).

[0254] References

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[0284] Example 3

[0285] Direct replacement of the CTAB / CTAC on Au nanospheres with citric acid or citrate ions. Demonstrated herein is the feasibility of directly replacing CTAB / CTAC on 10-nm Au spheres with citric acid or citrate ions at different pH. The exact form of citrate in an aqueous solution is dependent on the pH. Specifically, tri-citrate is the most abundant species when pH is above 7, and it progressively protonates to form bi-, mono-citrate, and eventually citric acid when the pH drops to 5.8, 4, and 2, respectively. Different from a tri-citrate ion with three negative charges, the other three species in lower valences would not cause aggregation to the Au nanospheres during a direct replacement process due to their weaker capability to attenuate the electrostatic repulsion among the nanospheres. This pH-dependent behavior of citrate was leveraged to directly replace the CTAB / CTAC by citric acid or citrate ions by controlling the pH involved.

[0286] In a standard protocol, an aqueous citric acid solution with a concentration of 10 mM (pH = 2.8) was prepared. The pH of the solution was then adjusted to 2, 4, and 5.8 by introducing different amounts of aqueous HC1 (0.1 M) and aqueous NaOH (0.1 M), see the table below for details. According to calculations, citric acid, mono- and bi-citrate are the most abundant species at pH = 2, 4, and 5.8, respectively. In the next step, the CTAB / CTAC-capped 10-nm Au spheres were introduced into the solution to initiate ligand replacement, and the exchange was allowed to proceed at 27 °C for 3 hours under magnetic stirring.

[0287] UV-vis spectra were recorded from the as-obtained mixtures to evaluate their colloidal stability. As expected, the LSPR peaks shared a similar position and full width at half maximum as the original suspension of Au nanospheres, indicating the absence of aggregation at each pH (Figure 7).

[0288] The Au nanospheres capped by citric acid or mono- / bi-citrate ions were then collected through centrifugation and the solid was re-dispersed in 0.2 mL of aqueous mono-citrate solution (50 mM, pH = 4) for storage. UV-vis spectrum was recorded from each storage solution after 24 h to check the stability. Again, the preservation of the LSPR peaks (Figure 8) indicates that the colloidal stability was well retained when the CTAB / CTAC on the surface was replaced by citric acid or mono- / bi-citrate ions.

[0289] The Au nanospheres were further characterized using infrared (IR) spectroscopy to confirm the exchange of the surface-bound CTAB / CTAC with citric acid or mono- / bi-citrate. Briefly, 0.1 mL of the storage solution was taken out, and the citric acid / citrate-capped Au nanospheres were collected by centrifugation. FT-IR spectra was then recorded from the solid samples (Figure 9). The Au nanospheres capped by citric acid / citrate exhibited strong doublet peaks in the region of 1400-1600 cm ( corresponding to the peaks of carboxylate group in citrate ions. In addition, there were two peaks at 1200 and 1800 cm corresponding to the carboxylic group of citric acid. Meanwhile, no peak was observed around 2900 cm for CTAB / CTAC. This result suggested that citric acid or mono- / bi-citrate ions could indeed replace the CTAB / CTAC on the surface of the Au nanospheres through a direct ligand exchange process.

[0290] Additionally, the ligand exchange was further investigated under different pH to see what pH gave the fastest replacement. The exchanging time was reduced from 3 h to 30 min and the FTIR spectra of the resultant citric acid / citrate-capped Au nanospheres were recorded (Figure 10). There was a sharp doublet at 2900 cm1for the sample when pH = 5.8, indicating incomplete replacement of surface-bound CTAB / CTAC at this pH and time. The peaks reduced in intensity when pH = 4 and eventually vanished under a lower pH of 2. The result showed that PH = 2 could give the fastest ligand exchange. Further studies will examine further modifications to reach a ligand exchange with an even shorter time.

[0291] Synthesis of Au nanospheres in the setting of one-shot rather than dropwise addition. By directly replacing the surface-bound CTAB / CTAC with citric acid / citrate, the procedure for obtaining the citric acid / citrate-capped Au nanospheres was greatly simplified since the deposition of fresh Au became unnecessary. However, scaling up the production for commercial application is still limited by the dropwise method involved in the synthesis of CTAB / CTAC-capped Au nanospheres with a diameter above 10 nm. In addressing this issue, the method was switched from dropwise to one-shot addition for the synthesis of Au nanospheres without compromising the quality. Briefly, the standard procedure for synthesizing of 10-nm Au spheres from Au cluster seeds outlined above was followed, but a more concentrated aqueous Au(III)solution (1.5 mM) was used. This gave 14-nm Au spheres as the product (Figure HA). Then, the 14-nm Au spheres were used as seeds for the synthesis of larger spheres. In a standard procedure, 2 mF of aqueous CT AC (100 mM) and 1.5 mL of aqueous HAuCU (0.25 mM) were mixed with 1.5 mL of aqueous AA (50 mM) at 27 °C under magnetic stirring. Then, 0.1 mL of the suspension of 14-nm Au spheres was injected into the solution to initial the growth of Au nanospheres. The reaction was allowed to proceed for 12 h at the same temperature, and the solid product was collected by centrifugation. The TEM image in Figure 1 IB indicates that the resultant nanospheres had an average diameter of 19.7 nm while the spherical shape was well preserved. These two experiments demonstrate that it is feasible to obtain Au nanospheres with diameters up to 19.7 nm without the involvement of dropwise addition. This new result opens the door to scale up the synthesis of CTAB / CTAC-capped and subsequently, citric acid / citrate- capped Au nanospheres.

[0292] Furthermore, it was discovered that using aqueous CT AC with lower concentration during the synthesis helped with the formation of Au nanospheres with a diameter of 20.5 nm in one-shot. Specifically, experiments were performed with aqueous CTAC with concentrations of 200, 130, 100, 70, and 0 mM. As shown in Figure 12A-Figure 12E, the as-obtained particles had the most spherical shapes when the concentration was 100 mM. The reduction in concentration led to less ligands bound on the surface, and thus facilitated the surface diffusion of atoms on the surface for the formation of spherical shape. However, further decreasing the concentration led to insufficient stabilization of nanoparticles. As a result, the particles started to form dimers and eventually aggregated when the concentration was dropped to 70 mM and 0 mM, respectively.

[0293] Example 4 - Direct Exchange of the Cetyltrimethylammonium Bromide / Chloride on Au Nanocrystals with Different Citrate Species: A Mechanistic Study of the Effects of pH and Facet Type

[0294] ABSTRACT: This work demonstrates an effective method for directly exchanging the toxic cetyltrimethylammonium bromide / chloride (CTAB / C) on Au nanocrystals with citrate species (Figure 13). The experimental and computational studies herein indicate that pH plays a vital role in the exchange process. At pH = 2, citric acid readily replaces the CTAB / C by forming a stronger bond with the Au surface. As pH is increased, the substitution of H+counterion by Na+weakens the binding strength of carboxylate group and thus hinders the exchange. Additionally, quantitative measurements and theoretical calculations indicate that Au nanospheres encased by high-index facets are advantageous over their octahedral and cubic counterparts, enclosed by { 111} and { 100} facets, respectively, for the exchange owing to the difference in binding strength. The mechanistic insights and experimental control should be extendible to other combinations of surface ligands and metal nanocrystals.

[0295] Introduction. Gold (Au) nanoparticles have found widespread use in biomedical applications, including sensing, imaging, therapy, and drug delivery [A1-A5]. They have been synthesized with many diverse shapes by manipulating the experimental parameters, including temperature, surface ligand, and the type and concentration of precursor or reducing agent, among others, typically involved in a colloidal synthesis [A6-A11], Among the parameters, the surface ligand plays a particularly important role by acting as a colloidal stabilizer to prevent aggregation during both synthesis and storage while also serving as a capping agent to help control the shape evolution [A12, A13]. However, certain ligands, as exemplified by cetyltrimethylammonium bromide / chloride (CTAB / C), adversely impact the intended biomedical application due to their inherent toxicity. To address this issue, ligand exchange has to be conducted to replace the original ligands with a more suitable one, without altering or compromising the properties of the nanocrystals [A14, A15].

[0296] Citrate species are widely considered the surface ligands of choice for Au nanoparticles to be applied to biomedicine due to their high biocompatibility and the ability to ensure superb colloidal stability [A16-A19]. Several protocols have been reported in the literature for effectively replacing the original ligands on Au nanoparticles with tri-citrate species [A14, A15]. For instance, a method involving the deposition of an ultrathin Ag layer on Au nanospheres to effectively replace the surface-bound CTAB / C with poly(vinylpyrrolidone) (PVP) was developed [A14]. Followed by etching of the Ag layer in the presence of sodium tri-citrate, tricitrate could be introduced to the surface without changing the size and optical properties of the Au nanospheres [A14]. It was further demonstrated that deposition of a monolayer of Au instead of Ag also resulted in effective ligand exchange without potentially contaminating the Au nanospheres with residual Ag [A15]. Indeed, both methods led to the synthesis of Au nanospheres with reduced cellular toxicity, but their involvement of multiple steps inevitably caused sample loss during the exchange process. The issue of sample loss motivated the search for a more effective method capable of achieving direct ligand exchange in a single step. So far, direct exchange of surface ligands with citrate species is still limited owing to two technical challenges. Firstly, only a few studies have been conducted to examine the binding strength of citrate species to Au surface and the explicit mechanism of ligand-surface coordination remains elusive [A20-A22]. This complicates the selection of an appropriate citrate species for direct ligand exchange. Secondly, most Au nanocrystals are synthesized with CTAB / C as the ligands [A23-A26], which confer a positive charge to the Au surface [A 14]. It is documented that directly mixing CTAB / C-capped Au nanocrystals with negatively-charged citrate species would induce particle aggregation due to the charge screening effect, significantly altering the optical properties of the sample [A27-A30].

[0297] Herein, a systematic study is reported to demonstrate the ability to directly exchange the CTAB / C on the surface of Au nanospheres with citric acid in an aqueous medium. After ligand exchange, different citrate species can be obtained on the surface by simply adjusting the pH of the dispersion medium. Integrated with computational studies, the binding between various citrate species and the surface of Au nanocrystals, a topic that remains elusive to the field of surface chemistry [A31], were further analyzed. Specifically, citrate species exist in different forms in an aqueous solution depending on the pH [A32]. As shown in Figure 23, four distinctive forms of citrate species bearing different charges are formed as the pH is varied: citric acid at pH = 2, mono-citrate with one counter cation at pH = 4, bi-citrate with two counter cations at pH = 5.8, and tri-citrate with three counter cations at pH = 8. Through experimental and density functional theoretical (DFT) studies involving Au nanocrystals with three different shapes, the effects of solution pH and facet type on the exchange of surface-bound CTAB / C with different citrate species were elucidated. The Au nanocrystals passivated by citrate species can find widespread use in biomedicine, including drug delivery, sensing, and imaging. The mechanistic insights can be extendible to exchange protocols involving other pairs of ligands and metal surfaces.

[0298] RESULTS AND DISCUSSION

[0299] Direct Ligand Exchange of the CTAB / C on Au Nanospheres with Citric Acid. The ligand exchange was conducted by directly adding an aqueous suspension of CTAB / C-capped Au nanospheres into aqueous citric acid at a final concentration of 10 mM. The pH of the mixture was set to 2 to ensure that the free citric acid remained in the fully protonated form. The extent of ligand exchange was monitored by leveraging the structural difference between the chemical species and characterizing the samples obtained at different stages of a standard process for ligand exchange using Fourier-transform infrared (FTIR) spectroscopy. As shown in Figure 14A, free citric acid exhibited two peaks at 1200 and 1700 cm whereas free sodium tricitrate gave two peaks at 1400 and 1600 cm Both peaks corresponded to the vibrational modes of carboxyl or carboxylate group (C-0 and C=O stretching, respectively). In the following discussion, these two sets of dual peaks are denoted v-COOH for carboxyl group and v- COO(Na+) for carboxylate group, respectively, with the ion in the bracket corresponding to the counterion. In contrast, the FTIR spectra of CTAB / C showed characteristic doublet peaks around 2900 cm1, corresponding to the stretching of CH2 group (V-CH2) in a long hydrocarbon chain. Figure 14A also shows the FTIR spectra recorded from the Au nanospheres obtained at different stages of ligand exchange. At t = 0 min, the spectrum (purple trace) only showed the V-CH2 peaks around 2900 cm indicating the presence of CTAB / C on the surface. Exchange then proceeded rapidly, with the peaks associated with CTAB / C starting to disappear while new dual peaks around 1200 and 1700 cm appeared in only 5 min (orange trace).

[0300] Despite their similar wavenumbers to those of free citric acids, the new dual peaks had to be assigned to those of surface-bound, deprotonated carboxylate group (with H+serving as the counterion) rather than carboxyl group. According to the results from DFT calculations, the deprotonated form of citric acid had a stronger binding to Au surface once the relatively weak O-H bond in the carboxyl group had been cleaved [A33]. As such, the carboxyl group was actually deprotonated, instead of remaining in the protonated form, when binding to Au surface. A detailed explanation of this binding mode, along with the exact binding energies, is provided in the last section. Accordingly, the carboxyl group was predicted to evolve into carboxylate accompanied by a H+counterion upon binding to Au surface (Figure 24A). In this configuration, the carboxylate group comprising C-0 and C=O gave dual peaks at 1200 and 1700 cm and these peaks should be denoted v-COO(H+) rather than v-COOH.

[0301] It is worth noting that the wavenumbers of the surface-bound carboxylate group differed from those reported in previous studies, where dual peaks were observed at 1400 and 1600 cm1[A14, A15]. The discrepancy could be attributed to the different counterions involved. Specifically, the counterion in the current study was H+, which was not expected to exert a significant impact on the binding between the carboxylate group and Au surface. In contrast, the Na+counterion in prior studies could withdraw electrons from the carboxylate group, weakening the C=O bond and thus causing a red shift, as illustrated in Figure 24B. Additionally, the Na+counterion was expected to promote resonance within the carboxylate group, leading to a blue shift for the C-0 bond. The shifts in wavenumbers were consistent with calculation results, where the C=O bond of the surface-bound citrate species was found to decrease in wavenumbers as the counterion was switched from H+to Na+(Figure 25). Taken together, the counterion of the carboxylate group binding to Au surface can have a major impact on the wavenumbers of the C- O and C=O stretching modes. The Na+counterion tends to weaken the binding between citrate species and Au surface, and a detailed explanation is given in the last section. For simplicity, the deprotonated form of surface-bound citric acid is still referred to as “citric acid” to reflect the dominant species in the dispersion medium, but it is actually -COO(H+).

[0302] A closer look was also taken at the FTIR spectra in the region of 2700-3000 cm1(Figure 14B). Weak v-CFh peaks were still observed at t = 5 min, indicating the existence of some residual CTAB / C on the surface and thus incomplete exchange at this point. As ligand exchange progressed, the v-CFh peaks decreased substantially in intensity and eventually disappeared at t = 30 min (red trace). The gradual vanishing of the V-CH2 peaks over time suggested that a sufficiently long period of time was needed to replace all the surface-bound CTAB / C with citric acid although exchange could be initiated in just a few minutes. As the exchange continued, more CTAB / C would be displaced, leading to complete ligand exchange in about 30 min.

[0303] Figure 26 shows a schematic illustration of the exchange process between the surfacebound CTAB / C and the free citric acid in solution. According to the literature, CTAB / C is supposed to bind to the surface of Au nanospheres in a Au-(Br / Cl)-N configuration [A31, A34- A36]. The tertiary ammonium head group of CTA+does not possess any lone pair electrons that can be donated to the Au atoms on the surface. Additionally, there is steric hinderance from the methyl groups connected to the nitrogen atom, preventing the ammonium group from binding to the Au surface effectively. In contrast, the negatively-charged halide ion with four lone pairs of electrons can readily adsorb on the surface of Au nanospheres through coordination and thus attract the ammonium group through a relatively strong electrostatic interaction. In such a configuration, the hydrophobic tail of CTA+points away from the Au surface, promoting the formation of a bilayer comprised of CTAB / C when the Au nanospheres are suspended in an aqueous solution.

[0304] Upon adding the suspension of Au nanospheres into an aqueous solution of citric acid, some of the surface-bound CTAB / C were predicted to desorb from the surface owing to the absence of free CTAC / B in the medium [A37, A38], generating voids in the bilayer. Such voids allowed the free citric acid in the solution to access the Au surface. As established by the DFT results below, citric acid bound to Au more strongly than CTAC / B did [A31], leading to the exchange of CTAB / C with citric acid on the Au surface. The replacement of some of the surface-bound CTAB / C by citric acid would destabilize the bilayer [A39], creating more voids to promote further ligand exchange. As a result, the CTAB / C were continuously replaced with citric acid, making the Au surface increasingly covered by citric acid. However, it should be pointed out that one cannot push the equilibrium described in Equation (1) completely to the right side, and thus a trace amount of CTAB / C was predicted to remain on the surface.

[0305] CTAB / Cadsorbed + citric acidfree CTAB / Cfree + citl'ic acidadsorbed (1)

[0306] Introducing a new ligand to the surface can alter the stability of a colloidal suspension. Specifically, the new ligands may attenuate the electrostatic repulsion among the nanocrystals when they bear a charge opposite to the original ligands, causing aggregation. The aggregation of Au nanocrystals is often accompanied by color changes to the suspension as a result of the broadening and red shift of the localized surface plasmon resonance (LSPR) peak [A40] . As shown by the UV-vis spectrum recorded from an aqueous suspension of the original CTAB / C- capped Au nanospheres (black trace in Figure 15), a sharp LSPR peak was observed at 521 nm and the suspension displayed a ruby-red color. The LSPR peak has been reported to broaden and red-shift upon adding aqueous sodium tri-citrate, together with a color change to purple, due to the occurrence of particle aggregation [A 14].

[0307] The colloidal stability of the Au nanospheres obtained through ligand exchange with citric acid was also examined using UV-vis spectroscopy. To maximize the colloidal stability, the resultant nanospheres were suspended in an aqueous solution of citric acid at a concentration of 50 mM (pH = 2) instead of water. This procedure was important to prevent the surface-bound citrate species from desorption and thus avoid particle aggregation [A37, A38]. The UV-vis spectrum recorded from an aqueous suspension of the citric acid-capped Au nanospheres (red trace in Figure 15) even showed a slightly narrower LSPR peak, together with the retention of peak position, when compared to the original CTAB / C-capped sample. This data suggested a slight improvement in colloidal stability for the Au nanospheres after the ligand exchange [A41], owing to the reduction in charge screening caused by the ionic species in the medium. Prior to ligand exchange, the CTAB / C-capped Au nanospheres were suspended in aqueous CTAC at a concentration of 20 mM, where the free CTA+and CL ions could attenuate the electrostatic repulsion among the particles due to charge screening. In comparison, the citric acid-capped Au nanospheres were suspended in aqueous citric acid at pH = 2, where the citric acid in the medium is essentially neutral in charge. The neutral citric acid lacked charges to screen and attenuate the electrostatic repulsion among the particles. On the other hand, the surface-bound citric acid was deprotonated, providing a strong enough repulsion among the particles to stabilize the colloidal suspension. Altogether, the citric acid-capped Au nanospheres showed slight improvement in colloidal stability relative to the original CTAB / C-capped sample. Colloidal stability is one of the key parameters in determining the functionality of Au nanocrystals [A42, A43], particularly in biomedical applications that require long-term storage and usage. To examine the colloidal stability, a UV-vis spectrum from the aqueous suspension of citric acid-capped Au nanospheres was also recorded after 24 h of storage at room temperature (blue trace in Figure 15). The colloidal stability was largely retained as the LSPR peak remained essentially unchanged between the freshly prepared and stored suspensions in terms of peak width and position. Despite slight broadening, the LSPR peak was still narrower than that of the aqueous suspension of CTAB / C-capped Au nanospheres, demonstrating excellent colloidal stability of the citric acid-capped Au nanospheres under ambient conditions.

[0308] One of the fascinating properties associated with the Au nanospheres is the possession of the narrowest LSPR peak among solid Au nanocrystals of other shapes (Figure 27) [A44, A45]. This is because the optical excitation is equivalent along all directions due to their small size and highly symmetric shape that only allow for one dipole resonance. Transmission Electron Microscopy (TEM) analysis was also employed to confirm the perseverance of spherical shape during the exchange process. Figure 16A shows a TEM image of the CTAB / C-capped Au nanospheres, with an average diameter of 9.7 + 0.2 nm. As shown in Figure 16B, both the size and spherical shape taken by the original Au nanospheres were retained during ligand exchange, confirming that the treatment with citric acid did not lead to surface migration of Au atoms nor oxidative etching and removal of atoms from the surface.

[0309] The preservation of spherical shape was further confirmed by high-angle annular dark field- scanning transmission electron microscopy (HAADF-STEM) analysis. Figure 16C and Figure 16D, shows two images recorded from different Au nanospheres in the same batch of sample, before and after the ligand exchange, respectively, along the [Oi l] zone axis. Both particles showed a nearly perfect spherical shape, containing similar steps, kinks, and high-index facets that are characteristic of a spherical particle. Taken together, the ligand exchange did not alter the spherical shape of the Au nanospheres, as supported by the consistent results obtained from UV-vis spectroscopy, TEM, and STEM analyses.

[0310] The Effect of pH on Ligand Exchange. As shown in Figure 23, citrate species can be found in multiple forms that bear different charges and numbers of counterions as a function of pH. Following the standard protocol, the pH of the mixture was also adjusted from 2 to 4, 5.8, and 8 by adding different amounts of aqueous NaOH (see the Experimental Section) to investigate the effect of pH and thus citrate speciation on the ligand exchange. The new pH values corresponded to the dominance of mono-citrate, bi-citrate, and tri-citrate with one, two, and three Na+counterions, respectively. FTIR spectroscopy was then used to analyze the identities of the surface ligands on the Au nanospheres obtained through ligand exchange using the standard protocol at the specific pH.

[0311] As shown in Figure 17, the surface-bound CTAB / C could only be fully replaced by citrate species in 30 min at pH = 2 (red trace), where citric acid was the dominant species in the medium.

[0312] At pH = 4, mono-citrate became the dominant species, and the FTIR spectrum (blue trace, Figure 17) displayed prominent v-COO(H+) peaks, together with v-COO(Na+) peaks in the region of 1400-1600 cm It should be noted that the Na+counterion in mono-citrate could alter the binding between one of the carboxylate groups and Au surface as illustrated in Figure 24B. Accordingly, two sets of peaks associated with the carboxylate groups containing H+and Na+counterions were observed in the FTIR spectrum (Figure 17). Notably, the v-COO(H+) peaks were stronger in intensity than those of v-COO(Na+) due to a 2: 1 ratio between -COO(H+) and - COO(Na+) in a mono-citrate molecule. Overall, the simultaneous presence of v-COO(H+) and v- COO(Na+) peaks confirmed that mono-citrate could also substitute CTAB / C and adsorb onto the surface of Au nanospheres during the ligand exchange process. However, weak doublet peaks were still observed around 2900 cm indicating that some of the surface-bound CTAB / C could not be replaced by mono-citrate. The co-existence of v-COO(H+), v-COO(Na+), and V-CH2 peaks confirmed that ligand exchange could not be completed in 30 min at pH = 4.

[0313] Ligand exchange did not proceed effectively at pH = 5.8 and 8, where bi-citrate and tricitrate, respectively, were the dominant species in the solution. Specifically, the v-COO(H+) peaks completely vanished while the v-COO(Na+) peaks decreased substantially in intensity compared to the mono-citrate sample, and V-CH2 peaks were still observed around 2900 cm The FTIR spectra suggested that the surface of Au nanospheres remained largely covered by CTAB / C, with negligible amounts of bi- or tri-citrate on the surface, indicating low efficiency in ligand exchange at pH = 5.8 and 8. The presence of weak v-COO(Na+) peaks could be ascribed to z) the replacement of a small portion of surface-bound CTAB / C with bi- or tri-citrate and / or z'z) the electrostatic trapping of some bi- or tri-citrate anions on the surface due to the attraction from the CTA+cation.

[0314] The different behaviors between various citrate species involved in the ligand exchange process can be attributed to their differences in binding strength to Au surface. Specifically, carboxylate groups accompanied by the H+counterion exhibited stronger binding to Au surface relative to CTAB / C, while replacing the H+with Na+greatly reduced the binding strength. The difference in binding strength favored ligand exchange at a low pH where citric acid and monocitrate were the dominating citrate species in the solution. To gain insights into the influence of Na+counterion on the binding strength of citrate species to the surface of Au nanospheres, UV-vis spectroscopy was used to monitor the adsorption / desorption behavior of citric acid and tri-citrate with three Na+counterions (denoted tri-citrate hereafter) on Au nanospheres upon dilution. Specifically, first Au nanospheres capped by citric acid and tri-citrate were prepared and subsequently suspended in aqueous citric acid (pH = 2) and aqueous sodium tri-citrate (pH = 7), respectively, at a concentration of 100 mM. The former sample was prepared in the current work. However, the same condition for ligand exchange was proven ineffective for the latter case, and a previously reported protocol that involved the deposition of a Au monolayer to replace the surface-bound CTAB / C with tri-citrate was used instead [A15]. To ensure a fair comparison, the concentration (6.2 x IO10particles / mL) and particle size (10 nm) was kept the same for both samples. The aqueous suspensions of both samples were then diluted to different volumes and changes to the LSPR peak width and position were analyzed to probe possible aggregation. The dilution of citric acid-capped Au nanospheres was performed with 0.1 M HC1 to maintain a constant pH of 2 for the suspension, thereby eliminating any possible structural changes to the surface-bound and free citric acid that could induce aggregation. For the tri-citrate-capped sample, it was diluted with water due to the involved pH of 7.

[0315] Figure 18A and Figure 18B show UV-vis spectra recorded from the two samples upon dilution. The LSPR peaks width and position were identical for both samples when the particles were freshly suspended in 100 mM aqueous citric acid or sodium tri-citrate (red trace in Figure 18A and Figure 18B, respectively), indicating good colloidal stability for the aqueous suspensions. For the citric acid-capped Au nanospheres, the width and position of their LSPR peak remained essentially unchanged when the suspension was diluted in volume by 2- and 10- fold (the blue and green traces in Figure 18A, respectively). However, a significant broadening of the LSPR peak was observed when the suspension was diluted by 100-fold (the orange trace in Figure 18A), indicating particle aggregation under this condition. In contrast, the LSPR peak associated with the aqueous suspension of tri-citrate-capped Au nanospheres exhibited notable broadening and red-shift in the case of only a 2- fold dilution (the blue trace in Figure 18B). Further dilution by 10- and 100-fold resulted in more pronounced broadening and more red-shift for the LSPR peak (the green and orange trace in Figure 18B, respectively), suggesting severe aggregation of the particles. Altogether, the citric acid-capped Au nanospheres demonstrated greater resistance against aggregation during dilution when compared with the counterpart involving tri-citrate.

[0316] The difference in aggregation behavior between the citric acid- and tri-citrate-capped Au nanospheres upon dilution can be explained using the Langmuir model [A37, A38]. According to this model (Figure 28), the ligands are predicted to desorb from the surface of Au nanospheres in a medium lacking free citric acid or tri-citrate (i.e., with C approaching zero), thereby reducing their surface coverage (0). Once the Avalue drops below a threshold for aggregation (^aggregation), the remaining ligands are no longer adequate to stabilize the nanospheres, causing aggregation. In the current study, the two samples of Au nanospheres, one capped by citric acid and the other by tri-citrate, had the same diameter and hence the same surface area. They should share the same Aggregation ■ As shown by the UV-vis spectra in Figure 18A-Figure 18B, the citric acid-capped nanospheres exhibited a retarded tendency toward aggregation upon dilution compared to their tri-citrate counterpart, suggesting that they reached Aggregation at a lower C (Figure 28). These trends further indicated that citric acid had a larger binding constant (A) in the Langmuir model than tri-citrate, implying stronger binding to Au surface. This result suggested that the Na+counterion tended to weaken the binding of carboxylate group to the surface of Au nanospheres. To understand the destabilizing effect of Na+counterion on the binding, DFT calculations were performed to elucidate the atomic-scale interactions between citrate species with different types of Au facets in the presence or absence of Na+counterion (see the next section).

[0317] It is worth pointing out that the current understanding of the influence exerted by Na+counterion on the binding of carboxylate groups to Au surface is still qualitative as the exact K values for the adsorption of citric acid and tri-citrate on Au surface cannot be found in the literature. At the moment, the Aggregation in the Langmuir model remains unknown, making it impossible to determine the exact K value by substituting the experimental data into the model. In addition, only a simple model built upon a homogeneous surface, where the adsorbed species do not dissociate and there is no interaction between the adsorbed ligands, can be used. Altogether, the conclusion regarding the binding strength of different citrate species to the Au surface is still qualitative rather than quantitative.

[0318] In addition to the unsuccessful replacement of CTAB / C by mono- or bi-citrate, gradual aggregation of the Au nanospheres was observed during ligand exchange as the pH was increased, as revealed by the broadening and red shift to the LSPR peak (Figure 29). Specifically, increasing the pH from 2 to 4 resulted in slight broadening of the peak while the peak position remained unchanged (blue trace). At pH = 5.8, a more significant broadening, together with a slight red shift was recorded (green trace). The changes to the LSPR peak intensified at pH = 8, where doubling of the peak width and a dramatic red shift of 100 nm were observed (orange trace). Overall, increasing the pH promoted the aggregation of Au nanospheres, which could be attributed to the presence of bi- and tri-citrate species in the medium that significantly screened and attenuated the electrostatic repulsion among the positively-charged CTAB / C-capped Au nanospheres. A similar scenario of aggregation was also reported when sodium tri-citrate was directly added into an aqueous suspension of CTAB / C- capped Au nanospheres [A14] .

[0319] Taken together, the exchange of the CTAB / C on Au nanospheres with different citrate species was only effective at a relatively low pH of 2, where citric acid was in dominance over mono-, bi-, and tri-citrate in the solution. Meanwhile, direct ligand exchange simply failed at pH = 5.8 and 8, where the ligands of interest became bi- and tri-citrate, respectively. The unsuccessful ligand exchange could be ascribed to the weakened binding of carboxylate groups to Au surface caused by the Na+counterion.

[0320] Quantitative Analysis of the Binding between Citric Acid and Different Au Facets.

[0321] The Au nanospheres exhibit a higher abundance of high-index facets, particularly {211}, {311 }, and {331 }, when compared to nanocrystals in other shapes, including { 111 }-capped octahedra and { 100}-capped cubes. The higher abundance of high-index facets seemed to play a critical role in facilitating the ligand exchange between surface-bound CTAB / C and citric acid. Using the standard protocol, ligand exchange was also extended to { 100} -capped cubic and { 111 }- capped octahedral Au nanocrystals of 20 nm in edge length (Figure 30A and Figure 30B). As shown by the TEM image in Figure 30A, some particles in the sample of cubic nanocrystals exhibited a bipyramidal or rod-like shape, likely due to the fast reduction kinetics involved in the growth and the templating effect arising from the singly- or multiply-twinned seeds. However, all these nanocrystals were enclosed by { 100} facets and hence the difference in shape was not expected to exert a major impact on the ligand exchange. The variations in projected profiles observed in Figure 30B could be attributed to octahedral nanocrystals with different orientations relative to the electron beam. Figure 30C and Figure 30D, shows TEM images of the nanocrystals after the ligand exchange for 30 min. The preservation of their original shapes indicated that the treatment with citric acid did not induce surface atomic migration or oxidative etching [A46] . As such, any observed alternations to the surface speciation could be attributed to the efficacy of ligand exchange.

[0322] Again, FTIR spectroscopy was employed to identify the ligands on the surface of the Au nanocrystals before and after the ligand exchange process. Firstly, FTIR spectra of the CTAB / C- capped Au nanocrystals with different shapes were obtained (Figure 19A), and the strong V-CH2 peaks around 2900 cm1confirmed the presence of CTAB / C on both samples. FTIR spectra from the nanocrystals was then recorded after ligand exchange, as depicted by the red and blue traces in Figure 19A for cubic and octahedral nanocrystals, respectively. Both spectra showed strong v- COO(H+) peaks around 1200 and 1700 cm implying the occurrence of ligand exchange. However, strong -CH2 peaks were still observed for both samples (Figure 19B), indicating the presence of residual CTAB / C on the surface due to incomplete ligand exchange. It is worth noting that the V-CH2 peaks in the FTIR spectrum of the octahedral nanocrystals after ligand exchange were stronger than those of the spherical and cubic samples, indicating the presence of more residual CTAB / C on the surface. Taken together, the extent of ligand exchange at t = 30 min decreased in the order of spheres > cubes > octahedra. The variations in ligand exchange on different nanocrystals could be attributed to the high-index facets exposed on the surface of spherical nanocrystals, and a detailed analysis can be found in the next section.

[0323] FTIR spectroscopy was also employed to achieve a quantitative comparison of the extents of ligand exchange on different types of nanocrystals. By applying the Beer-Lambert law, one can derive the concentration (c) of a compound using Equation (2): c = A / el (2) where A is the absorbance, e is the molar attenuation coefficient of the compound, and I is the optical path length. It should be pointed out that FTIR spectroscopy is commonly regarded as a qualitative method when applied to analyze the surface ligands on nanocrystals due to the involvement of attenuated total reflectance and thus uncertainty about the optical path length. In addition, the exact value of e for the ligand can hardly be found in literature. Consequently, it is challenging to know exactly how many ligand molecules are probed to give an accurate assessment of the coverage density of the ligands on the surface. This issue can be addressed by calculating the ratio between the absorbance of V-CH2 peaks and that of v-COO(H+) peaks.

[0324] Experimentally, a FTIR spectrum was firstly recorded from a solid mixture of citric acid and CTAC at the same molar number, and the ratio between the absorbance of v-COOH peak and that of V-CH2 peak was found to be 0.64 (Figure 31). This ratio was calculated using the absorbance at 1710 cm1(Ac=o), corresponding to the stretching of C=O group, and the absorbance of the peak at 2920 cm1(Acm) corresponding to the asymmetric stretching of CH2 group. Using this absorbance ratio and Equation (2), a simple equation relating the ratio of Ac-o to ACH2 recorded from nanocrystals with different shapes to the molar ratio of surface-bound citric acid to CTAB / C (c-citric acid / c-CTA+) was further derived, as shown in Equation (3) (see the Experimental Section):

[0325] Ac=o / CH2 = (3.2) • (3 / 15) • (c-citric acid / c-CTA+) (3)

[0326] The ratio of Ac=o to ACH2 for nanocrystals obtained at different stages of ligand exchange was then calculated (Figure 20). At t = 5 min, the ratio of absorbance for spherical (the black trace), cubic (the red trace), and octahedral (the blue trace) nanocrystals were 2.2, 2.2, and 0.9, respectively, which gave molar ratios of surface-bound citric acid to CTAB / C at 3.3, 3.3 and 1.4 for the samples with different shapes. The relatively low ratios confirmed the presence of a comparable amount of citric acid and CTAB / C on the particle surface, implying incomplete ligand exchange for all shapes within a short period of 5 min. At t = 30 min, the ratios of Ac=o to CH2 for the cubic and octahedral nanocrystals remained low at 1.9 and 1.1, respectively, corresponding to molar ratios of surface-bound citric acid to CTAB / C at 3.0 and 1.8. Such low values signified that most of the surface-bound CTAB / C were not replaced by citric acid in 30 min. In contrast, the ratio of Ac=o to Acm for Au nanospheres gradually increased to 4.7 at t = 30 min, corresponding to a molar ratio of surface-bound citric acid to CTAB / C at 7.4. This high ratio implied that the surface-bound CTAB / C was increasingly replaced by citric acid over time. As mentioned in Equation (1), the remaining small amount of CTAB / C on the surface could arise from the electrostatic trapping by citric acid on Au surface. Taken together, it can be concluded that only Au nanospheres exhibited an increase in the ratio of c-citric acid to c-CTAC, confirming that they were advantageous in enabling ligand exchange than cubic and octahedral counterparts. The efficacy of ligand exchange across different Au facets descended in the order of high-index facets > { 100 } > { 111 } . This trend could suggest a stronger binding of citric acid to high-index facets compared to low-index ones and this trend is supported by the results from a computational study in the next section.

[0327] DFT Calculations for Determining the Binding Energies of Surface Adsorbates. To better understand the strengths of interaction between the various adsorbates and Au surfaces relevant to the experiments herein, DFT calculations were performed. Specifically, citrate species with relevant molecular identities and CTAB / C were studied, all on different Au facets. To determine the appropriate facets to be studied, HAADF-STEM imaging was used to identify the facets exposed on the surface of Au nanospheres. Figure 32A shows the HAADF-STEM image recorded from an Au nanosphere projected along the

[0011] zone axis, as confirmed by the fast Fourier transform (FFT) pattern (Figure 32B). The nanosphere contained { 111 } and { 100} facets, as indicated by the green and red lines in Figure 32A, respectively. The surface also contained numerous steps and kinks, as marked by purple, yellow, and blue dots. To assign the high-index facets of a step or kink, its angle with (111) and (200) planes were measured (see Figure 32A for details), followed by comparing the measurements with the theoretical angles shown in Table 1A. Table lA-Table 1C details the assignments of the facets for steps and kinks, and they included {211}, {311 }, and {331 }, which are, hereafter, collectively termed high-index facets. It should be clarified that only a conservative estimate of the facet assignments could be performed due to three limitations. First, the actual nanosphere in the 3D space was rounded toward the projection axis, whereas the recorded STEM image was a 2D projection, leading to an underestimation of the assigned indices of the exposed facets. Second, the STEM image was captured along the

[0011] zone axis, and as a result some facets would be overshadowed. These included {210} and {310} facets that could only be observed along

[0001] zone axis. Third, the high-energy electron beam utilized in the STEM characterization might melt the surface of Au nanosphere, facilitating diffusion of atoms from high-index facets to low-index facets as the atoms on the latter had lower energies. Overall, the STEM analysis was expected to give fewer high-index facets than they actually existed.

[0328] Table 1A. Theoretical angles between the high-index facets, and between (111) or (200), as obtained from Figure 32C.

[0329] * The values were obtained by measuring the angles between the spots of hkl and 111 / 200 in

[0330] Figure 32C.

[0331] Table IB. The angles measured between the planes formed by the dots and between { 111} or

[0332] { 100} indicated by green and red line, respectively, in Figure 32A.

[0333] Table 1C. Assignment of the facets exposed on the surface of the Au nanosphere in Figure 32A.

[0334] *The results were obtained by matching the angles in Table 1A and Table IB. Attention was then turned to the results of the DFT calculations, technical details for which are provided below. The exchange correlation used in the calculations was PBE+D3. The binding energy of each adsorbate considered here on each Au facet (BEadsorbate) was evaluated using Equation (4):

[0335] BEadsorbate — Eps+adsorbate—Eps — Egas, (4) where Eps+adsorbate represents the total energy of the pristine slab exposing the respective Au facet with the adsorbate at its minimum energy configuration; Epsand Egasdenote the total energies of the respective pristine Au slab and the isolated adsorbate in the gas phase, respectively. In this analysis, citrate species resulting from the cleavage of the O-H bond in the carboxyl group of citric acid or its derivatives demonstrated enhanced binding to Au surface. However, when these citrate species were saturated with the appropriate number of Na+counterions, the resulting citrate species bound to the Au surface more weakly than citric acid to the same surface. Informed by the experimental characterizations of the Au nanocrystals used in this study, the interaction of adsorbed species was examined with the following Au facets: { 100}, { 111 }, {211 }, and {331 }. The adsorption of citric acid, all relevant sodium citrates, and the CTAB molecule was then studied on these four Au facets at the respective low coverage limit for each facet (1 / 32 monolayer (ML) for { 100}, 1 / 36 ML for { 111 }, and 1 / 45 ML for {211 } and {331 } facets, respectively), where the adsorbate-adsorbate interactions were expected to be minimal. The most stable binding configurations for each adsorbate on these Au facets are shown in Figure 21.

[0336] Figure 22 shows that citric acid bound more strongly to the high-index facets (e.g., {331 }, BEcitric add = -6.48eV) compared to lower-index facets ({ 100}, BEcitric acid = -5.80eV; { 111 }, BEcitric add = -4.34eV). In addition, a comparison of the binding energies of citric acid on each Au surface (red data points) with that of the CTAB molecule (purple data points) revealed that citric acid bound much more strongly to each Au surface than CTAB did, leading to a facile displacement of CTAB by citric acid, which was in good agreement with the experimental findings. Furthermore, the data presented in Figure 22 also suggested that as more carboxyl moieties in citric acid were replaced by sodium-carboxylate species, the interaction of the resulting species with each Au surface became weaker. This led to a smaller driving force for the displacement of CTAB by these citrate species with Na+counterion compared to the driving force for the displacement of CTAB by citric acid, a result rationalized the experimental observations discussed above. Finally, the results in Figure 22 also suggested that ligand exchange effectiveness, as reflected in the thermodynamic driving force for the CTAB displacement by citric acid and related citrate species, was maximized for the more open facets (e.g., {331 } and { 100}) and minimized for the closest packed (111) surface.

[0337] Conclusion. In summary, a facile method was demonstrated for directly replacing the toxic CTAB / C on Au nanospheres with citrate species by simply adjusting the pH of the aqueous medium used for ligand exchange. At pH = 2, citric acid rapidly replaces CTAB / C by forming stronger bonds with the Au surface. As the pH is increased, however, the H+counterion will be replaced by Na+, which, according to the experimental and computational studies, destabilizes the binding between carboxylate groups and Au surface, leading to very poor and practically unsuccessful ligand exchange. Quantitative FTIR analyses integrated with DFT calculations further indicate that citric acid binds more strongly to the high-index facets on Au nanospheres when compared with the low-index facets on cubic or octahedral Au nanocrystals. The insights into the binding mechanism of citrate with Au surface offer guidelines for achieving direct ligand exchange without eliciting particle aggregation. The experimental approach should be extendible to the other pairs of ligands and different metal surfaces.

[0338] Experimental Section

[0339] Chemicals and materials. Gold(III) chloride trihydrate (HAuCL SHaO, >99.9%), sodium borohydride (NaBE , 98%), ascorbic acid (AA, >99.0%), cetyltrimethylammonium bromide (CTAB, >99.0%), cetyltrimethylammonium chloride (CTAC, 25% in water), citric acid (>99.0%), trisodium citrate (>99.0%), HC1 (37%) and NaOH (>99.0%) were all obtained from Sigma- Aldrich and used as received. Deionized water with a resistivity of 18.2 MQ-cm at room temperature was used throughout the experiments.

[0340] Synthesis of 10-nm Au spheres capped by CTAC / B. A published protocol was followed to synthesize CTAC / B-capped Au nanospheres of 10 nm in diameter. In brief, Au clusters were firstly prepared by mixing 5 mL of aqueous CTAB (200 mM) and 5 mL of aqueous I IAUCU (0.5 mM) in a 20-mL glass vial, followed by the introduction of freshly-prepared 0.6 mL of aqueous NaBH4 (10 mM) in one shot at 27 °C. The mixture was placed on an orbital shaker at a speed of 270 rpm for 2 min and then kept undisturbed at 27 °C for at least 3 h to allow the NaBH4 to completely decompose. Meanwhile, 2 mL of aqueous CTAC (200 mM) and 2 mL of aqueous HAuCL (0.5 mM) were mixed in a separate 20-mL glass vial, followed by the one-shot injection of 1.5 mL of aqueous AA (100 mM) under magnetic stirring at a speed of 600 rpm and at 27 °C. Then, 50 p L of the aqueous suspension of Au clusters was introduced. The reaction was allowed to proceed at 27 °C for 15 min. The solid products were collected by centrifugation at 14500 rpm for 30 min and washed once with water. After the removal of supernatant, the particles were redispersed into 1 mL of aqueous CTAC (20 mM). Exchange of CTAC / B with citric acid on the surface of 10-nm Au spheres. In a standard protocol, a solution was firstly prepared by mixing 130 pL of aqueous citric acid (200 mM), 200 pL of aqueous HC1 (100 mM), and 2 mL of water in a 20-mL glass vial at a speed of 600 rpm at 27 °C. The pH of this solution was confirmed to be 2 by measuring with a pH meter. Subsequently, 200 pL of the aqueous suspension of Au nanospheres (6.22 X 1010particles per mL in 20 mM aqueous CT AC) was introduced in one shot. The ligand exchange was allowed to proceed at 27 °C for 30 min. The solid products were collected by centrifugation at 14500 rpm for 20 min, washed with water, and then re-dispersed into 500 pL of 50 mM aqueous citric acid (pH = 2) for storage. For FTIR measurement, the Au nanospheres capped with citric acid were collected by centrifugation at 14500 rpm for 30 min, washed with water, and then re-dispersed into water to exclude the possible effect from the citric acid solution.

[0341] For ligand exchange on cubic and octahedral Au nanocrystals, the Au nanocrystals with different shapes were firstly prepared by modifying the protocols reported in previous publications [A47, A48]. As for ligand exchange, 200 pL of the aqueous suspension of cubic and octahedral Au nanocrystals (at a concentration of 1.36 X 1012or 3.47 X 1012particles per mL in 20 mM aqueous CTAC) was used while all other parameters were kept the same as those in the case of nano spheres.

[0342] For ligand exchange at different pH, aqueous HC1 or NaOH was added prior to the introduction of Au nanospheres (see the table below for details). All other parameters were kept the same as those in the case of pH = 2.

[0343] Characterizations. Transmission electron microscopy (TEM) images were taken using a Hitachi HT7700 microscope operated at 120 kV. High-angle annular dark-field (HAADF) scanning TEM (STEM) imaging, as well as energy-dispersive X-ray spectroscopy (EDX) mapping and line-scanning, was conducted using an aberration-corrected Hitachi HD-2700200 kV STEM equipped with a Briiker Quantax 400 / S-STEM EDX detector. The metal contents of the samples were analyzed using an inductively-coupled plasma mass spectrometer (ICP-MS, NexION 300Q, PerkinElmer). All UV-vis spectra were recorded on a Cary 60 spectrometer (Agilent Technologies). All FTIR spectra were recorded on a Varian 640 IR spectrometer (Agilent Technologies).

[0344] Derivation of Equation 3. Since each CTAC molecule contains fifteen CH2 groups and each citric acid molecule contains three C=O groups, the ratio of A-C=O to A-CH2 can be derived based on Equation (2):

[0345] To calculate c-citric acid / c-CTA+, one has to find out the ratio of E-C=O to E-CH2. In the current study, this ratio was calculated according to the ratio of A-C=O to A-CH2 obtained in Figure 31 (0.64), as shown by Equation (S2):

[0346] 0.64 where c-CTAC cancelled out c-citric acid since the molar numbers were the same. The l- values also cancelled out in the numerator and denominator because of the use of a homogenous mixture of solid CTAC and citric acid. Consequently, the ratio of e-C=O to s-CFh was calculated to be 3.2. Subsequently, Equation (3) was derived by substituting the ratio of s-C=O to S-CH2 into Equation (SI):

[0347] Again, the Z-values were cancelled out in the numerator and denominator.

[0348] Computational details. Periodic DFT calculations were performed using the Vienna ab initio software package (VASP) [A49, A50]. The Perdew-Burke-Emzerhof (PBE) exchangecorrelation functional within the generalized gradient approximation (GGA) was employed [A51]. The long-range dispersive interactions which are poorly described by PBE alone, were accounted for using Grimme’s D3 method (PBE+D3) [A52, A53]. The projector augmented wave (PAW) method using a plane wave basis set was implemented to describe the ion-electron interactions [A54]. The Kohn-Sham electron eigenfunctions were expanded in plane- wave basis sets with an energy cutoff of 400 eV. Structures were optimized until the self-consistent energies and residual forces on all the constituent atoms became smaller than 10‘4eV and 0.02 eV / A. Convergence with respect to all computational parameters was checked. To determine the equilibrium geometries and total energies, the first Brillouin zone integration of each unit cell was sampled with a (3 x 3 x 1) Monkhorst-Pack mesh of k-points [A55]. Given, the large number of Au atoms per layer in the Au slabs, surface adsorption calculations, were performed on three-layer slabs infinitely repeated in a supercell geometry with the bottom two atomic layers of the metal slab fixed. Any pair of successive slabs in the z-direction was separated by a vacuum layer of varying thickness, which allows for at least 15A of separation to prevent the interaction between periodic images. Dipole correction was applied to all slab calculations [A56, A57]. To approximately match the number of atoms in each unit cell, cubic { 100} and octahedral { 111} surfaces were infinitely repeated in (4 x 4) and (6 x 6) supercell geometry, including 32 and 36 surface atoms per unit cell, respectively. For the {211} and {331} surfaces, a (5 x 3) supercell with 45 surface atoms was utilized. The calculated lattice constant for Au is

[0349] 4.100 A, which is in good agreement with the experimental value (4.078 A).

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[0407] In view of the described compositions and methods, herein below are described certain more particularly described aspects of the inventions. The particularly recited aspects should not, however, be interpreted to have any limiting effect on any different claims containing different or more general teachings described herein or that the “particular” aspects are somehow limited in some way other than the inherent meanings of the language and formulas literally used therein.

[0408] Example 1: A one-step ligand exchange method, the method comprising: contacting a gold particle capped with a toxic ligand with a solution, the solution comprising a less-toxic ligand, or contacting a solution comprising a gold particle capped with a toxic ligand with a less-toxic ligand, wherein the concentration of the less-toxic ligand in the solution and / or the pH of the solution is sufficient to cause the toxic ligand to be replaced by the less-toxic ligand at the surface of the gold particle.

[0409] Example 2: The method of any examples herein, particularly example 1, wherein the pH of the solution is selected to control the protonation of the less-toxic ligand, thereby controlling the binding strength of the less-toxic ligand to the gold particle.

[0410] Example 3: The method of any examples herein, particularly example 1 or example 2, wherein the pH is 7 or less, 4 or less, or 2 or less.

[0411] Example 4: The method of any examples herein, particularly examples 1-3, wherein the pH is from 2 to 4.

[0412] Example 5: The method of any examples herein, particularly examples 1-4, wherein the solution further comprises a gold salt, and the less-toxic ligand further serves as a reducing agent for the gold salt, such that the gold salt is reduced and deposited as a thin layer of gold onto the gold particle.

[0413] Example 6: A one-step ligand exchange method, the method comprising: contacting a gold particle capped with a toxic ligand with a solution, the solution comprising a gold salt and a less-toxic ligand, or contacting a solution comprising a gold particle capped with a toxic ligand with a gold salt and a less-toxic ligand, wherein the less-toxic ligand further serves as a reducing agent for the gold salt, such that the gold salt is reduced and deposited as a thin layer of gold onto the gold particle, wherein the concentration of the less-toxic ligand in the solution is sufficient to cause the toxic ligand to be replaced by the less-toxic ligand at the surface of the gold particle.

[0414] Example 7: The method of any examples herein, particularly example 6, wherein the method further comprises controlling the pH of the solution, wherein the pH of the solution is selected to control the protonation of the less-toxic ligand, thereby controlling the binding strength of the less-toxic ligand to the gold particle.

[0415] Example 8: The method of any examples herein, particularly example 6 or example 7, wherein the pH is 7 or less, 4 or less, or 2 or less.

[0416] Example 9: The method of any examples herein, particularly examples 6-8, wherein the pH is from 2 to 4.

[0417] Example 10: The method of any examples herein, particularly examples 5-9, wherein the thin layer of gold is an atomic monolayer.

[0418] Example 11: The method of any examples herein, particularly examples 5-10, wherein the gold salt comprises HAuCU.

[0419] Example 12: The method of any examples herein, particularly examples 5-11, wherein the concentration of gold salt is selected to control the thickness of the deposited layer.

[0420] Example 13: The method of any examples herein, particularly examples 5-12, wherein the concentration of the gold salt is selected such that the thin layer of gold has a thickness of 3 nm or less.

[0421] Example 14: The method of any examples herein, particularly examples 5-13, wherein the concentration of the gold salt is selected such that the thin layer of gold is an atomic monolayer.

[0422] Example 15: The method of any examples herein, particularly examples 1-14, wherein the solution is added dropwise.

[0423] Example 16: The method of any examples herein, particularly examples 1-14, wherein the solution is added in one shot.

[0424] Example 17: The method of any examples herein, particularly examples 1-16, the toxic ligand comprises cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTAC), dodecyltrimethylammonium bromide, dodecyltrimethylammonium chloride, octadecyltrimethylammonium bromide, octadecyltrimethylammonium chloride, or a combination thereof.

[0425] Example 18: The method of any examples herein, particularly examples 1-17, wherein the toxic ligand comprises cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTAC), or a combination thereof.

[0426] Example 19: The method of any examples herein, particularly examples 1-18, wherein the less-toxic-ligand comprises citric acid or a citrate (e.g., a mono-citrate, bi-citrate, tricitrate, or a combination thereof), such as a sodium citrate (e.g., sodium mono-citrate, sodium bi-citrate, sodium tri-citrate, or a combination thereof). Example 20: The method of any examples herein, particularly examples 1-19, wherein the less-toxic ligand comprises citrate, such as sodium citrate.

[0427] Example 21: The method of any examples herein, particularly examples 1-20, wherein the toxic ligand is more strongly bound to the gold particle relative to the less-toxic ligand.

[0428] Example 22: The method of any examples herein, particularly examples 1-21, wherein the gold particle has an average particle size of from 1 nm to 1000 nm.

[0429] Example 23: The method of any examples herein, particularly examples 1-22, wherein the gold particle has an average particle size of from 5 nm to 1000 nm.

[0430] Example 24: The method of any examples herein, particularly examples 1-23, wherein the gold particle has an average particle size of 5 nm to 150 nm.

[0431] Example 25: The method of any examples herein, particularly examples 1-24, wherein the gold particle has an average particle size of from 10 nm to 40 nm.

[0432] Example 26: The method of any examples herein, particularly examples 1-25, wherein the gold particle before and / or after the contacting step has a substantially spherical shape.

[0433] Example 27: The method of any examples herein, particularly examples 1-26, wherein the shape of the gold particle is preserved after the contacting step.

[0434] Example 28: The method of any examples herein, particularly examples 1-27, wherein the gold particle comprises a radioisotope, such as Au- 198 and / or Au- 199.

[0435] Example 29: The method of any examples herein, particularly examples 1-28, wherein the concentration of the less-toxic ligand is 200 mM or more.

[0436] Example 30: The method of any examples herein, particularly examples 1-29, wherein the method is a one pot method.

[0437] Example 31: The method of any examples herein, particularly examples 1-30, wherein the method is performed at room temperature.

[0438] Example 32: The method of any examples herein, particularly examples 1-31, wherein the method is completed in an amount of time of from 1 minute to 1 hour.

[0439] Example 33: The method of any examples herein, particularly examples 1-32, wherein the method is completed in an amount of time of from 1 minute to 30 minutes.

[0440] Example 34: The method of any examples herein, particularly examples 1-33, wherein the method does not result in agglomeration of the gold particles.

[0441] Example 35 : A gold particle made by the method of any examples herein, particularly examples 1-34.

[0442] Example 36: The gold particle of any examples herein, particularly example 35, wherein the gold particle after ligand exchange has decreased cell toxicity relative to the gold particle before ligand exchange.

[0443] Example 37: The method of any examples herein, particularly example 35 or example 36, wherein the gold particle after ligand exchange is colloidally stable for an amount of time of 24 hours or more.

[0444] Example 38: A composition comprising the gold particle of any examples herein, particularly examples 35-37.

[0445] Example 39: A pharmaceutical composition comprising the gold particle of any examples herein, particularly examples 35-37 and a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient, or a combination thereof.

[0446] Example 40: An assay comprising the gold particle of any examples herein, particularly examples 35-37, or the composition of any examples herein, particularly example 38 or example 39.

[0447] Example 41: The assay of any examples herein, particularly example 40, wherein the gold particle is further conjugated to a targeting ligand, such as an antibody, a nucleic acid probe, a molecular recognition element, or a combination thereof.

[0448] Example 42: A point of care test comprising the gold particle of any examples herein, particularly examples 35-37, or the composition of any examples herein, particularly example 38 or example 39.

[0449] Example 43: The point of care test of any examples herein, particularly example 42, wherein the point of care test comprises a pregnancy test.

[0450] Example 44: The point of care test of any examples herein, particularly example 42, wherein the point of care test comprises a test for a pathogenic microorganism.

[0451] Example 45: The point of care test of any examples herein, particularly example 42 or example 44, wherein the point of care test comprises a coronavirus test, such as a Covid-19 test.

[0452] Example 46: The point of care test of any examples herein, particularly example 45, wherein the point of care test comprises a cancer screening test.

[0453] Example 47 : A method of use of the gold particle of any examples herein, particularly examples 35-37, or the composition of any examples herein, particularly example 38 or example 39.

[0454] Example 48: The method of any examples herein, particularly example 47, wherein the method comprises using the gold particle or composition in biomedical imaging (e.g., as an imaging agent). Example 49: The method of any examples herein, particularly examples 47-48, wherein the method comprises using the gold particle or composition in photodynamic therapy, photothermal therapy, or a combination thereof.

[0455] Example 50: The method of any examples herein, particularly examples 47-49, wherein the method comprises using the gold particle or composition in radiotherapy, preferably wherein the gold particle includes a radioisotope, such as Au- 198 and / or Au- 199.

[0456] Example 51: The method of any examples herein, particularly examples 47-50, wherein the method comprises using the gold particle or composition as a sensor.

[0457] Example 52: The method of any examples herein, particularly examples 47-51, wherein the method comprises using the gold particle or composition for detection of biomarkers, infectious disease-related antigens, antibodies, or a combination thereof.

[0458] Example 53: The method of any examples herein, particularly examples 47-52, wherein the method comprises using the gold particle or composition for drug delivery.

[0459] Example 54: The method of any examples herein, particularly example 53, wherein the gold particle is further conjugated with a drug-carrying ligand.

[0460] Example 55: The method of any examples herein, particularly example 54, wherein the gold particle or composition further comprises a drug, the drug being conjugated to the drug-carrying ligand.

[0461] Example 56: The method of any examples herein, particularly examples 47-55, wherein the method comprises using the gold particle or composition as a catalyst.

[0462] Example 57: The method of any examples herein, particularly examples 47-56, wherein the method comprises using the gold particle or composition as a photosensitizer.

[0463] Example 58: A method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the gold particle of any examples herein, particularly examples 35-37, or the composition of any examples herein, particularly example 38 or example 39.

[0464] Example 59: The method of any examples herein, particularly example 58, wherein the disease comprises cancer.

[0465] Example 60: A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the gold particle of any examples herein, particularly examples 35-37, or the composition of any examples herein, particularly example 38 or example 39.

[0466] Example 61 : The method of any examples herein, particularly example 60, further comprising co-administering an anticancer agent to the subject. Example 62: A method of suppressing tumor growth in a subject in need thereof, the method comprising contacting at least a portion of the tumor with a therapeutically effective amount of the gold particle of any examples herein, particularly examples 35-37, or the composition of any examples herein, particularly example 38 or example 39.

[0467] Example 63: The method of any examples herein, particularly examples 60-62, further comprising irradiating the gold particle with electromagnetic radiation that overlaps with at least a portion of one or more of an absorption, emission, or excitation band of the gold particle.

[0468] Example 64: A method of imaging a cell or a population of cells within or about a subject, the method comprising administering to the subject an amount of the gold particle of any examples herein, particularly examples 35-37, or the composition of any examples herein, particularly example 38 or example 39, and detecting the gold particle or composition.

[0469] Example 65 : The method of any examples herein, particularly example 64, wherein the cell or population of cells is indicative of cancer.

[0470] Other advantages which are obvious and which are inherent to the invention will be evident to one skilled in the art. It will be understood that certain features and sub-combinations are of utility and may be employed without reference to other features and sub-combinations. This is contemplated by and is within the scope of the claims. Since many possible embodiments may be made of the invention without departing from the scope thereof, it is to be understood that all matter herein set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.

[0471] The methods of the appended claims are not limited in scope by the specific methods described herein, which are intended as illustrations of a few aspects of the claims and any methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative method steps disclosed herein are specifically described, other combinations of the method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.

Claims

CLAIMSWhat is claimed is:

1. A one-step ligand exchange method, the method comprising: contacting a gold particle capped with a toxic ligand with a solution, the solution comprising a less-toxic ligand, or contacting a solution comprising a gold particle capped with a toxic ligand with a less- toxic ligand, wherein the concentration of the less-toxic ligand in the solution and / or the pH of the solution is sufficient to cause the toxic ligand to be replaced by the less-toxic ligand at the surface of the gold particle.

2. The method of claim 1, wherein the pH of the solution is selected to control the protonation of the less-toxic ligand, thereby controlling the binding strength of the less-toxic ligand to the gold particle.

3. The method of claim 1 or claim 2, wherein the pH is 7 or less, 4 or less, or 2 or less.

4. The method of any one of claims 1-3, wherein the pH is from 2 to 4.

5. The method of any one of claims 1-4, wherein the solution further comprises a gold salt, and the less-toxic ligand further serves as a reducing agent for the gold salt, such that the gold salt is reduced and deposited as a thin layer of gold onto the gold particle.

6. A one-step ligand exchange method, the method comprising: contacting a gold particle capped with a toxic ligand with a solution, the solution comprising a gold salt and a less-toxic ligand, or contacting a solution comprising a gold particle capped with a toxic ligand with a gold salt and a less-toxic ligand, wherein the less-toxic ligand further serves as a reducing agent for the gold salt, such that the gold salt is reduced and deposited as a thin layer of gold onto the gold particle, wherein the concentration of the less-toxic ligand in the solution is sufficient to cause the toxic ligand to be replaced by the less-toxic ligand at the surface of the gold particle.

7. The method of claim 6, wherein the method further comprises controlling the pH of the solution, wherein the pH of the solution is selected to control the protonation of the less-toxic ligand, thereby controlling the binding strength of the less-toxic ligand to the gold particle.

8. The method of claim 6 or claim 7, wherein the pH is 7 or less, 4 or less, or 2 or less.

9. The method of any one of claims 6-8, wherein the pH is from 2 to 4.

10. The method of any one of claims 5-9, wherein the thin layer of gold is an atomic monolayer.

11. The method of any one of claims 5-10, wherein the gold salt comprises HAuCU.

12. The method of any one of claims 5-11, wherein the concentration of gold salt is selected to control the thickness of the deposited layer.

13. The method of any one of claims 5-12, wherein the concentration of the gold salt is selected such that the thin layer of gold has a thickness of 3 nm or less.

14. The method of any one of claims 5-13, wherein the concentration of the gold salt is selected such that the thin layer of gold is an atomic monolayer.

15. The method of any one of claims 1-14, wherein the solution is added dropwise.

16. The method of any one of claims 1-14, wherein the solution is added in one shot.

17. The method of any one of claims 1-16, the toxic ligand comprises cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTAC), dodecyltrimethylammonium bromide, dodecyltrimethylammonium chloride, octadecyltrimethylammonium bromide, octadecyltrimethylammonium chloride, or a combination thereof.

18. The method of any one of claims 1-17, wherein the toxic ligand comprises cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTAC), or a combination thereof.

19. The method of any one of claims 1-18, wherein the less-toxic-ligand comprises citric acid or a citrate (e.g., a mono-citrate, bi-citrate, tri-citrate, or a combination thereof), such as asodium citrate (e.g., sodium mono-citrate, sodium bi-citrate, sodium tri-citrate, or a combination thereof).

20. The method of any one of claims 1-19, wherein the less-toxic ligand comprises citrate, such as sodium citrate.

21. The method of any one of claims 1-20, wherein the toxic ligand is more strongly bound to the gold particle relative to the less-toxic ligand.

22. The method of any one of claims 1-21, wherein the gold particle has an average particle size of from 1 nm to 1000 nm.

23. The method of any one of claims 1-22, wherein the gold particle has an average particle size of from 5 nm to 1000 nm.

24. The method of any one of claims 1-23, wherein the gold particle has an average particle size of 5 nm to 150 nm.

25. The method of any one of claims 1-24, wherein the gold particle has an average particle size of from 10 nm to 40 nm.

26. The method of any one of claims 1-25, wherein the gold particle before and / or after the contacting step has a substantially spherical shape.

27. The method of any one of claims 1-26, wherein the shape of the gold particle is preserved after the contacting step.

28. The method of any one of claims 1-27, wherein the gold particle comprises a radioisotope, such as Au-198 and / or Au-199.

29. The method of any one of claims 1-28, wherein the concentration of the less-toxic ligand is 200 mM or more.

30. The method of any one of claims 1-29, wherein the method is a one pot method.

31. The method of any one of claims 1-30, wherein the method is performed at room temperature.

32. The method of any one of claims 1-31, wherein the method is completed in an amount of time of from 1 minute to 1 hour.

33. The method of any one of claims 1-32, wherein the method is completed in an amount of time of from 1 minute to 30 minutes.

34. The method of any one of claims 1-33, wherein the method does not result in agglomeration of the gold particles.

35. A gold particle made by the method of any one of claims 1-34.

36. The gold particle of claim 35, wherein the gold particle after ligand exchange has decreased cell toxicity relative to the gold particle before ligand exchange.

37. The method of claim 35 or claim 36, wherein the gold particle after ligand exchange is colloidally stable for an amount of time of 24 hours or more.

38. A composition comprising the gold particle of any one of claims 35-37.

39. A pharmaceutical composition comprising the gold particle of any one of claims 35-37 and a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient, or a combination thereof.

40. An assay comprising the gold particle of any one of claims 35-37 or the composition of claim 38 or claim 39.

41. The assay of claim 40, wherein the gold particle is further conjugated to a targeting ligand, such as an antibody, a nucleic acid probe, a molecular recognition element, or a combination thereof.

42. A point of care test comprising the gold particle of any one of claims 35-37 or the composition of claim 38 or claim 39.

43. The point of care test of claim 42, wherein the point of care test comprises a pregnancy test.

44. The point of care test of claim 42, wherein the point of care test comprises a test for a pathogenic microorganism.

45. The point of care test of claim 42 or claim 44, wherein the point of care test comprises a coronavirus test, such as a Covid-19 test.

46. The point of care test of claim 45, wherein the point of care test comprises a cancer screening test.

47. A method of use of the gold particle of any one of claims 35-37 or the composition of claim 38 or claim 39.

48. The method of claim 47, wherein the method comprises using the gold particle or composition in biomedical imaging (e.g., as an imaging agent).

49. The method of any one of claims 47-48, wherein the method comprises using the gold particle or composition in photodynamic therapy, photothermal therapy, or a combination thereof.

50. The method of any one of claims 47-49, wherein the method comprises using the gold particle or composition in radiotherapy, preferably wherein the gold particle includes a radioisotope, such as Au-198 and / or Au-199.

51. The method of any one of claims 47-50, wherein the method comprises using the gold particle or composition as a sensor.

52. The method of any one of claims 47-51, wherein the method comprises using the gold particle or composition for detection of biomarkers, infectious disease-related antigens, antibodies, or a combination thereof.

53. The method of any one of claims 47-52, wherein the method comprises using the gold particle or composition for drug delivery.

54. The method of claim 53, wherein the gold particle is further conjugated with a drugcarrying ligand.

55. The method of claim 54, wherein the gold particle or composition further comprises a drug, the drug being conjugated to the drug-carrying ligand.

56. The method of any one of claims 47-55, wherein the method comprises using the gold particle or composition as a catalyst.

57. The method of any one of claims 47-56, wherein the method comprises using the gold particle or composition as a photosensitizer.

58. A method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the gold particle of any one of claims 35-37 or the composition of claim 38 or claim 39.

59. The method of claim 58, wherein the disease comprises cancer.

60. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the gold particle of any one of claims 35-37 or the composition of claim 38 or claim 39.

61. The method of claim 60, further comprising co-administering an anticancer agent to the subject.

62. A method of suppressing tumor growth in a subject in need thereof, the method comprising contacting at least a portion of the tumor with a therapeutically effective amount of the gold particle of any one of claims 35-37 or the composition of claim 38 or claim 39.

63. The method of any one of claims 60-62, further comprising irradiating the gold particle with electromagnetic radiation that overlaps with at least a portion of one or more of an absorption, emission, or excitation band of the gold particle.

64. A method of imaging a cell or a population of cells within or about a subject, the method comprising administering to the subject an amount of the gold particle of any one of claims 35- 37 or the composition of claim 38 or claim 39, and detecting the gold particle or composition.

65. The method of claim 64, wherein the cell or population of cells is indicative of cancer.

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