Patent Description:
Bioorthogonal catalysis has enabled a wealth of non-natural chemical transformations in complex living environs, such as cross-coupling and protective groupcleavage reactions for biomolecular labeling and protein/prodrug activation, broadening the understanding and interrogation of biological processes. The implementation of this class of chemistry mainly relies on abiotic transition metals (TMs) including Pd, Au, Ru, Ir and Cu, which are typically categorized into two formulations, metal complexes and metal nanoparticles. Because of their unpredicted toxicity, lack of target ability and limited stability, the application of metal complexes has been largely restricted to bacterial and cellular studies. With the aim to ameliorate the potential toxicity of TMs, researchers have shifted their attention to supported TM nanoparticles, mostly on resin beads, to modulate heterogenous bioorthogonal catalysis both inside and outside cells. By further transplanting resin-supported TMs into zebrafish embryos, orthogonal conversion of exogeneous substrates has been demonstrated, but the further application of such formulated TMs requires elaborate and complicated surgeries. Although progress has been made in improving the possibility of bioorthogonal catalysis in vivo by using injectable TMs, concerns still exist over how to withdraw TMs after treatment, potential metal toxicity, immunogenicity and unexpected catalysis caused by nonspecific deposition, not to mention the requirements for disparate dosage optimization of TMs and substrate molecules. In this line of reasoning, a bioorthogonal catalytic device that can work in a minimally invasive and spatially controlled manner would provide vast versatility for manipulating abiotic chemistry in higher-level living entities.

<CIT> discloses a bioorthogonal deprotection method for preparing heterocyclic compounds by bond cleavage using palladium. The method has general application in the field of biological synthetic chemistry. Compounds, such as prodrugs, which are useful in such methods are also provided. While this provides technical advances, improvements remain desirable.

Disclosed herein are microneedle-based devices (e.g., patches) that are a simple and robust device for catalyzing bioorthogonal chemistry both in vitro and in vivo. Here, a catalytically active microneedle-based patch device was manufactured made of polyvinyl alcohol (PVA) matrix with TiO<NUM> nanosheet-supported palladium nanoparticles (Pd-TNSs) as the nanofillers. The incorporation of the Pd-TNSs greatly enhanced the mechanical performance of the microneedles in the dry glass state, conferring on them sufficient strength to pierce into skin in a minimally invasive manner. Once placed in aqueous environment (e.g., within the skin tissue), the microneedles turned into a swollen hydrogel state, forming microporous structures. This construction has three levels of structural hierarchies, namely the three-dimensional needle array, the open micropores within in each needle matrix and the highly exposed Pd-TNSs surface in the network, which facilitate the diffusion of caged molecules to contact with the Pd nanoparticles and thereby promoted their activation. Remarkably, by using a mouse melanoma model, local intratumor activation of a systemically administered prodrug, N-(allyloxycarbonyl)doxorubicin, was demonstrated, which not only enabled increasing of dosing but also limited the side effects towards organs and tissues far away. Of note, the rich hydrogen bonds in the nanocomposites conferred the microneedle tips enough mechanical toughness in the hydrate state and made it easy to remove the whole patch, without leaving potentially hazardous transition metals within the body or inducing inflammation.

The invention provides a patch for in vivo biorthogonal catalysis applications in mammalian tissue includes a base or substrate having a plurality of microneedles extending away from the surface of the base, wherein the plurality of microneedles are formed from a polyvinyl alcohol (PVA) matrix with TiO<NUM> nanosheet-supported palladium nanoparticles (Pd-TNSs) dispersed therein. To use the patch, the patch is placed on living tissue of mammal such that the plurality of microneedles penetrate the tissue. This may be, for example, skin tissue although the patch may be applied to other tissue types. A prodrug that is administered to the mammal is then catalyzed by the Pd-TNSs in the patch device into a therapeutic agent or drug. In one embodiment, the prodrug comprises N-(allyloxycarbonyl)doxorubicin (allocDOX) and the produced therapeutic agent or drug is doxorubicin (DOX). Further developments are according to dependent claims <NUM>-<NUM>.

A therapeutic system according to claim <NUM> is also provided that includes the microneedle patch and the prodrug which is delivered to the mammalian subject. Further developments are according to dependent claims <NUM>-<NUM>.

While the prodrug used in the experiments was N-(allyloxycarbonyl)doxorubicin (alloc-DOX), it should be appreciated that other allyloxycarbonyl-modified prodrugs may also be used in a similar manner.

<FIG> illustrates plan view of a patch <NUM> (also referred to herein as PT-MNs <NUM>) that may be used for in vivo biorthogonal catalysis applications in mammalian tissue <NUM> (<FIG>). The patch <NUM> includes a base or substrate <NUM> that includes a plurality of microneedles <NUM> that extend or project from the substrate <NUM>. The patch <NUM> may in some embodiments be partly or entirely biodegradable. However, in other preferred embodiments, the patch <NUM> is not made to be biodegradable and is instead removed from the site of application on the tissue <NUM>. Removal of the patch <NUM> ensures that any potentially hazardous or potentially toxic transition metals in the patch <NUM> are not present at the site of application. The term biodegradable in the context of a biodegradable patch <NUM> refers to the base or substrate <NUM> and/or the microneedles <NUM> being formed from a material that is biodegradable. The plurality of microneedles <NUM> generally extend or project in a perpendicular direction from a surface of the base or substrate <NUM>. The plurality of microneedles <NUM> may be arranged in a regular repeating array as illustrated in <FIG> or, alternatively, they may be arranged in a random pattern or array of microneedles <NUM>. In one embodiment, the plurality of microneedles <NUM> that are formed on the base or substrate <NUM> may have substantially similar shapes and sizes. However, in other embodiments, the plurality of microneedles <NUM> may have different shapes and/or sizes. For example, the perimeter region of the array or field of microneedles <NUM> that extend from the base or substrate <NUM> may be longer or have different shapes than those in the central region of the patch <NUM> to better secure the patch <NUM> to site of application.

In one particular embodiment, the microneedles <NUM>, as their name implies, have a needle-like shape. For example, the microneedles <NUM> may include a sharpened tip <NUM> (seen in <FIG>) that aid in penetrating the epidermal layer of the skin tissue <NUM> (seen in <FIG>), although the tip <NUM> may not perfectly sharp. For example, the tip <NUM> may have small radius (e.g., around <NUM>) but still provide enough sharpness for tissue penetration. The patch <NUM> may also be used with other types of tissue <NUM> beyond skin. This may include other organ tissues <NUM> beyond skin. The tissue <NUM> may be healthy in some embodiments while in other embodiments the tissue <NUM> may be diseased. The length (L) of the microneedles <NUM> may vary although typically the microneedles <NUM> extend less than about <NUM> from the base or substrate <NUM> (<FIG>). A typical length of the microneedles <NUM> is about <NUM> to about <NUM>,<NUM>, although the dimensions may extend outside this range. In experiments described herein, the height of the microneedles <NUM> was <NUM>,<NUM>. The base <NUM> of the microneedle <NUM> is wider than the tip <NUM>. Typically, the base <NUM> of the microneedle <NUM> may have a diameter or width (W) that is less than about <NUM> (e.g., <NUM> base and a height of around <NUM>,<NUM>) (<FIG>). The particular dimensions and shape(s) of the microneedles <NUM> are controlled by the particular construction of the mold that is used to form the patch <NUM>, which is described more in detail below. The spacing or pitch of the microneedles <NUM> may also vary. In the experiments described herein, an array of <NUM> × <NUM> microneedles <NUM> were formed with <NUM> center-to-center spacing. It should be appreciated that a wide variety of arrangements of microneedles <NUM> and spacing may be used in connection with the patches <NUM> described herein.

Still referring to <FIG>, the base or substrate <NUM> which holds the microneedles <NUM> may be optionally bonded or otherwise adhered to a backing material <NUM> (e.g., through the use of an adhesive, chemical linking, or the like). The backing material <NUM> may be made from a woven fabric, a plastic material such as polyvinylchloride, polyethylene, or polyurethane, or latex. The backing material <NUM> may be flexible so that the patch <NUM>, when applied, can conformally cover the tissue <NUM> (seen in <FIG>). Optionally, the backing material <NUM> may include an adhesive material <NUM> that covers all or a portion of the tissuefacing surface of the backing material <NUM>. For example, adhesive may be formed on the backing material <NUM> around the periphery of the base or substrate <NUM> or the backing material <NUM> so that the base or substrate <NUM> may be secured in place to the surface of the tissue <NUM>. The adhesive material <NUM> aids in securing the patch <NUM> to the tissue <NUM>. The adhesive material <NUM> may include resins (e.g., vinyl resins), acrylates such as methacrylates epoxy diacrylates. Of course, in other embodiments, the patch <NUM> may not have a backing material <NUM>.

The base or substrate <NUM> and the microneedles <NUM> may be relatively rigid in a substantially dry state. Because of this, in one alternative embodiment, multiple sub-patches may be integrated into the backing material <NUM> to make the final patch <NUM>. This may be useful for large coverage areas or curved surfaces that may pose a risk of breakage to the base or substrate <NUM>. The various sub-patches, while generally rigid, are still able to conform to the surface of the tissue <NUM> (e.g., <FIG>) due the flexible backing material <NUM> which enables bending of the overall patch <NUM>. Because individual sub-patches are smaller in size these do not experience significant bending stresses which would otherwise cause a larger, more rigid structure to break in response to bending and/or manipulation. Bending or flexing can occur within the backing material <NUM> between the locations of where the sub-patches are located (e.g., between the rows and columns of sub-patches). While the patch <NUM> may be in a substantially dry state upon application to tissue <NUM>, the patch <NUM> including the microneedles <NUM> will become hydrated and swell in response to exposure to the aqueous fluid of the tissue <NUM>. In addition, by being in a dry state at the time of application, the microneedles <NUM> can more easily penetrate into the tissue <NUM>.

The microneedles <NUM> may have a number of different shapes and configurations including, for example, a pyramid, cone, cylindrical, tapered tip, canonical, square base, pentagonal-base canonical tip, side-open single lumen, double lumen, and side-open double lumen. The plurality of microneedles <NUM> swell upon breaching or penetrating the biological barrier and absorbing fluid from the surrounding tissue <NUM>. The microneedles <NUM> may swell from about <NUM>% to about <NUM>% (wt. The microneedles <NUM> swell and, in one embodiment, form a flexible material. In some embodiments, the microneedles <NUM> are also biodegradable and dissolve over time. In one embodiment, with reference to <FIG>, the plurality of microneedles <NUM> are formed from polyvinyl alcohol (PVA) matrix with TiO<NUM> nanosheet-supported palladium nanoparticles (Pd-TNSs) <NUM> used as a nanofillers. With reference to <FIG>, the nanofiller <NUM>, in one preferred embodiment, includes thin TiO<NUM> nanosheets (TNSs) <NUM> that have one or more surfaces populated with metallic nanoparticles <NUM>. <FIG> illustrates X-ray diffraction (XRD) pattern of the TiO<NUM> nanosheets. The intense reflection at 2θ = <NUM>° was ascribed to the lamellar structure with layer spacing of ~<NUM>. <FIG> illustrates UV-Vis absorption spectra of the obtained TiO<NUM>(B) nanosheets. The strong absorption of TiO<NUM>(B) nanosheets in the UV range were then leveraged for photodepositing palladium nanoparticles <NUM>.

The metallic nanoparticles <NUM> function as a catalyst. While palladium (Pd) nanoparticles <NUM> are the focus of one particular embodiment, it should be understood that other metal-based nanoparticles <NUM> may also be used. These include, other transition metal catalysis and alloys and/or metal complexes of the same (e.g., gold (Au), ruthenium (Ru), iridium (Ir), or copper (Cu)). The base or substrate <NUM> of the patch may also be made from the same or different materials as the microneedles <NUM>. The PVA serves as a matrix material and entraps Pd-TNSs <NUM> within the pore walls of the matrix material.

To use the patch <NUM>, in one embodiment and with reference to <FIG>, the patch <NUM> is applied to tissue <NUM> by applying gentle force to insert the microneedles <NUM> into the tissue <NUM>. The microneedles <NUM> penetrate the tissue <NUM> upon application of the patch <NUM>. The patch <NUM> may be applied to the target tissue <NUM> to be treated. This may include diseased tissue <NUM> in one embodiment (e.g., cancerous tissue as one example). In another embodiment, the patch is applied to healthy tissue <NUM>. The patch <NUM> may also be applied to a location that is remote from the tissue <NUM> to be treated. For example, the patch <NUM> may be applied to skin tissue <NUM> even though the desired target tissue <NUM> to be treated by the therapeutic agent or drug may is not the skin tissue <NUM>. An optional adhesive may also be used to secure the patch <NUM> the tissue <NUM>. The microneedles <NUM> are then exposed to a prodrug by systemic and/or local delivery (e.g., injection such as intraperitoneally). The patch <NUM> is preferably delivered prior to administration of the prodrug and some elapsed time may be needed (e.g., tens of minutes to an hour or more) to allow for swelling of the microneedles <NUM>. The prodrug that is administered to the mammal is then catalyzed by the Pd-TNSs <NUM> in the patch <NUM> into a therapeutic agent or drug which then leaves the microneedles <NUM> into the surrounding tissue <NUM>. This is schematically illustrated in <FIG>. In one embodiment, the prodrug is N-(allyloxycarbonyl)doxorubicin (alloc-DOX), it should be appreciated that other allyloxycarbonyl-modified prodrugs may also be used in a similar manner. In addition, other prodrugs with good water solubility could be delivered, for example, intravenously. However, the prodrug alloc-DOX is too hydrophobic and was thus injected intraperitoneally.

Synthesis of Pd-TiO<NUM> Nanofillers. Ultrathin TiO<NUM> nanosheets (TNSs) <NUM> were prepared by solvothermal hydrolysis of TiCl<NUM> in ethylene glycol and utilized as the support for growth of Pd catalysts (<FIG>). <FIG> shows a representative transmission electron microscopy (TEM) image of the obtained TNSs <NUM> and they were wrinkled due to the surface tension, which is often observed for two-dimensional materials like graphene. As indicated by infrared spectroscopy and thermalgravimetric analysis, the TNSs <NUM> were covered with ethylene glycol (~<NUM> w. By a photochemical process, Pd nanoparticles <NUM> with average size ~<NUM> were deposited over the surface of the TNSs <NUM> (Pd-TNSs, <FIG>), where the distribution of Pd, Ti and O in the nanocomposites was revealed by elemental mapping analysis (<FIG>, <FIG>) and the Pd content was determined to be about <NUM> w. % by inductively coupled plasma mass spectrometry (ICP-MS). Notably, no obvious change was observed in either the morphology of the nanosheets <NUM> or the amount of surface-attached ethylene glycol (<FIG>) after the deposition of Pd. Such two-dimensional structures with high exposed Pd surface would be favorable for mass transfer during catalysis. Furthermore, the chemical state and short-range structure of Pd in the Pd-TNSs hybrid nanofiller <NUM> was studied by X-ray absorption near-edge structure (XAENS) and extended X-ray absorption fine structure spectra (EXAFS) at Pd K-edge, respectively. Compared with PdO, the XANES of Pd-TNSs showed closer edge position to Pd foil, indicating dominant form of metallic Pd (<FIG>). This is further confirmed by the existence of strong Pd-Pd bonding in Pd-TNSs, as-revealed by Fourier transformed-EXAFS (<FIG>). However, different from the Pd foil, Pd-O bonding in the region of <NUM> to <NUM>Å was also observed in Pd-TNSs <NUM>, which suggested that the photo-reduced Pd nanoparticles <NUM> anchored onto the TNSs support mainly via Pd-O bonds.

The performance of Pd-TNSs <NUM> for mediating bioorthogonal catalysis was examined using bis-N,N'-allyloxycarbonyl-caged rhodamine <NUM> (alloc-RhB <NUM>, <FIG>) as the substrate in an isotonic media, PBS buffer (pH <NUM>). As shown in <FIG> upon rhodamine <NUM> (RhB <NUM>) was released by catalytic cleavage of the caging group, green fluorescence gradually increased for solutions containing both Pd-and substrate. The conversion was determined to be <NUM>% by using the standard curve (<FIG>). By contrast, no generation of fluorescence was detected for control groups with either catalysts or substrate omitted over the same time. The catalytic nature of the reaction was confirmed by using sub-stoichiometric amounts of Pd, although a longer time, <NUM>, was required to reach the similar conversion efficiency (<FIG>). Also, to demonstrate that the conversion was directed by heterogenous catalysis, the Pd-TNS <NUM> were dispersed in PBS buffer for <NUM> first and then the suspension was centrifugated. Afterwards, the upper supernatant was mixed with alloc-RhB <NUM> and incubated for <NUM>, which failed to give detectable fluorescent RhB <NUM> (<FIG>). Fabrication of Catalytically Active Microneedle Patches. To make a device for mediating bioorthogonal catalysis, microneedle array patches <NUM> made of PVA incorporated with Pd-TNS nanofillers <NUM> (~<NUM> wt%) were prepared. Generally, the wherein the Pd-TNS nanofillers <NUM> (i.e., TiO<NUM> nanosheet-supported metallic nanoparticles) comprise between about <NUM> wt% and about <NUM> wt% of the patch <NUM>. The microneedle-based patch <NUM> with Pd-TNS nanofillers <NUM> is also referred to herein as PT-MNs <NUM>. PVA was chosen as the microneedle matrix because of its unique character of forming microcrystalline regions as physical crosslinkers, non-toxic nature and good compatibility with metal oxide particles. The microneedles <NUM> were arranged in <NUM>×<NUM> array (<FIG>), and each one was conical in shape with a tip radius of <NUM>, a height of <NUM>, a base diameter of <NUM> and a center-to-center space of <NUM> (<FIG>). The overall incorporation of Pd-TNS <NUM> inside the microneedle <NUM> was revealed by time-of-flight secondary ion mass spectrometry (<FIG>). Moreover, SEM image of the longitudinal section of one microneedle <NUM> showed that the Pd-TNS <NUM> were homogeneously dispersed inside the PVA matrix (<FIG>). Measurement of the fracture force of the microneedles <NUM> gave a value of <NUM> N/needle, which was about <NUM>-fold higher than that of pure PVA microneedles (<FIG>). This increase could be attributed to the good dispersion of Pd-TNS <NUM> inside the matrix (<FIG>) and the interfacial interactions between TNS <NUM> and the hydroxyl groups on PVA backbone (<FIG>), which reinforced the strength of PVA composites. Importantly, such enhanced strength facilitates skin insertion.

To study the catalytic efficacy of this patch <NUM>, the microneedles <NUM> were immersed in a homemade chamber <NUM> as best illustrated in <FIG> (also seen in <FIG>) filled with alloc-RhB <NUM>-contained PBS buffer solution (FIGS. Like the case of free Pd-TNS studied above, RhB <NUM> were gradually released with the assistance of PT-MNs <NUM>, whereas no obvious fluorescence was detected for control groups utilizing blank PVA MNs (<FIG>-iii). Meanwhile, the swelling behavior of the microneedles <NUM> were studied by an optical microscope, which revealed that the length and width of the microneedles <NUM> increased quickly after immersed within water and reached plateaus within <NUM> (<FIG>). After further lyophilizing the swelled microneedles <NUM>, three-dimensional network with pores ranging from several to tens of microns were formed both on the surface and inside the needle matrix with Pd-TNSs <NUM> entrapped within the pore walls (<FIG>). Such hierarchical porous structure is beneficial for facilitating the transport of substrate/product molecules to contact with/leave the catalytic sites and thereby promoting their conversion. At the same time, the Pd content in the aqueous part was measured by ICP-MS, where about <NUM> w. % of Pd was leaked during the whole reaction time, suggesting the stability of the patch <NUM> (<FIG>). Such stability could be due to the interaction between the Pd and TNSs support and the interaction between TNS <NUM> and PVA, which together held the Pd catalyst inside the PVA matrix. This conclusion is further supported by two additional experiments. One is to remove the PT-MNs <NUM> in the midterm, where no further increase in fluorescence intensity was detected (<FIG>-ii). The other one showed that about <NUM> w. % of Pd was released over the same soaking time when nonsupported free Pd nanoparticles (<FIG>) were used as the putative nanofillers (<FIG>).

PT-MNs mediated bioorthogonal catalysis in vitro. Having characterized the catalytic activity of PT-MNs <NUM> in mediating profluorophore activation in solution, their potency was studied in mediating bioorthogonal catalysis in cellular culture. B16F10 cells were incubated in culture medium containing <NUM> alloc-RhB <NUM>, within which the microneedles <NUM> of PT-MNs <NUM> were immersed in the extracellular space. The cells were then analyzed by flow cytometry and confocal microscope at different time points. As indicated by flow cytometry (<FIG>), the fluorescence of the whole cell population increased in a time-dependent fashion. While for control groups in the absence of PT-MNs <NUM> or alloc-RhB <NUM>, no fluorescence evolved over all the time (<FIG>). Consistent with the flow cytometry results, confocal laser microscopy allowed to visualize the gradually increased green fluorescence inside the cells treated with PT-MNs <NUM> and alloc-RhB <NUM> combination (<FIG>), but no distinct fluorescence was seen for the control groups (<FIG>). These results demonstrated that PT-MNs <NUM> could efficiently implement bioorthogonal catalysis in vitro to trigger the uncage of alloc-RhB <NUM>. The fluorophores produced in the extracellular medium promoted their diffusion into the entire cell population. In addition, importantly, similar catalytic trend could be replicated with HepG2 (<FIG>) and 4T1 cells (<FIG>), suggesting the generality of the device to mediate bioorthogonal catalysis in different extracellular environments.

In vitro prodrug activation by PT-MNs. Next, prodrug activation by PT-MNs <NUM> for cancer therapy was investigated. With the aim of confining cytotoxicity to the tumor site while diminishing damage toward normal tissues <NUM> during treatment, various bioliable prodrugs have been developed by leveraging the metabolic or physiological aberrations within cancerous tissues. Yet, their efficiency is often hampered by the heterogeneity nature of tumors. In this regard, bioorthogonal catalysis would provide an alternative promising strategy for spatial and temporal activation of prodrugs. For benchmarking, N-(allyloxycarbonyl)doxorubicin (alloc-DOX) was synthesized as a bioorthogonal prodrug (<FIG>). Doxorubicin (DOX) is therapeutic agent/drug that is clinically used for cancer treatment which acts by binding DNA and eliciting enzyme-mediated strand breakage. Still, its full deployment is limited by side effects such as cardiotoxicity, myelosuppression, nausea, and vomiting. By caging the primary amine with allylcarbamate group, alloc-DOX (Kalloc-DOX= <NUM>×<NUM><NUM> M-<NUM>) showed a significantly lower binding affinity toward calf thymus DNA than DOX (KDOX= <NUM>×<NUM><NUM> M-<NUM>), as revealed by fluorescence titration method (<FIG> and <FIG>). This weaker DNA binding ability of the alloc-DOX could be due to that the reduction in positive charge of the daunosamine moiety reduced the stability of DNA-drug intercalating complex. Meanwhile, the decaging of alloc-DOX catalyzed by Pd-TNSs <NUM> in solution was proved by high performance liquid chromatography (HPLC) analysis (<FIG>). Such masking strategy would afford a less toxic prodrug while allow activation by bioorthogonal catalysis.

Next, a cell viability assay using B16F10 cells was carried out to study the toxicity profiles of alloc-DOX, DOX and alloc-DOX/PT-MNs <NUM> combination at different concentrations from <NUM> to <NUM>. Compared to parent DOX which showed elevated toxicity as its concentration increased, alloc-DOX displayed much lower antiproliferative activity (<FIG>). It was calculated that alloc-DOX (IC<NUM> = <NUM>) was nearly <NUM>-times less toxic than DOX (IC<NUM> = <NUM>). In contrast, the introduction of PT-MNs <NUM> into the culture medium containing alloc-DOX led to a distinct decrease in cell viability at each concentration with an IC<NUM> of <NUM>, which was comparable to the trend manifested by DOX. In parallel, similar results were observed when the same experiments were performed with other cell lines, such as HepG2 (<FIG>) and 4T1 cells (<FIG>).

Analysis of the culture medium immersed with PT-MNs <NUM> by HPLC verified the extracellular generation of DOX (<FIG>). Furthermore, by dually staining the cells with Annexin V-APC (allophycocyanin) and SYTOX Green and analyzing with flow cytometry (<FIG>), a high proportion (<NUM>%) of the cell population treated with alloc-DOX/PT-MNs <NUM> combination were Annexin V-APC positive, similar to the group treated with DOX (<NUM>%). Meanwhile, no significant apoptotic cell death was observed in the control groups treated with alloc-DOX or PT-MNs <NUM> alone. Additionally, different from control cells which grow exuberantly, cells in the groups treated with alloc-DOX/PT-MNs combination or DOX showed extensive apoptotic DNA fragmentation, as revealed by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay (<FIG>). All these results agree well with the features of apoptosis triggered by DOX, corroborating that the cytotoxicity induced by the alloc-DOX/PT-MNs <NUM> combination was due to the bioorthogonal generation of DOX in cell culture. Moreover, it is worth mentioning that the PT-MNs <NUM> at zero prodrug concentration showed no toxicity towards the cells after different time exposure (<FIG>) and negligible leaked Pd was detected at the end (<FIG>), which could be attributed to the stability of the patch <NUM> as discussed above.

In vivo prodrug activation by PT-MNs for cancer therapy. To evaluate the in vivo tumor inhibition performance by the bioorthogonal PT-MNs/alloc-DOX combination, an anticancer study was undertaken using a B16F10 mouse melanoma model. First, the ability of PT-MNs <NUM> to penetrate skin tissue <NUM> was evidenced by pressing the patch <NUM> with a gentle force against the skin <NUM> over the melanoma tumor (<FIG>). It could be seen that the needle <NUM> matrix swelled into three-dimensional porous networks after removing from mice (<FIG>), such changes in structure could help to alleviate mass transport barriers during the bioorthogonal catalysis. Meanwhile, to decide which the dose of alloc-DOX could be given for treatment, the in vivo toxicity of alloc-DOX was determined by measuring the weight changes of mice and compared with those of mice treated with DOX (<FIG>). Intraperitoneal (i. ) injection of alloc-DOX up to <NUM>/kg every three (<NUM>) days led to weight loss of ~<NUM>% meanwhile serious weight loss and death were caused by DOX when its dose was over <NUM>/kg, suggesting the low toxicity of the prodrug in vivo. Based on these data, <NUM>/kg alloc-DOX and <NUM>/kg DOX were applied in the following antitumor studies.

Next, B16F10 tumor-bearing mice were randomly divided into five groups and treated with alloc-DOX/PT-MNs <NUM> combination, DOX, alloc-DOX, PT-MNs <NUM> (without prodrug) and PBS, respectively, once every <NUM> days. DOX, alloc-DOX and PBS were administered by i. Before starting injection of prodrugs, PT-MNs <NUM> were inserted into the tumor site from the surface skin and fixed there for one (<NUM>) hour to allow the needles <NUM> to swell thoroughly. Bioluminescence imaging and tumor volumes were recorded to visualize tumor growth and evaluate the therapy efficiency (<FIG>). As shown in <FIG>, like the control group treated with PBS, no delayed tumor growth was observed for groups treated with alloc-DOX or PT-MNs <NUM> alone, indicating that neither the prodrug nor the catalytic alone had antitumor effect. Moderate inhibitory outcome was observed in the DOX-treated group, but the tumors grew quickly after DOX administration was stopped. By significant contrast, the alloc-DOX/PT-MNs <NUM> combination suppressed tumor growth much better, leading to the smallest tumors in mice after four (<NUM>) times treatment. In addition, no obvious body weight fluctuation was observed for the mice in all groups (<FIG>), indicating the low side effects of the prodrug, PT-MNs <NUM> device and their combination. Importantly, the TUNEL assay verified apoptotic signals only in the groups treated with alloc-DOX/PT-MNs <NUM> and DOX, implying similar tumor cell killing mechanism (<FIG>).

To gain further insights into these treatment results, the concentrations of drug/prodrug in various tissues, including tumor, plasma, and other major organs, versus time after different treatments were analyzed. As shown in <FIG>, no DOX was detected in all tissues in the group treated with alloc-DOX alone, indicating the stability of the prodrug against unexpected activation by high-level biological metabolic processes. In comparison with the group treated with DOX, there was more alloc-DOX than DOX in all tissues, which was due to that the dose of prodrug was allowed to be given higher. As for the alloc-DOX/PT-MNs <NUM> combination-treated group, the profiles of alloc-DOX concentration in plasma and other normal organs were similar to those in the group treated with alloc-DOX alone. However, the generation of DOX within the tumor tissues from alloc-DOX in the presence of PT-MNs <NUM> was validated, and, notably, the concentration of DOX was much higher at <NUM> and <NUM> post i. injection than that of DOX-treated group. Also, importantly, there was no obvious leakback of the generated DOX from tumor site to the blood stream or other major organs. This might be attributed to the three-dimensional configuration of PT-MNs <NUM> that facilitated the uniform diffusion of the generated DOX within tumors and thereby promote their uptake by cancer cells. The small amount of DOX measured in some tissues might originate from the DOX generated by the part of microneedles <NUM> that stride between the tumor area and the skin. Collectively, it can be concluded that the accumulation of alloc-DOX inside tumors and its locoregional activation by the bioorthogonal catalytic patch <NUM> restored the drug's pharmacological property to combat tumors from within while minimizing side toxicity towards distant organs and tissues.

Lastly, but importantly, the PT-MNs <NUM> could be withdrawn without notable damage in the hydrated state after treatment (<FIG>), which could be attributed that the hydrogen bonds between PVA chains and the interfacial interaction between TNS <NUM> and PVA conferred the swollen PT-MNs <NUM> with enough mechanical strength. Analysis of the Pd content in the PT-MNs-inserted tumor tissue part, plasma, or other major organs showed no differences when compared with the blank control groups, suggesting that the stability of the patch <NUM> did not allow catalyst leakage in living systems (<FIG>). Such complete removal of bioorthogonal catalysts in the form microneedle devices would relieve the concern of transition metal toxicity. Moreover, the temporal microholes gradually resealed over <NUM> after removing the microneedles <NUM> (<FIG>), which would reduce the chance of infection there. Hematoxylin and eosin (H&E) staining of the major organs revealed that no noticeable damage or inflammation for any group (<FIG>). And, the levels of factors, such as TNF-α, IFN-y and IL-<NUM>, did not differ from those in the blank control group injected with PBS, indicating no inflammation was induced (<FIG>).

A microneedle-based catalytic patch <NUM> device and its application are disclosed for mediating bioorthogonal transformation of biologically innocuous substances into active therapeutic agents or drugs in vitro and in vivo. The patch <NUM> was built upon a polyvinyl alcohol matrix integrated with palladium nanoparticle-deposited TiO<NUM> nanosheets <NUM> as the nanofillers <NUM>, where the favorable interfacial interactions give the microneedles <NUM> high mechanical strength in a dry state for skin penetration and good stability in hydrated state against unexpected transition metal leakage. As such, this bioorthogonal patch <NUM> device showed high stability, good biocompatibility, easy removability and could effectively convert inert substrates into their parental states in aqueous solution, extracellular space and a selected area of tissue. The patch <NUM> enabled activation of a caged anticancer drug at tumor site and restoration of its pharmacological properties in a spatiotemporally controlled manner, by which off-targeted activation or dose-dependent side effects towards organs or tissues in distance were mitigated. This paves the way for locally interrogating the biological processes of a defined area of interest in high-level livings with bioorthogonal chemistry.

The development of readily implantable and removable microneedle-based patch <NUM> supports the transition towards safer bioorthogonal catalytic chemistry without metal deposition or causing inflammation. Either metal complexes or particulate metals still faces challenges of noncontrolled metal release, non-specific absorption during circulation, uncertain metal toxicity or clearance by the biological systems, which not only affects their own effectiveness but also can trigger off-targeted prodrug activation. Also, additional labor is needed to optimize the time lag between the sequential administration of catalysts and prodrugs, study the biological distribution of catalysts, and surgically transplant and/or withdraw the catalysts at the end. All these issues imply that achieving transition metalcatalyzed conversions of exogenous substrates within living systems is challenging. Conversely, by integrating microneedle array patches <NUM> with biorthogonal functionality, microneedle patches <NUM> are designed for extremely localized bioorthogonal chemistry to a specific area of tissue, further strengthening the orthogonality of abiotic chemistry. Moreover, such confined nature only requires minimally invasive transdermal application, the operation of which can be conducted with simple training. Along with the being expanded development of microneedles and bioorthogonal concept, it can be naturally envisioned that the device or patch <NUM> is modular and scalable, where the transition metal catalysts, the mediated chemical reactions (prodrugs), and the microneedle matrix and array patterns can be flexibly modulated and optimized for treating a variety of diseases, not only associated with skin, but also for other organs and tissue types.

Claim 1:
A patch (<NUM>) for in vivo biorthogonal catalysis applications in mammalian tissue comprising:
a base or substrate (<NUM>) having a plurality of microneedles (<NUM>) extending away from the surface of the base (<NUM>), wherein the plurality of microneedles (<NUM>) comprise a polyvinyl alcohol (PVA) matrix having TiO<NUM> nanosheets (<NUM>) populated with metallic nanoparticles (<NUM>) dispersed therein.