Liposome-templated mesoporous nanostructures of noble metals and methods for preparation and their use

Abstract

Liposomes are leveraged to function as sacrificial templates for the growth of mesoporous nanostructures constructed with noble metals and other metals. Such templates allow for programmability for the specific distribution of metals and their properties while generating mesopores within the formed nanostructures. Ascorbic acid or other reductant is introduced to the sacrificial template for the reduction of metallic precursors. Once metallic precursors are added, the reductant finishes the reduction reaction to form the metallic nanostructures. Such nanostructures can have outside-in mesopores and pore diameters in the range of 10 nm to 50 nm.

Description

LIPOSOME-TEMPLATED MESOPOROUS NANOSTRUCTURES OF NOBLE METALS AND METHODS FOR PREPARATION AND THEIR USECROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 625,546 filed January 26, 2024, the entire disclosure of which is incorporated herein by reference.FIELD OF THE INVENTION

[0002] This application and the inventions disclosed herein relate to porous noble metal nanoparticles and nanostructures made therefrom.BACKGROUND OF THE INVENTION

[0003] In recent years, there has been fast-growing interest and attention paid to noble metal (e.g., gold (Au), palladium (Pd), platinum (Pt), rhodium (Rh), etc.)-based nanomaterials. With the ready modulation of their morphology and aspect ratios as well as their surface chemistry, these nanomaterials afford unique traits such as remarkable photoelectric properties, high drug loading capacities, good stability and low biotoxicity. Among the various nanostructures, the porous ones, especially those with outside-in mesopores, are of particular interest because of the capabilities of large mesopores and tunable porosity, providing abundant surface and interior binding sites for various molecules and a better realization of the desirable physicochemical properties than solid nanostructures. In this regard, diverse strategies have been explored to synthesize mesoporous metallic structures, including electrochemical dealloying, soft- or hard-templating, and solution-based synthesis. By comparison, soft- templating typically affords a more favorable means to synthesize porous nanostructures with relatively large mesopore distributions (from 1 nm to 40 nm), monodisperse size, better structural control, and high compositional purity. However, most of the current noble metalbased porous nanomaterials are prepared by using cytotoxic reagents as the soft templates, such as cetyltrimethyl ammonium bromide (CTAB), oleylamine, and sodium borohydride, significantly restricting their wide adoption for biomedical utilities. Meanwhile, current successes have only demonstrated select metals, such as platinum and palladium. Moreover, very limited progress has been made with respect to other metals, such as gold, to generate the outside-in porous nanostructures. This is partially due to a much faster deposition rate of gold than platinum or palladium, leading to rapid growth of gold primarily along the dominant channels of the templates, but with insufficient or no growth in those micropores. Therefore, a green yet robust methodology that could produce stable outside-in mesoporous nanostructuresfrom single noble metals or even multiple combined ones with programmable control of their spatial distribution is an outstanding design goal.

[0004] Recently, it was found that positively charged terminal ammonium functional groups (-NH3+) of biocompatible liposomes prepared from phospholipids such as 1,2-dipalmitoyl-sn- glycero-3 -phosphocholine (DPPC) are primarily located on the surface of liposomal spheres (+15 mV in zeta potential measurement). As such, the negatively charged noble metallic precursor species (e.g., [AuCU]’ or [PtCk]2') would preferably distribute and locate in the “corona halo” of the liposomes via electrostatic interactions.SUMMARY OF THE INVENTION

[0005] Based on the assumption that negatively charged noble metallic precursor species would preferably distribute and locate in the “corona halo,” the possibility of forming aggregated liposome networks hinged by noble metallic species during the reduction process and subsequently guiding the formation of 3D mesoporous structures of noble metals within the inter-liposome space upon complete reduction was investigated.

[0006] Broadly speaking, a mesoporous nanostructure can be formed by first forming a liposome-based network and then growing the mesoporous nanostructure by using the liposome-based network as a template before removing the liposome-based network, leaving the mesoporous nanostructure.

[0007] An embodiment of the present invention uses liposome-based networks as soft templates to establish a facile yet effective synthesis strategy for preparing monodispersed outside-in mesoporous nanostructures with programmable and spatially controlled deposition of monometallic (Au, Pd and Pt), bimetallic (AuPd, AuPt, AuRh, PtRh, and PdPt), and trimetallic (AuPdRh, AuPtRh, and AuPdPt) (FIG. 1) elements. During the synthesis, biocompatible liposomes (i.e., building blocks of the soft template) and ascorbic acid (AA) (a reducing agent) are employed to control the morphology of the template and allow for selective growth of various metal struts on the boundary of lipid bilayers of the aggregated liposomes upon AA reduction (FIG. 1).

[0008] During the synthesis of metallic nanostructures, the soft liposome templates and ascorbic acid (AA) (or another reducing agent) are employed to control the mesopores (i.e., selective growth of various metal struts of ground zero metals on the boundary of aggregated liposomes upon AA reduction). This enables programmability, which herein refers to the possibility of controlling the metallic composition of each layer and its spatial organization within the as-prepared nanostructures, and such programmability is achieved by selecting thesequential order of adding the metallic precursors into the reaction. Thus, ascorbic acid (or another reductant) has two noted functions in soft-templating synthesis of metal nanostructures, not only decreasing the size of the liposomes but also serving as a reductant to facilitate conversion of the metal precursor into metal desired for the final product. Alternatively, it can be described as a partial reduction reaction during the hydrating phase (i.e., formation of the template) which is completed in forming the final product, since the reductant is present during both the hydration and final reaction steps.

[0009] After removal of the liposome templates, the as-prepared mesoporous nanostructures exhibit a unique outside-in porous structure, with controllable pore size, and composition of metal layers. Advantages of this synthesis strategy include: (1) broad applicability for diverse noble metals such as Au, Pd, Pt, Rh as well as their double or triple combinations, (2) good biocompatibility by excluding cytotoxic chemicals during the process, (3) simple setup of the entire synthesis process under mild conditions and in a short duration, and (4) high controllability of the nanostructure size via manipulating the metal precursor concentration and the reaction time and the high spatial organization of different metals via distinct reducing potentials and sequential addition to the reaction.

[0010] As demonstrated, such noble metal mesoporous nanostructures endow some essential and unique capabilities. For example, they typically exhibited surface plasmon resonance (SPR) in the visible to near-infrared range and showed a tunable photothermal effect. Due to unoccupied d- orbitals, these nanostructures also inherently showed catalytic activity. The spatially tunable deposition of selected metals would ensure the optimal exposure of reactants to the metal for efficient biocatalysis via the electronic and surface strain effects. Meanwhile, the large open mesopores of such nanostructures offer sufficient surface area to accommodate more therapeutics at a high loading capacity, while promising the potentials for co-delivery of multiple drugs and sequentially responsive release.

[0011] A potential configuration involves placing gold (Au) as the core of the mesoporous nanostructure, while other metals, such as palladium (Pd), platinum (Pt), or rhodium (Rh), form a shell or occupy the outer surfaces thereof. Such a design is advantageous for catalytic applications where Au acts as a core metal to achieve selected specific reactions. For example, the localized surface plasmon resonance (LSPR) properties of Au core enable efficient photocatalytic processes under visible light, which are valuable for applications such as water splitting for hydrogen production or CO2 reduction. Additionally, in the reaction of selective oxidation of alcohols (e.g., benzyl alcohol to benzaldehyde), gold catalysts demonstrate a higher activity and selectivity compared to platinum, which can often cause overoxidation orthe production of unwanted byproducts. The outer mesoporous shell of other metals can enhance the catalytic activity while providing further stability to the nanostructures, remaining to be versatile for a wide range of catalytic applications.

[0012] Taken together, a liposome-based aggregate template made in accordance with embodiments of the present invention would effectively guide the formation of mesoporous noble metal nanostructures in a simple, reproducible, programmable, and green fashion. It can address bottlenecks in a multitude of applications in the biomedical regime, including biocatalysis, biosensors, bioimaging, and disease therapy.

[0013] Precise control over the order of metal deposition of and its spatial orientation within the mesoporous nanostructures allow for the maximization of the catalytic activities of different metal elements. A gradient configuration could also be developed where the metal composition gradually changes from one metal to another across the mesoporous structure. In other words, relative concentrations of gold and / or other noble metals would vary as a function of spatial position. For instance, this might involve a gradient of gold to palladium or platinum, allowing for tailored catalytic properties across different zones of the metallic materials.

[0014] Catalytic therapy based on the nanozymes is an emerging strategy for tumor therapy. However, its therapeutic outcomes are considerably limited by many factors in the tumor microenvironment (TME), such as hypoxia, immunosuppression, and insufficient endogenous hydrogen peroxide (H2O2) levels. Herein, a new layer-by-layer trimetallic nanozyme (Au@Pt@Rh) with large mesopores is developed for immunoregulation and enhanced tumor nanocatalytic therapy. Such outside-in porous Au@Pt@Rh nanozyme exhibits both intrinsic peroxidase (POD)- and catalase (CAT)-like activities under acidic TME, which can catalyze H2O2 into hydroxyl radicals (-OH) and oxygen (O2), respectively. Meanwhile, Au@Pt@Rh- enabled photothermal hyperthermia improves the POD- and CAT -mimic activities. With the assistance of homologous tumor cell membrane (CM), the TGF-P inhibitor (LY2157299, LY) is successfully loaded and retained in the large mesopores of Au@Pt@Rh-CM. LY- Au@Pt@Rh-CM nanocomposites can improve tumor immunosuppressive microenvironment through polarizing the macrophages from M2 to Ml, and thus induce the regeneration of H2O2 to promote the catalytic activities. Both in vitro and in vivo results demonstrate that LY- Au@Pt@Rh-CM is able to effectively reprogram immunosuppressive TME, relieve the tumor hypoxia, produce highly toxic -OH, and therefore yield increased therapeutic efficiency upon laser irradiation of tumors. Therefore, the present invention provides a promising strategy for immunomodulatory enhanced tumor catalytic therapy based on polymetallic nanozymes.

[0015] Herein, a new layer-by-layer trimetallic nanozyme (Au@Pt@Rh) with large mesopores is developed for immunoregulation-enhanced tumor nanocatalytic therapy. Such outside-in porous Au@Pt@Rh nanozyme exhibits both intrinsic peroxidase (POD)- and catalase (CAT)- like activities under acidic TME, which can catalyze H2O2 into hydroxyl radicals (-OH) and oxygen (O2), respectively. Meanwhile, Au@Pt@Rh-enabled photothermal hyperthermia improves the POD- and CAT -mimic activities. Homologous tumor cell membrane (CM), the TGF-P inhibitor (LY2157299, LY), when successfully loaded and retained in the large mesopores of Au@Pt@Rh-CM, LY-Au@Pt@Rh-CM nanocomposites, can improve tumor immunosuppressive microenvironment through polarizing the macrophages from M2 to Ml, and thus induce the regeneration of H2O2 to promote the catalytic activities. Both in vitro and in vivo results demonstrate that LY-Au@Pt@Rh-CM is able to effectively reprogram immunosuppressive TME, relieve the tumor hypoxia, produce highly toxic -OH, and therefore yield increased therapeutic efficiency upon laser irradiation of tumors. Therefore, the present invention provides a promising strategy for immunomodulatory enhanced tumor catalytic therapy based on polymetallic nanozymes. This soft-templating synthesis method can also be adapted to synthesize similar nanostructures from other metals, such as Ru, Ir, Co and Cu, upon slight modification.

[0016] An object of the present invention is the reduction of unwanted toxicity and side effects for medical and pharmaceutical applications. This can be done via exclusion of any cytotoxic chemicals from the synthesis process to provide a unique green method to prepare the nanostructures without concerns of toxicity and side effects arising as a result of the involvement of cytotoxic chemicals in traditional metal nanoparticle synthesis methods. Such a green synthesis method would be highly beneficial for further use of such nanostructures in medical and pharmaceutical formulae or applications.

[0017] It is a further object of the present invention to increase the efficiency for catalytic reactions. Specifically, the unique porous nanostructures of mesoporous metal nanoparticles provide high surface areas, significantly increasing the active sites available for catalytic reactions. As such, the increased surface area can further improve the catalytic efficiency of nanocatalysts compared to those solid nanoparticles.

[0018] Yet another object of the present invention is to increase the drug loading capacity of porous nanostructures. Specifically, the large open mesopores of such nanostructures offer sufficient surface area to accommodate more therapeutics and large therapeutic molecules such as protein at the high loading capacities. This could allow for co-delivery of multiple drugs and sequentially responsive release.

[0019] A not-necessarily-final object of the present invention is to provide tunability of surface plasmon resonance to near-infrared windows (i.e., both I and II windows) for improved tissue penetration depth. Mesoporous nanostructures exhibit surface plasmon resonance (SPR) in the visible to near-infrared ranges and have shown a tunable photothermal effect. The long SPR absorption wavelength range allows for the use of NIR-window II light, which can significantly increase the tissue penetration depth.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Reference will be made to color in discussing the figures with the understanding that such figures are embodied in color in the corresponding figures of the sources incorporated by reference hereinbelow. For a more complete understanding of the present invention, reference is made to the following figures, in which:

[0021] FIG. 1 is a schematic illustration of liposome-templated programmable and green synthesis of mesoporous metal nanostructures;

[0022] FIG 2A shows SEM and TEM micrographs of monometallic (Au, Pd and Pt) mesoporous nanostructures;

[0023] FIG. 2B shows SEM micrographs, TEM micrographs, and elemental mapping of bimetallic (AuRh, AuPt, AuPd, PdPt, and PtRh) mesoporous nanostructures;

[0024] FIG. 2C shows SEM micrographs, TEM micrographs and element mapping of trimetallic (AuPtRh, AuPdRh, and AuPdPt) mesoporous nanostructures;

[0025] FIG. 3 A is a schematic illustration of liposome-templated Pt mesoporous nanostructure growth at different reaction periods;

[0026] FIG. 3B is a series of micrographs illustrating cryo-TEM of liposome-templated Pt mesoporous nanostructure growth at different reaction periods;

[0027] FIG. 3C is a schematic illustration of Pt mesoporous nanostructures sampled at different reaction times (1 h, 2 h, 3 h, 6 h, and 12 h, respectively);

[0028] FIG 3D is a series of TEM micrographs of Pt mesoporous nanostructures sampled at different reaction times (1 h, 2 h, 3 h, 6 h, and 12 h, respectively);

[0029] FIG. 3E is a schematic illustrating optimized geometries of DPPC:[PtCle]2' binding modes: point and planar;

[0030] FIG 3F is a schematic illustrating optimized geometries of 2DPPC:ion binding modes for 2DPPC:[PtCl6]2';

[0031] FIG. 3G is a table showing DPPC:ion binding conformations (units: bond length in A and energies in kcal / mol), wherein column a) is the average of three hydrogens pointingtowards Cl’, column b) is the average of three terminal methyl groups, column c) is the average of all CL’ involved in binding, and column d) is relative energy compared to the point mode;

[0032] FIG. 3H is a table showing DPPCion binding energies (units: kcal per mol);

[0033] FIG. 4A is a schematic showing the photothermal ability of noble metal-based mesoporous nanostructures;

[0034] FIG. 4B is a heating curve of monometallic nanostructures compared to DI water only under 808-nm laser irradiation (1 W / cm2);

[0035] FIG. 4C is a heating curve of bimetallic nanostructures compared to DI water only under 808-nm laser irradiation (1 W / cm2);

[0036] FIG. 4D is a heating curve of trimetallic nanostructures compared to DI water only under 808-nm laser irradiation (1 W / cm2);

[0037] FIG. 4E is a series of photothermal images of the aqueous solutions containing noble metal-based mesoporous nanostructures under 808-nm laser irradiation (1 W / cm2, 300 s);

[0038] FIG. 4F is a schematic of the catalytic ability of noble metal-based mesoporous nanostructures to decompose H2O2;

[0039] FIG. 4G is a graph showing measured catalytic reaction velocity on monometallic nanostructures as a function of H2O2 concentrations;

[0040] FIG. 4H is a graph showing measured catalytic reaction velocity on bimetallic nanostructures as a function of H2O2 concentrations;

[0041] FIG. 41 is a graph showing measured catalytic reaction velocity on trimetallic nanostructures as a function of H2O2 concentrations;

[0042] FIG. 4J is a schematic of drug loading into noble metal-based mesoporous nanostructures;

[0043] FIG. 4K is a UV-vis-NIR absorption spectra of DOX-loaded mesoporous AuPtRh nanostructures from different drug-loading concentrations upon removal of excessive free drug molecules;

[0044] FIG. 4L is a comparison of the photothermal-conversion efficiency (PCE), catalytic kinetic parameters, and drug loading capacity of various nanostructures;

[0045] FIG. 5 A is a schematic diagram of a typical proton exchange membrane fuel cell using mesoporous nanostructures as the catalysts;

[0046] FIG. 5B is a schematic illustration of a typical rotating disk electrode setup for an oxygen reduction reaction (ORR) performance test;

[0047] FIG. 5C illustrates rotating disk electrode (RDE) polarization curves of the as-prepared solid Pt and mesoporous nanostructures of Pt, AuPt, or AuPtRh in O2 saturated 0.1 mol / L HCIO4 at 10 mV / s and 1,600 rpm;

[0048] FIG. 5D illustrates RDE curves of mesoporous AuPtRh catalysts before cycling and after 10,000 cycles, which were measured in O2 saturated 0.1 mol / L HCIO4 solution at 10 mV / s and 1,600 rpm;

[0049] FIG. 5E illustrates CV curves of as-prepared solid Pt and mesoporous nanostructures of Pt, AuPt, or AuPtRh in Ar-saturated 0.1 mol / L HCIO4 solutions at 50 mV / s and 100 rpm;

[0050] FIG. 6A is a photograph of a liposome suspension;

[0051] Fig. 6B is TEM images of liposomes;

[0052] Fig. 6C is a graph showing size distribution of the as-prepared liposomes;

[0053] FIG. 7A is a TEM image and the corresponding EDS line scan of bimetallic AuRh porous nanostructures;

[0054] FIG. 7B is a TEM image and the corresponding EDS line scan of bimetallic AuPd porous nanostructures;

[0055] FIG. 7C is a TEM image and the corresponding EDS line scan of bimetallic AuPt porous nanostructures;

[0056] FIG. 7D is a TEM image and the corresponding EDS line scan of bimetallic PtPd porous nanostructures;

[0057] FIG. 7E is a TEM image and the corresponding EDS line scan of bimetallic PtRh porous nanostructures;

[0058] FIG. 7F is a TEM image and the corresponding EDS line scan of trimetallic AuPtPd porous nanostructures;

[0059] FIG. 7G is a TEM image and the corresponding EDS line scan of trimetallic AuPtRh porous nanostructures;

[0060] FIG. 7H is a TEM image and the corresponding EDS line scan of trimetallic AuPdRh porous nanostructures;

[0061] FIG. 8 is a series of selected-area electron diffraction characterizations, showing electron diffraction patterns recorded from various porous nanostructures;

[0062] FIG. 9A is a series of TEM and HRTEM micrographs of mesoporous AuPtRh nanostructures, in which the zoom-in examination revealed the presence of crystal lattice (round broken circles);

[0063] FIG. 9B shows wide-angle XRD patterns of the mesoporous trimetallic AuPtRh nanostructures;

[0064] FIG. 10A shows Brunauer-Emmett-Teller (BET) specific surface areas and pore sizes of mesoporous Au, AuPt, and AuPtRh nanostructures.

[0065] FIG. 10B shows an N2 adsorption / desorption isotherm and the corresponding pore size distribution (inset) of Au nanostructures;

[0066] FIG. 10C shows an N2 adsorption / desorption isotherm and the corresponding pore size distribution (inset) of AuPt nanostructures;

[0067] FIG. 10D shows an N2 adsorption / desorption isotherm and the corresponding pore size distribution (inset) of AuPtRh nanostructures;

[0068] FIG. 11 A is a schematic of a preparation process, in which additional Au precursor was added into the reaction upon the formation of porous AuPt nanostructures. More specifically, after the initial addition of HAuCh and EEPtCk into the liposome / AA suspension and reacting for 12 h, additional HAuCh was added into the reaction again;

[0069] FIG. 1 IB is composed of TEM images, STEM images, and element mappings of mesoporous AuPtAu nanostructures;

[0070] FIG. 12 is a schematic illustration of the chemical structure of DPPC;

[0071] FIG. 13(A) is a series of schematic illustrations of optimized geometries of DPPC:[PdCl4]“ binding modes: 13A(1), point, 13A(2), edge, and 13A(3), planar; and optimized geometries of DPPC: [PtCE]2-binding modes: 13A(4) point, and 13A(5) planar;

[0072] FIG. 13B is a table showing DPPC:ion binding conformations (units: bond length in A and energies in kcal / mol), showing: a) average of three hydrogens pointing towards Cl“ , b) average of three terminal methyl groups, c) Average of all Cl“ involved in binding, and d) relative energy compared to the point mode;

[0073] FIG. 13C illustrates optimized geometries of 2DPPC:ion binding modes for 13C(1), 2DPPC:[PdCl4]2-, 13C(2), 2DPPC:[AuCl4]-, 13C(3), 2DPPC:[PtCl6]2-, and 13C(4), 2DPPC:[RhCl6]3-.

[0074] FIG. 13D is a table showing DPPC:ion binding energies (units: kcal / mol);

[0075] FIGS. 14(A)-(C) are schematic diagrams illustrating initial binding modes of DPPGfPtCk]2-for (A) point, (B) edge, and (C) planar;

[0076] FIG. 15A shows optimized geometries of 2DPPC:[PdCl4]“ planar binding conformations in a trans orientation;

[0077] FIG. 15B shows optimized geometries of 2DPPC:[PdCl4] planar binding conformations in a cis orientation;

[0078] FIG. 16A is a series of TEM images of porous Pt nanostructures prepared with liposome templates;

[0079] FIG. 16B shows a size distribution of porous Pt nanostructures prepared with liposome templates;

[0080] FIG. 16C is a series of TEM images of Pt nanoparticles prepared without liposome templates;

[0081] FIG. 16D is a size distribution of Pt nanoparticles prepared without liposome templates;

[0082] FIG. 17A is a schematic illustrating the growth process of the trimetallic AuPtRh nanostructures;

[0083] FIG. 17B is a series of TEM images, STEM images, and element mapping images of mesoporous trimetallic AuPtRh nanostructures sampled at different reaction time points (5 min, 30 min, 2 h, and 12 h, respectively);

[0084] FIG. 18 is a UV-vis-NIR absorption spectra of mesoporous trimetallic AuPtRh nanostructures sampled from different reaction time points (5 min, 15 min, 30 min, 2 h, and 12 h, respectively);

[0085] FIG. 19 is a graph illustrating cell viability measurements of HUVECs upon incubation with monometallic (Au, Pd, and Pt), bimetallic (AuPt, AuPd, AuRh, PdPt, and PtRh), or trimetallic (AuPtRh, AuPdPt, and AuPdPt) mesoporous nanostructures for 24 h, wherein data are expressed as mean ± SD (n=6);

[0086] FIG. 20A is a UV-vis-NIR absorption spectrum of monometallic nanostructures;

[0087] FIG. 20B is a UV-vis-NIR absorption spectrum of bimetallic nanostructures;

[0088] FIG. 20C is a UV-vis-NIR absorption spectrum of trimetallic nanostructures;

[0089] FIG. 21 A shows a photothermal conversion analysis, illustrating a time-resolved photothermal effect of the aqueous solution (100 pg / mL) of monometallic porous nanostructures with 808 nm laser irradiation (1 W / cm2) for 600 s, and then cut off;

[0090] FIG. 2 IB shows a photothermal conversion analysis, illustrating a plot of cooling time versus negative natural logarithm of the temperature driving force obtained from the cooling stage of FIG. 21 A;

[0091] FIG. 21C shows a photothermal conversion analysis, illustrating a time-resolved photothermal effect of the aqueous solution (100 pg / mL) of bimetallic porous nanostructures with 808 nm laser irradiation (1 W / cm2) for 600 s, and then cut off;

[0092] FIG. 2 ID shows a photothermal conversion analysis, illustrating a plot of cooling time versus negative natural logarithm of the temperature driving force obtained from the cooling stage of FIG. 21C;

[0093] FIG. 2 IE shows a photothermal conversion analysis, illustrating a time-resolved photothermal effect of the aqueous solution (100 pg / mL) of trimetallic porous nanostructures with 808 nm laser irradiation (1 W / cm2) for 600 s, and then cut off.

[0094] FIG. 21F shows a photothermal conversion analysis, illustrating a plot of cooling time versus negative natural logarithm of the temperature driving force obtained from the cooling stage of FIG. 2 IE;

[0095] FIG. 22A is a double-reciprocal plot to determine the kinetic constants of monometallic nanostructures for H2O2 decomposition;

[0096] FIG. 22B is a double-reciprocal plot to determine the kinetic constants of bimetallic nanostructures for H2O2 decomposition;

[0097] FIG. 22C is a double-reciprocal plot to determine the kinetic constants of trimetallic nanostructures for H2O2 decomposition;

[0098] FIG. 23 is a table showing composition and size distributions of as-prepared bimetal and trimetal mesoporous nanostructures;

[0099] FIG. 24 is a table showing selected geometric parameters of DPPC models (units: bond lengths in A and angles in degrees, a) Average for all three terminal methyl groups of the used DPPC models; b) Average of the shortest three hydrogens pointing towards Cl’; c) Carbons of terminal methyl groups connected to N);

[0100] FIG. 25 is a table showing selected NPA charges of DPPC models (units in e): a) average for all three carbons of terminal methyl groups connected to N; b) Average of three hydrogens pointing towards Cl’; c) Average of all three terminal methyl groups connected to N; d) Average of all Cl’ involved in binding);

[0101] FIG. 26 is a table showing correlation of photothermal conversion, catalytic activity, and drug loading capacity with mesopore size of AuPt nanostructures;

[0102] FIG. 27 is a schematic illustration of hyperthermia and immunomodulation-enhanced tumor catalytic therapy for Ly-Au@Pt@Rh-CM;

[0103] FIG. 28A is a schematic illustration for the synthesis process of LY-Au@Pt@Rh-CM layer-by-layer nano-system;

[0104] FIG. 28B is a representative SEM image of Au@Pt@Rh;

[0105] FIG. 28C is a representative TEM image of Au@Pt@Rh;

[0106] FIG. 28D is a representative TEM image of Au@Pt@Rh-CM;

[0107] FIG. 28E is an SDS- PAGE protein analysis of pure CM, Au@Pt@Rh, and Au@Pt@Rh-CM;

[0108] FIG. 28F is an STEM-HAADF image of Au@Pt@Rh nanozymes and the corresponding area-elemental mapping;

[0109] FIG. 28G is a table showing the mass ratios of Au, Pt, and Rh in Au@Pt@Rh nanostructures measured by ICP-MS;

[0110] FIG. 28H is a UV-vis-NIR absorption spectra of LY, Au@Pt@Rh-CM, and LY- Au@Pt@Rh-CM;[OHl] FIG. 281 is a graph illustrating the concentration-dependency of the photothermal effect of Au@Pt@Rh-CM nanostructures under 1208 nm laser irradiation (1 W / cm2);

[0112] FIG. 28J is a series of infrared thermal images of Au@Pt@Rh-CM nanostructures at different concentrations under 1208 nm laser irradiation (1 W / cm2);

[0113] FIG. 29A is a schematic illustration of POD- and CAT -mimic catalytic activity of Au@Pt@Rh nanozymes;

[0114] FIG. 29B is a graph showing the absorbance of Au@Pt@Rh-CM (100 pg / mL), H2O2 (40 mM), and MB (5 pg / mL) in PBS buffer solutions of different pH (5.0, 6.5 and 7.4);

[0115] FIG. 29C is the UV-vis absorption spectra of oxTMB under different treatments in pH 6.5 PBS buffer, wherein the inset shows the corresponding color changes of the different solutions;

[0116] FIG. 29D is a graph showing the time-dependent absorbance of oxTMB at 652 nm upon the addition of Au@Pt@Rh nanozyme (100 pg / mL) and various H2O2 concentrations (0, 5, 10, 20, 40, 60, and 80 mM) at 50 °C;

[0117] FIG. 29E is a graph showing steady-state kinetic analysis for Au@Pt or Au@Pt@Rh at 25, 37 or 50 °C, wherein the inset shows the color change of TMB after different treatments;

[0118] FIG. 29F is a Lineweaver-Burk plotting for Au@Pt or Au@Pt@Rh at 25, 37, or 50 °C;

[0119] FIG. 29G is a tabular comparison of the POD-like catalysis kinetic parameters of Au@Pt or Au@Pt@Rh at room or elevated temperature;

[0120] FIG. 29H is a graph showing time-dependent O2 production after the addition of Au@Pt@Rh (100 pg / mL) and H2O2 (40 mM) in PBS buffer solutions with different pH values (5.0, 6.5, and 7.4);

[0121] FIG. 291 is a graph showing time-dependent O2 production after the addition of Au@Pt@Rh (100 pg / mL) and various H2O2 concentrations (0, 5, 10, 20, 40, 60, and 80 mM);

[0122] FIG. 30A is a series of TEM images showing the intracellular localization of Au@Pt@Rh-CM in LA795 cells after incubation for 6 h;

[0123] FIG. 30B is a schematic illustration of the hyperthermia-enhanced enzyme-mimic activities of Au@Pt@Rh-CM nanozyme in LA795 cells;

[0124] FIG. 30C is a fluorescence image of ROS production in LA795 cells after treatment with: (cl) Untreated, (c2) LY-Au@Pt@Rh-CM, (c3) LY-Au@Pt@Rh-CM plus 1208 nm laser irradiation, (c4) LY-Au@Pt@Rh-CM in the presence of H2O2, and (c5) LY-Au@Pt@Rh- CM in the presence of 1208 nm laser irradiation and H2O2;

[0125] FIG. 30D is a fluorescence image of OH generation in LA795 cells after treatment with: (dl) Untreated, (d2) LY-Au@Pt@Rh-CM, (d3) LY-Au@Pt@Rh-CM plus 1208 nm laser irradiation, (d4) LY-Au@Pt@Rh-CM in the presence of H2O2, and (d5) LY-Au@Pt@Rh- CM in the presence of 1208 nm laser irradiation and H2O2;

[0126] FIG. 30E is a semi-quantification of ROS fluorescence intensity in LA795 cells after various treatments;

[0127] FIG. 30F is a semi-quantification of OH fluorescence intensity in LA795 cells after various treatments;

[0128] FIG. 30G(l)-30G(5) are series of fluorescence image of O2 level in LA795 cells in hypoxia (N2 atmosphere) after various treatments: (1) Control, (2) LY-Au@Pt@Rh-CM, (3) LY- Au@Pt@Rh-CM + 1208 nm laser irradiation, (4) LY-Au@Pt@Rh-CM + H2O2, and (5) LY-Au@Pt@Rh-CM + 1208 nm laser irradiation + H2O2;

[0129] FIG. 30H is a graph showing the viability of LA795 cells after treatment with LY, Au@Pt@Rh-CM, and LY-Au@Pt@Rh-CM, respectively, wherein data are expressed as mean ± SD (n = 6);

[0130] FIG. 301 is a graph showing the viability of LA795 cells after various treatments, wherein data are expressed as mean ± SD (n = 6);

[0131] FIG. 30J is a series of fluorescence images of live / dead LA795 cells after different treatments: (j l) Untreated, (j2) LY- Au@Pt@Rh-CM, (j3) LY-Au@Pt@Rh-CM under 1208 nm laser irradiation, (j4) LY-Au@Pt@Rh-CM in the presence of H2O2, and (j5) LY- Au@Pt@Rh-CM in the presence of 1208 nm laser irradiation and H2O2, wherein Live cells are stained green with calcein-AM, and dead cells are stained red with PI;

[0132] FIG. 31A is a schematic illustration of the hyperthermia-enhanced enzyme-mimic activities of LY-Au@Pt@Rh-CM nanozymes for immunomodulation-improved tumor catalytic therapy in 3D tumor spheres;

[0133] FIG. 3 IB is a series of photographs after treatment of LY-Au@Pt@Rh-CM nanozymes with various groups, including the 1) control, 2) LY, 3) Au@Pt@Rh-CM, 4) LY-Au@Pt@Rh-CM, 5) Au@Pt@Rh-CM plus 1208 nm laser irradiation, and 6) LY-Au@Pt@Rh-CM plus 1208 nm laser irradiation at different days, wherein **p < 0.01;

[0134] FIG. 31C is a graph showing corresponding 3D tumor sphere volumes of LY- Au@Pt@Rh-CM nanozymes after treatment with various groups, including the (1) control, (2) LY, (3) Au@Pt@Rh-CM, (4) LY-Au@Pt@Rh-CM, (5) Au@Pt@Rh-CM plus 1208 nm laser irradiation, and (6) LY-Au@Pt@Rh-CM plus 1208 nm laser irradiation at different days, wherein **p < 0.01;

[0135] FIG. 3 ID is a series of immunofluorescence images of -OH in 3D tumor spheres (red: •OH, blue: nuclei);

[0136] FIG. 3 IE is a series of immunofluorescence images of HIF-la in 3D tumor spheres (green: HIF-la, blue: nuclei);

[0137] FIG. 3 IF is a series of immunofluorescence images of macrophages’ distribution in 3D tumor spheres (red: Ml macrophages, green: M2 macrophages, blue: nuclei);

[0138] FIG. 32A is a schematic illustration showing the therapeutic process of LA795 -tumor- bearing mice;

[0139] FIG. 32B is a graph showing biological distribution of Au, Pt, Rh in tumors at different time points after tail intravenous injection of Au@Pt@Rh-CM, wherein data are expressed as mean ± SD (n = 3);

[0140] FIG. 32C is a series of photothermal images of LA795-tumor-bearing mice exposed to NIR-II laser irradiation (1208 nm, 1 W cm’2) after intravenous injection with PBS and LY- Au@Pt@Rh-CM, respectively;

[0141] FIG. 32D is a graph showing the time-dependent volume changes of LA795 tumors in mice from different groups, wherein data are expressed as mean ± SD (n = 5), *p < 0.05, **p < 0.01, ***p < 0.001;

[0142] FIG. 32E is a photograph showing tumors harvested from different treatment groups after 8 doses treatments (n = 5);

[0143] FIG. 32F is a graph showing average weight of tumors harvested from different treatment groups after 8 doses treatments (n = 5), wherein data are expressed as mean ± SD (n = 5), *p < 0.05, **p < 0.01, ***p < 0.001;

[0144] FIG. 32G is a graph showing the time-dependent body weight changes of mice from different groups, wherein data are expressed as mean ± SD (n = 5), *p < 0.05, **p < 0.01, ***p < 0.001;

[0145] FIG. 32H is a series of images showing H&E staining of tumor slices after different treatments;

[0146] FIG. 321 is a series of representative fluorescence images of DHE in tumor tissues from mice after different treatments (red: DHE, blue: nuclei);

[0147] FIG. 32J is a series of representative fluorescence images of HIF-la in tumor tissues from mice after different treatments (green: HIF-la, blue: nuclei);

[0148] FIG. 33A is a schematic diagram of TME regulation by LY-Au@Pt@Rh-CM macrophage cells in LA795 tumor analyzed by flowcytometry;

[0149] FIG. 33B is a schematic diagram of TME regulation by F4-80+Ly6C+macrophage cells in LA795 tumor analyzed by flowcytometry;

[0150] FIG. 33C is a schematic diagram of TME regulation by F4-80+CD86+macrophage cells in LA795 tumor analyzed by flowcytometry;

[0151] FIG. 33D is a schematic diagram of TME regulation by F4-80+CD80+macrophage cells in LA795 tumor analyzed by flowcytometry;

[0152] FIG. 33E is a schematic diagram of TME regulation by F4-80+CD206+macrophage cells in LA795 tumor analyzed by flow cytometry;

[0153] FIG. 33F is a quantitative analysis of F4-80+Ly6C+macrophages cells, wherein the data are shown as mean ± SD (n = 3). *P < 0.05, **P < 0.01;

[0154] FIG. 33G is a quantitative analysis of F4-80+CD86+macrophages cells, wherein the data are shown as mean ± SD (n = 3). *P < 0.05, **P < 0.01;

[0155] FIG. 33H is a quantitative analysis of F4-80+CD80+macrophages cells, wherein the data are shown as mean ± SD (n = 3). *P < 0.05, **P < 0.01;

[0156] FIG. 331 is a quantitative analysis of F4-80+CD206+macrophages cells, wherein the data are shown as mean ± SD (n = 3). *P < 0.05, **P < 0.01;

[0157] FIG. 33J is a series of immunofluorescence images of Ml and M2 macrophages in tumor tissues (red: Ml-like macrophages, green: M2-like macrophages);

[0158] FIG. 33K is a series of charts showing CD4+CD8+T cells in LA795 tumor tissues analyzed by flow cytometry;

[0159] FIG. 34A is a graphical size distribution of Au@Pt@Rh;

[0160] FIG. 34B is a graphical size distribution of Au@Pt@Rh-CM;

[0161] FIG. 35 A is an N2 adsorption / desorption isotherm of porous Au@Pt@Rh nanostructures;

[0162] FIG. 35B is a pore-size distribution curve, corresponding to FIG. 35 A, of porous Au@Pt@Rh nanostructures;

[0163] FIG. 36 illustrates the wide-angle XRD pattern of the trimetallic Au@Pt@Rh nanostructures;

[0164] FIG. 37 is an image showing the SAED pattern obtained from the single Au@Pt@Rh nanostructure;

[0165] FIG. 38 is the energy-dispersive X-ray spectroscopy of Au@Pt@Rh nanostructures;

[0166] FIG. 39A is a TEM micrograph of layer-by-layer Au@Pt@Rh nanostructures;

[0167] FIG. 39B is a graph showing EDS line scan signals corresponding to FIG. 39 A;

[0168] FIG. 39C is an individual EDS line scan signal for Au;

[0169] FIG. 39D is an individual EDS line scan signal for Pt;

[0170] FIG. 39E is an individual EDS line scan signal for Rh;

[0171] FIG. 40A is a TEM image of AuPtRh nanoparticles prepared without liposome templates;

[0172] FIG. 40B is a size distribution of AuPtRh nanoparticles prepared without liposome templates;

[0173] FIG. 41A is a graph showing zeta potentials of Au@Pt@Rh, pure CM, and Au@Pt@Rh-CM, shown as mean ± SD (n = 3);

[0174] FIG. 4 IB is an FTIR spectra of Au@Pt@Rh, pure CM, and Au@Pt@Rh-CM;

[0175] FIG. 42 A is a graph showing the time-resolved photothermal effect of aqueous Au@Pt@Rh nanostructures (100 pg / mL) with 1208 nm laser irradiation (1 W / cm2) for 600 s, and then cut off with stable temperature;

[0176] FIG. 42B is a graph showing the photothermal performance of Au@Pt@Rh nanostructures and the corresponding linear relationship between -Ln (9) and time (s);

[0177] FIG. 42C is a series of photothermal curves of Au@Pt@Rh nanostructures aqueous solution at a concentration of 100 pg / mL over four laser on / off cycles (1208 nm, 1 W / cm2);

[0178] FIG. 43 A is a size distribution of LY-Au@Pt@Rh-CM;

[0179] FIG. 43B is a graph showing size and PDI of an LY-Au@Pt@Rh-CM nanosystem, respectively dispersed in l x PBS at 0, 7, and 14 day as determined by DLS (n = 3);

[0180] FIG. 44A is UV-vis absorption spectra of LY with different concentrations (1, 2, 4, 6, 8, 10, 12, 14, 16, and 18 pg / mL);

[0181] FIG. 44B is a concentration-dependent standard curve for LY absorption;

[0182] FIG. 44C is a graph showing the release behavior of LY from LY-Au@Pt@Rh-CM with or without 1208 nm laser irradiation (1 W / cm2) at 0, 3, 8, 15, and 30 min, wherein data are shown as mean ± SD (n = 3);

[0183] FIG. 45 A is a TEM image of Au;

[0184] FIG. 45B is a TEM image of Au@Pt;

[0185] FIG. 45C is a TEM image of Au@Pt@Rh;

[0186] FIG. 46A is a graph showing POD-like activity of Au@Pt@Rh in buffers with different pH values;

[0187] FIG. 46B is a graph showing POD-like activity of Au, Au@Pt, Au@Rh, Pt@Rh, and Au@Pt@Rh nanoparticles;

[0188] FIG. 47A is a graph showing time-dependent absorbance of oxTMB at 652 nm upon the addition of Au@Pt (100 pg / mL) and a series of different concentrations of H2O2 at 25 °C;

[0189] FIG. 47B is a graph showing time-dependent absorbance of oxTMB at 652 nm upon the addition of Au@Pt@Rh (100 pg / mL and a series of different concentrations of H2O2 at 25 °C);

[0190] FIG. 47C is a graph showing time-dependent absorbance of oxTMB at 652 nm upon the addition of Au@Pt@Rh (100 ug / mL) and a series of different concentrations of H2O2 at 37 °C;

[0191] FIG. 47D is a UV-vis absorption spectra of oxTMB for Au@Pt@Rh-CM and LY- Au@Pt@Rh-CM;

[0192] FIG. 48 is a calibration curve of absorbance at 240 nm as a function of the concentration ofH2O2;

[0193] FIG. 49 is a graphical semi-quantification of hypoxia dye fluorescence intensity in LA795 cells treated with LY-Au@Pt@Rh-CM under laser irradiation (1208 nm, 1 W / cm2, 3 min), wherein fluorescence images of cells stained with hypoxia dye for oxygen level as recorded from more than five images for each group were analyzed using Image J (NIH) and data are shown as mean ± SD. *P<0.05, **P<0.01;

[0194] FIG. 50 is a chart illustrating the viability of HUVEC cells after treatment with LY, Au@Pt@Rh-CM, and LY-Au@Pt@Rh-CM, wherein data are expressed as mean ± SD (n = 6);

[0195] FIG. 51 is a fluorescence image of live / dead LA795 cells after H2O2 only treatments, wherein live cells are stained green with calcein-AM, and dead cells are stained red with PI;

[0196] FIG. 52A is a series of images corresponding to OH staining of 3D tumor spheroids treated with various treatments (blue color indicates cell nucleus, red color indicates OH);

[0197] FIG. 52B is a semi -quantification of OH fluorescence intensity analyzed by ImageJ (NIH) from each set of five images, wherein the data are shown as mean ± SD. *P<0.05, **P<0.01, ***P<0.001;

[0198] FIG. 53A is a series of images corresponding to HIF-la staining of 3D tumor spheroids treated with various treatments (blue color indicates cell nucleus, green color indicates HIF- la);

[0199] FIG. 53B is a semi-quantification of HIF- la fluorescence intensity analyzed by ImageJ (NIH) from each set of five images, wherein the data are shown as mean ± SD, *P<0.05, **P<0.01;

[0200] FIG. 54A is a series of immunofluorescence images of Ml and M2 macrophages in 3D tumor spheroids after various treatments (red: Ml macrophages, green: M2 macrophages);

[0201] FIG. 54B is a semi-quantification of the ratio of Ml to M2 macrophages fluorescence intensity analyzed by ImageJ (NIH) from each set of five images of FIG. 54A, the data being shown as mean ± SD, *P<0.05, **P<0.01, ***P<0.001;

[0202] FIG. 55A is a chart showing biological distribution of Au in major organs at different time points after tail intravenous injection;

[0203] FIG. 55B is a chart showing biological distribution of Pt in major organs at different time points after tail intravenous injection;

[0204] FIG. 55C is a chart showing biological distribution of Rh in major organs at different time points after tail intravenous injection;

[0205] FIG. 56A is a graph showing levels of the ALP liver function marker in mice before treatment (control) and after treatment with LY-Au@Pt@Rh-CM + 1208 nm laser irradiation at 8 and 16 days, wherein data are shown as mean ± SD (n = 3);

[0206] FIG. 56B is a graph showing levels of the ALT liver function marker in mice before treatment (control) and after treatment with LY-Au@Pt@Rh-CM + 1208 nm laser irradiation at 8 and 16 days, wherein data are shown as mean ± SD (n = 3);

[0207] FIG. 56C is a graph showing levels of the AST liver function marker in mice before treatment (control) and after treatment with LY-Au@Pt@Rh-CM + 1208 nm laser irradiation at 8 and 16 days, wherein data are shown as mean ± SD (n = 3);

[0208] FIG. 56D is a graph showing levels of the BUN kidney function marker in mice before treatment (control) and after treatment with LY-Au@Pt@Rh-CM + 1208 nm laser irradiation at 8 and 16 days, wherein data are shown as mean ± SD (n = 3);

[0209] FIG. 56E is a graph showing levels of the CR kidney function marker in mice before treatment (control) and after treatment with LY-Au@Pt@Rh-CM + 1208 nm laser irradiation at 8 and 16 days, wherein data are shown as mean ± SD (n = 3);

[0210] FIG. 56F is a graph showing levels of the LDH kidney function marker in mice before treatment (control) and after treatment with LY-Au@Pt@Rh-CM + 1208 nm laser irradiation at 8 and 16 days, wherein data are shown as mean ± SD (n = 3);

[0211] FIGS. 57A-57L are graphs showing routine blood data of mice before treatment (control) and after treatment with LY-Au@Pt@Rh-CM + 1208 nm laser irradiation at 8 and 16 days, wherein data are shown as mean ± SD (n = 3);

[0212] FIG. 58 is a series of images showing histologic analysis of major organs including heart, liver, spleen, lung, and kidney from mice before treatment (control) and after treatment with LY-Au@Pt@Rh-CM + 1208 nm laser irradiation;

[0213] FIG. 59A is a DHE staining of LA795 tumor treated with various treatments (blue color indicates cell nucleus, red color indicates DHE);

[0214] FIG. 59B is a semi-quantification DHE fluorescence intensity analyzed by ImageJ (NIH) from each set of five images, wherein data are shown as mean ± SD, *P<0.05, **P<0.01, ***P<0.001;

[0215] FIG. 60A is a HIF-la staining of LA795 tumors from various treatment groups;

[0216] FIG. 60B is a semi-quantification of HIF-la fluorescence intensity analyzed by ImageJ (NIH) from each set of five images, wherein the data are shown as mean ± SD, *P<0.05, **P<0.01, ***P<0.001;

[0217] FIG. 61 A is a series of immunofluorescence images of Ml and M2 macrophages in tumor tissues after various treatments, (red: Ml macrophages, green: M2 macrophages);

[0218] FIG. 61B is a semi-quantification of the ratio of Ml to M2 macrophages’ fluorescence intensity analyzed by ImageJ (NIH) from each set of five images, wherein the data are shown as mean ± SD. *P<0.05, **P<0.01;

[0219] FIG. 62A is a quantitative analysis of CD4+T cells, wherein the data are shown as mean ± SD (n = 3), *P<0.05, **P<0.01; and

[0220] FIG. 62B is a quantitative analysis of CD8+T cells, wherein the data are shown as mean ± SD (n = 3), *P<0.05, **P<0.01.DETAILED DESCRIPTION OF THE INVENTION

[0221] The following disclosure is presented to provide an illustrative description of the general principles of the present invention and is not meant to limit, in any way, the inventive concepts contained herein. Moreover, the particular features described in this section can be used in combination with the other described features in each of the multitude of possible permutations and combinations contained herein.

[0222] All terms defined herein should be afforded their broadest possible interpretation, including any implied meanings as dictated by a reading of the specification as well as anywords that a person having skill in the art and / or a dictionary, treatise, or similar authority would assign thereto.

[0223] Further, it should be noted that, as recited herein, the singular forms “a”, “an”, “the”, and “one” include the plural referents unless otherwise stated. Additionally, the terms “comprises” and “comprising” when used herein specify that certain features are present in that embodiment, however, this phrase should not be interpreted to preclude the presence or addition of additional steps, operations, features, components, and / or groups thereof.

[0224] All examples and conditional language recited herein are intended for pedagogical purposes to aid the reader in understanding the principles of the invention and the concepts contributed thereby to furthering the relevant art and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents as well as equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure.

[0225] FIG. 1 is a schematic illustration of liposome-templated programmable and green synthesis of mesoporous metal nanostructures. Colloidal liposomes can serve as the soft template for the synthesis of mesoporous structures made of monometal, bimetal or trimetal. Accordingly, representative monometallic (3 types), bimetallic (5 types), and trimetallic (3 types) mesoporous nanostructures can be accordingly designed and prepared.

[0226] Templating can be useful for the preparation of noble metal mesoporous nanostructures. In an embodiment, liposome templates can be prepared using the thin-film hydration method with modification. l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol at a molar ratio of 55:45 can be dissolved in chloroform (i.e., an organic solvent) to form a homogeneous phospholipid solution which can then be placed in a high vacuum rotary evaporator to remove the residual organic solvent at a temperature, which can range from 30 and 70 degrees (e.g., 55 degrees), forming a dry phospholipid film. The lipid film can be hydrated (e.g., at 1 mg / mL) with an aqueous ascorbic acid (AA) solution at a concentration between 0.1 M and 0.5 M (e.g., 0.3 M) to create a phospholipid suspension. Alternative reducing agents to AA, such as sodium citrate, sodium borohydride, or citric acid, could also be used, provided there is a stable interaction between the metal precursor and the liposome template complex. This step can be followed by sonication at a controlled temperature (e.g., 55 degrees) to generate unilamellar liposomes. By choosing different reducing agents, the surfacecharge of produced nanostructures can be modulated, providing another degree of control. The unilamellar liposomes can then be separated by centrifugation (e.g., at 13,000 rpm for 15 min at 15 degrees) to remove aggregates or impurities, resulting in substantially uniform unilamellar liposomes (i.e., a uniform liposome template).

[0227] To initiate the formation of mesoporous nanostructures from the above-described liposome template containing reducing agent, 0.4 mL of metal precursor solution such as FhPdCU, HAuCU 3H2O, FhPtCle 3H2O, NasRhCk can be added dropwise into the supernatant liposomes produced above (e.g., 1 mL) and reacted for 12 hours in order to enable the complete reduction of metal ions to metal and the resulting formation of nanostructures with the assistance of the liposome template. This process is carried out under mild reaction conditions (e.g., room temperature up to approximately 60°C) to ensure the control of the reduction reaction with precise metal deposition in between the inter-liposomal space. The concentration ratio of precursor-to-liposome typically ranges from 1.36:2 to 1.36: 15.

[0228] Different metal precursors have their own preferred reaction temperature. For example, room temperature is suitable for Au precursor, while 50-60°C is better for Pt precursor, which can reduce its reaction time from 2 h to 12 h while maintaining comparable morphology. For the synthesis of bimetallic or trimetallic hybrid nanoparticles, mixtures of different metal precursors can be introduced into the liposome solution containing the reducing agent. The adding sequence of metal precursors into the reaction can determine the spatial distribution of individual metals within the mesoporous nanostructures. During the reaction, the order of adding the precursors and the adjustment of reaction temperature are both helpful for forming the mesoporous nanostructures with spatially controlled distribution of individual metal elements. For example, to prepare Au@Pt nanostructures, Au precursor is added first to the reaction and then Pt precursor is added afterwards.

[0229] The excessive reactants and liposome templates can then be removed by centrifugation that can be between 5,000 and 15,000 rpm (e.g., at 10,000 rpm for 10 min) and the final nanostructures may be rinsed with deionized (DI) water three times. Obtained mesoporous nanostructures can then be stored at 4 degrees for further use. Further information can be found in the publications entitled “Liposome-templated Green Synthesis of Mesoporous Metal Nanostructures with Programmable Composition for Biomedical Application” and “A Large- Pore Mesoporous Au@Pt@Rh Trimetallic Nanostructure with Hyperthermia-Enhanced Enzyme-mimic Activities for Immunomodulation-Improved Tumor Catalytic Therapy,” which are herein incorporated by reference in their entirety, including any supplemental materials, and made a part of the present application for all purposes.

[0230] It is noted that depending on the reducing agent, the process can either be continuous or divided into separate steps for template synthesis and metal nanostructure synthesis. As above, in the case of using ascorbic acid as the reducing agent, during the template synthesis, ascorbic acid is incorporated in DPPC liposome synthesis. The nanostructure (product) synthesis reaction does not begin until the metal precursor is added to the liposome suspension. The product synthesis involves mixing the template with the metal precursor to initiate the growth of the nanoframeworks for the nanostructures.

[0231] As a result of the presence of mesopores and programmable / spatial control of distribution of varying metals in the nanostructures, novel and unusual features are identified with the mesoporous nanostructures and the synthesis method. Compared to the prior art, nanostructures made in accordance with embodiments of the present invention have higher surface areas, better biocompatibility due to the green synthesis procedures, broader applicability of its synthesis methods, easier scale-up for cost-effective production, and higher controllability. Precise control over the order of metal deposition and their spatial positioning within the mesoporous nanostructures allow for the maximization of the catalytic activities of different metal elements.

[0232] Further functionalization of the nanostructures is possible. For instance, the nanostructures can undergo PEGylation via the strong thiol-metal interactions, wherein the thiolated PEG is used, to enhance the hydrophilic properties of nanostructures for high stability and long circulation in the blood stream. Additionally, they can be functionalized with biological molecules, such as antibodies, aptamers, or peptides, through thiol-metal interactions or EDC-NHS coupling reactions. This modification enables the nanostructures to possess cell -targeting capabilities. Furthermore, these nanostructures can serve as contrast agents for enhanced diagnostic purposes, such as in photoacoustic imaging. Due to their inherent photothermal (plasmonic) properties, the nanostructures loaded with thermo- responsive initiator molecules can also facilitate photothermal gelation of hydrogel for specific applications like photothermal bioprinting, synthesis of wound healing hydrogel or creation of supportive scaffold under skin.

[0233] The high surface area of nanostructures with the presence of outside-in pores with controllable pore sizes and metal layer compositions in accordance with embodiments of the present invention affords the mesoporous metal nanostructures a much higher surface area than those with solid metal nanoparticles or nanoshells / nanocages.

[0234] Good biocompatibility results from the green synthesis procedures of the present invention. Exclusion of cytotoxic chemicals from the synthesis process assures good biocompatibility of the as-prepared mesoporous noble metal nanostructures.

[0235] The synthesis method of the present invention has a broad applicability, which can be applied to diverse noble metals such as Au, Pd, Pt, Rh, and their combinations.

[0236] The simple synthesis setup, mild synthesis conditions and a short synthesis duration of the present invention allow for relatively easy scale-up for mass production.

[0237] There is a high controllability of nanostructures in terms of their size, composition, and spatial organization via modulating the metal precursor concentration and reaction time (size), and via distinct reducing potentials and sequential addition to the reaction (composition and organization), respectively.

[0238] The present invention can be leveraged in biomedical applications. For instance, the synthesis of noble metal mesoporous nanostructures with only biocompatible liposomes (i.e., the building blocks of the soft template) and ascorbic acid (AA) (a reducing agent) via selective growth of various metal struts on the boundary of lipid bilayers of aggregated liposomes upon AA reduction yields the as-prepared nanostructures with good biocompatibility for medical applications. Some examples are provided below.

[0239] Photothermal therapy: Mesoporous noble metal nanostructures exhibit strong surface plasmon resonance (SPR) in the visible to near-infrared range and showed a tunable photothermal effect depending on the light wavelength and the irradiation duration. As such, mesoporous noble metal nanostructures can serve as promising photothermal reagents for photothermal therapy, in which the photonic energy is converted to heat for killing cancer cells or infectious pathogens. In photothermal therapy, mesoporous noble metal nanostructures (with near-infrared absorption) are introduced into the body and then selectively accumulate in cancer cells, to which near-infrared light is exposed to elevate the local temperature for cell destruction. Photodynamic therapy is also a possible application of the present invention.

[0240] Drug delivery carrier: Due to the presence of large mesopores and their structural stability, mesoporous noble metal nanostructures could be loaded with therapeutic molecules within the mesopores to be used for drug delivery. The large open mesopores offer sufficient surface area to accommodate more therapeutics at a high loading capacity. Such nanostructures can also be functionalized with the sealing layer such as polyethylene glycol (PEG) to contain the therapeutic molecules in the pores until they are needed, while promising the potential for co-delivery of multiple drugs and sequentially responsive release. Depending on the nature of the cargo, such nanostructures can be formulated to treat various cancers or infectious diseases,or release growth factors / cytokines for tissue regeneration, or deliver the plasmid / mRNA / microRNA for cell engineering.

[0241] Mesopores for therapeutic loading and conjugation with targeting moieties to improve the efficacy can also be developed. In biomedical applications such as cancer therapy, anticancer therapeutics (large molecules or small molecules) can be loaded into the mesopores of the nanostructures and then conjugated with targeting moieties on the nanostructure surfaces to improve the specificity for treatment.

[0242] Due to the existence of unoccupied d orbitals in noble metals, mesoporous noble metal nanostructures display strong catalytic activity. Spatially tunable deposition of selected metals with mesopores allows for optimal exposure of reactants to the metal for efficient catalysis such as oxidation, reduction, and hydrogenation via electronic and surface strain effects. Mesoporous noble metal nanostructures can be added into the reactors to catalyze desired chemical reactions due to increased surface area. Mesoporous noble metal nanostructures are particularly useful for the reduction of toxic pollutants and production of fine chemicals. Compared to solid nanoparticles or larger particles, such mesoporous nanostructures would exhibit much higher catalytic capacity, which consequently reduces the amount of metal needed to catalyze the reaction. Meanwhile, due to the use of noble metals, they would also be easy to reactivate upon the completion of the reaction. Some examples are listed below:

[0243] Fuel cells: As an example, mesoporous noble metal nanostructures could be used as catalysts in fuel cells. Oxygen reduction reactions (ORR) have notoriously sluggish kinetics that severely limit the performance of fuel cells. That is why highly effective and stable electrocatalysts are needed to boost the reaction. Mesoporous metal nanostructures with programmable composition and monodispersed outside-in mesoporous nanostructures increasing the atom utilization efficiency therefore have noticeably enhanced the mass activities. Promoting mass activity of noble metal-based materials is beneficial for future large- scale commercialization.

[0244] Electrocatalysts in manufacturing of ammonia: The Haber-Bosch process is the dominant industrial procedure for manufacturing ammonia (N2 + 3H2 — >2NHs) but suffers from the low conversion ratio (ca. 10%-l 5%) because of unfavorable chemical equilibrium and strict reaction conditions (such as high temperature and high pressure). In fact, electrocatalysts play an important role in ammonia yield of the nitrogen reduction reaction (NRR). Among various electrocatalyst materials, noble metals, especially rhodium (Rh), are on the top list of the volcano diagrams of Skulason and possess the most active surfaces for NRR. Thus, mesoporous Rh or hybrid noble metal nanostructures with high surface area would potentially serve asefficient electrocatalysts for the Haber-Bosch process. Another potential application of the present invention is acting as an artificial catalase-like enzyme.

[0245] Photocatalysis in environmental applications: Mesoporous metal nanostructures are being studied for their potential use in photocatalytic reactions, wherein they facilitate the degradation of pollutants. For example, Pt-based photocatalysts can efficiently improve the decomposition of phenol in the presence of UV light. In photocatalytic applications and environmental remediation, mesoporous noble metal nanostructures exposed to pollutants facilitate degradation and removal of pollutants from the environment.

[0246] The present invention may also be useful for bioimaging and sensing applications.

[0247] Biosensors: Mesoporous noble metal nanostructures can be used as the sensing probes in biosensors, wherein they detect changes of the electrical signals generated by biological systems. They are particularly useful in detecting changes in the concentration of glucose, lactate, and other biochemicals. In biosensing and imaging applications, mesoporous metal nanostructures can be incorporated into devices. That is, incorporation of functionalized nanostructures into biosensor devices to improve the sensitivity and selectivity to the targeted molecules or analytes for detection.

[0248] Surface enhanced Raman spectroscopy: Mesoporous gold nanostructures themselves contain hot spots with highly localized electromagnetic fields that can amplify Raman signals of nearby analyte molecules. As such, mesoporous gold nanostructures can be used as the contrast agents for SERS-based Raman imaging, as well as for NIR imaging and OCT.

[0249] Photoacoustic imaging: Mesoporous gold nanostructures could be used as a promising tool for photoacoustic imaging (e.g., as imaging contrast agents). Mesoporous metal nanostructures have large surface areas, providing more binding sites for targeting ligands, and subsequently enabling their targeted accumulation in specific cells or tissues. Meanwhile, the presence of porous structures within the nanostructures results in strong surface plasmon resonance (SPR) in visible to near-infrared wavelength ranges, and they display a high efficiency in converting light energy into heat, which in turn generates acoustic waves. Photoacoustic imaging uses the physics principles of optical and acoustic (ultrasonic) waves to generate high contrast images. Mesoporous metal nanostructures could be used as contrast enhancing agents in such photoacoustic imaging applications.

[0250] Near-infrared imaging: Mesoporous gold nanostructures exhibit strong surface plasmon resonance (SPR) in the visible to near-infrared wavelength ranges. Depending on the light wavelength and irradiation duration, mesoporous gold nanostructures exhibit tunable photothermal effects. As a result, the laser irradiation can induce the generation of localizedheat, enabling the detection and imaging of regions of interest (Rol) accumulated with mesoporous gold nanostructures through IR thermal imaging.

[0251] CT imaging: The high atomic number of noble metals provides excellent X-ray attenuation. Thus, mesoporous noble metal nanostructures can strongly scatter and absorb X- rays, and consequently offer contrast for computed tomography images. Conventional iodine- based contrast agents have a short blood circulation time, while mesoporous noble metal nanostructures show prolonged circulation times. Excitingly, mesoporous noble metal nanostructures can be further functionalized or coated with various molecules to improve stability, biocompatibility, and targeting capacity, thereby making them attractive candidates for enhancing the contrast within targeted regions in CT scans.

[0252] Mesoporous noble metal nanostructures and their applications may attract the broader interest of industries and government agencies. For instance, pharmaceutical companies may be interested in the potential uses of mesoporous metal nanostructures for therapeutic delivery to achieve high efficacy (e.g., in cancer therapy or immune therapy). Such noble metal nanostructures have multi-fold advantages compared to other nanocarriers.

[0253] Companies that manufacture medical devices, such as diagnostic tools (biosensors and imaging agents) and drug delivery systems (including inhalers and transdermal patches) may also have an interest in the use of mesoporous metal nanostructures.

[0254] Companies engaged in the development of clean energy technologies and fuel cells may benefit from the utilization of mesoporous metal nanostructures as catalysts for fuel cell reactions and hydrogen production.

[0255] Government agencies and industries responsible for environmental protection could be interested in the use of mesoporous metal nanostructures for photocatalytic degradation of pollutants and other environmental remediation applications.

[0256] A not-necessarily-final application is relevant to research laboratories: research laboratories are often at the forefront of research into new materials and technologies and may have interest in mesoporous metal nanostructures considering their physical and chemical properties and potential applications.

[0257] Recently, it was found that positively charged terminal ammonium functional groups (-NH3+) of biocompatible liposomes prepared from phospholipids such as 1,2-dipalmitoyl-sn- glycero-3 -phosphocholine (DPPC) are primarily located on the surface of liposomal spheres (+15 mV in zeta potential measurement). As such, the negatively charged noble metallic precursor species (e.g., [AuCU] or [PtCk,]2) would preferably distribute and locate in the “corona halo” of the liposomes via electrostatic interactions. Based on this assumption, thereis the possibility of forming aggregated liposome networks hinged by noble metallic species during the reduction process and subsequently guiding the formation of 3D mesoporous structures of noble metals within the inter-liposome space upon complete reduction.Example 1:

[0258] Overview: the use of liposome-based networks as soft templates is proposed to establish a facile yet effective synthesis strategy for preparing monodispersed nanostructures with large outside-in mesopores (approximately 20 nm to 40 nm in diameter) and universal and spatially controlled deposition of monometallic (Au, Pd, and Pt), bimetallic (AuPd, AuPt, AuRh, PtRh, and PdPt), and trimetallic (AuPdRh, AuPtRh, and AuPdPt) (FIG. 1). This can potentially be achieved without sacrificing the bioactivity of such nanostructures. During the synthesis, biocompatible liposomes (i.e., building blocks of the soft template) and ascorbic acid (AA) (a reducing agent) are employed to control the morphology of the template and allow for selective growth of various metal struts on the boundary of lipid bilayers of the aggregated liposomes upon AA reduction (FIG. 1). The metallic struts are nanoframes formed in between the interliposomal space, and they are composed of ground-zero metals (Au°, Pt°, Pd° and Rh°) with free electrons that exhibit catalytic and plasmonic properties. They are formed as metallic precursors are attracted and trapped in between the liposomes via the chelation with liposomes. Due to the high chelating stability, the metal precursors are attracted and trapped to the space of adjacent liposomes and then first reduced to ground zero metal, similar to “hinges,” which serves as the nucleating point for further growth of the metal struts along the interliposomal space to form the nanostructures. Ascorbic acid located within the liposomes acts as a reducing agent, converting the metal ions into ground zero metal (metal0) to form the struts. The assembled metallic struts form monodispersed nanostructures featuring large external mesopores (~40 nm). Following the order of sequentially adding metallic precursors into the reaction, nanostructures with spatially controlled deposition of bimetallic (AuPd, AuPt, AuRh, PtRh, PdPt), and trimetallic (AuPdRh, AuPtRh, AuPdPt) configurations can be produced besides those of monometallic (Au, Pd, Pt) ones.

[0259] The final product of the metallic mesoporous nanostructures can then be obtained through centrifugation to remove excess or unreacted reagents. The synthesis method itself is environmentally friendly and very straightforward without the need for complex setups.

[0260] After the removal of the liposome templates, the as-prepared mesoporous nanostructures exhibit a unique outside-in porous structure with controllable pore size and composition of the metal layers. Along with demonstrating the effectiveness of liposome- templated synthesis of various mesoporous nanostructures, the possible mechanism of how theliposome templates guided the formation of such nanostructures was investigated through time sectioning of the synthesis process and computational modeling to monitor various stages of nanostructure growth and evaluate the chemical interaction energy between template and metal precursor at the enthalpy level. Several advantages can be identified with this synthesis strategy, including: 1) broad applicability for diverse noble metals such as Au, Pd, Pt, and Rh as well as their double or triple combinations, 2) good biocompatibility by excluding cytotoxic chemicals during the process, 3) simple setup of the entire synthesis process under a mild condition and in a short duration, and 4) high controllability of the nanostructure size via metal precursor concentration and reaction time, and the spatial organization of different metals via distinct reducing potentials and sequential addition to the reaction. As demonstrated, such noble metal mesoporous nanostructures endow some essential and unique capabilities. For example, they typically exhibited surface plasmon resonance (SPR) in visible to near-infrared ranges and showed a tunable photothermal effect. Due to unoccupied d orbitals, these nanostructures also inherently showed catalytic activity. The spatially tunable deposition of selected metals would ensure the optimal exposure of reactants to the metal for efficient biocatalysis via the electronic and surface strain effects. Meanwhile, the large open mesopores of such nanostructures offer sufficient surface area to accommodate more therapeutics at a high loading capacity, while promising the potential for co-delivery of multiple drugs and sequentially responsive release. Taken together, liposome-based aggregate templates would effectively guide the formation of mesoporous noble metal nanostructures in a simple, reproducible, universal, and green fashion.

[0261] Universal and Facile Synthesis of Various Outside-in Mesoporous Nanostructures: To guide the fabrication of outside-in mesoporous metal nanostructures with controllable pore size, the liposome-based soft templates were first prepared from DPPC and cholesterol using the modified thin-film hydration method of “Hyperthermia-Mediated Local Drug Delivery by a Bubble-Generating Liposomal System for Tumor-Specific Chemotherapy” by Chen et al, ACS Nano 2014, 8, 5, 5105-5115, the entire contents of which paper are incorporated herein by reference. In addition to DPPC, DPPE (l,2-Dipalmitoyl-sn-glycero-3- phosphorylethanolamine), and / or DSPE (l,2-distearoyl-sn-glycero-3-phosphoethanolamine) could also be used. During the process of preparing liposomes, an AA aqueous solution at a high concentration (300 mM) was used to hydrate the thin lipid films in order to enhance their dispersion in aqueous solution, decrease their hydrodynamic size and form a phospholipid suspension. In fact, the high AA concentration typically having a low pH (~5) is expected to reduce the size of liposomes. FIGS. 6A-6C illustrate liposome template characterizations,wherein the obtained liposome / AA aqueous solution appeared to be clear and transparent (FIG. 6A), containing spherical liposomes (FIG. 6B) with a relatively small size of 42.4 ± 2.2 nm (FIG. 6C). Aqueous solutions of metal precursors at a constant concentration (e.g., 15 mM of EfePtCk) were added dropwise into the liposome / AA suspension under continuous stirring. The metal precursor ions were quickly trapped into the interfaces in between neighboring liposomes to form a “DPPC-metal ion-DPPC” complex with a special conformation as a result of the electrostatic interactions between metal ions and the ammonium groups of DPPC. Upon AA reduction, the metal precursor, e.g., [PtCk,]2was reduced to Pt° nucleus and gradually grew along the interfaces of liposome aggregates into a porous nanostructure (see FIG. 1). Depending on the combinations of metal pre-cursors, various monometallic (e.g., Au, Pt, Pd), bimetallic (e.g., AuPt, AuPd, AuRh), or trimetallic (e.g., AuPtPd, AuPtRh, AuP-dRh) mesoporous nanostructures were selectively synthesized through this approach.

[0262] Both scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses confirmed successful synthesis of the spherical porous nanostructures (FIG. 2A-2C). SEM examinations of the morphology of as-prepared nanostructures made of single or multiple metals revealed rather uniform sizes and shapes with well-defined mesopores across the outer surface (FIGS 2A-C). The mean diameters of these nanoparticles ranged from approximately 100 nm to approximately 200 nm (FIG. 23), appropriate for the needs of many biomedical applications, e.g., prolonged circulation in the blood and enhanced extravasation into tumors. Such large mesopores can better accommodate therapeutic molecules with different sizes (i.e., large or small molecule drugs). Further TEM visualization to evaluate the interior structures of these nanostructures demonstrated the presence of consistent outside-in porous structures (FIGS. 2A-C). 3D electron tomography inspection of porous Pt nanostructures reconstructed from tilted series further confirmed the distribution of interconnected mesopores throughout the interior of nanostructures. The outside-in porous layers could be tuned to be composed of single or multiple metals, depending on the precursor combinations and their reduction rate. Considering the distinction of their intrinsic surface energy, reduction potentials, and thermodynamic stability, synthesis of Au nanostructures via chemical reduction of Au precursor ([AuCU] ) typically required a much shorter reaction time than other metals investigated, and the reduction time followed the sequence: Au < Pd < Pt < Rh. Thus, in the case of coexistence of HAuCU with other metal precursors such as [PtCU]2-, [PdCU]2-, [RhCk,]3in the solution, a porous Au core would form first; then other metals were attracted to the surface of Au core by sharing the electron cloud between Au° and liposome / metal precursor nanocomplexes and gradually assembled into multi-layered porousshells upon reduction. The elemental mapping results (FIGS. 2B, C) confirmed such phenomena and verified the presence of diverse porous layers, demonstrating the generality and programmability of this synthesis strategy. Taking AuPtRh mesoporous nanostructure as a representative example, elemental mapping images showed that Pt and Rh were evenly distributed in the outer porous shell, while Au was located in the interior core (FIG. 2C). To better distinguish the spatial distribution of individual metal elements (Au, Pt, Rh) in the outside-in layered mesoporous AuPtRh nanostructures, line-scan profiling was performed to the high angle annular dark field scanning transmission electron microscopic (HAADF-STEM) images (FIG. 7H upper panel). Once again, the analyses (FIG. 7H) confirmed the compartmented distribution of different elements, i.e., Au in the interior core and Pd and Rh in the outer shell along with the presence of pores throughout the nanostructures and the separation of individual metal elements. It is worth noting that the morphology of mesoporous PtRh nanostructures shown in TEM images is somewhat different from others, which may come from the larger mismatch of the Pt and Rh theoretical lattice (3.1%) in comparison to other bimetal combinations (e.g., 0.77% for Pd-Pt), restricting the epitaxial growth of PtRh, and eventually evolving into nondense structures. Selected-area electron diffraction (SAED) characterization indeed showed a high crystallization of the AuPtRh nanostructures despite their mesoporous structures (FIG. 8). FIGS. 11A and 11B are characterizations of the crystallization behavior of representative AuPtRh mesoporous nanostructures. The wide-angle X-ray diffraction (XRD)-based examination of AuPtRh nanostructures revealed five characteristic diffraction peaks of Au, Pt, and Rh (111), (200), (220), (311) and (222) (FIG. 9A), consistent with above SAED patterns. Altogether, a random aggregation of crystal grains rather than oriented growth is suggested in the AuPtRh nanostructures. Based on the Pt / Rh (111) diffraction peak (FIG. 9B), the size of crystalline grains was calculated to be 6.1 nm using the Scherrer equation, which was similar to the size of 5.5 nm as determined on the TEM images of the mesoporous shell. As measured by the inductively coupled plasma mass spectrometry (ICP-MS) (FIG. 23 f), the weight fraction of Au, Pt, and Rh in the AuPtRh nanostructures was 49.92, 35.18, and 14.90%, respectively, which partially corresponded to the initial molar fraction of precursors and the reduction rate of the reaction. Similarly, the successful preparation of nanostructures with other metals was also confirmed for the presence of controllable outside-in mesopores and uniform particle size distribution. FIGS. 10A-10D are characterizations of pore size and surface area of the representative mesoporous nanostructures. As determined in N2 adsorption-desorption isotherms, the representative mesoporous nanostructures of Au, AuPt, and AuPtRh possess specific surface area up to 120.1,117.3, and 103.9 m2g respectively (FIGS. 10A-10D) and the respective mesopore sizes are 42.54, 40.85, and 38.19 nm, notably larger than other noble metal-based mesoporous nanoparticles reported previously. All the above results demonstrate the versatility and robustness of the liposome-templating method in synthesis of outside-in mesoporous nanostructures with large and controllable pore size and universal metal layers from mono-, bi- , and tri-noble metals. Theoretically, this approach can be extended to fabricate more metal combinations such as quad- or quint-metals as long as they can complex with DPPC. Furthermore, the different metal layers can also be programmed by sequentially adding the metal precursors into the reaction. As a demonstration, AuPtAu mesoporous nanostructures were synthesized by forming the AuPt first and then adding additional Au precursor ([AuCU] ) into the reaction to form new Au porous layers. This is illustrated in FIG. 11A-B, which are structural characterizations of the AuPtAu nanostructures prepared by sequential addition of metal precursors into reaction

[0263] Synthesis Mechanism Investigation of the Liposome-Templated Growth of Mesoporous Nanostructure: To gain an insightful understanding of the liposome-templating synthesis, it would be beneficial to monitor the synthesis process in a time-resolved manner. Compared to other noble metals (e.g., Pd and Au), a prolonged chemical reduction time would be needed to synthesize Pt nanostructures due to its relatively higher surface energy and thermodynamic stability and lower reduction potentials. Hereby, the growth process of porous Pt nanostructures was chosen as the model to investigate the synthesis mechanism (FIGS 3 A- D). Upon addition of Pt precursor (15 mM ^PtCk) into the as-prepared liposome / AA solution (~40 nm in diameter, FIG. 6A-6C), the synthesis was initiated.

[0264] In recognition of the determinant role of the spatial organization of noble metal precursors within liposome templates in defining the growth of final nanostructures, the quantum molecular modeling study was performed using [N(Me)3(CH2CH2OH)]+, the structure of which is shown in FIG. 12, to represent the positively charged terminal group of the large DPPC molecule and interact with the entire precursor ions (no truncations). In the quantum molecular chemistry study, [N(Me)3(CH2CH2OH)]+ (right structure of FIG. 12) was used as the model to represent the positively charged terminal group of the large DPPC molecule (left structure of FIG. 12) to interact with the metal precursor ions in order to facilitate the simulation calculation. As such, the interaction energy at the enthalpy level was investigated.

[0265] FIGS. 13A-13D pertain to the quantum molecular modeling study of the liposome- templated growth of mesoporous nanostructure discussed herein. The 1 : 1 molar ratio ofDPPC:ion models was investigated with various possible binding modes. Different from [PdCU]2and [AuCU] ions, which have a square planar geometry, and can bind to DPPC via one, two, or four CD (called point, edge, or planar binding modes, respectively, see FIG. 13 A), octahedral [PtCk,]2(including [RhCD] ) only leads to a stable point and planar modes via one and three CD (FIG. 3E and FIG. 13A, and FIG. 14) while the edge binding via two CD automatically optimized to the planar mode. Among all the optimized structural models of four metal ions, the planar mode is of the lowest energy (Figure 3G and FIG. 13B). This suggests that the planar mode, due to the presence of most CD for strongest electrostatic interactions, is the most favorable mode for all 1 : 1 ratio DPPC don models, which was used in the subsequent studies of 2: 1 ratio DPPGion interface models. Results of all four metal ion interface models show significantly enhanced binding strengths compared to 1 : 1 models by 103, 53, 52, and 76% for [PdCU]2-, [AuCU]", [PtCU]2", [RhCU] respectively (see FIG. 13D). Here, [PtCU]2is used as an example to further illustrate this effect. Two possible arrangements of CD with respect to the positions of terminal methyl groups are proposed. The calculations show that DPPC: [PtCU]2with three CD located between three hydrogens of terminal CH3 of DPPC have slightly lower energies by 0.31 kcal moD1than that with three CD overlapping with hydrogens. Therefore, the former conformation is used to simulate the interface binding of [PtCU]2in between two DPPC molecules in order to determine whether the interface binding exhibits stronger interactions. Although the terminal hydroxyl groups of two DPPC molecules can have cis or trans orientations (see FIGS. 15 A, B), the large spatial distance between the two groups (0...0 distances of ~16 A) makes these orientations of insignificant energy differences to influence the binding strength analysis. Therefore, the trans orientation is used for 2DPPC:ion models. All the optimized geometries of 2DPPC:ion binding models are shown in FIG. 3F and FIG. 13C. As shown in FIG. 3H and FIG. 13D, the 2DPPC:[PtCle]2binding significantly increases the binding strength by 52% compared to DPPC : [PtCU]2, which are most likely due to the increased number of electrostatic interactions between CD and positively charged terminal hydrogens of DPPC in the 2DPPC:[PtCle]2model. The almost linear N-M-N (N is from DPPC and M is the metal) angle (179.90, see FIG. 24) indicates head-to-head binding in 2DPPC:[PtCle]2. Notably, the interface binding is more favorable. Since the calculation is just for one [PtCU]2", with more such 2DPPC:[PtCle]2interactions, as in the experimental system, the interface binding strength can be significantly increased.

[0266] With [PtCU]2trapped in the interface of two liposomes, Pt° reduced by AA started to aggregate into the small Pt nanostrips, the mesopore rudiment of a final mesoporous Pt nanostructure (see the time-section result of 1 h reaction, FIGS 3A-D). As the reaction timeextended, more such small Pt nanorings developed along the interfaces of aggregated liposomes and began to form complex 3D architecture (see the results harvested at 2 h and 3 h, Figure 3B, D). When the reaction time increased to 6 h, the interconnected mesopores were clearly visible, and beyond this point, no significant change in morphology and mesoporous structure was identified except for the increase of particle size. After 12 h, well -dispersed porous Pt nanostructures with uniform shape and size were obtained. A comparison was also performed to affirm the necessity of liposome templates in the formation of mesoporous Pt nanostructures (FIG. 16A-D). In the absence of liposomes, only solid Pt nanoparticles with a broad size range were formed (FIG. 16C).

[0267] FIGS. 17A-B show time-resolved growth of AuPtRh. To assure a similar growth course to other noble metals occurred, especially those of multiple ones, a time-resolved investigation of the growth of a selected trimetallic AuPtRh nanostructure (FIG. 17A) was also performed. As shown in FIG. 17B, porous gold nanoparticles were formed at the very early reaction time (5 min). Surprisingly, Pt° was also seen at this time, much faster than that with mono Pt nanostructures (FIG 3B), which might attribute to the accelerated development of Pt crystals by Au seeds. With the time prolonged, the Pt layer continued to grow, but with no significant morphology and size change of the Au core. When the reaction reached 2 h, a noticeable amount of Rh° attached onto the surface of Pt layer to form the outermost layer of AuPtRh nanostructure. UV-vis-NIR absorption spectra were also used to monitor the growth process (FIG. 18) with the characteristic peak of gold nanoparticles at the early reaction and then with broadband absorption at 12 h.

[0268] Quantum Molecular Modeling Study: More details on the atomic level binding geometries and properties of the noble metal ions within the liposome interface are given in what follows.

[0269] The 1 : 1 ratio of DPPC:ion models with various possible binding modes were firstly examined. For [PdC14]2' and [AuC14]- ions which have square planar geometry, three models were studied with DPPC binding via one CF, two CT, and four CT, called point, edge, and planar binding modes, respectively, as shown in FIG. 13A items (l)-(3) for [PdCh]2'. In the optimized structures, the average distances between the negatively charged CF ion(s) and partially positively charged hydrogens ( — HO.2 e) of terminal methyl groups in the DPPC models are around 2.81-2.96 A (see FIG. 13 A), which are close to and a little bit shorter than the sum of van der Waal’s radii of CF and H bond (2.95 A). These results suggest that the interactions are primarily of an electrostatic nature, but close to van der Waals interactions. With more CF involved in the binding, the interaction becomes stronger from point, to edge,and then to planar (FIG. 13B). As such, the planar binding mode was used for [PdCU]2’ and [AuCU]’ ions in this investigation.

[0270] The DPPC binding modes for octahedral [PtCU]2’ and [RhCU]3’ ions with initial point, edge, and planar conformations were also studied, see FIG. 14. However, only the point and planar modes (FIG. 3E) can be successfully obtained with the planar mode being of ~ 1 kcal / mol lower energy (FIG. 13B), while the edge binding was optimized automatically to the planar mode. This suggests that the planar mode, due to the presence of more negatively charged CT ions compared to the other two modes, thus has stronger electrostatic interactions and is also the most favorable mode for [PtCU]2’. In this case, there are two possible arrangements of CT ions with respect to the positions of terminal methyl groups. Calculations showed that DPPC:[PtCle]’ and DPPC:[RhCle]2’ models with three CF ions locating between three hydrogens of terminal CH3 groups of DPPC have slightly lower energies by 0.31 and 0.03 kcal / mol than models with three CF overlapping with these three hydrogens. So, the former conformation is used in the subsequent discussion for [PtCU]2’ and [RhCU]3’ containing models.

[0271] With the most favorable conformations for 1 : 1 ratio DPPC:ion bindings, the 2: 1 ratio (i.e., 2DPPC:ion) was studied to simulate an interface binding environment with the ion in the middle of this sandwich structure and the two DPPC models on the two ends, which were used to investigate if these kinds of 2:1 interface binding models exhibited stronger interactions. Although the two terminal hydroxyl groups from the two DPPC models can have different orientations (e.g. cis or trans; see FIGS. 15A,B), the large spatial distances between these two groups (O. . .0 distances of- 16 A) makes these orientations of insignificant energy differences (<= 0.16 kcal / mol for both 2DPPC:[PdCU]2’ and 2DPPC:[AuCU]’ models). Therefore, the trans orientation was used for 2DPPC:ion models here. All the optimized geometries of 2DPPC:ion binding models are shown in FIG. 13C.

[0272] As shown in FIG. 13D, the use of 2: 1 ratio binding significantly increases the binding strength (i.e., lowers the binding energy, which was calculated by using the complex’s energy minus the energies of all components) by 103%, 53%, 52%, and 76%, respectively, for [PdCU]2’ [AuCU]’, [PtCU]2’, [RhCU]3’ ions, which is likely due to the increased number of electrostatic interaction pairs between CF ions and positively charged terminal hydrogens in such 2:1 binding models versus the 1 : 1 models. The almost linear (>177°) N-M-N (N is from DPPC and M is the metal) angles indicate head-to-head binding for 2DPPC models. Overall, these results clearly show that the interface binding is more favorable due to the involvement of more electrostatic interactions between negatively charged ions and positively chargedDPPCs. Since the calculations are just for one ion, it is expected that with more ions having such 2DPPC:ion interactions, the interface binding strength will be significantly increased and favored, as observed experimentally.

[0273] Photothermal, Catalytic, and Drug Loading Performance of Porous Metallic Nanostructures: For potential utility in biomedical applications, good biocompatibility of these nanostructures is beneficial. Hence, cytocompatibility of these nanostructures (200 jig mL1) was tested with primary human healthy cells (i.e., human umbilical vein endothelial cells (HUVECs)), and the cell viability after 24-h incubation with various nanostructures was evaluated by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As shown in FIG. 19, negligible cytotoxicity was seen with all nanostructures tested, indicating their superior cytocompatibility for normal cells. In view of the unique characteristics of obtained mesoporous noble metal nanostructures, such as the SPR absorption in the near-infrared (NIR) range, catalytic efficiency, large mesopores, and high surface area, these characteristics were selectively evaluated based on their photothermal effect under laser irradiation, catalytic capacity of decomposing hydrogen peroxide (H2O2), and drug loading capacity of doxorubicin (DOX) as examples of their potentials in biomedical applications (FIGS. 4A, F, J). First, the UV-vis-NIR spectra of obtained nanostructures were established. Interestingly, all the synthesized porous nanostructures (i.e., monometallic, bimetallic, and trimetallic) exhibited strong broadband absorption (FIG. 20 A), similar to a black body. With that said, these porous nanostructures could effectively convert the absorbed photonic energy from a broad spectrum of wavelengths including NIR into heat. Thus, further tests were conducted to measure the photothermal efficacy of these porous nanostructures. Respective aqueous solutions of these nanostructures (0.1 mg mL1in deionized (DI) water) were irradiated with an 808 nm laser at a power intensity of 1 W cm2. Time-dependent temperature changes were then recorded using a digital thermocouple thermometer. As noted in FIGS. 4B- D, after 600 s irradiation, the temperature of all the nanostructure solutions increased dramatically, significantly higher than that of DI water. Photothermal images further confirmed the heat generated within these solutions (FIG. 4E). The temperature of porous Au nanostructure solution was measured at 56.8 °C, slightly higher than that of Pd (52.4 °C) and Pt (47.5 °C) ones. Such a difference most likely comes from the higher plasmonic absorption of Au nanostructures at 800 nm wavelength (FIG. 20A). Compared to monometallic nanostructures, bimetallic and trimetallic solutions showed a much higher temperature increase, especially for AuPdPt nanostructures, reaching as high as 73.7 °C by 600 s, a result of the combined photothermal effects from different metals. Based on FIGS. 21A-F, thephotothermal-conversion efficiency (PCE) of different nanostructures was calculated. As shown in Figure 4L, a higher PCE was achieved with trimetallic nanostructures (AuPdPt > AuPtRh > AuPdRh) followed by bimetallic ones (PdPt > AuPt > PtRh > AuRh > AuPd) and then monometallic ones (Au > Pt > Pd). Of them, AuPdPt nanostructures exhibited the best PCE, as high as 69.22%. As noted, such mesoporous nanostructures show a higher PCE than those commonly used plasmonic metal nanoparticles such as nanospheres (22.9%), nanoshells (25%), or nanorods (22%). To further elaborate how the size of mesopores affects the PCE, solutions of AuPt nanostructures with comparable particle sizes and morphologies but varying mesopore sizes (16.21 nm prepared by other methods, versus 40.85 nm) (FIG. 26) were similarly irradiated with 808-nm laser and then the temperature increase was measured. As calculated, negligible improvement in PCE was seen with the large mesoporous nanostructures, suggesting both sizes of mesopores provide comparably effective light contact area for the enhanced photothermal performance. Apparently, these noble metal-based mesoporous nanostructures with high NIR absorption are well-suited as the photothermal agents for photothermal therapy of solid tumors. Furthermore, the obtained PCE of various nanostructures can serve as an indicative reference to rationally select those potent photothermal agents for future uses.

[0274] Enzyme-mediated catalysis, as an omnipresent phenomenon in living organisms, is closely involved in regulating various physiological processes. Under certain pathophysiological conditions, occurrence / kinetics of nanocatalytic reactions can be altered and compromised, leading to unwanted accumulation of cytotoxic molecules such as H2O2 in tumorous or inflammatory tissues. In this regard, endogenously triggered catalytic therapy has been accordingly developed as an effective therapeutic modality. Establishing evidence has highlighted the enzyme-like catalysis of H2O2 decomposition by noble metals; thus, the catalytic capacity of various porous nanostructures was evaluated using H2O2 as the substrates (FIG. 4F). Steady-state kinetics of H2O2 decomposition by porous nanostructures was established by measuring the initial decomposition rates as a function of H2O2 concentration using the Goth method. As reported, the catalytic process typically follows a Michaelis-Menten reaction model. Based on the corresponding Lineweaver-Burk plots (FIGS. 4G-L and FIGS. 22A-22C, two key kinetic parameters (i.e., Michaelis-Menten constants (Km) and maximum reaction velocity (Vmax)), were calculated (FIG. 4L). In a catalytic reaction, Km represents the enzyme affinity for substrate (low Km indicates a high affinity). Among all three monometallic porous nanostructures, Pt ones displayed the highest Vmax value, mainly due to platinum’s unsaturated d electron orbitals (i.e., 5d96sl) to facilitate the interactions withmolecules containing nonbonded electron pairs. Of bimetallic nanostructures, PtRh had the lowest Km, but the highest Vmax value, implying its strong catalytic capability. A similar trend was also seen with trimetallic nanostructures, in which AuPtRh had the highest catalytic activities. Obviously, much improved catalytic capacity is measured with bimetallic or trimetallic nanostructures over monometallic nanostructures, which may attribute to the s-p-d hybridization and intra-atomic charge redistribution, leading to an increased abundance of transferable d electrons close to the Fermi level and consequently enhancing the catalysis. Interestingly, the mesopore size (between 16.21 nm and 40.85 nm) seemingly offered comparable catalytic efficacy in H2O2 decomposition (only a slightly lower Km was seen with large mesopores, FIG. 26) probably due to sufficient surface areas from both sizes of mesopores for the desired catalytic activities. Overall, the high catalytic efficiency of these noble metal nanostructures with large mesopores is of great benefit in catalytic therapy or effective modulation of the local microenvironment for better tissue regeneration.

[0275] The presence of large mesopores (e.g., 40 nm) in these noble metal nanostructures can offer more surfaces and interior void space to accommodate biomolecules of different sizes, implying their suitability as vehicles to deliver therapeutics. DOX, a model drug with broad anticancer activity and relatively stable fluorescence for in vitro and in vivo tracking, was particularly chosen to evaluate the drug loading capacity of various porous nanostructures (FIG. 4J). To better trap DOX within the mesopores without leakage, the surface of nanostructures was also coated with SH-PEG. As noted in the UV-vis-NIR spectra of DOX- loaded AuPtRh nanostructures (FIG. 4K), increase of the DOX characteristic peak as a function of drug concentrations confirmed the successful payload of DOX while exhibiting the concentration-dependent loading capacity. As shown in FIG. 4L, all the as-prepared porous nanostructures yielded the drug loading capacity in the range of 205%-310% (w / w), noticeably higher than other DOX-loaded nanocomposites such as nanoshells (~8%), nanosheets (38%), and other meso-porous nanomaterials (78%). To better correlate the pore size with drug loading efficiency, DOX was also loaded into AuPt porous nanostructures but with distinct pore sizes (16.21 nm vs 40.85 nm). As expected, a much higher DOX loading capacity was achieved with the large mesopores (FIG. 26), indicating the large pores indeed favor the access of therapeutic molecules. This high drug-loading capacity would enable the delivery of the desirable therapeutic amount with the reduced need of nanostructures while achieving the high therapeutic efficacy with individual therapeutic-loaded nanostructures.

[0276] Besides their potential in biomedical applications, the possible use of such mesoporous nanostructures was explored in electrocatalysis (FIGS. 5A-5E). As demonstrated, allmesoporous nanostructures (Pt, AuPt, and AuPtRh) exhibited noticeably improved electrocatalytic activities compared to solid Pt nanoparticles. Particularly, trimetallic AuPtRh mesoporous nanostructures have shown the highest oxygen reduction reaction (ORR) activity, which might come primarily from the large electrochemically active surface area (ECSA) and strong electronic interaction between different metals (more details are provided in the Supporting Information).

[0277] Conclusions: a green and controllable synthesis strategy has been demonstrated for the fabrication of outside-in porous nanostructures with large mesopores from noble metals. This strategy has been successfully applied to synthesize monometallic, bimetallic, and trimetallic nanostructures. With the use of only biocompatible liposomes as the templates and AA as the reductant, along with other unique characteristics, the as-synthesized nanostructures can be easily adopted for biomedical applications. Taking mesoporous AuPtRh nanostructures as an example, its superior photothermal effect (67.94% of PCE) can significantly boost the efficiency in photothermal therapy of diseases. Meanwhile, the layered mesoporous nanostructures can offer a promising means to co-deliver multiple drugs loaded in different porous layers and achieve temporal-spatial release in response to pH and light irradiation, as well as the possibility of cascading the catalysis.

[0278] Inspired by their elevated electrocatalytic activities, such mesoporous noble metallic nanostructures may see extended applications in other fields such as energy storage and sensors. In recognition of the catalytic activity of nonprecious metal -based (such as Ni, Co, Fe, Mo, etc.) nanoparticles, such a template-guided strategy can also be used to synthesize mesoporous catalysts from those metal precursors in the future.

[0279] Sample Preparation: Liposome templates were prepared using the thin-film hydration method with modification. Briefly, DPPC and cholesterol at a molar ratio of 55:45 were dissolved in chloroform and then placed in a high vacuum rotary evaporator to remove the residual organic solvent at 55 °C. The lipid film was hydrated (1 mg / mL) with an aqueous AA solution (0.3 M) via sonication at 55 °C. The unilamellar liposomes were then separated by centrifugation at 13,000 rpm for 15 min at 15 °C. 0.4 mL of metal precursor solution such as H2 PdCL, HAuCL AH2O, H2 PtCL 3H2O, or Nas RhCle was added dropwise into the above supernatant liposomes (1 mL) and reacted for 12 h. For the synthesis of bimetallic or trimetallic hybrid nanostructures, mixtures of different metal precursors were introduced into the liposome solution containing the reducing agent. Excessive reactants and liposome templates were removed by centrifugation at 10,000 rpm for 10 min and the final nanostructures were rinsedwith DI water three times. Obtained mesoporous nanostructures were stored at 4 °C for further use.

[0280] Characterization: SEM images were obtained with a Zeiss Auriga FIB-SEM. TEM images were obtained with an FEI Titan Themis 200 TEM at an acceleration voltage of 200 kV. UV-vis-NIR absorption spectra were performed with a Shimadzu UV-3600 spectrophotometer. To obtain cryo-TEM images, copper mesh grids (Lacey Carbon Film 300 mesh, Copper, Ted Pella) were glow discharged for 20 s. The cryogenic sample was plunged into liquid ethane with a typical volume of 5 pL. The as-prepared samples were stored in a liquid nitrogen tank for later use. Single tilt cryogenic tomography holder was cooled down to below 90 K under liquid nitrogen before loading with the samples. N2 adsorption-desorption isotherms were recorded using a Micromeritics 3Flex analyzer at the temperature of 77 °K. The samples were degassed under a vacuum at 150 °C for 6 h, and the Brunauer-Emmett- Teller (BET) method was utilized to calculate the specific surface areas and pore sizes.

[0281] Computational Calculation: All calculations were done using the Gaussian 16 program. The models were subject to full geometry optimization and subsequent frequency calculations using the hybrid HF-DFT method mPWlPW91 with the basis set including the effective core potential (ECP) basis SDD for metal elements and 6-311G++(2d,2p) for all other elements. This method provided useful results from previous computational calculations of those molecules containing the same or similar late-transition metals. Optimization and frequency calculations were conducted with water as the solvent used for tests, simulated by using the self-consi stent reaction field method of the PCM approach. The frequency analysis was used to verify the nature of the stationary points on the respective potential energy surfaces and to provide enthalpies at room temperature and 1 atm. Natural Population Analysis (NPA) charges were also calculated as implemented in Gaussian 16.

[0282] Evaluation of Photothermal Effect, Catalase-Like Activity, and Drug Loading Efficiency: To determine the photothermal effect, a 808 nm multimode pump laser (Shanghai Connect Fiber Optics Co. Ltd.) was used as the irradiation source. Nanostructures dispersed in DI water at a concentration of 100 pg mL1were exposed to 808 nm laser irradiation with a power intensity of 1 W / cm2. The temperature and thermal images at different time durations were measured and recorded with a two-channel data logger thermometer and an infrared camera (FLIR TM A325SC camera), respectively. The PCE (rj) was calculated using the equation: q = [hs

[0283] To determine the catalytic competence of nanostructures, catalase-like activity assays were performed at 37 °C using the Goth method. To determine its Km and Vmax, 0.02 mL of the as-prepared nanostructure solution (0.2 mM) was added into 0.1 mL prewarmed phosphate buffer solution (67 mM) containing H2O2 and kept at 37 °C for 1 min. Then, 0.1 mL ammonium molybdate solution (Mo = 240 mM) was added to stop the catalase-like reaction. Absorbance of the yellow complex of molybdate and H2O2 was measured at 405 nm using a micro-plate reader (Synergy Hl, BioTek). The Km and Vmax of the nanostructures were calculated using the Lineweaver-Burk plot.

[0284] To evaluate the drug loading capacity of nanostructures, a common chemotherapeutic drug, DOX was chosen as the model. Various nanostructures (0.2 mg mL1) were respectively mixed with DOX at a concentration of 1 mg mL1in phosphate buffer solution (pH 8.0). After stirring at room temperature for 12 h, 2 mg mL1of SH-PEG (MW = 2000, Sigma) was added to trap the DOX molecules within the pores without leakage, and excessive unbounded DOX was removed by centrifugation at 10,000 rpm for 10 min. The DOX-loaded nanostructures were rinsed several times with DI water. To calculate the DOX loading capacity, supernatants of the DOX loading solution upon removal of DOX-loaded nanostructures were analyzed with a fluorescence spectrophotometry for non-loaded DOX. The DOX loading capacity was calculated according to the formula: Drug loading capacity (%) = (mass of drug within the nanostructures / mass of nanostructures)* 100%.

[0285] Electrocatalysis Performance Evaluation: A typical three-electrode system utilizing a rotating disc electrode (RDE) device was used for electrochemical evaluation. The nanostructure-coated glassy carbon electrode served as the working electrode while graphite rod was used as the counter electrode and Hg / HgO electrode as the reference electrode. All the potentials were reported with respect to a reversible hydrogen electrode (RHE). The ORR activity was tested using an electrochemical workstation in 0.1 mol L1HCIO4 electrolyte at room temperature. Cyclic voltammetry (CV) curves were obtained by scanning the potentials at 50 mV s ' in Ar-saturated HCIO4 solutions. Polarization curve was obtained in Ch-saturated solutions at 1600 rpm using a scanning rate of 10 mV s ' . The stability tests were performed by applying 10,000 CV cycles in Ch-saturated solutions.

[0286] Fuel cells have garnered significant interest as a promising technology for sustainable energy sources. The efficiency of catalysts used for the oxygen reduction reaction (ORR) is crucial to determine the operational longevity and performance of fuel cells. Currently, noble metal (such as Pd, Pt, or Rh)-based catalysts remain irreplaceable to improve the sluggish cathodic ORR kinetics. In this regard, the ORR performance of the present mesoporousnanostructures of representative mono-, bi- and tri -metal composition (z.e., Pt, AuPt, AuPtRh) was examined in Ch-saturated 0.1 mol / L HCIO4 solutions as shown in FIGS. 5A-B. To better evaluate the effects of mesopores and composition of mesoporous nanostructures on ORR performance, solid Pt nanoparticles with an identical size (z.e., 146 nm) were included as controls. The respective ORR polarization curves of all four types of nanostructures were recorded at 10 mV / s and 1,600 rpm and summarized in FIG. 15C. Notably, solid Pt nanoparticles exhibited the lowest ORR activity. As noted, the half-wave potential of mesoporous Pt nanostructures (0.787 V) is 41 mV higher than that of solid Pt nanoparticles (0.746 V), confirming the contribution of porous structure to the superior electrocatalytic activity for ORR. Furthermore, the trimetallic AuPtRh mesoporous nanostructures show an even higher half-wave potential (0.795V), mainly due to the incorporation of Au and Rh. The strong electronic interaction between different metals leads to a downshifting of the Pt d-band center with a simultaneously enhanced adoption of reactants and reduced binding energy of ORR intermediates, thereby further enhancing the ORR activity. The limiting current of AuPtRh mesoporous nanostructures was close to 6 mA / cm2, much higher than that of Pt (4.26 mA / cm2) or AuPt (4.60 mA / cm2) mesoporous nanostructures. This observation again suggests a more facile mass transfer of oxygen into the mesoporous AuPtRh nanocatalyst, which consequently results in a higher utilization efficiency of the active sites. Apart from the catalytic activity, electrochemical stability is another essential factor to determine the quality of a catalyst. The ORR durability of mesoporous AuPtRh nanostructures was tested within a polarization range of 0-1.1 V. As shown in the polarization curve (FIG. 5D), the ORR activity of mesoporous AuPtRh nanostructures remains as high as 95.7% after 10,000 cycles.

[0287] To further evaluate the superiority of mesopores and polymetallic composition for ORR, cyclic voltammograms (CV) of solid Pt, and mesoporous nanostructures (Pt, AuPt, and AuPtRh) in Ar-saturated 0.1 M HCIO4 solution were recorded (FIG. 5E). Compared to solid Pt, the reduction current peaks of metal oxides from all mesoporous nanostructures are enhanced. This observation further affirms that mesoporous structure exhibits a better electrochemical catalytic activity compared to solid Pt, which may result from the availability of more active sites due to increased surface area. To confirm this, the electrochemically active surface area (EC SA) of various nanostructures was calculated. From the hydrogen desorption region, the ECSA can be calculated using the following equation: _0.21where “QH” represents the charge for hydrogen desorption (mC / cm2), “[MN]” is the loading density of nanostructures on the glassy carbon electrode (mg / cm2), and the number “0.21” represents the charge required to oxidize the monolayer H2 adsorbed on the nanocatalyst surface. The respective EC SA of Pt, AuPt, and AuPtRh mesoporous nanostructures was 98.69, 102.98, and 101.64 m2 / g, much higher than that of solid Pt nanoparticles (85.97 m2 / g). Clearly, with the same loading density of nanostructures, mesoporous nanostructures provided much higher active surfaces for catalytic reaction, thus leading to a higher ORR activity.Example 2:

[0288] Sample preparation: Liposome templates were prepared using the thin-film hydration method with modification. l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC) and cholesterol at a molar ratio of 55:45 were dissolved in chloroform and then placed in a high vacuum rotary evaporator to remove the residual organic solvent at 55 °C. The lipid film was hydrated (1 mg / mL) with an aqueous ascorbic acid (AA) solution (0.3 M) via sonication at 55 °C. The unilamellar liposomes were then separated by centrifugation at 13,000 rpm for 15 min at 15 °C. 0.4 mL of metal precursor solution such as EhPdCU, HAuCU 3H2O, EhPtCk 3H2O, NasRhCL was added drop-wise into the above supernatant liposomes (1 mL) and reacted for 12 h. For the synthesis of bimetallic or trimetallic hybrid nanoparticles, mixtures of different metal precursors were introduced into the liposome solution containing the reducing agent. The excessive reactants and liposome templates were removed by centrifugation at 10,000 rpm for 10 min and the final nanostructures were rinsed with deionized (DI) water three times. (FIG. 1)

[0289] Porous metallic nanostructures characterization: Scanning electron microscopic (SEM) images were obtained with a Zeiss Auriga FIB-SEM and Transmission electron microscopic (TEM) images were obtained with an FEI Titan Themis 200 TEM at an acceleration voltage of 200 kV. Both the TEM and SEM confirmed successful synthesis of the spherical porous nanostructures. (FIG. 2A) The elemental mapping results (FIGS. 2B, 2C) confirmed such phenomena and verified the presence of diverse porous layers, demonstrating the generality and programmability of this synthesis strategy. Taking AuPtRh mesoporous nanostructure as a representative example, elemental mapping images showed that Pt and Rh evenly distributed in the outer porous shell while Au located in the interior core (FIG. 2C).

[0290] The synthesis process was monitored in a time-resolved manner to gain an insightful understanding of the liposome-templating synthesis. FIGS. 3A-3D pertain to a mechanisticinvestigation of the liposome-templated growth of mesoporous nanostructure. The growth process of porous Pt nanostructures was chosen as the model to investigate the synthesis mechanism because of its prolonged chemical reduction time (FIGS. 3A-D). Upon addition of Pt precursor (15 mM EhPtCk) into the as-prepared liposome / AA solution, the synthesis was initiated. With [PtCk]2' trapped in the interface of two liposomes, Pt° reduced by AA started to aggregate into the small Pt nanostrips, the mesopore rudiment of a final mesoporous Pt nanostructure (see the time-section result of 1 h reaction, FIGS. 3A-D). As the reaction time extended, more such small Pt nanorings developed along the interfaces of aggregated liposomes and began to form complex 3D architecture (see the results harvested at 2 h and 3 h, FIG. 3B, 3D). When the reaction time increased to 6 h, the interconnected mesopores were clearly visible and beyond this point, no significant change in morphology and mesoporous structure was identified except for the increase of particle size. After 12 h, well -dispersed porous Pt nanostructures with uniform shape and size were obtained.

[0291] Photothermal, catalytic, and drug loading performance of porous metallic nanostructures: UV-vis-NIR absorption spectra were performed with a Shimadzu UV-3600 spectrophotometer. All the synthesized porous nanostructures (monometallic, bimetallic, and trimetallic) exhibited strong broadband absorption. With that said, these porous nanostructures could effectively convert the absorbed photonic energy from a broad spectrum of wavelengths including NIR into heat. Thus, the photothermal efficacy of these porous nanostructures was evaluated. 808-nm multimode pump laser (Shanghai Connect Fiber Optics Co. Ltd.) was used as the irradiation source. Respective aqueous solution of these nanostructures (0.1 mg / mL in deionized (DI) water) was irradiated with the 808-nm laser at a power intensity of 1 W / cm2. Time-dependent temperature changes were then recorded using a digital thermocouple thermometer. As noted in FIGS. 4B-4D, after 600 s irradiation, the temperature of all the nanostructure solutions increased dramatically, significantly higher than that of DI water. Photothermal images further confirmed the heat generated within these solutions (FIG. 4E). The temperature and thermal images at different time durations were measured and recorded with a two-channel data logger thermometer and an infrared camera (FLIR TM A325SC camera), respectively. Compared to monometallic nanostructures, bimetallic and trimetallic solutions showed a much higher temperature increase, especially for AuPdPt nanostructures, reaching as high as 73.7 °C by 600 s, a result of the combined photothermal effects from different metals. As shown in FIG. 4L, a higher photothermal-conversion efficiency (PCE) was achieved with trimetallic nanostructures (AuPdPt > AuPtRh > AuPdRh) followed by bimetallic (PdPt > AuPt > PtRh > AuRh > AuPd) and then monometallic (Au > Pt > Pd) ones. Of them,AuPdPt nanostructure exhibited the best PCE, as high as 69.22%. As noted, such mesoporous nanostructures show a higher PCE than those commonly used plasmonic metal nanoparticles such as nanospheres (22.9%), nanoshells (25%), or nanorods (22%). Apparently, these noble metal-based mesoporous nanostructures with high NIR absorption are well-suited photothermal agents for photothermal therapy of solid tumors. Furthermore, the obtained PCE of various nanostructures can serve as an indicative reference to rationally select those potent photothermal agents for future uses.

[0292] Establishing evidence has highlighted the enzyme-like catalysis of H2O2 decomposition by noble metals; thus, the catalytic capacity of various porous nanostructures was evaluated using H2O2 as the substrates (FIG. 4F). To determine the catalytic competence of nanostructures, catalase-like activity assays were performed at 37 °C using the Goth method. To determine its Michaelis-Menten constant (Km) and maximum reaction velocity (Vmax), 0.02 mL of the as-prepared nanostructure solution (0.2 mM) was added into 0.1 mL prewarmed phosphate buffer solution (67 mM) containing hydrogen peroxide (H2O2) and kept at 37 °C for 1 min. Then, 0.1 mL ammonium molybdate solution (Mo = 240 mM) was added to stop the catalase-like reaction. Absorbance of the yellow complex of molybdate and H2O2 was measured at 405 nm using a micro-plate reader (Synergy Hl, BioTek). The Km and Vmax of the nanostructures were calculated using the Lineweaver-Burk plot.

[0293] To evaluate the drug loading capacity of nanostructures, a common chemotherapeutic drug, doxorubicin (DOX) was specifically chosen as the model. Various nanostructures (0.2 mg / mL) were respectively mixed with DOX at a concentration of 1 mg / mL in phosphate buffer solution (pH 8.0). After stirring at room temperature for 12 h, 2 mg / mL of SH-PEG (MW=2000, Sigma) was added to trap the DOX molecules within the pores without leakage and excessive unbounded DOX was removed by centrifugation at 10,000 rpm for 10 min. The DOX-loaded nanostructures were rinsed several times with DI water. To calculate the DOX loading capacity, the supernatants of the DOX loading solution upon removal of DOX-loaded nanostructures were analyzed with a UV-vis spectrometer for non-loaded DOX and the absorbance at 488 nm was recorded to determine the DOX concentration remaining in the loading solution. The DOX loading capacity was calculated according to the formula: Drug loading (DL%) = (mass of drug within the nanostructures / mass of nanostructures)* 100%. As shown in FIG. 4L, all the as-prepared porous nanostructures yielded the drug loading capacity in the range of 205-310% (w / w), noticeably higher than other DOX-loaded nanocomposites such as nanoshells (~ 8%), nanosheets (38%), and other mesoporous nanomaterials (78%).

[0294] Electrocatalysis performance evaluation: A typical three-electrode system utilizing a rotating disc electrode (RDE) device was used for electrochemical evaluation. The nanostructure-coated glassy carbon electrode served as the working electrode while graphite rod was used as the counter electrode and Hg / HgO electrode as the reference electrode. All the potential was reported with respect to a reversible hydrogen electrode (RHE). The ORR activity was tested using an electrochemical workstation in 0.1 mol / L HCIO4 electrolyte at room temperature. The limiting current of AuPtRh mesoporous nanostructures was close to 6 mA / cm2, much higher than that of Pt (4.26 mA / cm2) or AuPt (4.60 mA / cm2) mesoporous nanostructures. This observation again suggests more facile mass transfer of oxygen into the mesoporous AuPtRh nanocatalyst, which consequently results in a higher utilization efficiency of the active sites. (FIG. 5C) Apart from the catalytic activity, electrochemical stability is another useful parameter to determine the quality of a catalyst. The ORR durability of mesoporous AuPtRh nanostructures was tested within a polarization range of 0-1.1 V. As shown in the polarization curve (FIG. 5D), the ORR activity of mesoporous AuPtRh nanostructures remains as high as 95.7% after 10,000 cycles. Cyclic voltammetry (CV) curves were obtained by scanning the potentials at 50 mV / s in Ar-saturated HCIO4 solutions. The polarization curve was obtained in 02-saturated solutions at 1600 rpm using a scanning rate of 10 mV / s. The stability tests were performed by applying 10,000 CV cycles in 02-saturated solutions. Compared to solid Pt, the reduction current peaks of metal oxides from all mesoporous nanostructures are enhanced. This observation further affirms that mesoporous structure exhibits a better electrochemical catalytic activity compared to solid Pt, which may result from the availability of more active sites due to increased surface area. The respective electrochemically active surface areas (ECSA) of Pt, AuPt, and AuPtRh mesoporous nanostructures was 98.69, 102.98, and 101.64 m2 / g, which is much higher than that of solid Pt nanoparticles (85.97 m2 / g). (FIG. 5E)Example 3: Large-pore mesoporous Au@Pt@Rh trimetallic nanostructure with hyperthermia- enhanced enzyme-mimic activities

[0295] Tumor catalytic therapy, a strategy that employs enzymes to initiate catalytic reactions and further modulate biological tumor microenvironment (TME) for generating antitumor effects, has attracted considerable interest in recent years. To achieve the effective tumor catalytic therapeutic outcomes and avoid adverse biotoxicities, pathological and chemical hallmarks of TME have been exploited to provide distinct stimulation to initiate the catalytic reactions. Differing from normal tissue circumstances, TME is characterized by localized mild acidosis intracellular stroma (pH ~ 6.0) and hypoxia environment (pO2 < 2.5 mmHg), whichare mainly caused by the burgeoning metabolic demands and exuberant aerobic glycolysis of tumor. Moreover, the intratumoral disproportionation of super anion radicals induced by superoxide dismutase leads to the overproduction of hydrogen peroxide (H2O2, -50-100 pM), which can be catalyzed into highly toxic hydroxyl radicals (-OH) or oxygen (O2) by peroxidase (POD) and catalase (CAT) activity, respectively, to produce antitumor effect. Such intrinsic hallmarks of solid tumor offer a distinct condition for chemical catalytic reactions and thus endow the tumor catalytic therapy with high specificity and low biotoxicity.

[0296] Besides the above features, tumor-associated macrophages (TAMs) in TME often display an immune-suppressive (i.e., M2-like) phenotype, facilitating tumor growth and promoting resistance to therapy. Nevertheless, such M2 macrophages are highly plastic and can be re- educated into the anti-tumorigenic Ml-like phenotype, which can not only ameliorate the immunosuppression, but can also increase the H2O2 level within TME to further enhance the efficiency of tumor catalytic therapy. In addition, the above-mentioned tumor hypoxia plays a supportive role during the M2-polarized TAMs recruitment and induction to significantly decrease immunotherapy effect. While the hypoxia condition can be relieved by catalase-like activity of enzymes in turn to inhibit the M2-like polarization of TAMs. Taking above into consideration, a specific counter-measure to regulate the immune functions of macrophages via M2-to-Ml repolarization to improve the tumor catalytic therapy effect would be beneficial.

[0297] During the tumor catalytic therapy process, enzymes are the necessary initiators for catalytic reactions that determine the therapeutic efficiency. Many strategies have been explored to utilize the natural enzymes (e.g., glucose oxidase, catalase, and lactate oxidase) for tumor catalytic therapy. Despite their efficient catalysis performance, foreseeable challenges with such natural enzymes, such as selective stability, poor reusability, and strict physiological conditions limit the development of more robust enzyme-like materials. As the prospective alternatives for natural enzymes, nanozymes, which refer to the nanoscale materials with intrinsic enzyme like properties, have been extensively explored for tumor therapy due to their high stability, tunable catalytic activities, and facile synthesis.

[0298] In an early demonstration, a bimetallic nanozyme of Au@Rh with solid Au core and mesoporous Rh shell was developed to catalyze EECE-to-Ch decomposition and modulate the hypoxic TME. Other recent efforts have also been made to fabricate noble metals (such as Au, Pd, Pt, and Rh etc.) based nanozymes to decompose H2O2 into the highly toxic -OH by POD mimics or H2O and O2 by CAT mimics. More importantly, noble metal nanoalloys such as bimetallic, trimetallic, and even polymetallic alloys, exhibited higher performance for catalyticreactions than the monometallic nanoparticles. In addition, these noble metals-based nanostructures can also act as near-infrared (NIR)- absorbing nanomaterials to generate localized hyperthermia via the NIR light excited ion resonance on the surface of metals for tumor photo-thermal therapy (PTT), which are helpful in overcoming the temperature limitation of catalytic therapy. Built on the established evidence, it was hypothesized that the noble metal-based trimetallic nanomaterials could effectively and continuously catalyze -OH and O2 production from endogenous H2O2 in the TME, and consequently ameliorate local hypoxia-induced immunosuppression to enhance tumor catalytic therapy.

[0299] In this example, the development of a large-pore mesoporous Au@Pt@Rh trimetallic nanozyme is described as well as the further formulated nano-therapeutic reagent, (i.e., LY- Au@Pt@Rh-CM nanocomposites), by loading the TGF-P inhibitor (LY2157299, LY) in the mesopores and coating with cancer cell membrane (CM), for immunoregulation enhanced nanocatalytic tumor therapy (FIG. 27). Adoption of such trimetallic core-shell nanocrystals with outside-in large mesopores (~38 nm) would allow for the optimal exposure of reactants to three metals for efficient catalysis via electronic and surface strain effects. In addition, the large pore size and surface area could also increase the loading efficiency of the therapeutics. LY, as an ideal small molecule inhibitor of immunosuppressive cytokine TGF-P, which has entered Phase I clinical trial, was utilized to induce the polarization of the macrophages from M2 to Ml. The morphological, physicochemical, and biological properties of LY- Au@Pt@Rh-CM were investigated by solution, cell, and animal-based studies to comprehensively evaluate the POD and CAT catalytic ability, photothermal conversion performance, M1 / M2 reprogramming efficiency, CD4+ / CD8+ cells regulation capacity, tumor therapeutic effects, and biosafety. As recognized, such LY-Au@Pt@Rh- CM nanosystems exhibited several unique features, such as 1) Au@Pt@Rh-CM with inherent POD-like activity can decompose the endogenous H2O2 into toxic -OH to kill tumor cells, while its CAT-like activity can catalyze H2O2 into O2 to overcome tumor hypoxic condition to further regulate immunity microenvironment; 2) Large porous cavities of Au@Pt@Rh combined with the trapping function of CM can increase the loading efficiency of LY while stabilizing its activity during biological transport without early release; 3) Due to homologous binding capability of tumor CM, selective tumor accumulation of Au@Pt@Rh-CM leads to effective delivery of LY to tumor site for immunomodulation-improved tumor catalytic therapy specificity; 4) The increased O2 and LY level within the TME can raise the amount of Ml macrophages to further promote the generation of H2O2, and in turn improving the catalytic efficiency of the nanozymes; 5) Broadband NIR absorption of Au@Pt@Rh-CM like a blackbody enables theexcitation with high tissue penetration NIR laser (i.e., 1200 nm) for hyperthermia generation, which favors the catalytic effect of nanozymes. Along with successful demonstration of such outside-in porous Au@Pt@Rh trimetallic nanostructures for immunomodulation- enhanced tumor nanocatalytic therapy, the present embodiment of the present invention also offers the concept to enable design of other polymetallic nanoplatforms with varying attributes for tumor treatment.Synthesis and Characterization of Au@Pt@Rh-CM Nanozyme

[0300] To fabricate mesoporous Au@Pt@Rh trimetallic nanostructures with large pores, the modified liposome-templating method detailed above was similarly adopted. In this method, the metal precursor can bring more liposomes together to form aggregated liposome networks via the electrostatic interaction between AuCl4' , PtCl6' , or RhCl6' and the -N(CH3)3+of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and subsequently guide the formation of mesopores within the inter-liposome space. Firstly, liposomes were prepared by using the DPPC / cholesterol as lipids and ascorbic acid (AA) aqueous solution (300 mM) as the hydration medium. In the obtained liposome template solution, AA can act as the reducing agent to reduce HAuCU, HPtCle, and NasRhCk into Au, Pt, and Rh respectively. Given that the different surface energy (Rh > Pt > Au), chemical reduction of Rh from its precursor salts normally requires prolonged reaction times and higher reaction temperature than Pt or Au, based on such reduction differences and the electron cloud between Au° and other metal precursors, the trimetallic layer-by-layer nanostructures of Au@Pt@Rh with porous Au core, secondary middle Pt layer, and outermost Rh shell were synthesized accordingly (FIG. 28A). Both scanning electron microscope (SEM) and transmission electron microscopy (TEM) were employed to confirm the formation of desired nanostructures. SEM examination clearly revealed that the particles were fairly uniform in size and shape with well-defined mesopores throughout the outer surface (FIG. 28B). Through dynamic light scattering (DLS) measurement, the mean diameter of Au@Pt@Rh was analyzed as 122.4 nm (FIG. 34A), which is beneficial for the enhanced accumulation into tumor via the enhanced permeability and retention (EPR) effect. Further careful TEM observation of Au@Pt@Rh revealed the consistent presence of an outside-in porous structure (FIG. 28C). As evidenced in N2 adsorptions-desorption isotherms, such Au@Pt@Rh mesoporous nanostructures possess a large pore volume up to 0.16 cm3g-1(FIG. 35 A), which provide the sufficient cavity space for active agent loading. Meanwhile, the pore size is about 38.01 nm (FIG. 35B), which is larger than most common mesoporous nanoparticles, successfully overcoming their traditional inability to carry large molecules. In the wide-angle X-ray diffraction (XRD) pattern ofAu@Pt@Rh, five characteristic diffraction peaks of Au, Pt, and Rh (111), (200), (220), (311) and (222) are observed respectively (FIG. 36), which is consistent with the results of the selected area electron diffraction (SAED) pattern (FIG. 37). These peaks and concentric rings of spots indicate the random growth of crystal grains in the Au@Pt@Rh nanoparticles, rather than the oriented aggregation. In the energy-dispersive X-ray spectrum, the Au, Pt, and Rh peaks were clearly observed, demonstrating the three-metal composition of Au@Pt@Rh nanoparticles (FIG. 38). Energy-dispersive X-ray spectroscopy (EDS) elemental mapping further confirmed the exact distribution of Au, Pt, and Rh elements in one particle: Au is the core, Pt is located in the sub-outer layer, and Rh is located in the outermost layer (FIGS. 28F and FIG. 39). Via the inductively coupled plasma mass spectrometer (ICP-MS), the mass ratios of Au, Pt, and Rh in Au@Pt@Rh were also precisely measured as 50.2 %, 32.9 %, and 16.9%, respectively (FIG. 28G). To confirm the necessity of liposome templates in guiding the formation of such porous trimetallic nanostructures, parallel synthetic procedures were also performed. Indeed, in the absence of liposomes, only solid AuPtRh nanoparticles with the size of 91.3 nm were formed (FIG. 40). Overall, these results indicate that the liposome template- mediated mesoporous Au@Pt@Rh trimetallic nanostructures with large pores were successfully prepared.

[0301] To endow the Au@Pt@Rh nanoparticles with a homologous targeting effect and trap the drug molecules within the mesopores, it was attempted to coat these porous nanostructures with the cancer CM. The LA795 lung cancer CM solution was obtained by lysing the cells with hypotonic lysate and collecting the supernatant after centrifugation. Under the action of ultrasound, the LA795 tumor CM fragments were uniformly coated on the surface of Au@Pt@Rh nanoparticles via the mechanical force. From the TEM image, a layer of CM on the surface of the nanostructure was indeed observed (FIG. 28D). The sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) experiment result revealed the similar protein bands of Au@Pt@Rh-CM with purified CM, verifying the presence and effective retention of protein contents of the Au@Pt@Rh nanoparticles and portending the unique biological functions of homologous cell membranes (FIG. 28E). After coating with tumor CM, the size of the nanoparticles increased from 122.4 to 141.8 nm (FIG. 34B), indicating the thickness of CM layer was about 9.7 nm. Moreover, due to the negatively charged CM (-35.2 mV), the zeta potential of these nanoparticles was decreased from 17.9 to -23.2 mV after CM coating (FIG. 41 A). From Fourier transform infrared (FTIR) spectra, the characteristic peaks of amide bonds, phosphate, and carbohydrate regions of CM in Au@Pt@Rh-CM (FIG. 4 IB) were observed, further confirming the successful attachment of CM on the Au@Pt@Rh nanostructures.

[0302] Because of the unique surface plasmon resonance (SPR) phenomenon, noble metalbased nanoparticles can usually convert the light into heat to apply for PTT treatment of diseases. Therefore, the photoabsorption behavior of the prepared Au@Pt@Rh-CM, nanostructures was evaluated by performing the UV-vis-NIR spectra. Interestingly, these nanoparticles displayed strong broadband absorption and covered both NIR I and II biological windows (FIG. 28H), like a blackbody, indicating that Au@Pt@Rh-CM nanostructures could effectively convert the absorbed photonic energy from a very broad spectrum of wavelengths into heat. Thus, the 1208 nm laser was chosen in the second NIR (NIR-II) biowindow as the light source, which shows deeper tissue penetration (>2 cm vs ~1 cm) and higher maximum permissible exposure (MPE) to skin (1 W / cm2vs 0.33 W / cm2, American National Standard for Safe Use of Lasers, ANSI Z136.1-2007) than that in the first NIR (NIR-I) region. To evaluate the photothermal conversion effect, a series of concentrations (0-200 pg / mL) of Au@Pt@Rh- CM nanoparticles were continuously irradiated by the 1208 nm laser with a power density of1 W / cm2for 360 s. During the irradiation process, it was noticed that the aqueous solution of Au@Pt@Rh-CM nanoparticles exhibited a concentration- and time-dependent temperature rise (FIGS. 281, 28 J). At a concentration of 100 pg / mL, the temperature rose to 47.4 °C only after2 min irradiation, which is high enough to regulate tumor vascular perfusion, lymphocyte trafficking, innate and adaptive immune function, and improve the catalytic efficiency of nanozymes via hyperthermia. The photo-thermal conversion efficiency of Au@Pt@Rh-CM nanostructures under 1208 nm laser irradiation was calculated as 41.74 % based on the data in FIGS. 42A, 42B, which is higher than other porous monometallic or poly-metallic nanoparticles, such as porous hollow palladium nanoparticles (36.9%), or PEG-CuS-Au-MnO2nanoparticles (28%). In addition, the unattenuated four cycles of the heating and cooling process under 1208 nm laser irradiation suggests the photostability of these Au@Pt@Rh-CM nanostructures (FIG. 42C). As such, the continuously stable hyperthermia produced by the Au@Pt@Rh-CM under NIR-II laser irradiation is expected to improve subsequent tumor catalytic- immunological therapy efficacy.

[0303] The large mesopores in Au@Pt@Rh can offer more space to accommodate active agents for drug delivery. Meanwhile, the CM could also act as gatekeeper to trap drug molecules inside the channel of pores. Here, the TGF-P inhibitor LY (LY2157299) was loaded into Au@Pt@Rh-CM to improve the immunosuppressive microenvironment. The reason why this drug was chosen is that the TGF-P is associated with the expression of M2 macrophages, which are over-produced in the tumor environment and further contribute to tumor progression. To demonstrate the successful loading of LY, UV-vis absorption spectrum was performed. Asshown in FIG. 28H, an obvious peak at about 239 nm was observed for LY-Au@Pt@Rh-CM, which was attributed to the characteristic peak of LY, suggesting the presence of LY in Au@Pt@Rh-CM nanostructures. After the LY loading, the particle size of LY- Au@Pt@Rh- CM was determined to be 142 nm (FIG. 43A), which is almost unchanged compared with Au@Pt@Rh-CM. In addition, when suspended in 1 x PBS, LY-Au@Pt@Rh-CM showed negligible changes in particle size and poly dispersity index (PDI) within 14 days, indicating the excellent stability of this CM coated LY-Au@Pt@Rh-CM nanosystem (FIG. 43B). According to the standard curve of LY (FIG. 44 A, 44B), the drug loading efficiency of LY- Au@Pt@Rh-CM was calculated as 80.43 %, which is much higher than other previously reported nanostructures. Subsequently, NIR-II laser-triggered LY release from LY- Au@Pt@Rh-CM was spectrometrically measured. As shown in FIG. 44C, weak drug release was observed in solution without laser irradiation, suggesting the efficiency of CM on Au@Pt@Rh surfaces for retention of LY within the mesopores. Upon addition of laser irradiation, rapid release of LY from Au@Pt@Rh-CM was observed, which may be attributed to the degradation of CM by laser induced heat.

[0304] FIGS. 29A-29I are a characterization of POD and CAT enzyme-like activities of Au@Pt@Rh nanozyme. Based on the evidence that the metal Pt and Rh exhibited the CAT and POD activity of decomposing H2O2, it was therefore hypothesized that the mesoporous trimetallic Au@Pt@Rh-CM can effectively catalyze the H2O2 into O2 and -OH (FIG. 29A). To confirm this, typical colorimetric analysis based on methylene blue (MB) and 3, 3', 5,5'- tetramethyl- benzidine (TMB) was firstly used to investigate the POD-mimic activity of Au@Pt@Rh-CM nanozyme. Under the POD-like effect, the produced -OH from decomposition of H2O2 can degrade the MB indicator. As shown in FIG. 29B, in the presence of H2O2 and Au@Pt@Rh-CM, the content of MB in the pH 5.0 and 6.5 PBS buffer decreased by 36.02 % and 55.30 %, respectively, while no significant change was found in the pH 7.4 PBS buffer. Such results suggest that the Au@Pt@Rh-CM nanozyme can effectively catalyze the decomposition of H2O2 into -OH under acidic conditions. This pH dependent POD activity was also further confirmed by the TMB (FIG. 46A), which can be oxidized by the generated •OH to form the blue-colored oxTMB with typical characteristic absorbances at 370 and 652 nm. Due to the weak acid tumor microenvironment, the subsequent detection of the production of oxTMB with various treatments was measured in pH 6.5 PBS buffer using UV-vis absorption spectroscopy (FIG. 29C). The TMB alone and TMB + H2O2 group showed no obvious absorbance and color changes, indicating no oxidation reaction occurred. Aftertreatment with Au@Pt@Rh-CM nanozyme, an obvious blue color of mixed solution with two characteristic absorbance peaks at 370 and 652 nm could be observed, demonstrating the intrinsic POD-mimic activity of Au@Pt@Rh-CM. Previous findings highlighted the superior catalytic capabilities of multiple metallic structures to their monometallic or bimetallic counterparts. To elaborate the superiority of trimetallic Au@Pt@Rh, mesoporous structures, Au and Au@Pt nanoparticles with comparable sizes were also synthesized under the same liposome-templating procedure and used to evaluate their catalytic efficiency (FIGS. 45A-C). In the presence of TMB, H2O2, and monometallic Au nanoparticles, negligible absorbance can be observed (FIG. 46B), indicating the invalid POD-like activity of single Au based porous nanostructures. Though the significant peaks can be found after treatment with Au@Pt, Au@Pt, and Pt@Rh, the absorbance is still lower than that of Au@Pt@Rh, suggesting the stronger catalytic capability of trimetallic nanozyme than bimetallic ones. In order to further evaluate the superior catalytic performance of Au@Pt@Rh, the steady-state catalytic kinetics were tested in a reaction system containing different H2O2 concentrations (5, 10, 20, 40, 60, and 80 mM), TMB (416 x 10'3mM), and Au@Pt@Rh or Au@Pt nanoparticles (100 pg / mL) at room temperature. In contrast to the bimetallic Au@Pt group, the Au@Pt@Rh shows higher time-dependent and H2O2 concentration-dependent absorbance intensity (FIGS. 46A-B), further confirming the improved catalytic capability of trimetallic nanozyme. Due to the photothermal response of the Au@Pt@Rh nanozyme, the effect of local heat on the catalytic performance was also evaluated. As shown in FIG. 29D, the group at 50 °C displays higher absorbance intensity than the groups at 37 and 25 °C (FIG. 47B-47C), which proves that hyperthermia can enhance the production of -OH through use of Au@Pt@Rh nanozyme. In addition, this enzyme-like activity was not affected after the loading of LY (FIG. 47D).

[0305] Subsequently, the initial reaction rates of the production of -OH were calculated based on the Beer-Lambert law, and then were plotted against the corresponding concentrations of H2O2 and fitted with Michaelis-Menten saturation curve (FIG. 29E). From the inset images in FIG. 29E, the color changes of different groups after adding various H2O2 concentrations can be visually observed. In addition, according to the data in the linear double-reciprocal plot (Lineweaver-Burk plot) in FIG. 29F, fundamental enzyme kinetic parameters including maximum reaction velocity (Vmax) and Michaelis-Menten constants (Km) were calculated (FIG. 29G). The results showed that the Vmax value for Au@Pt@Rh nanostructures at 50 °C was higher than those at 37 and 25 °C, implying improved catalytic capability from the hyperthermia. In the comparison between Au@Pt and Au@Pt@Rh, it was noticed that the Km value of Au@Pt@Rh nanostructures was lower than that of Au@Pt, while its Vmax value washigher than that of Au@Pt, confirming the higher catalytic activity of trimetallic nanozymes than bimetallic nanostructures. Unsurprisingly, the porous Au@Pt@Rh nanostructures exhibited a strong affinity for H2O2 and high POD-like activity in the heated condition.

[0306] To examine the CAT activity of Au@Pt@Rh nanozyme to catalyze H2O2 into O2, the production of oxygen in real time was monitored through dissolved oxygen instrument. A large and comparable O2 generation amount could be observed in both neutral (pH 7.4) and weak acid (pH 6.5) environment (FIG. 29H), verifying the CAT -like effect of Au@Pt@Rh on decomposing H2O2 to generate O2 under tumor acidic microenvironment. According to the standard absorption curve of H2O2 (FIG. 48), the consumption of H2O2 by Au@Pt@Rh is 59.2 % after 15 min of reaction. In addition, the generation of O2 catalyzed by Au@Pt@Rh complied with an H2O2 concentration- and time-dependent manner (FIG. 291). Such a phenomena of H2O2 consumption and O2 generation proves the ability of Au@Pt@Rh-CM to ameliorate the local hypoxic conditions of the tumor. As expected, the above results suggest that Au@Pt@Rh-CM with intrinsic POD-mimic and CAT-like activities can act as a promising H2O2-responsive nanozyme for tumor catalytic therapy.In Vitro Cell-Killing Effect of LY-Au@Pt@Rh-CM Nanozyme

[0307] Encouraged by the excellent solution-based performance of LY-Au@Pt@Rh-CM nanozyme, the cellular level photothermal-enhanced catalysis and immunomodulation therapeutic efficacy was evaluated. Initially, the cellular phagocytosis behavior of the Au@Pt@Rh-CM nanozymes by LA795 lung adenocarcinoma tumor cells was examined under TEM. FIGS. 30A-J relate to in vitro assessments for photothermal -enhanced tumor nanocatalytic therapeutic efficiency of the LY-Au@Pt@Rh-CM nanosystems. As shown in FIG. 30A, a majority of mesoporous Au@Pt@Rh-CM nanostructures could be observed in cytosolic vesicles, which may be ascribed to the homologous targeting effect of cell membranes in the surface of these trimetallic nanoparticles.

[0308] Next, the hyperthermia-improved production of -OH and O2 catalyzed by LY- Au@Pt@Rh-CM nanozymes was investigated in the LA795 cells (FIG. 30B). To better mimic the TME with endogenous H2O2, 100 pM H2O2 was supplemented to the culture medium. The intracellular -OH level was firstly measured by the 2', 7' dichlorofluoresceindiacetate (DCFH- DA), which can be oxidized by reactive oxygen species (ROS) to generate green fluorescence. As shown in FIGS. 30C and 30E, weak green fluorescence was detected in cells treated with LY-Au@Pt@Rh-CM plus laser irradiation, with no detectable fluorescence in control and LY- Au@Pt@Rh-CM treated groups. While, in presence of H2O2 and LY-Au@Pt@Rh-CM, strong green fluorescence was observed in cells either treated with or without laser irradiation,indicating that the enhanced ROS was mainly generated from the H2O2. Considering that DCFH-DA can react with all ROS including H2O2, MitoROSTM OH580 with good selectivity toward -OH than other species (e.g., H2O2 or singlet oxygen) was used to evaluate the -OH generation. In comparison to negligible fluorescence in the untreated or LY-Au@Pt@Rh-CM treated groups, LY-Au@Pt@Rh-CM plus laser irradiation (1 W / cm2, 3 min) yielded some red fluorescence, but still relatively weak (FIG. 30D(d3) vs FIG. 30D(dl), 30D(d2)). However, upon addition of H2O2, much stronger red fluorescence (FIG. 30D(d4), 30D(d5) vs 30D(d3)) was observed in both laser irradiation treated and untreated groups, -11.9 and 17.5 times of the control group (FIG. 30F), respectively, suggesting that the hyperthermia could improve the H2O2 decomposition to generate -OH through the catalytic effect of LY-Au@Pt@Rh-CM nanozyme. For FIGS. 30E-F, Imaged (NIH) was used to analyze these fluorescence images recorded from more than five images for each group. Data are shown as mean ± SD. *P < 0.05, **P <0.01.

[0309] To evaluate the intracellular CAT-like effect of LY-Au@Pt@Rh-CM nanozymes, the LA795 cells were cultured in a hypoxia condition (N2 atmosphere). The intracellular O2 level was measured using a hypoxia dye as an oxygen indicator, which can be quenched to decrease its red fluorescence in the presence of O2. As shown in FIGS. 30G(l)-30G(5), LA795 cells treated with LY-Au@Pt@Rh-CM plus H2O2 either in the absence or presence of laser irradiation displayed the comparable low fluorescence compared to other groups (control, LY- Au@Pt@Rh-CM, and LY- Au@Pt@Rh-CM + 1208 nm laser irradiation) in the hypoxia condition. Semi-quantification of the fluorescence intensity using Imaged revealed that the control group was -10.4 times that of LY-Au@Pt@Rh-CM + H2O2 + laser group (Fig. S16), indicating the CAT-like catalytic capability of cellular LY-Au@Pt@Rh-CM in O2 generation from H2O2. The biocompatibility of LY-Au@Pt@Rh-CM and the in vitro cell killing efficacy to LA795 cells via photothermal -enhanced tumor catalytic therapy were investigated using 3- (4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. Initially, to evaluate the biocompatibility of the prepared nanozyme, the cell viability of human umbilical vein endothelial cells (HUVECs) and LA795 cells incubated with LY-Au@Pt@Rh-CM or Au@Pt@Rh-CM was evaluated. As shown in FIG. 30H and FIG. 50, no obvious cytotoxicity was observed after HUVEC and LA795 cells incubated with various groups, indicating the high biosafety of Au@Pt@Rh-CM and LY-Au@Pt@Rh-CM for normal and tumor cells. However, after addition of H2O2 and NIR-II (1208 nm) laser irradiation, 78.92 % cancer cell killing efficiency was achieved, which is higher than the nanozyme plus H2O2 (36.93 %) or laser (69.52 %) treated groups. Meanwhile, above 90 % cell viability was observed in the H2O2,laser irradiation, or Au@Pt@Rh-CM only treated groups. Clearly, the high cell-killing efficiency is mainly from the synergistic effect of photothermal and catalytic therapy. These MTT results were further confirmed by live / dead staining of LA795 cells under various treatments (FIG. 30J and FIG. 51). Once again, upon 1208 nm laser irradiation addition of exogenous H2O2 to the LA795 cells incubated with LY-Au@Pt@Rh-CM, all dead cells was observed, in contrast to partial and identical cell death in nanozyme plus either H2O2 or laser irradiation groups. The above results demonstrate the high therapeutic efficacy of LY- Au@Pt@Rh-CM-caused photothermal-enhanced tumor nano-catalytic therapy, in which local hyperthermia and highly toxic -OH could synergistically induce the apoptosis of LA795 cells.

[0310] As a classical three-dimensional (3D) tumor cell culture model in vitro, multicellular tumor spheroids have attracted extensive attention as an effective model for biological study due to their closer growth morphology and functions to the real in vivo tumor and better ability to mimic the real situation of drug-tumor interaction. Thus, a multicellular tumor spheroid model was established via coculture of LA795 tumor cells and M2-like macrophages to investigate the in vitro tumor inhibition effect of LY-Au@Pt@Rh-CM (FIG. 31 A). FIGS. 31 A- F show antitumor effects of LY-Au@Pt@Rh-CM nanozymes on an in vitro multicellular 3D tumor spheres model. As shown in FIG. 3 IB, the 3D multicellular tumor spheroids in the control group grew rapidly over time. Compared with the control group at day 13, the volume in the groups of LY, Au@Pt@Rh-CM, LY-Au@Pt@Rh-CM, and Au@Pt@Rh- CM + laser irradiation decreased by 1.47, 1.62, 2.16, and 4.41 times, respectively (FIG. 31C). While, after treatment with LY-Au@Pt@Rh-CM Maser irradiation, most of tumor cells were killed at day 13 and the volume was reduced by 18.92 times compared to those at day 3. These results imply the superiority of LY-Au@Pt@Rh-CM in tumor growth inhibition caused by the synergetic effect of Au@Pt@Rh-CM and LY.

[0311] To investigate the therapeutic mechanism of LY-Au@Pt@Rh-CM, the immunofluorescencestaining was performed on 3D tumor spheroids with different treatments. First of all, the intracellular -OH generation was investigated by using the probe of MitoROSTM OH580 as the indicator, which can react with -OH to produce red fluorescence. As shown in FIGS. 3 ID and FIG. 52, an obvious red fluorescence can be found for LY- Au@Pt@Rh-CM + laser irradiation treated tumor spheroids, indicating the high POD-like activity of LY-Au@Pt@Rh-CM to generate cytotoxic -OH. In addition, the intracellular O2 level was evaluated by detecting the expression of hypoxia-inducible factors- 1 a (HIF- la). FIG.3 IE and FIGS. 53A-B showed the undetectable green fluorescence in the LY-Au@Pt@Rh- CM plus laser group, indicating that the hypoxic conditions of TME were effectively relieved through the high CAT-like activity of LY-Au@Pt@Rh-CM under the laser irradiation. To explore the regulation of the immune microenvironment by LY-Au@Pt@Rh-CM, the distribution of Ml and M2 macrophages was compared after different treatments. As shown in FIG. 3 IF and FIGS. 54A-B, few Ml macrophages were observed in control group. Compared with LY, Au@Pt@Rh-CM, LY-Au@Pt@Rh-CM, or Au@Pt@Rh-CM plus laser groups, a significantly increased ratio of Ml to M2 macrophages was observed for the LY-Au@Pt@Rh- CM plus laser group, demonstrating that LY, Au@Pt@Rh-CM, and laser irradiation synergistically induced the polarization from M2 to Ml macrophage, improving the immunosuppressive microenvironment.In Vivo Anti-Tumor Effect and TME Immunomodulation

[0312] Encouraged by ideal in vitro photothermal-enhanced catalysis-immunomodulation therapeutic outcomes, the in vivo antitumor efficacy of LY-Au@Pt@Rh-CM was assessed on LA795-tumor-bearing mice (FIG. 32A). FIGS. 32A-J show in vivo photothermal -enhanced tumor immunomodulation-catalytic combination therapy of LY-Au@Pt@Rh-CM against LA795 tumor xenografts. Due to the investigation of nano active agents, biodistribution is a consideration for possible tumor therapeutic application. The biodistribution of LY- Au@Pt@Rh-CM nanozyme in tumor-bearing mice was firstly evaluated before the assessment of its therapeutic effect by analyzing the Au, Pt, and Rh content in tumor and major organs (heart, liver, spleen, lung, and kidney) via ICP-MS. As displayed in FIG. 32B and FIGS. 55A- 55C, the LY-Au@Pt@Rh-CM nanozymes were mainly distributed in the liver and spleen due to uptake by the reticuloendothelial system. The relative distributed amounts of LY- Au@Pt@Rh-CM nanozymes in the tumor site were 3.79 %, 7.47 %, 15.53 %, and 11.83 %, respectively, at 2, 6, 12, and 24 h after intravenous injection. Following 3 minutes of irradiation with a 1208 nm laser, the temperature can attain 43.9 °C, which is sufficient to induce apoptosis in tumor cells. All Au, Pt, and Rh contents in the tumors reached their maximum after 12 h injection, indicating that the LY-Au@Pt@Rh-CM nanozymes could effectively accumulate into the tumor site due to the homogenous targeting action from the surface CM and EPR effect.

[0313] Recognizing the high accumulation of LY-Au@Pt@Rh-CM in tumor tissues, efforts were made to utilize such nanozymes for tumor therapy. Tumor-bearing mice were randomly divided into 6 groups (n = 5): 1) control (PBS), 2) LY, 3) Au@Pt@Rh-CM, 4) LY- Au@Pt@Rh-CM, 5) Au@Pt@Rh-CM + 1208 nm laser, 6) LY-Au@Pt@Rh-CM + 1208 nm laser. Mice in groups 5 and 6 were exposed to 1208 nm laser irradiation at the time of 12 hpost-injection of nanozymes based on the findings of tumor tissue accumulation amount (FIG. 32B). As measured, the temperature of tumor sites on mice treated with LY-Au@Pt@Rh-CM dramatically increased to 41.6 °C after 3 min NIR-II laser irradiation (1208 nm, 1 W / cm2) (FIG. 32C), which is sufficient for tumor PTT and improving the nanocatalytic therapy. Since no significant temperature elevation (37.4 °C) was observed in control group (PBS only), the safety of the power of 1 W / cm2 at 1208 nm was confirmed. During the therapeutic process, the tumor volume and body weight of each mouse were measured every two days. As shown in FIG. 32D, tumor growth was suppressed to a certain degree for those mice treated with LY only (immunoregulation therapy), Au@Pt@Rh-CM (catalytic therapy), LY- Au@Pt@Rh-CM (immunoregulation-enhanced catalytic therapy), and Au@Pt@Rh-CM + laser (PTT improved catalytic therapy). As expected, the highest tumor inhibition efficiency was observed with LY- Au@Pt@Rh-CM plus 1208 nm laser irradiation, in which tumors were significantly suppressed after 16 days of treatment. Such superior tumor therapeutic efficacy was further confirmed by the visualized photographs (FIG. 32) and relevant average weights of the excised tumors (FIG. 32F). To better observe cell-level intratumor changes, hematoxylin and eosin (H&E) staining was carried out on resected tumors, showing significant necrosis of tumor cells and substantial reduction of nucleus for the LY-Au@Pt@Rh-CM plus laser group as opposed to other groups (FIG. 32H). The in vivo antitumor outcome confirmed the synergetic effect of photothermal- improved immunoregulation-catalysis therapy enabled by the LY-Au@Pt@Rh-CM nanozyme.

[0314] Along with the therapeutic assessment, the biosafety of LY- Au@Pt@Rh-CM was also evaluated through multiple analyses to maximize the use of animals. As monitored, during the entire treatment, the average body weight of the mice in each group showed no apparent changes, suggesting negligible adverse effects after injection with LY-Au@Pt@Rh-CM (FIG. 32G). As shown in the results of blood biochemical analysis (FIG. 56A-F), the values of liver and kidney function markers, including alanine transaminase (ALT), alkaline phosphatase (ALP), aminotransferase (AST), blood urea nitrogen (BUN), creatinine (CR), and lactate dehydrogenase (LDH), were all within the normal range at the treatment of day 8 and 16. Meanwhile, routine blood analysis of LY-Au@Pt@Rh-CM-treated mice for 16 days also confirmed the maintenance of normal ranges for twelve blood indexes, indicating blood biosafety of LY-Au@Pt@Rh-CM (FIG. 57). In addition, hematoxylin-eosin (H&E) staining of major organs collected from the mice after treatment with LY-Au@Pt@Rh-CM plus laser irradiation displayed no obvious signs of tissue damage or inflammatory injury (FIG. 58), implying unnoticeable potential toxicity of LY-Au@Pt@Rh- CM to major organs. The aboveanalyses therefore suggest good in vivo biocompatibility of this LY-Au@Pt@Rh-CM nanozyme, which is promising for future applications.

[0315] To better understand the therapeutic mechanism of LY-Au@Pt@Rh- CM, immunofluorescence staining of the excised tumors was performed. Initially, ROS generation was investigated by using the probe of dihydroethidium (DHE), which can be oxidized into ethidium bromide by ROS to produce red fluorescence. As shown in FIG. 321 and FIG. 59A- B, strong red fluorescence was observed in tumors treated with LY- Au@Pt@Rh-CM and 1208 nm laser irradiation or LY-Au@Pt@Rh-CM alone, confirming the high level of ROS generation. In addition, the capability of LY-Au@Pt@Rh-CM in relieving tumor hypoxia was evaluated by using hypoxia-inducible factor (HIF)-la (tagged with green fluorescence) as the indirect indicator of hypoxic microenvironment. Notably, LY-Au@Pt@Rh-CM combined with laser irradiation could effectively alleviate the hypoxic conditions of TME, displaying the lowest green fluorescence in contrast to other groups (FIG. 32J and FIG. 60A). Semi quantitative analysis of the fluorescence intensity using Imaged revealed that the control group was ~5.2 times that of the LY- Au@Pt@Rh-CM plus laser irradiation group (FIG. 60B), demonstrating the photothermal -enhanced CAT -like catalytic performance of Au@Pt@Rh- CM in O2 generation.

[0316] FIGS. 33A-33K relate to in vivo immunomodulation in TME caused by LY- Au@Pt@Rh-CM. In order to explore the TME immunoregulation induced by LY- Au@Pt@Rh-CM (FIG. 33 A), the multicolor flow cytometry analysis and immunofluorescence staining were performed on tumor tissues with different treatments. Quantitative analysis by flowcytometry showed the significantly increased presence of Ml-like macrophages (F4- 80+Ly6C+, F4-80+CD86+, and / F4-80(-|-)+ CD80+) and reduced quantities of M2 macrophages (F4-80+ CD206+) in the LY-Au@Pt@Rh-CM + laser treated group (FIG. 33B), which suggested that LY-Au@Pt@Rh-CM can successfully skew TAMs away from the M2 phenotype to a tumor-inhibiting Ml phenotype. As shown in FIG. 33 J and FIGS. 61 A-B, large numbers of Ml-like macrophages with red fluorescence and few M2-like macrophages with green fluorescence were found in the tumor region of the LY-Au@Pt@Rh-CM + laser treated group in contrast to other groups, indicating the occurrence of macrophages polarization had shifted from M2- to Ml-like subsets. As reported, Ml-like macrophages exerted cytotoxic effects are implicated in collaboration with the activation of T cells. Thus, the infiltration of T lymphocytes within the TME was also analyzed (FIG. 33K and 62A-B). In comparison to negligible percentage changes in other groups (LY or Au@Pt@Rh-CM), LY- Au@Pt@Rh- CM and Au@Pt@Rh-CM plus laser groups yielded some increases for both T-helper cells(CD4+) and cytotoxic T lymphocytes (CD8+). However, after the treatment of LY- Au@Pt@Rh-CM plus laser irradiation, these ratios were further increased, indicating its ability to reeducate the immunosuppressive TME. Due to the supportive role of tumor hypoxia in M2- polarized TAMs recruitment and immunosuppression, the improved immunosuppressive TME in the LY-Au@Pt@Rh-CM plus laser irradiation group could be ascribed to the relieved tumor hypoxia induced by Au@Pt@Rh-CM, and was further enhanced by the TGF-P inhibition effect ofLY.Conclusion

[0317] In summary, a TCG-P inhibitor loaded layer-by-layer trimetallic porous nanozyme (LY-Au@Pt@Rh-CM) with hyperthermia-enhanced POD- and CAT-mimic activity for efficient immunomodulation-improved tumor catalytic therapy in the NIR-II window was developed. These porous Au@Pt@Rh nanostructures with large mesopores exhibit a high drug loading and superior catalytic performance to decompose H2O2 into highly toxic -OH and O2 to kill tumor cells and relieve the hypoxic TME, respectively. Meanwhile, with the assistance of homologous tumor CM, TGF-P inhibitor LY loaded Au@Pt@Rh nano-structures preferably accumulate in the tumor site to produce their antitumor effect. Moreover, Au@Pt@Rh-enabled photothermal hyperthermia in the NIR-II region significantly enhanced the POD- and CAT- mimic activities. Both in vitro cellular studies and in vivo animal evaluations confirmed that LY-Au@Pt@Rh-CM can effectively alleviate tumor hypoxia, produce the highly toxic -OH, reprogram the immuno-suppressive TME, and therefore yield increased therapeutic efficiency upon laser irradiation of tumors. Overall, the reported findings provide a new route to synthesize a trimetallic multilayer nanozyme with photo-thermal, drug loading, and catalytic capacity for hyperthermia / immunomodulation-enhanced tumor catalytic therapy. In addition, these special triphasic layer-by-layer nanocrystals may achieve heterogeneous catalytic effects with the possibility of individually tailoring the layers with the different metals for desired catalytic potential. Furthermore, such mesoporous nanostructures with large pore space and multilayers are also promising for co-delivery of multiple drugs in different sites and realizing sequential responsive release.Materials

[0318] Tetrachloroauric acid trihydrate (HAuC14-3H2O), sodium hexa-chlororhodate(III) (Na3RhC16), AA, cholesterol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chloroplatinic acid (H2PtC16) was obtained from Chemical Technology Co., Ltd. (Tianjin, China). l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC) was purchased from AVTPharmaceutical Tech CO., Ltd. (Shanghai, China). LY2157299 was obtained from Shanghai Yuanye Biological Technology Co., Ltd. (Shanghai, China). Tri chloromethane was purchased from Fuchen Chemical Reagent Co., Ltd. (Tianjin, China). H2O2 was obtained from Jiangtian Chemical Technology Co., Ltd. (Tianjin, China). DCFH-DA probe, [Ru(dpp)3]2+C12 probe, and reactive oxygen species test kits were purchased from Biyuntian Biotechnology Co., Ltd. (Shanghai, China). Calcein / PI live-dead test kits were purchased from Beijing solarbio science & technology Co., Ltd. (Beijing, China). All chemicals used were at least of analytical reagent grade and used without further purification.Preparation of Au@Pt@Rh Nanostructures

[0319] Au@Pt@Rh was prepared according to simple and green template methods with some modifications. Firstly, DPPC and cholesterol were dissolved in chloroform at a molar ratio of 55:45, and then the chloroform was removed in vacuum. At room temperature, the obtained lipid film was hydrated with 300 mM AA aqueous solution and sonicated for 20 min. The liposome solution was centrifuged at 13,000 rpm for 15 min at 15 °C, and the supernatant was collected as the template for subsequent synthesis of Au@Pt@Rh nanoparticles.

[0320] Mesoporous Au@Pt@Rh nanostructures were synthesized using the above-obtained liposomes as the soft templates. Typically, 0.15 mL of HAuC14-3H2O solution (5 mM), 0.15 mL of H2PtCl6solution (15 mM), and 0.15 mL of NasRhCL aqueous solution were added into 1 mL of liposome solution in sequence. Then the mixed solution was kept in a water bath at 60 °C for 12 h. During the reaction, color of the solution rapidly changed from milk white into blue gray and slowly into black. The liposome templates and unreacted residue were removed by centrifugation at 10,000 rpm for 15 min and washed with water for 3 times. Obtained Au@Pt@Rh nanoparticles were stored at room temperature for future use.Extraction of LA795 Cancer CM

[0321] A795 lung adenocarcinoma cancer cells grown to 90% were digested with trypsin and centrifuged at 1000 rpm for 5 min. The obtained cell precipitation at the bottom was washed twice with PBS. Then the cells were resuspended with the hypotonic lysate buffer containing the membrane protein extraction kit. After placing them in an ice bath for 30 min, the suspended cell solutions were sonicated with a cell breaker for 5 s. Upon centrifugation at 6000 rpm for 5 min at 4°C, the supernatant was further centrifuged at 13,000 rpm for 10 min to collect the cell membrane fragments. The extracted cell membrane solution was stored at -80°C for subsequent experiments.Preparation of LY-Au@Pt@Rh-CM Nanostructures

[0322] The selected drug LY (0.25 mg) and Au@Pt@Rh nanoparticles (0.1 mg) were dissolved in methanol (2 mL) and stirred at room temperature for 12 h. After that, the methanol was removed by a rotary evaporator. For CM coating, 1 mL of cell membrane solution was put into the above mixture and sonicated for 20 min. The product was centrifuged at 13,000 rpm for 15 min and washed with 20 vol% dimethylsulfoxide-water solution three times to remove the unpacked drug and un-coated CM. The obtained LY-Au@Pt@Rh-CM was preserved in water for further use.

[0323] To determine the loading efficiency of LY in Au@Pt@Rh-CM, the non-trapped LY collected in the supernatant was determined using a multi-detection microplate reader (SpectraMaxi3x, Molecular devices) according to the absorbance at 239 nm. Drug loading efficiency(%) =((weight of loaded LY) / (total weight of nanoparticles)) x 100.Characterization

[0324] TEM images, element distribution mapping, and EDS were obtained with a FEI Talos F200S field emission high resolution TEM. SEM images were obtained by Hitachi S-4800- SEM. The contents of metal ions were measured by ICP-MS (Optima 8300, PE, USA). UV- vis-NIR absorption spectra were obtained from an Analytik Jena Specord 250 plus UV-vis- NIR spectrophotometer. The zeta potential and particle size were determined using the Malvern Zetasizer Nano ZEN3600 instrument. The stability test was examined by mixing 100 pg of LY- Au@Pt@Rh-CM with 1 mL of 1 x PBS. And the size distributions of this nanosystem were measured at 0, 7, and 14 days. FTIR spectra were recorded in a range of 400- 4000 cm'1at a 0.5-4 cm'1resolution with a Bruker Vertex 80 V spectrometer. The XRD spectra were recorded using an X-ray diffractometer at 40 kV and a scan rate of 5° (29) / min (scan range: 30-90°). SDS-PAGE was used to verify the integrity of the CM composition in Au@Pt@Rh-CM. Typically, Au@Pt@Rh, Au@Pt@Rh-CM, and pure CM were separated by electrophoresis in 10 % gel (80 voltage, 1 h) with the loading amount of 40 pg. After the bands were completely separated, they were then stained with coomassie bright blue for 1 h, decolorized with decolorizing solution and photographed.Photothermal Performance of Au@Pt@Rh-CM

[0325] Photothermal Performance of Au@Pt@Rh-CM in the NIR-II Bio- window: Au@Pt@Rh-CM nanoparticles solutions at different concentrations (0, 25, 50, 100, 150, and 200 pg / mL) were irradiated by 1208 nm laser (1 W / cm2). Temperature and images within 360 s were recorded by a thermometer and an infrared thermal imager, respectively.

[0326] The formula for calculating the photothermal conversion coefficient of these nanomaterials is:> (Tmax 'Tsurr) Qor' ~ i(i-io-A1208) where h is the heat transfer coefficient, S is the surface area of the container. Tmax is the maximum steady temperature and Tsurr is the environmental temperature. The laser power for irradiation is 1 W / cm2. QO is heat dissipated from the light absorbed by the solvent and container.LY release from LY-Au@Pt@Rh-CM

[0327] To evaluate the release behavior of LY from nanostructures, LY-Au@Pt@Rh-CM was incubated with PBS buffers at 37 °C under stirring. At the time points of 3, 8, 15, and 30 min, the nanoparticles solution was irradiated with or without 1208 nm laser (1 W / cm2), respectively, and then centrifuged. The absorbance of LY released into the supernatant at the designated time points was detected by the microplate reader and the LY concentration was calculated according to the established standard curve.Pod-like and CAT-like Activity of Au@Pt@Rh-CM

[0328] To test the POD-like effect of the nanoparticles, MB (5 pg / mL), H2O2 (40 mM), and Au@Pt@Rh-CM (100 pg / mL) were dispersed in buffer solutions (1 mL) with different pH values (5.0, 6.5, and 7.4). The absorbance at 665 nm at different time points was recorded by the micro-plate reader. Next, the POD-like activity of Au@Pt@Rh-CM was further assessed by using TMB as the substrate in the presence of H2O2 under different pH buffers (5.0, 6.5, and 7.4). The spectra of oxTMB were recorded through the UV-vis spectrophotometer. For kinetic assay, the experiments were carried out in 100 pL PBS buffer (pH = 6.5) containing 100 pg / mL Au@Pt@Rh, 416 pM TMB, and a series of concentrations of H2O2 range from 0 to 80 mM at the temperature of 25 or 50 °C. To evaluate catalytic efficiency, the Vmax and Km values were calculated according to the following equations:Fo= Vmax[S] / (Km + [S])1 Km 1 1— = - x - 1 -FoVmax [S] Vmax where Vo represents the initial reaction velocity, Vmax represents the maximal reaction velocity, [S] represents the initial concentration of substrates, and Km represents the Michaelis-Menten constant.

[0329] To test the CAT-like effect of the nanoparticles, Au@Pt@Rh-CM and H2O2 with a final concentration of 100 pg / mL and 40 mM, respectively, were added into 10 mL of PBS buffer with various pH (5.0, 6.5, and 7.4). At different reaction times, the oxygen production was monitored by a dissolved oxygen meter. After 15 min, the above mixed solution was centrifuged, the absorbance of remaining H2O2 at 240 nm was recorded using the microplate reader, and the consumed H2O2 amount was evaluated according to the standard curve. In addition, to confirm the generation of O2 catalyzed by Au@Pt@Rh-CM complies with an H2O2 concentration- and time-dependent manner, the produced O2 amount was assessed by the dissolved oxygen meter. In detail, the Au@Pt@Rh- CM (100 pg / mL) was mixed with the various concentrations of H2O2 (0, 5, 10, 20, 40, 60, 80 mM) in the buffer solution with pH 6.5. The O2 generation was monitored at different reaction times.In Vitro Distribution of Au@Pt@Rh-C

[0330] LA795 cells were cultured at 37 °C and 5 % CO2 in 1640 culture medium (Gibco, Grand Island, New York) containing 10 % fetal bovine serum (FBS, Sijiqing, Hangzhou, China) and 1 % antibiotics for 48 h. Then, the cells were seeded into 6-well plates (1 x 106 cells per well) and incubated with Au@Pt@Rh-CM (100 pg / mL) for 4 h. The resulting cells were trypsinized, washed with PBS, and centrifuged to obtain cell pellets. The obtained pellets were fixed with 2.5 % glutaraldehyde, post-fixed with osmium tetroxide, and dehydrated with a series of graded ethanol. Finally, the pellets were embedded in epoxy and cut into thin sections (60 nm) for TEM observation.Detection of Intracellular ROS and -OH Production

[0331] To detect the production of intracellular ROS, LA795 cells seeded in 6-well plates were incubated with 50 pg / mL LY-Au@Pt@Rh-CM for 6 h. After treatment with or without the 1208 laser irradiation (1 W / cm2, 3 min) in the absence or presence of H2O2 (100 pM, pathophysiological concentration of natural tumor environment), the resulting cells were stained with the ROS probe DCFH-DA (10 pM) for 25 min and observed under the confocal microscope. In addition, the intracellular -OH production effect was studied using MitoROSTM OH580 as the indicator of -OH radical under the above similar procedure.Detection of Intracellular O2 Production

[0332] The intracellular production of O2 was investigated using the hypoxia (Red) dye in the ROS-ID kit. As the increase of the intracellular O2 concentration happened, the intensity of red fluorescence of hypoxia dye deceased. Briefly, LA795 cells were incubated with 50 pg / mL LY-Au@Pt@Rh-CM for 6 h in the N2 atmosphere. Then the cells were rinsed with PBS andincubated with hypoxia probe for 25 min. After the nuclei were stained with DAPI, the fluorescence images were obtained by the confocal microscope.In Vitro Antitumor Performance

[0333] The HUVEC and LA795 cells were seeded on a 96-well plate and cultured overnight, and different concentrations of sample solutions (LY, Au@Pt@Rh-CM, LY-Au@Pt@Rh-CM with the concentration of 6.25, 12.5, 25, 50, 100, and 200 pg / mL) were added to each well. The NIR-II laser (1208 nm, 1 W / cm2) duration time is 3 min. After 24 h of culture, the medium was removed and washed three times with PBS. Next, 20 pL of MTT was added to each well and incubated at 37 °C for 4h. Finally, 200 pL of DMSO was added to each well to extract the formazan, and absorbance at 490 nm was recorded by a microplate reader. In addition, the cytotoxicity of cells under other different treatments was measured through the same procedure.

[0334] Live and dead staining using calcein-AM / PI was also performed to investigate the in vitro antitumor activity of LY-Au@Pt@Rh-CM. Briefly, LA795 cells seeded into 6-well plates were treated with LY-Au@Pt@Rh- CM, LY-Au@Pt@Rh-CM plus H2O2 (100 pM), LY - Au@Pt@Rh-CM in the presence of 1208 nm laser irradiation (1 W / cm2, 3 min), LY- Au@Pt@Rh-CM plus H2O2 (100 pM) plus 1208 nm laser irradiation (1 W / cm2, 3 min). Then the cells were stained with calcein-AM / PI and placed under a confocal microscope for viewing. Formation and Detection of Multicellular Tumor Spheres

[0335] The LA795 and raw 264.7 macrophage cells mixed at a ratio of 3: 1 (1.0 x 103 cells in total) were cultured at 37°C and 5 % CO2 to form multicellular 3D tumor spheres. After 3 days, the spheres were respectively treated with PBS, LY, Au@Pt@Rh-CM, Au@Pt@Rh-CM with 1208 nm laser irradiation, and LY-Au@Pt@Rh-CM, LY-Au@Pt@Rh-CM with 1208 nm laser irradiation. The NIR-II laser (1208 nm, 1 W / cm2) duration time is 3 min. After that, half of the old medium was replaced with fresh medium every day, and the diameters of spheres in each group were recorded every 2 days. Tumor spheres were analyzed by immunofluorescence staining and observed with a fluorescence microscope. -OH activity was assessed by using MitoROSTM OH580. The interior O2 level of the 3D tumor spheres was evaluated by using Anti-mouse HIF-la and secondary antibody (Alexa fluor488 conjugated goat anti-mouse IgG antibody). Ml and M2 macrophages were analyzed by using CD80 Rabbit Polyclonal antibody with goat anti-rabbit IGg / TRITC secondary antibody and CD206 Mouse Monoclonal antibody with goat anti-mouse IGg / FITC secondary antibody, respectively.In Vivo Therapeutic Effect of LY-Au@Pt@Rh-CM

[0336] Male Balb / c mice (5-6 weeks old, ~20 g) were purchased from Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China) and maintained under standard housing conditions. All animal experiments were performed in accordance with the guidelines evaluated and approved by the ethics committee of Hebei University of Technology. Subcutaneous LA795 tumors were generated by subcutaneous injection of LA795 cells (2 x 106) suspended in 100 pL of PBS on the back of each male Balb / c mouse. When the tumor volume reached ~ 120 mm3, the mice were randomly divided into 6 groups (n = 5) as follows: (1) PBS, (2) LY, (3) Au@Pt@Rh-CM (1 mg / mL, 100 pL), (4) LY-Au@Pt@Rh-CM (1 mg / mL, 100 pL), (5) Au@Pt@Rh-CM (1 mg / mL, 100 pL) + 1208 nm laser irradiation, (6) LY-Au@Pt@Rh-CM (1 mg / mL, 100 pL) + 1208 nm laser irradiation. The temperature of mice bodies from the PBS and LY- Au@Pt@Rh-CM treated groups were monitored within 3 min by an infrared thermal imager after the tumor site was irradiated with 1208 nm laser (1 W / cm2) at 12 h post-injection of nanostructures. During the therapeutic process, the mice received various treatments via i.v. injection every 2 days. The tumor volume and body weights of the mice were recorded every 2 days up to 16 days. Tumor volume was calculated by the following formula: tumor volume = length x width2 / 2. By the time of sacrificing, the tumors and major organs of mice were collected for H&E and immunofluorescencestaining. Blood was also collected for biochemical and routine blood tests.In Vivo Distribution of LY-Au@Pt@Rh-CM

[0337] The mice were injected with LY-Au@Pt@Rh-CM (1 mg / mL, 100 pL) through the tail vein and killed at different time intervals (2, 6, 12, and 24 h). The tumor and major organs were dissected, weighed, and dissolved in aqua regia. The Au, Pt, and Rh amounts distributed in the tissues were quantitatively analyzed by ICP-MS and calculated as percent injected dose per gram tissue (%ID / g).Immunofluorescence Staining

[0338] After therapy, the mice from different groups were sacrificed, and the tumors were harvested and sliced. To study the expression of HIF-la, thin frozen sections were stained by anti-mouse HIF-la. After incubation with primary antibody for 1 h, the sections were incubated with secondary antibodies (Alexa fluor 488 conjugated goat anti mouse IgG antibody, diluted 1 :200, Jackson Inc.), and then examined with a fluorescence microscope. To evaluate the ROS levels in tumor tissues, DHE (20 mM, Invitrogen) was employed for immunofluorescence staining. In order to evaluate the Ml and M2 macrophages levels in tumor tissues, CD80 rabbit polyclonal antibody with goat anti-rabbit IGg / TRITC secondary antibody and CD206 mousemonoclonal antibody with goat anti-mouse IGg / FITC secondary antibody were used to stain Ml and M2 macrophages, respectively.In Vivo Flowcytometry Assay in Tumor Issue

[0339] After therapy, the tumor tissues of mice from different groups were digested into single cell suspensions and washed twice with PBS. The obtained cells were stained with PE- conjugated anti-CD3, FITC- conjugated anti-CD4, APC-conjugated anti-CD8, FITC- conjugated anti- F4-80, APC-conjugated anti-CD206, PE-conjugated anti-CD80, PE- conjugated anti-CD86, PE-conjugated anti-Ly6C, respectively, and then analyzed by the flowcytometry.Statistical Analysis

[0340] All quantitative results were obtained from at least three samples. Graphic creation and statistical data analysis were performed using an Origin 8.0. Comparisons of two groups were made with an unpaired Student’s t-test. Statistical significance was set at ns: not significant, * p< 0.05, **p < 0.01, ***p < 0.001.

[0341] It will be understood that the embodiments described herein are merely exemplary and that a person skilled in the art may make many variations and modifications without departing from the spirit and scope of the invention. All such variations and modifications are intended to be included within the scope of the invention.

Claims

CLAIMS1. A method for producing a mesoporous nanostructure, comprising the steps of: mixing phospholipids and cholesterol to form a first mixture; dissolving the first mixture in an organic solvent; evaporating the first mixture to remove residual organic solvent and form a lipid film; hydrating the lipid film with a reductant; sonicating the lipid film and the reductant to form unilamellar liposomes; separating the unilamellar liposomes from the lipid film via centrifugation; adding metal precursor solution to the unilamellar liposomes; reacting the metal precursor solution with the unilamellar liposomes to form a product; and removing excess reactants and liposome templates from the product via centrifugation.

2. The method of Claim 1, wherein the organic solvent comprises chloroform.

3. The method of Claim 1, wherein the reductant comprises aqueous ascorbic acid.

4. The method of Claim 3, wherein the ascorbic acid has a concentration of approximately 300 mM.

5. The method of Claim 1, wherein the reductant comprises a compound selected from the group consisting of: sodium citrate, citric acid and sodium borohydride.

6. The method of Claim 1, wherein the reductant facilitates formation of the product during said reacting step.

7. The method of Claim 1, wherein said hydrating step includes partially reducing the liquid film with the reductant.

8. The method of Claim 1, wherein the metal precursor solution comprises a noble metal.

9. The method of Claim 8, wherein the noble metal comprises gold or platinum.

10. The method of Claim 1, wherein the first mixture is a homogenous phospholipid suspension.

11. The method of Claim 1, wherein said evaporating step is performed via a vacuum rotary evaporator.

12. The method of Claim 11, wherein said evaporating step is performed at a temperature in the range of 30 and 70 °C.

13. The method of Claim 1, wherein the reductant is introduced at a concentration ranging from 0.1 M to 0.5 M during said hydrating step.

14. The method of Claim 1, wherein the unilamellar liposomes are substantially uniform.

15. The method of Claim 1, wherein the product is formed with monodispersed nanostructures.

16. The method of Clam 15, wherein the monodispersed nanostructures have diameters in a range from approximately 100 nm to approximately 200 nm.

17. The method of Claim 15, wherein the monodispersed nanostructures comprise outside-in mesopores approximately 20-40 nm in diameter.

18. The method of Claim 1, wherein the metal precursors comprise at least one compound selected from the group consisting of: JLPdCh, HAuCh 3H2O, JLPtCle 3H2O, and NasRhCk19. The method of Claim 18, wherein said adding step is conducted in a dropwise manner.

20. The method of Claim 1, wherein said reacting step is conducted at a temperature ranging from room temperature to approximately 60 °C.

21. The method of Claim 1, wherein the reductant is present during both said hydrating step and said reacting step.

22. The method of Claim 21, wherein said reacting step includes completing reduction of the metal precursor using the reductant.

23. The method of Claim 1, wherein said reaction step is conducted with a ratio of from about 1.36: 15 to about 1.36:2 of liposome concentration relative to metal precursor concentration.

24. The method of Claim 1, wherein the metal precursors include multiple metals.

25. The method of Claim 24, wherein said adding and reacting steps are conducted for each metal of the multiple metals by introducing each metal of the multiple metals in sequence.

26. The method of Claim 25, wherein said reacting step is conducted at a temperature optimized for each metal of the multiple metals.

27. The method of Claim 1, wherein said reacting step includes chelating the metal precursors to form metallic struts, comprising ground zero metals, in interliposomal space of said liposome templates.

28. The method of Claim 27, wherein the ground zero metals are bimetallic.

29. The method of Claim 27, wherein the ground zero metals are trimetallic.

30. The method of Claim 1, further comprising the step of enhancing the product via PEGylation.

31. The method of Claim 1, further comprising the step of functionalizing the product with biological molecules.

32. The method of Claim 31, wherein the product is adapted for drug delivery.

33. A method for preparing a mesoporous nanostructure, comprising the steps of: forming a liposome-based network; growing a mesoporous nanostructure by using the liposome-based network as a template; and removing the liposome-based network, leaving the mesoporous nanostructure.

34. The method of Claim 33, wherein said forming step includes introducing ascorbic acid.

35. The method of Claim 34, wherein the ascorbic acid is present during said growing step.

36. The method of Claim 33, wherein said removing step includes removing excess reactants and unused liposome templates through centrifugation and is conducted following said reacting step.

37. A mesoporous nanostructure, comprising: a gold core; anda shell formed by other noble metals, cooperating with said gold core to form said mesoporous nanostructure, such that said other noble metals occupy outer surfaces of said mesoporous nanostructure.

38. The mesoporous nanostructure of Claim 37, adapted for use as a photocatalytic catalyst.

39. The mesoporous nanostructure of Claim 37, wherein said mesoporous nanostructure forms a metal gradient, such that relative concentrations of said other noble metals and / or gold vary as a function of spatial position.

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