SELF-THERAPEUTIC NANOPARTICLE FOR ENHANCED TOPICAL DELIVERY TO SKIN KERATINOCYTES AND TREATING SKIN INFLAMMATION
20230048258 · 2023-02-16
Inventors
Cpc classification
A61K47/60
HUMAN NECESSITIES
A61K9/0014
HUMAN NECESSITIES
International classification
A61K47/69
HUMAN NECESSITIES
A61K47/60
HUMAN NECESSITIES
Abstract
A composition of an anti-psoriatic drug and methods of applying the anti-psoriatic drug for transdermal delivery of nanoparticles and entry into skin cells are provided. The composition of the anti-psoriatic drug includes a core having at least one gold nanoparticle, a shell of polyethylene glycol (PEG) strands conjugated to the core, and a plurality of alkyl groups conjugated to the shell of PEG strands. Moreover, a chain length of the plurality of alkyl groups, chain loading of the plurality of alkyl groups, or a diameter of the core is configured to optimize a distribution of the composition in the skin cells. The distribution may include skin permeability or an entry into keratinocytes. Further, methods of modulating effectiveness of the anti-psoriatic drug for inhibiting development of a psoriasis phenotype or for treatment of the psoriasis phenotype are provided.
Claims
1. A composition for transdermal delivery of nanoparticles and entry into skin cells, the composition comprising: a core comprising at least one gold nanoparticle; a shell comprising a plurality of polyethylene glycol (PEG) strands conjugated to the core; and a plurality of alkyl functional groups conjugated to the shell of PEG strands.
2. The composition of claim 1, wherein chain lengths of the plurality of alkyl groups are configured to be in a range of 1-24 carbons.
3. The composition of claim 1, wherein chain loading of the plurality of alkyl groups is configured to be in a range of 0 mol %-50 mol %.
4. The composition of claim 3, wherein the chain loading of the plurality of alkyl groups is configured to be about 30 mol %.
5. The composition of claim 1, wherein a diameter of the core is configured to be in a range of 1-10 nm.
6. The composition of claim 5, wherein the diameter of the core is configured to be about 3 nm.
7. The composition of claim 1, wherein an overall diameter of the composition is configured to be smaller than 15 nm and greater than 0.0 nm.
8. The composition of claim 4, wherein the 30 mol % chains loading is octadecyl group loading.
9. The composition of claim 1, wherein the composition is configured for entry into keratinocytes.
10. A method of modulating effectiveness of an anti-skin inflammation agent for inhibiting development of a skin inflammation phenotype or treating a skin inflammation phenotype, comprising steps of: applying the anti-skin inflammation agent concurrently with a toll-like receptor (TLR) 7/8 ligand, for a period of time onto skin of a subject who is at risk for developing a skin inflammation phenotype or who is exhibiting a skin inflammation phenotype; wherein the anti-skin inflammation agent comprises a core comprising at least one gold nanoparticle, a shell comprising a plurality of polyethylene glycol (PEG) strands conjugated to the core, and a plurality of alkyl groups conjugated to the shell of PEG strands.
11. The method of claim 10, wherein the core has a diameter of about 3 nm.
12. The method of claim 10, wherein the plurality of alkyl groups has a chain loading of about 30 mol % octadecyl group loading.
13. The method of claim 10, wherein chain lengths of the plurality of alkyl groups are configured to be in a range of 1-24 carbons.
14. The method of claim 10, comprising the steps of: topically applying the anti-skin inflammation agent subsequent to topically applying a toll-like receptor (TLR) 7/8 ligand for a period of time onto skin of a subject exhibiting a skin inflammation phenotype.
15. The method of claim 14, wherein the core has a diameter of about 3 nm.
16. The method of claim 14, wherein the plurality of alkyl groups has a chain loading of about 30 mol % octadecyl group loading.
17. The method of claim 14, wherein chain lengths of the plurality of alkyl groups are configured to be in a range of 1-24 carbons.
18. The method of claim 10, wherein the anti-skin inflammation agent is configured to downregulate genes enriched in downstream of one or more inflammatory pathways.
19. A method of preparing an alkyl-terminated, PEG-coated AuNPs (alkyl.sub.x%-PEG-AuNPs) composition, comprising: mixing an aqueous suspension of AuNP solution with HS-PEG.sub.1000-C.sub.nH.sub.2n+1 having a molecular weight of 1000 Da and HS-PEG.sub.1000-OCH.sub.3 having a molecular weight of 1000 Da by keeping a total PEG concentration at 10 PEG molecules per nm.sup.2 of AuNP surfaces to be coated; keeping PEGylation reactions for 1 hour with sonication; purifying resultants of the reactions by multiple rounds of centrifugal filter filtration for a predetermined period of time; and resuspending the resultants by deionized water.
20. A method of transdermal delivery of nanoparticles and entry into skin cells, comprising the step of: administering a therapeutically effective amount of a composition to skin of a subject, the composition comprising: a core comprising at least one gold nanoparticle; a shell comprising a plurality of polyethylene glycol (PEG) strands conjugated to the core; and a plurality of alkyl functional groups conjugated to the shell of PEG strands.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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BRIEF DESCRIPTION OF THE SEQUENCES
[0072] SEQ ID NO: 1 Forward primer for Mouse Il17a
[0073] SEQ ID NO: 2 Reverse primer for Mouse Il17a
[0074] SEQ ID NO: 3 Forward primer for Mouse Il17f
[0075] SEQ ID NO: 4 Reverse primer for Mouse Il17f
[0076] SEQ ID NO: 5 Forward primer for Mouse Il12b
[0077] SEQ ID NO: 6 Reverse primer for Mouse Il12b
[0078] SEQ ID NO: 7 Forward primer for Mouse Il1b
[0079] SEQ ID NO: 8 Reverse primer for Mouse Il1b
[0080] SEQ ID NO: 9 Forward primer for Mouse Tnf
[0081] SEQ ID NO: 10 Reverse primer for Mouse Tnf
[0082] SEQ ID NO: 11 Forward primer for Mouse GAPDH
[0083] SEQ ID NO: 12 Reverse primer for Mouse GAPDH
DETAILED DISCLOSURE OF THE INVENTION
[0084] The embodiments of subject invention show a non-steroidal and topical solution for treating psoriasis. The interactions of the alkyl-PEG-AuNPs with skin, the self-therapeutic property of AuNPs against psoriasis, and the favorable therapeutic outcome are also discussed below.
[0085] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. As used herein, the singular forms “a,” “am,” and “the” are intended to include the plural forms as well as the singular forms, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, steps, operations, elements, and/or components, but do not prelude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups thereof.
[0086] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one having ordinary skill in the art to which this invention pertains. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
[0087] When the term “about” is used herein, in conjunction with a numerical value, it is understood that the value can be in a range of 90% of the value to 110% of the value, i.e. the value can be +/−10% of the stated value. For example, “about 1 kg” means from 0.90 kg to 1.1 kg.
[0088] In describing the invention, it will be understood that a number of techniques and steps are disclosed. Each of these has individual benefits and each can also be used in conjunction with one or more, or in some cases all, of the other disclosed techniques. Accordingly, for the sake of clarity, this description will refrain from repeating every possible combination of the individual steps in an unnecessary fashion. Nevertheless, the specification and claims should be read with the understanding that such combinations are entirely within the scope of the invention and the claims.
[0089] A platform termed “alkyl-PEG-AUNP” that comprises a gold nanoparticle (AuNP) core, a shell of polyethylene glycol (PEG) strands, and alkyl groups at the periphery is provided. Alkyl groups are commonly found in the form of hydrophobic excipients or cream in many skincare products or therapeutic ointments. Based on the in vitro and in vivo experiments, it is proved that alkylation of the NP surface can significantly enhance the delivery of AuNPs to Kera 308 cells, primary epidermal cells, and mouse skin.
[0090] The effects of alkyl chain length, alkyl chain loading, or Au core size such as Au core diameter on the distribution of alkyl-PEG-AuNP in both healthy mice and Imiquimod (IMQ) induced psoriatic mice are also investigated.
[0091] In certain embodiments, the alkyl chain length is in a range of 1-24 carbons, the alkyl chain loading is in a range of 0%-50%, and Au core size/diameter is in a range of 1-10 nm. The results show that alkyl-PEG-AUNP with a longer alkyl chain, a higher alkyl loading, and a smaller size exhibit higher skin permeability and entry to keratinocytes when compared to methoxy-PEG-AuNPs.
[0092] The anti-psoriatic potential of alkyl-PEG-AuNPs (with 30% octadecyl group loading and a 3 nm Au core, termed “octadecyl.sub.30%-PEG-Au.sub.3 NPs”) in the “prevention mode” by concurrently applying IMQ and alkyl-PEG-AuNPs for a certain period of time, for example, 6 days is also investigated. Remarkably, it is found that the octadecyl.sub.30%-PEG-Au.sub.3 NPs, without drug loading, exhibit anti-psoriatic inflammation efficacy by inhibiting the development of psoriasis phenotype and significantly reducing epidermis thickness, proliferative keratinocytes, and CD3.sup.+ T cell infiltration, and psoriasis-related cytokines. RNA sequencing analysis demonstrates that topical treatment with octadecyl.sub.30%-PEG-Au.sub.3 NPs leads to a significant downregulation of the cytokine-cytokine receptor interaction and IL-17 inflammatory pathway.
[0093] To evaluate the translational potential of alkyl-PEG-AuNPs, a “treatment mode” is employed by establishing psoriasis in a mouse IMQ model for a certain period of time, for example, 6 days before topically applying certain doses, for example, 4 doses of octadecyl.sub.30%-PEG-Au.sub.3 NPs. During the treatment period, both the octadecyl.sub.30%-PEG-Au.sub.3 NPs and the Daivobet® ointment (a commercial anti-psoriasis ointment) show similar levels of anti-psoriatic efficacy when compared to PBS treated mice. At the end of treatment, octadecyl.sub.30%-PEG-Au.sub.3 NPs treated skin appear normal and smooth, while Daivobet® ointment treated skin show wrinkles and hair loss.
Embodiment One: Alkyl-Terminated Gold Nanoparticles for Enhanced Topical Delivery to Skin Keratinocytes
[0094] The delivery platform, termed “Au.sub.x@PEG-alkyl.sub.y % NP”, contains a gold core, a shell of PEG strands, and a fraction of alkyl groups at the periphery. The platform provides the advantage of modularity, allowing for flexible adjustment of the length and density of alkyl group to be attached.
[0095] First, by screening a series of sub-15 nm alkylated NPs that bear different lengths and loadings of alkyl chains, it is proved that alkylation promotes the in vitro uptake of NPs by immortalized keratinocytes as well as primary epidermal cells isolated from both healthy mice and psoriatic mice.
[0096] Next, by topically applying un-alkylated NPs with various Au core sizes onto the skin of healthy mice and psoriatic mice, it shows that the optimal core size for epidermal delivery is about 3 nm.
[0097] Further, screening is performed to identify that sub-15 nm alkylated NPs with longer alkyl chains and higher alkyl loadings enter epidermal cells in both healthy skin and psoriatic skin more abundantly, showing that Au.sub.3@PEG-octadecyl.sub.30% NP is the top performer. As a result of these comprehensive studies on skin-nano interaction, Au.sub.3@PEG-octadecyl.sub.30% NP is chosen for downstream efficacy studies.
Materials and Methods
1.1.1 Synthesis of HS-PEG.SUB.1000.-Alkyl
[0098] The carboxyl group of the bifunctional PEG linker, HS-PEG.sub.1000-COOH (JenKem Technology), is activated by 1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide (Sigma)/N-hydroxy succinimide (Sigma) (EDC/NHS) chemistry, HS-PEG1000-COOH (0.1 μmol), EDC (0.5 μmol), NHS (0.5 μmol), and trimethylamine (0.5 μmol) (TAE) (Sigma) is dissolved in 1 mL of freshly distilled dichloromethane (DCM) and vortexed for 2 hours. Next, 0.5 μmol of hexylamine (H.sub.2N—C.sub.6H.sub.13) (Sigma), dodecylamine (H.sub.2N—C.sub.12H.sub.25) (Sigma), or octadecylamine (H.sub.2N—C.sub.18H.sub.37) (Alfa Aesar), initially dissolved in 0.5 mL of DCM, is added in excess to the 0.1 μmol of activated PEG linker, followed by vortexing overnight. The reaction mixture is added dropwise into cold diethyl ether and then centrifuged at 3000×g for 3 minutes. The precipitated PEG product is washed with cold diethyl ether for 5 more times with sonication and briefly dried. Finally, the product is dialyzed against methanol for 1 week, against 0.1% acetic acid in water for 1 day, and against deionized water for 1 more day. Successful conjugation of alkylamine to the bifunctional PEG linker is confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) (Bruker).
1.1.2 Synthesis of Gold NPs (Au.sub.x NPs) with Different Core Sizes (x)
[0099] AuNPs can be prepared by various methods including chemical, sonochemical, or photochemical routes..sup.7 The most common chemical route is precipitation of the AuNPs in an aqueous solution obtained from a dissolved gold precursor, for example, HAuCl.sub.4, by a reducing agent such as sodium citrate, ascorbic acid, sodium boron hydride, or block-copolymers. Whereas in most cases another stabilizing agent is required to inhibit agglomeration or further growth of the NPs, some reducing agents, for example, sodium citrate and block copolymers also act as stabilizers. The classical citrate method of reduction of a gold precursor with sodium citrate in aqueous solution near the boiling point.sup.8 is one of the most reliable synthesis pathways. Using the combination of small-angle X-ray scattering and X-ray absorption near-edge spectroscopy analysis, Polte et al. proposed a four-step mechanism of AuNP formation.sup.9 as shown in
[0100] The size of the final gold NPs is tunable by changing both the activity of the gold precursor and the reducing power of the reductants or their ratios. Generally, a strong reductant such as sodium borohydride results in the formation of small gold NPs and a weak reductant such as citrate results in the formation of large particles. Moreover, the temperature and time duration of the reaction also have effects on the NP size.
[0101] To synthesize AuNPs of about 3 nm in diameter (Au.sub.3 NPs), 100 μL of 50 mM HAuCl.sub.4 (Sigma) and 129 μL of 38.8 mM sodium citrate (1% w/v) (Alfa Aesar) is added into 20 mL of Nanopure water. The mixture immediately turns brownish orange after injection of 0.6 mL of 0.1 M freshly prepared NaBH.sub.4 (Sigma) under rapid stirring. The resultant solution is stirred for 30 minutes and incubated at 60° C. for another 30 minutes. To synthesize AuNPs of 6 nm in diameter (Au.sub.6 NPs), 50 mL of 0.01% (w/v) HAuCl.sub.4 is brought to boil in a 125 mL flask for 6-7 minutes. A reducing agent containing 1 mL of 38.8 mM sodium citrate (1% w/v) and 225 μL of freshly prepared 1% (w/v) tannic acid (Sigma) is injected into the boiling solution under vigorous stirring. The solution immediately changes to dark violet and wine red in a few seconds. The mixture is kept boiling for another 5 minutes before being cooling down to the room temperature. AuNPs of 13 nm in diameter (Au); NPs) is synthesized by the Frens' method.sup.13. 50 mL of 1 mM HAuCl.sub.4 is brought to boil, followed by a rapid injection of 5 mL of 38.8 mM sodium citrate under vigorous stirring. The reaction is stopped after 15 minutes.
1.1.3 Preparation of Alkyl-Terminated, PEG-Coated AuNPs (Au.sub.x(PEG-Alkyl.sub.y % NPs)
[0102] The citrate-capped gold cores are negatively charged such that they maintain colloidal stability in aqueous solution by electrostatic repulsion. However, in high-ionic-strength solvents (for example, saline), the gold cores aggregate due to electrostatic screening of the negative charges around the gold cores by the positive salt ions (for example, Na), NP aggregation is a nearly irreversible process in which the particles attach to one another as shown in
[0103] First, an aqueous suspension of citrate-capped Au.sub.x NPs (x=3 nm, 6 nm, or 13 nm) is mixed with y mol % of HS-PEG.sub.1000-alkyl and (100−y) mol % of HS-PEG.sub.1000-OCH.sub.3 (JenKem Technology) by keeping the total PEG concentration at 10 PEG molecules per nm.sup.2 of AuNP surface to be coated. All PEGylation reactions last for 1 hour with sonication, followed by purification by 5 rounds of centrifugal filtration (Amicon® Ultra-15, MWCO: 50 k Da) at 4,000 g for 15 minutes and resuspension in deionized water. For confocal fluorescence imaging, cyanine 5 (Cy5)-labeled Au.sub.3-PEG-alkyl.sub.y % NPs are also prepared. An aqueous suspension of citrate-capped Au; NPs is mixed with y mol % of HS-PEG.sub.1000-alkyl, (99−y) mol % of HS-PEG.sub.1000-OCH.sub.3, and 1 mol % of HS-PEG.sub.1000-Cy5 (JenKem Technology) by keeping the total PEG concentration at 10 PEG molecules per nm.sup.2 of AuNP surface to be coated. 1 ml % of Cy5 loading is selected because it does not severely affect the physiochemical properties (for example, hydrodynamic sizes and zeta potentials) and the cellular uptake properties of the NPs. All PEGylation reactions last for 1 hour with sonication in the dark, followed by purification and resuspension in deionized water.
1.1.4 Physicochemical Characterization of Au.sub.x@PEG-alkyl.sub.y % NPs
[0104] The concentration of the NPs is determined by spectrophotometry (Agilent Cary 5000) based on the Beer-Lambert's law and the molar extinction coefficient of AuNPs at 450 nm (Au.sub.3 NPs: 1.49×10.sup.6 M.sup.−1 Au.sub.6 NPs: 1.26×10.sup.7 M.sup.−1 cm.sup.−1; Au.sub.3 NPs: 1.39×10.sup.8 M.sup.−1 cm.sup.−1). Hydrodynamic diameters (HD) and ζ-potentials are measured by the DelsaMax PRO light-scattering analyzer (Beckman Coulter), For HD measurements, the NPs are suspended in PBS buffer. For ζ-potential analysis, the NPs are suspended in 1 mM KCl. The values represent mean+SD from three independent measurements. The AuNPs are visualized by TEM at a voltage of 100 kV (Hitachi H7700).
1.1.5 Quantification of PEG Strands on AuNPs
[0105] The density of PEG strands attached to the AuNP surface in terms of number of PEG strands per NP or per nm.sup.2 is determined based on the thiol depletion approach using Ellman's assay (5,5-dithiobis (2-nitrobenzoic acid)) (JenKem Technology). After PEGylation of AuNPs of different sizes, the supernatant containing free alkyl-PEG-SR strands are obtained after centrifugation. Then, 20 μL of concentrated supernatant is added to 100 μL of assay buffer (for example, 1 mM EDTA and 0.1 M sodium phosphate dibasic; pH=8). 50 μL of detection buffer (for example, 0.5 mg/mL Ellman's reagent formulated in assay buffer) is added into the mixture. Known concentrations of Au.sub.x@PEG-alkyl.sub.y % NPs are used as standards. The reaction is allowed to proceed for 10 minutes and an absorbance at 412 nm is recorded by a UV-absorbance plate reader (Thermo Scientific Multiskan GO). The PEG amount on the AuNP surface is calculated by subtracting the PEG amount in the supernatant from the total PEG amount initially added. The PEG density is calculated by dividing the number of PEG strands by the surface area of AuNPs.
1.1.6 Water/1-Octanol Partitioning of NPs
[0106] 0.5 mL of 1-octanol (Sigma) and 0.5 mL, of Nanopure water containing Au.sub.x@PEG-alkyl.sub.y % NPs (50 ppm) are added into a glass vial and mixed at 40 rpm using a tube revolver (Thermo Scientific). After 24 hours, the concentration of the NPs remaining in the aqueous phase is determined by LIV-vis spectroscopy (Agilent Cary 5000).
1.1.7 Uptake of NPs by Immortalized Keratinocytes
[0107] Mouse Kera-308 keratinocytes (Cell Lines Service) are cultured in complete DMEM [DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco)] and maintained at 37° C. and in an environment with 5% CO.sub.2. Cells are pre-seeded in 24-well plates until the cell population reached about 80% confluence. During the uptake experiment, the cells are incubated with 300 nM Au.sub.3@PEG-alkyl.sub.y % NPs or equivalently, 50 ppm Au, formulated in 0.3 mL of OptiMEM (Gibco) per well for 24 hours. It is noted that alkyl.sub.y % includes methoxy, hexyl.sub.10%, dodecyl.sub.10%, octadecyl.sub.10%, hexyl.sub.30%, dodecyl.sub.30%, and octadecyl.sub.30%. After that, the cells are rinsed with phosphate-buffered saline (PBS) twice and trypsinized (0.25% Trypsin-EDTA, Gibco) for cell counting by a hemocytometer. Cell pellets are collected by centrifugation at 500 g for 10 minutes for further ICP-MS analysis of the Au content associated with the cells.
1.1.8 Removal of Hair for In Vivo Studies
[0108] All procedures followed the guidelines stipulated by the Animal Experimentation Ethics Committee at The Chinese University of Hong Kong. The dorsal hairs (about 3 cm×3 cm in area) of Balb/c mice (male or female, 6-8 weeks old) are shaved using an electronic shaver (Hair clipper A2, Jiamei) and treated with a thin layer of hair remover spray foam (about 1 g, Dimples) under anesthesia. The mice are intraperitoneally injected with ketamine (100 mg/kg) and xylazine (10 mg/kg) to achieve anesthesia. After 10 minutes, the hair remover spray foam is wiped by cotton and the skin is rinsed by PBS-soaked cotton for 3 times. Then, the depilated mice are used for further distribution or efficacy experiments. At the end of the experiments, the animals are sacrificed by cervical dislocation under anesthesia.
1.1.9 Isolation of Primary Epidermal Cells from Healthy Mouse Skin
[0109] After hair removal, the dorsal skin is immediately isolated after sacrifice. Freshly isolated skin is chopped into slices of 10 mm.sup.2 in area and then digested with 2 mL of neutral protease (LS02109, 2.4 U/mL, Worthington) in a 35 mm cell culture dish with the epidermis side up for 3 hours at 37° C. to detach the epidermis from the tissue. The epidermis is digested by adding 0.2 mL of 0.25% Trypsin-EDTA (Gibco) for 20 minutes. Then, the cells are filtered by passing the cell suspension through a cell strainer (40 μm, Falcon) and collected by centrifugation at 500×g for 10 minutes.
1.1.10 Uptake of NPs by Primary Epidermal Cells
[0110] Three healthy mice are sacrificed for harvesting the fresh primary epidermal cells. After resuspending the cells in OptiMEM and combining them into one single tube of about 7.2 mL in total volume, they are seeded in an entire 24-well plate of wells at a seeding density of about 4×10.sup.5 cells per well. Next, the cells are incubated with Au.sub.3@PEG-alkyl.sub.y % NPs (alkyl.sub.y %: methoxy, hexyl.sub.10%, dodecyl.sub.10%, octadecyl.sub.10%, hexyl.sub.30%, dodecyl.sub.30%, octadecyl.sub.30%) formulated with 150 μL of OptiMEM, such that the final concentration of Au.sub.3@PEG-alkyl.sub.y % NPs is 300 nM or equivalently 50 ppm. After 24 hours, the cell pellets are collected by centrifugation at 500×g for 10 minutes, rinsed with PBS for 3 times, and collected for ICP-MS analysis.
1.1.11 ICP-MS Analysis
[0111] The cell pellets collected from in vitro cellular uptake experiments, or the isolated epidermis collected from in vivo distribution and efficacy experiments are digested by adding 0.25 mL of aqua regia [68% HNO.sub.3: 37% HCl=1:3 (v/v)] overnight at room temperature. The composite layer of skin dermis and hypodermis (D+HD) and other internal organs excised from mice are fully digested in 1 mL of aqua regia for 2-3 days. The lysate is diluted to 10 mL by adding matrix solution (for example, 2% HCl, 2% HNO.sub.3) with 10 ppb indium as internal standard, followed by passing through a 0.2 μm hydrophilic syringe filter for ICP-MS analysis (Agilent 7900).
1.1.12 Biodistribution of NPs on Healthy Mouse Skin
[0112] After hair removal, Balb/c mice are randomly divided into various treatment groups. A piece of gauze (15 mm×25 mm), pipetted with 200 μL of Au.sub.x@PEG-alkyl.sub.y % NP solution with about 3 μM Au.sub.3@PEG-alkyl.sub.y % NPs, about 500 nM Au.sub.6@PEG-alkyl.sub.y % NPs, or about 50 nM Au.sub.13@PEG-alkyl.sub.y % NPs in PBS; or equivalently 500 ppm Au for all three Au core sizes, is applied to the shaved area of healthy mice, followed by covering the NP-applied area with a piece of Tegaderm (3M) adhesive dressing for 24 hours. After that, the gauze is removed, and the treated skin is rinsed with PBS-containing cotton for 3 times. The mice are then sacrificed for harvesting the skin. The harvested NP-containing skin (for example, 15 mm×25 mm) is cut into slices, treated with neutral protease for 3 hours to separate the epidermis from the composite layer of dermis and hypodermis. Then, the epidermis is treated with trypsin for 20 minutes and filtered by a cell strainer (40 μm) to obtain a single primary epidermal cell suspension. Then, the cell pellets are collected by centrifugation at 500 g for 10 minutes. It is noted that the collected epidermal cell pellet is denoted as isolated epidermis. Next, the isolated epidermis and H+HD composite layer are digested by adding 0.25 mL and 1 mL of aqua regia, respectively, for ICP-MS quantification. The collected NP-containing skins are also processed to prepare samples for TEM and histological analysis (see procedures below) to elucidate the cellular-level distribution of NPs.
1.1.13 TEM Imaging
TEM Imaging of the Isolated Epidermal Cells
[0113] The cell pellets are fixed with glutaraldehyde (for example, 2.5% in phosphate buffer, pH=7.2-7.4, J&K Scientific) at room temperature for 2 hours and stained by osmium tetroxide [2%, Electron Microscopy Sciences (EMS)] for 1 hour. Blocks are washed by phosphate buffer (pH=7.2-7.4) for 3 times, then gradually dehydrated in increasing ethanol gradients and propylene oxide. The blocks are embedded in Epon 812 resins (EMS) and polymerized at 55° C. for 48 hours. Ultrathin sections of about 70 nm in thickness are deposited onto 200-mesh copper grids (EMS) and stained with 4% uranyl acetate (in 50% methanol/water, EMS) and Reynolds lead citrate (Sigma) for observation under TEM at a beam voltage of 100 kV (Hitachi H7700).
TEM Imaging of the Whole Skin Tissue
[0114] NP-treated skin tissue blocks (about 1 mm×1 mm×3 mm) are fixed with glutaraldehyde (2.5% in phosphate buffer, pH=7.2-7.4) at 4° C. overnight. It is noted that the use of heavy metal stains, for example, osmium, uranium, and lead, is omitted to increase the contrast between the small AuNPs of 3 nm and the tissue structures. Instead, skin blocks are gradually dehydrated in increasing ethanol gradients and propylene oxide, followed by embedding in Epon 812 resins and polymerization at 55° C. for 48 hours. Ultrathin sections of about 70 nm in thickness are deposited onto 200-mesh copper grids (EMS) for observation under TEM at a beam voltage of 100 kV (Hitachi H7700). As a negative control, untreated skin tissue blocks are also prepared using the above protocol for TEM imaging.
[0115] The cell pellets are fixed with glutaraldehyde (2.5% in phosphate buffer, pH=7.2-7.4, J&K Scientific) at room temperature for 2 hours and stained by osmium tetroxide [2%, Electron Microscopy Sciences (EMS)] for 1 hour. Blocks are washed by phosphate buffer (pH=7.2-7.4) for 3 times, then gradually dehydrated in increasing ethanol gradients and propylene oxide. The blocks are embedded in Epon 812 resins (EMS) and polymerized at 55° C. for 48 hours. Ultrathin sections of about 70 nm in thickness are deposited onto 200-mesh copper grids (EMS) and stained with 4% uranyl acetate (in 50% methanol/water, EMS) and Reynolds lead citrate (Sigma) for observation under TEM at a beam voltage of 100 kV (Hitachi H7700).
1.1.14 Histology
[0116] Tissues are fixed in 10% buffered formalin (3.7% w/v) for 24 hours and then stored in PBS (0.1 M, pH=7.5) at 4° C. until tissue dehydration. Fixed tissues are dehydrated in ethanol, cleared in xylene, and embedded in paraffin blocks. Paraffin-embedded tissues are cut into sections of 5 μm thick using a rotary microtome (Leica RM 2235) and mounted on Superfrost Plus™ Adhesion microscope slides (Thermo Scientific). Paraffin-embedded tissue slides of 5 μm thick are deparaffinized in xylene (5 min 3 times) and rehydrated through a series of ethanol (100%, 90%, 70%; 3 min×2 times at each ethanol concentration) as well as deionized water (Milli Q) (5 min×5 times). Then, the tissues are counterstained by hematoxylin and eosin (Sigma) for 2 minutes and 1 minute, respectively. The stained sections are dehydrated in ethanol, cleared in xylene, and mounted with DPX mountant (Sigma) for visualization and photograph under a Ti-E motorized inverted fluorescence microscope (Nikon) in bright-field mode.
1.1.15 Silver Enhancement Staining
[0117] Deparraffinized and rehydrated tissue sections of 5 μm thick are stained by the Silver Enhancement Kit for Light and Electron Microscopy (Nanoprobes). The silver enhancement solutions, A (enhancer) and B (initiator), are mixed at a 1:1 ratio immediately before use. A drop of the mixture of about 50 μL is applied to the tissue section for 15 minutes in the dark. The tissue sections are rinsed with Milli Q water (3 minutes×3 times), followed by immunofluorescence staining or hematoxylin and eosin staining.
1.1.16 Confocal Immunofluorescence (IF) to Determine Cellular-level Distribution of NPs
[0118] Silver-enhanced tissue sections undergo antigen retrieval by a microwave-based antigen retrieval technique.sup.17. After immersing in citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH=6.0), the slides are heated in a microwave oven for 3 minutes under high power at about 95-100° C. and for 20 more minutes under low power. After cooling in the heated solution for 30 minutes, the slides are washed in distilled water twice, then rinsed in PBS for 5 minutes. Next, the slides are blocked with 2.5% normal horse serum (Vector Laboratories) for 2 hours at room temperature and incubated with 50 μL of primary antibodies [1× prediluted AE1/AE3 (ab961, Abcam), 5 μg/mL for CD207 (Langerin; 14-2073-82; Thermo Scientific), and 2.5 μg/mL for CD3 (ab16669; Abcam)] formulated in horse serum overnight at 4° C. After rinses with PBS, the sections are stained with secondary antibody [5 μg/mL Alexa Fluor 532-conjugated goat anti-mouse secondary (A11002; Thermo Scientific) for AE1/AE3, 5 μg/mL Alexa Fluor 647-conjugated goat anti-rat secondary (A21247; Thermo Scientific) for CD207, and 5 μg/mL Alexa Fluor 532-conjugated goat anti-rabbit secondary (A10520; Thermo Scientific) for CD3] at room temperature for 1 hour. Next, the slides are stained by 4′,6-diamidino-2-phenylindole (1 μg/mL DAPI; D9542; Sigma-Aldrich) for 10 min, washed in PBS for 3 times and in distilled water twice, and mounted with Antifade Mountant (P36980; Thermo Scientific). Slides are visualized under a confocal laser scanning microscope (TCS SP8, Leica). The excitation wavelengths of DAPI, Alexa Fluor 532, and Alexa Fluor 647 are 405 nm, 532 nm, and 647 nm, respectively. The emission wavelength ranges of DAPI, Alexa Fluor 532, and Alexa Fluor 647 are 415-500 nm, 542-650 nm, and 657-700 nm, respectively. Images are taken at the same laser settings for each fluorophore type (DAPI, Alexa Fluor 532 or Alexa Fluor 647). For reflectance imaging to detect the Au cores, the samples are imaged under a Leica SP8 microscope in the reflectance mode with a 20× objective under the excitation of an argon laser at 488 nm and the emission wavelength range is 483-493 nm. All reflectance images are taken at the same laser settings irrespective of the fluorophore type. Fluorescent images and reflectance images are taken between frames. The overlay images and mean fluorescent intensity (MFI) of the reflected signal (from Au core) in the region of cells stained positively by the fluorescent dye (e.g., CD3.sup.+ cells, CD207.sup.+ cells, and AE1/AE3.sup.+ cells) are processed by the Image J software.
1.1.17 Animal Disease Model of Psoriasis
[0119] Balb/c mice between 6 and 8 weeks of age are randomly divided into various treatment groups after dorsal hair removal on Day 0. Each mouse receives a daily topical dose of 62.5 mg of commercially available IMQ cream (Aldara, 5% w/w) on the depilated back (3 cm×3 cm in area) for 6 consecutive days. On Day 7, the psoriasis disease model is established for further studies such as primary epidermal cell uptake and biodistribution of NPs.
1.1.18 Primary Psoriatic Epidermal Cells Isolation and Uptake of NPs
[0120] Balb/c mice between 6 and 8 weeks of age are depilated on Day 0 and daily receive topical application of 62.5 mg of IMQ cream on the dorsal skin (3 cm×3 cm) for 6 consecutive days (Day 1 to Day 6). On Day 7, the psoriatic skin is harvested for epidermal cells isolation via the same method as that of healthy mice. Then, the primary epidermal cells (from 1 psoriasis mouse), freshly seed in a 24-well plate at a density of about 4×10.sup.5 cells per well, are incubated with 0.3 mL of 300 nM Au.sub.3@PEG-alkyl.sub.y % NPs (alkyl.sub.y %; methoxy, hexyl.sub.10%, dodecyl.sub.10%, octadecyl.sub.10%, hexyl.sub.30%, dodecyl.sub.30%, octadecyl.sub.30%; or equivalently 50 ppm Au) in OptiMEM. After 24 hours, the cell pellets are collected and washed by PBS for 3 times for ICP-MS measurements.
1.1.19 Biodistribution of NPs in Psoriatic Mouse Skin
[0121] When IMQ-induced psoriasis is established on Day 7, the mice are randomly divided into different groups (n=3-4 mice per group) for topically applying 200 μL of Au.sub.x@PEG-alkyl.sub.y % NPs (about 3 μM Au.sub.3@PEG-alkyl.sub.y % NPs, about 500 nM Au.sub.6@PEG-methoxy NPs, or about 50 nM Au.sub.13@PEG-methoxy NPs in PBS; or equivalently 500 ppm Au for all three Au core sizes). For Au.sub.3@PEG-alkyl.sub.y % NPs, 10 mol % octadecyl, 30 mol % hexyl, 30 mol % dodecyl, 30 mol % octadecyl, and 50 mol % octadecyl groups are selected for the PEG coating. Detailed experimental procedures of topical application of NPs and further analysis are the same as that of the healthy mice. For Au.sub.6@PEG-alkyl.sub.y % and Au.sub.13@PEG-alkyl.sub.y % NPs, only NPs with 30 mol % octadecyl groups are tested. Au.sub.x@PEG-methoxy NPs serve as control for each size. Detailed experimental procedures of topical application of NPs and further analysis are the same as that of the healthy mice.
1.1.20 Intradermal Distribution of NPs in Fluorescence Mode
[0122] Skin harvested from mice treated with Cy5-labeled NPs is cut into slices (3 mm×10 mm), frozen in Shandon Cryomatrix frozen embedding medium (Thermo Fisher Scientific) and sliced into cryosections of 20 μm thick. After fixing in cold acetone at −20° C. for 20 minutes, the slides are rinsed with PBS and stained by 4′,6-diamidino-2-phenylindole (1 μg/mL DAPI; D9542; Sigma-Aldrich) for 10 minutes. After being rinsed with PBS for 3 times and with distilled water twice, the stained slides are mounted with Antifade Mountant (P36980; Thermo Scientific) and visualized under a confocal laser scanning microscope (TCS SP8, Leica) at identical imaging settings. The excitation wavelengths of DAPI and Cy5 are 405 nm and 647 nm, respectively. The emission wavelength ranges of DAPI and Cy5 are 415-500 nm and 657-700 nm, respectively.
1.2 Results
1.2.1 Preparation and Characterization of Thiol-PEG-Alkyl Ligand
[0123] The bifunctional alkyl (hexyl, dodecyl, octadecyl), and thiol-terminated PEG strands (HS-PEG-alkyl) for the surface passivation of AuNPs are prepared. First, the carboxyl group of a bifunctional PEG (MW: about 1000) linker is activated by EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/NHS (Nthydroxysuccinimide) chemistry. Then, HS-PEG-NHS ester reacts with the amine group of the alkylamine to form HS-PEG-alkyl as shown in
[0124] After dialysis, MALDI-TOF is used to characterize the molecular weight of the product as shown in
1.2.2 Preparation and Characterization of Alkyl-Terminated, PEG-Coated AuNPs
[0125] A library of alkyl-terminated AuNPs with an overall hydrodynamic diameter smaller than 20 nm for topical delivery in vivo is established, because NP with a diameter larger than 25 nm cannot permeate the epidermis. Specifically, citrate-capped AuNPs is coated with a core diameter of 3 nm, 6 nm, or 13 nm with a mixture of y mol % of HS-PEG.sub.1000-alkyl strands that bear hexyl, dodecyl, or octadecyl groups and (100−y) mol % of HS-PEG.sub.1000-methoxy as shown in
[0126] It is confirmed that the loading of PEG strands by Ellman's assay and the colloidal stability of the Au.sub.x@PEG-alkyl.sub.y % NPs in water and UV-Vis spectrophotometry, respectively as shown in Table 1 and
TABLE-US-00001 TABLE 1 Hydrodynamic size, ζ-potential, PEG loading, and colloidal stability in PBS of Au.sub.x@PEG-alkyl.sub.y % NPs. ζ- HD size after potential No. of PEG HD size incubation in 1 strands/nm.sup.2 in water in PBS mM KCl of AuNPs Sample (nm) .sup.a (37° C., 24 h) (nm) (mV) surface Citrate-capped Au.sub.3 NPs 6.43 ± 0.23 (0.24) 226.03 ± 15.59 (0.23) −17.81 ± 4.23 NA Au.sub.3@PEG-methoxy NPs 9.45 ± 0.10 (0.19) 9.75 ± 0.32 (0.16) −3.52 ± 0.37 5.73 ± 0.03 Au.sub.3@PEG-hexyl.sub.10% NPs 9.70 ± 0.21 (0.14) 9.62 ± 0.41 (0.19) −3.79 ± 0.14 5.56 ± 0.09 Au.sub.3@PEG-hexyl.sub.30% NPs 9.98 ± 0.32 (0.26) 11.20 ± 0.32 (0.31) −2.84 ± 0.65 5.07 ± 0.14 Au.sub.3@PEG-dodecyl.sub.10% NPs 10.96 ± 0.42 (0.2) 11.45 ± 0.67 (0.24) −3.63 ± 1.05 5.59 ± 0.13 Au.sub.3@PEG-dodecyl.sub.30% NPs 12.64 ± 0.51 (0.33) 13.93 ± 0.41 (0.34) −1.89 ± 0.99 5.12 ± 0.22 Au.sub.3@PEG-octadecyl.sub.10% NPs 12.71 ± 0.58 (0.22) 12.86 ± 0.53 (0.32) −4.73 ± 1.53 5.6 ± 0.1 Au.sub.3@PEG-octadecyl.sub.30% NPs 13.30 ± 0.45 (0.35) 14.5 ± 0.61 (0.33) −1.65 ± 0.87 5.14 ± 0.15 Citrate-capped Au.sub.6 NPs 10.87 ± 0.06 (0.21) 80.57 ± 1.80 (0.57) −19.51 ± 3.93 NA Au.sub.6@PEG-methoxy NPs 13.67 ± 0.23 (0.17) 13.52 ± 0.36 (0.15) −3.89 ± 0.14 5.44 ± 0.32 Au.sub.6@PEG-octadecyl.sub.10% NPs 14.20 ± 0.26 (0.14) 14.78 ± 0.43 (0.18) −1.35 ± 0.41 4.87 ± 0.11 Au.sub.6@PEG-octadecyl.sub.30% NPs 14.87 ± 0.06 (0.23) 15.55 ± 0.37 (0.31) −3.74 ± 1.25 6.07 ± 0.62 Citrate-capped Au.sub.13 NPs 16.40 ± 0.08 (0.26) 374.77 ± 30.38 (0.04) −30.28 ± 2.36 NA Au.sub.13@PEG-methoxy NPs 18.65 ± 0.35 (0.24) 18.90 ± 0.45 (0.23) −5.74 ± 0.37 4.92 + 0.08 Au.sub.13@PEG-octadecyl.sub.10% NPs 19.25 ± 0.17 (0.22) 19.85 ± 0.37 (0.25) −3.35 ± 0.38 3.98 ± 0.88 Au.sub.13@PEG-octadecyl.sub.30% NPs 19.87 ± 0.06 (0.21) 22.47 ± 0.66 (0.28) −4.51 ± 0.73 3.36 ± 1.24 .sup.a Reported data represent mean ± SD from three independent measurements of Z-average sizes. Numbers in parentheses refer to the polydispersity index (PDI).
[0127] By dynamic light scattering (DLS), after loading citrate-capped AuNPs with different percentages (0-30%) of alkyl chain of various lengths (1-18 carbons), the hydrodynamic sizes of Au.sub.3@PEG-alkyls % NPs, Au.sub.6@PEG-alkyl.sub.y %, NPs, and Au.sub.13@PEG-alkyl.sub.y % NPs are observed to be about 9.5-13.3 nm, about 13.7-14.9 nm, and about 18.7-19.9 nm, respectively as shown in Table 1.
[0128] Since Phosphate Buffered Saline (PBS) is used as the solvent for the in vivo experiments, the stability of NPs in PBS is verified. After 24 hours incubation in PBS at 37° C., DLS is used to test the hydrodynamic size of NPs as shown in Table 1. It is noted that citrate-capped Au.sub.x NPs tend to aggregate in PBS by exhibiting obvious color change and a significant increase in size; while the color and hydrodynamic sizes of Au.sub.x@PEG-alkyl.sub.y %NPs do not show obvious change, demonstrating their colloidal stability in PBS buffer by the coating of a dense layer of PEG.sub.1000 strands. OptiMEM with reduced serum is selected as the culture medium for in vitro studies. It is confirmed that the Au.sub.x@PEG-alkyl.sub.y % NPs remain colloidally stable upon incubation in OptiMEM or PBS for 24 hours as shown in Table 2.
TABLE-US-00002 TABLE 2 Hydrodynamic sizes and stability of Au.sub.3@PEG-alkyl.sub.y % NPs in OptiMEM. HD size HD size after (nm) .sup.a before incubation Sample incubation (37° C., 24 h) (nm) Au.sub.3@PEG-methoxy NPs 11.0 ± 0.8 (0.4) 15.1 ± 0.1 (0.5) Au.sub.3@PEG-hexyl.sub.10% NPs 11.3 ± 1.4 (0.5) 13.1 ± 0.6 (0.5) Au.sub.3@PEG-hexyl.sub.30% NPs 9.7 ± 0.3 (0.5) 12.0 ± 1.8 (0.5) Au.sub.3@PEG-dodecyl.sub.10% NPs 10.7 ± 2.6 (0.4) 11.8 ± 0.4 (0.5) Au.sub.3@PEG-dodecyl.sub.30% NPs 9.5 ± 1.0 (0.5) 12.4 ± 1.6 (0.5) Au.sub.3@PEG-octadecyl.sub.10% NPs 11.1 ± 0.8 (0.5) 15.5 ± 1.6 (0.5) Au.sub.3@PEG-octadecyl.sub.30% NPs 13.5 ± 1.1 (0.5) 16.0 ± 0.7 (0.5) .sup.a Reported data represent mean ± SD from three independent measurements of Z-average sizes. Numbers in parentheses refer to the polydispersity index (PDI).
[0129] Prior to animal studies, it is further confirmed that the collection of Au.sub.x@PEG-alkyl.sub.y % NPs does not induce severe cytotoxicity to Kera-308 cells after 24 hours of incubation as shown in
[0130] When loaded with 30 mol % alkyl group, the calculated mass percentage of alkyl group on the total PEG coating is only 2.49%, 4.83%, and 7.05% in Au.sub.x@PEG-hexyl.sub.30% NPs, Au.sub.x@PEG-dodecyl.sub.30% NPs, and Au.sub.x@PEG-octadecyl.sub.30% NPs, respectively, which is much lower than that in other lipid-based NPs (for example, 28%), due to the effects of the functional group, not the effects of hydrophobicity. To prove that the NPs are overall hydrophilic, the 1-octanol/water partitioning experiment is conducted as shown in
[0131] Referring to 7B, the results from the experiment show that even for the NPs bear 30% octadecyl at the PEG periphery, the NPs are mainly dispersed in the water phase, with limited transferring to the 1-octanol phase. However, when octadecyl loading exceeds 40%, the AuNPs gradually partition to 1-octanol. The results indicate that when octadecyl loading exceeds 30%, the Au.sub.3@PEG-octadecyl.sub.y % NPs are less stable and less hydrophilic.
[0132] Therefore, attaching excessive octadecyl chains to the AuNPs causes NP agglomeration due to interparticle hydrophobic interaction as shown in
[0133] On the same day as the colloidal stability experiments, artificial sweat is freshly prepared according to the standard protocol (EN 1811:2011) by mixing urea (0.1 wt %; TCI), NaCl (0.5 wt %; J&K Scientific), and DL-lactic acid (0.1 wt %; Sigma) in deionized water. The pH of the solution is adjusted with 1 M sodium hydroxide to a final pH of 6.5±0.05. After 24 hours of incubation, the hydrodynamic (HD) sizes of all tested AuNPs do not show significant increase, indicating the stability of alkylated AuNPs as shown in Table 3.
TABLE-US-00003 TABLE 3 HD sizes of AuNPs after incubation in artificial sweat for 24 h. Citrate- Citrate- Citrate- Au.sub.13@ capped Au.sub.3@PEG- Au.sub.3@PEG- capped Au.sub.6@PEG- capped PEG- Au.sub.3 methoxy octadecyl.sub.30 Au.sub.6 methoxy Au.sub.13 methoxy NPs NPs NPs % NPs NPs NPs NPs NPs HD size 789 ± 47 11.7 ± 0.3 17.0 ± 0.7 1131 ± 93 15.6 ± 0.4 1856 ± 503 21.2 ± 0.3 (nm)
1.2.3 Surface Alkylation Enhances the Uptake of Au.SUB.3 .NPs by Immortalized Keratinocyte and Primary Epidermal Cells.
[0134] It has been reported that gold NPs of 25 nm in size with an outer hydrophilic coating of PEG strands of about 5000 Da exhibit enhanced uptake by Kera-308 mouse keratinocytes in vitro upon modification with minute amounts of alkyl groups. The issue of whether such effect of alkylation is also applied to AuNP with a smaller core is investigated and the uptake of Au.sub.3@PEG-alkyl.sub.y % NPs (300 nM, 0.3 mL) by Kera-308 cells after 24 hours of NPs incubation in OptiMEM is studied.
[0135] ICP-MS results show that the cellular uptake of Au.sub.3@PEG-alkyl.sub.y % NPs is increased with increasing alkyl chain length and loadings as shown in
[0136] The primary epidermal cells (mainly keratinocytes) are further isolated from the dorsal skin of healthy Balb/c mice and incubated them with Au.sub.3@PEG-alkyl.sub.y % NPs (300 nM, 0.3 mL) for 24 hours. Similar to Kera-308 cells, ICP-MS data shows that the uptake of Au.sub.3@PEG-alkyl.sub.y % NPs by primary cells increases with alkyl chain length and loadings. For example, the uptake of Au.sub.3@PEG-octadecyl.sub.30% NPs by the primary cells is about 12.1-fold (p<0.0001) higher than Au.sub.3@PEG-methoxy NPs. The in vitro data have proved that surface alkylation of Au.sub.3 NPs with longer alkyl chain length and higher alkyl loading promotes the NP uptake by both immortalized and primary keratinocytes.
[0137] The interactions between alkyl.sub.x %-PEG-Au.sub.6 NPs and the Kera 308 cell line (mouse keratinocytes) are studied, and then the results of investigation applied to primary epidermal cells and Balb/c mice skin are shown in
[0138] In one embodiment, Kera-308 cells are first, incubated with dodecyl.sub.x %-PEG-Au.sub.6 NPs (for example, 50 ppm; formulated in OptiMEM) that contain 0%, 1%, 2%, 4%, 10%, or 20% dodecyl group for 8 hours. By inductively coupled plasma mass spectrometry measured (ICP-MS), the association of dodecyl.sub.x %-PEG-Au.sub.6 NPs by Kera-308 cells increases with increasing alkyl amounts as shown in
[0139] Referring again to
[0140] Specifically, compared to NPs loaded with methoxy group only, the cellular uptake of dodecyl.sub.x %-PEG-Au.sub.6 NPs loaded with 10% and 20% dodecyl is enhanced by about 23-fold and about 27-fold, respectively. It is note that 10% is selected as the minimal alkyl loading percentage for further study because alkyl loading more than 10% leads to more drastic cellular uptake.
1.2.4 Distribution of Au.sub.3@PEG-alkyl.sub.y % NPs in Healthy Mouse Skin
[0141] The effects of Au core size, alkyl chain length, and loading on the biodistribution of Au.sub.x@PEG-alkyl.sub.y % NPs in healthy Balb/c mice are studied. Au.sub.x@PEG-methoxy NPs or Au.sub.x@PEG-alkyl.sub.y % NPs (500 ppm, 200 μL) are topically applied onto shaved mouse dorsal skin. It is noted that the same gold mass of AuNPs with different core sizes is applied to the mouse skin. After 24 hours, the treated skin is rinsed then harvested for further analysis. To quantify the Au content in different skin layers, the harvested skin is treated with neutral protease to isolate the epidermis from the dermis and hypodermis as shown in
[0142] The ICP-MS results show that Au.sub.x@PEG-alkyl.sub.y % NP with a longer alkyl chain, higher alkyl loading density exhibit higher keratinocytes entry and skin permeability among all sizes as shown in
[0143] To further visualize the biodistribution of AuNPs in the healthy skin at the tissue level, the locations of topically applied AuNPs are tracked by silver staining enhancement using confocal reflectance imaging. Representative confocal images of
[0144] TEM imaging is used to confirm the internalization of Au.sub.3@PEG-octadecyl.sub.30% NPs as shown in
[0145]
[0146] To examine the intradermal distributions of AuNPs in healthy skin at cell level, the locations of AuNPs in skin sections are tracked by confocal reflectance imaging upon silver staining enhancement. Representative confocal images of
1.2.5 Cellular Distribution of Au.sub.3@PEG-alkyl.sub.y % NPs in Psoriatic Skin of IMQ-induced Mouse Model
[0147] It is noted that the structures of psoriatic skin are different from these of healthy skin (for example, psoriatic skin has impaired SC with scars), the interaction between NPs and psoriatic skin are studied to enhance the anti-psoriasis nanomedicine design. Balb/c mice (6-8 weeks) are daily treated with IMQ (62.5 mg of IMQ cream, with 3.125 mg of the active compound) on shaved back for 6 days, then a series of skin inflammatory reactions such as scaling and thickening of skin patches are induced on Day 7, matching the phenotype of psoriasis. The same cell isolation procedures are used in the healthy cellular uptake experiment to harvest primary cells from psoriatic epidermis as shown in
[0148] To investigate the effect of NP size on delivery to psoriatic epidermis, Au.sub.x@PEG-methoxy NPs (x=3, 6, 13) are topically applied onto the psoriatic skin of IMQ-treated mice for 24 hours and the NP distributions in the different skin layers and epidermal cells are examined. ICP-MS data of
[0149] The effect of alkylation on delivery to psoriatic epidermis is then investigated by topically applying Au.sub.3@PEG-alkyl.sub.30% NPs (hexyl, dodecyl) and Au.sub.3@PEG-octadecyl.sub.y % NPs (y=0, 10, 30, or 50) to IMQ-treated mice for 24 hours. By ICP-MS, Au.sub.3@PEG-octadecyl.sub.30% NPs accumulate more abundantly in the epidermis than Au.sub.3@PEG-alkyl.sub.y % NPs with shorter alkyl chain length and lower octadecyl loading as shown in
[0150] Further, the epidermal accumulation of Au.sub.3@PEG-octadecyl.sub.50% NPs is lower than that of Au.sub.3@PEG-octadecyl.sub.30% NPs, probably because enhanced hydrophobic interactions of the former NP lead to a larger NP size (for example, 36.4 nm) that disfavors skin permeation. These results convincingly demonstrate that there exists an optimal loading of octadecyl chains of 30 mol %, in the design of NPs for best topical delivery to the epidermis. In addition, because psoriatic skin has multiple layers of keratinocytes (unlike the single layer in healthy skin), the probability of finding Au.sub.3@PEG-octadecyl.sub.30% NPs in the epidermis and keratinocytes is higher in psoriatic skin; meanwhile the accumulation of Au.sub.3@PEG-octadecyl.sub.30% NPs in dermis and hypodermis is lower in psoriatic skin as shown in
[0151] Confocal immunofluorescence images show that NPs tend to accumulate in keratinocytes of the middle epidermis than in the flattened keratinocytes of the upper layer in psoriatic skin as shown in
[0152] To validate the label-free confocal reflectance imaging data, Cyanine 5 (Cy5)-labeled Au.sub.3@PEG-methoxy NPs and Cy5-labeled Au.sub.34 PEG-octadecyl.sub.30% NPs are prepared for tracking their intradermal distributions by confocal fluorescence imaging.
[0153] It is revealed that such Cy5-labeled NPs exhibit similar hydrodynamic sizes, zeta potentials, and cellular uptake properties as their nonfluorescent versions as shown in
[0154] After applying the Cy5-labeled NPs to the skin of psoriatic mice for 24 hours, Cy5 fluorescence detected in the epidermis of mice treated with Au.sub.3@PEG-octadecyl.sub.30% NPs is stronger than those treated with Au.sub.3@PEG-methoxy NPs. The confocal fluorescence imaging data of
1.2.6 Biodistribution after Topical Application of Au.sub.3@PEG-alkyl.sub.y % NPs
[0155] After the study of the intradermal distributions of topically applied NPs, the biodistributions of these NPs in blood and internal organs are further investigated. According to the ICP-MS measurements, there is very limited Au content (less than 2 ng) detected in major internal organs and blood after topical application of NPs to both healthy mice and mice with psoriasis for 24 hours as shown in
[0156] To further understand the biodistribution of topically applied Au.sub.3@PEG-allyl.sub.y % NPs, the concentration of applied NPs is increased by 10 times (30 μM, 200 μL) and the Au content in different organs and blood is checked after 24 hours. However, no NPs are detected in the blood or major internal organs of both healthy mice and mice with psoriasis as shown in
1.2.7 Effects of Excipient
[0157] The NPs into PBS may be suspended for topical delivery without the aid of an excipient. Further, a commercial hand cream is used as the excipient for topical delivery of Au.sub.3@PEG-methoxy NPs. Specifically, 50 μL of Au.sub.3@PEG-methoxy NPs (12 μM) are mixed with 50 mg of hand cream, then this resultant AuNP-containing cream is applied onto shaved dorsal skin of mouse, covering an area of 15 mm 25 mm (same as the size of gauze). After 24 hours, the skin is rinsed with PBS for 3 times then harvested for analysis. ICP-MS results show that with the help of cream, the AuNPs penetrate through the skin and enter internal organs and blood, without showing any significant enhancement in epidermis accumulation when compared to the negative PBS control as shown in
[0158] It is shown that sub-15 nm NPs can cross the SC upon topical application and when attached with octadecyl chains, enhanced accumulation in the epidermis and entry to the keratinocytes for healthy mice and psoriasis mouse models are shown, without the use of excipients.
[0159] First, by screening a series of sub-15 nm alkylated NPs that bear different lengths and loadings of alkyl chains, it is proved that allylation promotes the in vitro uptake of NPs by immortalized keratinocytes as well as primary epidermal cells isolated from both healthy mice and psoriatic mice. Next, by topically applying un-alkylated NPs with various Au core sizes onto the skin of healthy mice and psoriatic mice, it is shown that the optimal core size for epidermal delivery is about 3 nm (the optimal overall size is <15 nm). The sub-15 nm size of Au.sub.3@PEG-octadecyl.sub.30% NPs allows penetration of the SC and accumulation in the epidermis without the aid of excipients, and an optimal loading of octadecyl groups (30 mol %) maintains the colloidal stability of Au cores in skin while boosting their uptake by epidermal cells. Despite the structural differences in psoriatic skin, the total absolute Au content for the permeation of Au.sub.3@PEG-octadecyl.sub.30% NPs in the psoriatic skin is similar to that in the healthy skin. Lastly, very limited Au content is detected in major internal organs and blood after topical application of NPs to both healthy mice and mice with psoriasis for 24 hours.
[0160] These in vivo data not only improve the cellular-level understanding of the bio-nano interactions of NPs with psoriatic skin, but also enable optimization of rational design of bionanomaterials to overcome the delivery bottleneck to keratinocytes based on surface alkylation or more broadly, functional group engineering.
Embodiment Two: Alkyl-Terminated Gold Nanoparticles as a Self-Therapeutic Treatment for Psoriasis
[0161] The anti-psoriasis potential of the alkylated gold NPs, which consists of a 3 nm gold core, a shell of 1000 Da PEG strands, and octadecyl chains attached to 30% of the PEG strands (Au.sub.3@PEG-octadecyl.sub.30% NP) is studied. When being applied onto the skin of IMQ-induced mice (an established model of psoriasis), the NPs may penetrate the SC and enter keratinocytes. As a result, concurrently applying the NPs with IMQ inhibits psoriasis. Further, the NPs can treat psoriasis as effectively as standard betamethasone-calcipotriol therapy, with significantly reduced skin wrinkling and hair loss. No NPs are found in major organs and no cutaneous or systemic toxicity is observed in the animals four weeks post-application. The work presents a simple, safe, and effective alternative for treating psoriasis.
2.1 Materials and Methods
2.1.1 Efficacy Evaluation in “Prevention Mode”
[0162] Balb/c mice between 6 and 8 weeks of age are depilated on Day 0 and divided into 4 treatment groups (n=8 mice per group). From Day 1 to Day 6, all groups of mice receive daily topical application of the IMQ cream. 30 minutes post-application of the IMQ cream, the middle dorsal area is topically applied daily with either (i) no additional treatment (denoted “IMQ only”), (ii) Au.sub.3@PEG-octadecyl.sub.30% NPs (200 μL of PBS containing 6 μM NPs pipetted into a piece of gauze with an area of 15 mm×25 mm; or equivalently 1000 ppm Au) (denoted “IMQ+Au.sub.3@PEG-octadecyl.sub.30% NPs”), (iii) Au.sub.3@PEG-methoxy NPs (200 μL of PBS containing 6 μM NPs pipetted into a piece of gauze; or equivalently 1000 ppm Au) (denoted “IMQ+Au.sub.3@PEG-methoxy NPs”), or (iv) free PEG strands (967105, M.W: about 1000, J&K Scientific) (200 μL of 1.2 mg/mL PEG in PBS, the same amount of PEG found in Au.sub.3@PEG-methoxy NPs) (denoted “IMQ+free PEG”), all with a target coverage area of 15 mm×25 mm on the psoriatic skin. The treated area is then covered with a Tegaderm film (3M).
[0163] The severity of psoriatic skin lesions is assessed daily using the modified Psoriasis Area and Severity Index (PASI) scores, a composite score for the degree of erythema, scaling, and thickness on a scale of 0 to 4. Specifically, 0 indicates no symptoms, 1 indicates mild, 2 indicates moderate, 3 indicates severe, and 4 indicates very severe. The body weight is also monitored daily. On Day 7, mice are sacrificed. The treated skin area is then rinsed with PBS for three times, harvested, and chopped into slices of 3 mm×10 mm in area for further analysis. Next, whole blood is collected from the mouse heart via an intracardiac puncture with 25 G needle under anaesthesia before animal sacrifice. Other organs are collected after sacrifice for further analysis. The group size of each treatment group is calculated according to Dunnett's formalism (see the section “Sample Size Calculation” below).
2.1.2 Synthesis and Characterization of Polythymidine Coated AuNPs (Au.SUB.3.@T12 NPs)
[0164] Standard reagents for solid-state synthesis of oligonucleotides are purchased from GeneParma. DNA phosphoramidites are purchased from Hongene. Thiolated DNA oligonucleotides with 12 repeating thymidines (HS-T12) are synthesized by a MerMade 12 Oligonucleotide synthesizer (LGC Bioautomation) based on manufacturer-recommended cleavage and deprotection protocols. All oligonucleotides are purified by a high-performance liquid chromatography instrument (Agilent 1260) equipped with a reverse-phase PLRP-S 300 Å 8 μm column (Agilent). Triethylammonium acetate (TEAA) buffer (0.03 M) is run with a 2%/min gradient of 100% CH.sub.3CN at a flow rate of 3 mL/min while monitoring the absorbance of nucleic acid at 260 nm and 280 nm. After HPLC purification, the oligonucleotides are lyophilized and stored at −20° C. for future use.
[0165] Then, 300 μL of 200 μM thiolated DNA oligonucleotides (HS-T12) are incubated in 300 μL of 40 mM Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) for 1 hour. To modify citrate-capped Au.sub.3 NPs with DNA, 10 mL of 300 nM Au.sub.3 NPs is mixed with 600 μL HS-T12/TCEP mixture and manually shaken for a few seconds and tuned to 0.01% sodium dodecyl sulfate (SDS) and 1×Tris-acetate-EDTA (TAE) buffer. After incubating the solution at room temperature for 20 minutes, NaCl solution is sequentially added to the NP solution at time intervals of 30 minutes until a final concentration of 0.3 M is reached to achieve dense coverage of the NP surface with DNA oligonucleotides. The NP product is obtained by dialyzing the reaction mixture against Nanopure water (Barnstead, Thermo Fisher) by 5 rounds of centrifugal filtration (Amicon® Ultra-15, MWCO: 10 k Da) at 4,000 g for 15 minutes and resuspension in deionized water. The loading of oligonucleotides are then quantified by a reported method.sup.32. Specifically, 20 mol % of cyanine 5 (Cy5)-conjugated SH-T12 is mixed with 80 mol % of SH-T12 to synthesis DNA coated AuNP, then DNA is chemically displaced from the NP surface using DTT. The displacement is achieved by adding equal volumes of oligonucleotide-functionalized gold NPs and 1.0 M DTT in 0.18 M PB at pH 8.0. The oligonucleotides are released into solution during an overnight incubation and the gold precipitate is removed by centrifugation. To determine oligonucleotide concentration, 100 μL of supernatant is placed in a 96-well plate and the fluorescence is compared to a standard curve. During the fluorescence measurement, the fluorophore is excited at 650 nm and the emission is collected at 670 nm. The DNA loading is calculated by dividing the number of DNA strands by the number of AuNPs.
2.1.3 Efficacy Evaluation in “Treatment Mode”
[0166] Balb/c mice between 6 and 8 weeks of age are depilated on Day 0 and divided into 3 treatment groups (n=7 mice per group). Each group daily receives topical application of 62.5 mg of IMQ cream for 6 consecutive days (Day 1 to Day 6). From Day 7 to Day 10, the mice are daily treated with either (i) PBS (200 μL of liquid pipetted into a piece of gauze), (ii) Betamethasone and Calcipotriol (BC) ointment (20 mg of cream spread as a thin layer), or (iii) Au.sub.3@PEG-octadecyl.sub.30% NPs (200 μL PBS containing 6 μM NPs pipetted into a piece of gauze; or equivalently 1000 ppm of Au), all with a target coverage area of 15 mm×25 mm on the psoriatic skin. The treated area is then covered with a Tegaderm film. The PASI scores and body weight are measured daily from Day 7 to Day 10. Mice are sacrificed on Day 11. The treated skin area is rinsed with PBS for three times, harvested, and chopped into slices of 3 mm 10 mm in area for further analysis. Whole blood and other organs are also collected for further analysis. The group size of each treatment group is calculated according to Dunnett's formalism (see the section “sample size calculation” below).
2.1.4 Sample Size Calculation
[0167] For in vivo efficacy studies, the group size of each treatment is calculated according to Dunnett's formalism..sup.33 Dunnett's test is a multiple comparison procedure that compares the efficacy of each treatment group with the same control group. Here, “H0: All treatment groups are equivalent to the control group” is tested against “H1: There exists one group that is superior to the control group”. Specifically, the treatment groups and the control group are compared in a way that (i) the chance of committing type 1 error is <5% and that (ii) our comparison is of power 80%. Dunnett's formalism states that p=√{square root over (N)}δ/σ, p is the correlation coefficient that depends on N. There are 3 and 2 treatment groups in the “prevention mode” (excluding the IMQ only control group) and the “treatment mode” (excluding the PBS control group), respectively. Thus, p equals 4.30 and 4.05 for the “prevention mode” and the “treatment mode”, respectively. If the superior treatment group gives an outcome (5) of 1.5 standard deviation (a) better than the control group, the required N is determined to be (4.30/1.5).sup.2 for the “prevention mode” and (4.05/1.5).sup.2≈7 for the “treatment mode”.
2.1.5 Immunohistochemistry (IHC) Staining for Efficacy Evaluation
[0168] For each treated or control mouse, two small slices of skin tissue, each of 3 mm×10 mm in area and at least 3 mm apart from each other in the original harvested skin, are embedded into separate paraffin blocks and sectioned. The two slices from each mouse are IHC stained with two different markers, CD3 and Ki67 (see below). Deparraffinized and rehydrated tissue sections are immersed in citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH=6.0) and heated in the microwave oven for 3 minutes under high power (about 95-100° C.) and for 20 more minutes under low power. After cooling in the heated solution for 30 minutes, the slides are washed in distilled water twice, then rinsed in PBS for 5 minutes. Next, the slides are blocked with 2.5% normal horse serum (Vector Laboratories) for 2 hours at room temperature and incubated with 50 μL of primary antibodies [2.5 μg/mL for Ki67 antibody (ab15580; Abcam) or 2 μg/mL for CD3 antibody (ab16669; Abcam)] formulated in horse serum are incubated overnight at 4° C. After rinses with PBS, the sections are treated with 3% H.sub.2O.sub.2 (Merck Millipore) for 10 minutes, rinsed again, and incubated with about 50 μL of secondary antibodies (NP-7401-50; ImmPRESS HRP Polymer detection Kit; Vector Laboratories) for 30 minutes. The sections are developed using 3,3′-diaminobenzidine (DAB) enzyme substrate (ImmPACT™ DAB, Vector Laboratories) for 4 minutes. All slides are counter-stained with Mayer's hematoxylin for 2 minutes, washed in distilled water, dehydrated in ethanol, cleared in xylene, and mounted with DPX mountant (Sigma). Sections are for visualization and photograph under a Ti-E motorized inverted fluorescence microscope (Nikon) with the bright-field mode.
2.1.6 Image Analysis for Efficacy Evaluation
[0169] For each section-containing slide, 3 pictures (1.2 mm×0.9 mm; 10× under bright field) are taken such that 6 pictures in total are counted for analysis of each mouse. To measure the thickness of epidermis (from the stratum granulosum to the epidermal-dermal junction) and the thickness of whole skin (from epidermis to hypodermis-muscle junction), the area of epidermis or whole skin in each picture is measured by the ImageJ software and divided the area by the length of skin in each picture (typically 1.2 mm long). Similarly, the number of Ki67.sup.+ cells in basal epidermis, the number of CD3.sup.+ cells in the whole skin, and the number of hair follicles in the whole skin are counted and divided by the length of skin in each picture. The averaged value of 6 pictures per mouse is displayed using a stacked bar chart with scatter plot points.
2.1.7 Levels of Inflammatory Cytokines in Skin Homogenates for Efficacy Evaluation
[0170] After removing 40 mg of skin tissue from each treated mouse, the skin tissue is treated with 1.6 mL of tissue protein extraction reagent (78510; Thermo Scientific) supplemented with 1× protease inhibitor cocktail (78430; Thermo Scientific). The samples are homogenized by using a Tissue-Tearor homogenizer (BioSepc Products) on ice and centrifuged at 15000 rpm and 4° C. for 10 minutes to remove any tissue debris. 200 μL aliquots of the supernatant are used for measuring the concentrations of IL-17A, IL-12/23 p40, and IL-1β by using the Mouse IL-17 DuoSet ELISA kit (DY421-05; R&D Systems), ELISA MAX™ Deluxe Set Mouse IL-12/IL-23 p40 (431604; BioLegend), and ELISA MAXIM Deluxe Set Mouse IL-1β (432604; BioLegend) per the manufacturer's instructions.
2.1.8 Determination of Levels of ALT, AST and BUN in Blood for Safety Evaluation
[0171] 500 μL of whole blood is collected from the mouse heart via an intracardiac puncture (using a 25-Gauge needle) under anaesthesia before animal sacrifice. The whole blood is left to sit for 30 minutes at room temperature and then centrifuged at 2,000×g for 10 min at 4° C. Next, about 200 μL of blood serum is collected from the supernatant for measuring the concentrations of alanine aminotransferase (ALT, a marker for liver function), aspartate aminotransferase (AST, a marker for liver function) and blood urea nitrogen (BUN, a marker for kidney function) at the PathLab Medical Laboratories.
2.2 Results
[0172] 2.2.1 Improvement of the IMQ-induced Psoriatic Skin Condition in the “Prevention mode” by Topical Application of Au.sub.3@PEG-octadecyl.sub.30% NPs.
[0173] To determine whether Au.sub.3@PEG-octadecyl.sub.30% NP can inhibit the development of psoriasis, psoriasis is induced by concurrently appling IMQ and the NPs for 6 consecutive days as shown in
[0174] Compared to the healthy skin, the skin of the IMQ control mice displayed significantly higher psoriasis area and severity index (PASI). It is observed that the treatment with the alkyl-PEG-AuNPs can ameliorate inflammation as indicated by the reduction of scales and induration, while free PEG molecules cannot prevent the development of psoriasis. After 6 days, treatment with octadecyl.sub.30% PEG-Au.sub.3 NPs can even smoothen and thin the psoriatic skin with barely visible scales, bringing the appearance of the skin close to that of the normal skin.
2.2.1.1 Efficacy Evaluation of Au.sub.3@PEG-octadecyl.sub.30% NPs
[0175] Gross examination show mice treated with IMQ only displayed the expected symptoms of psoriatic inflammation as shown in
[0176] On Day 7, the treated skin is harvested from all groups for histological examination and immunohistochemical staining. Hyper proliferating keratinocytes are stained with Ki67 marker.sup.34 and infiltrating T cells are stained with CD3 marker. Concurrent application of Au.sub.3@PEG-octadecyl.sub.30% NPs and IMQ inhibit psoriatic phenotypes, including parakeratosis, acanthosis, hyperproliferation of keratinocytes and infiltration of T cells as shown in
[0177] IMQ is known to induce systemic inflammation and splenomegaly, the weights of spleens from all groups are measured and normalized to body weights. The normalized spleen weights of psoriatic animals (IMQ only) increase as expected from 0.43% at Day 0 (before treatment) to 1.02% on Day 7 after IMQ treatment. Notably, the mean normalized spleen weights of animals treated with IMQ and Au.sub.3@PEG-methoxy NPs (0.78%) or animals treated with IMQ and Au.sub.3@PEG-octadecyl.sub.30% (0.65%) NPs do not increase to the level of the psoriatic animals, indicating that NP treatment inhibits splenomegaly in IMQ-treated animals as shown in
[0178] The results demonstrate that un-alkylated Au.sub.3@PEG-methoxy NPs can also inhibit inflammation associated with psoriasis. Although less effective than alkylated NPs, Au.sub.3@PEG-methoxy NPs lead to the significant reduction in mean epidermal thickness (37.2%), population of Ki67.sup.+ cells (38.7%), CD3.sup.+ cells (33.1%), and the cytokine levels of IL-17 (35.5%), CL-12/23 p40 (44.5%) and IL-1β (30%), suggesting that the therapeutic function of the NPs comes from the gold core, and alkyl chain can improve the efficacy by enhanced delivery to epidermal cells. In addition, the amounts of psoriasis-related cytokines, IL-17, IL-12/23 p40, and IL-1β, in octadecyl.sub.30%-PEG-Au.sub.3 NPs treated skin are significantly reduced by 41.7%, 47.9%, and 35%, respectively.
2.2.1.2 Evaluation of Efficacy of Free PEG-octadecyl.SUB.30% .Mixture, Free PEG Strands, and PBS Solvent
[0179] To understand the effects of each component in the NP formulation, the effects of free PEG-octadecyl.sub.30% mixture, free PEG strands, and PBS in the prevention mode are further evaluated. During the experiment, it is observed that PEG strands can moisturize the treated skin aera but may not inhibit psoriasis.
[0180] After being treated in the prevention mode, free PEG-octadecyl.sub.30% mixture, free PEG strands, and PBS buffer treated skin do not show obvious improvement when compared to IMQ only treated skin. By histological analysis, free PEG-octadecyl.sub.30% mixture, free PEG strands, and PBS buffer treated groups do not significantly reduce epidermis thickness and number of proliferative keratinocytes and CD3.sup.+ T cells as shown in
2.2.1.3 Efficacy of Another Type of Au.sub.3-based NP
[0181] Au.sub.3 NPs are further coated with polythymidine segments instead of PEG to form “spherical nucleic acids” (termed Au.sub.3@T12 NPs). The loading of polythymidine is 17.16±0.13 strands per Au.sub.3 NP. By DLS, the hydrodynamic size of Au.sub.3@T12 NPs is 11.97±0.57 nm, which is similar to that of Au.sub.3@PEG-alkyl.sub.y % NPs. After incubation in PBS for 24 hours, the hydrodynamic size (14.03±1.81 nm) does not show significant change, confirming the stability of Au.sub.3@T12 NPs.
[0182] Topically application of IMQ cream in conjunction with Au.sub.3@T12 NPs (200 μL of PBS containing 6 μM NPs) in accordance with the “prevention mode”, Au.sub.3@T12 NPs exhibit anti-psoriasis efficacy, leading to significant reduction in mean epidermis thickness (34.3%) and population of Ki67 cells (24.5%) as shown in
[0183] DEGs cluster heat map shows highly similar profile for both types of AuNPs treatment in upregulated (red) and downregulated (blue) genes when compared to IMQ control as shown in
2.2.1.4 Biodistribution of Au.sub.3@PEG-alkyl.sub.y % NPs After the Treatment in Prevention Mode
[0184] Apart from efficacy, the biodistributions of Au.sub.3@PEG-alkyl.sub.y % NPs at the end of the prevention mode are also assessed. ICP-MS data show that both Au.sub.3@PEG-methoxy NPs and Au.sub.3@PEG-octadecyl.sub.30% NPs mostly accumulate in the skin layer with limited uptake by internal organs as shown in
2.2.1.5 Acute Toxicity Evaluation of Au.sub.3@PEG-Alkyl.sub.y % NPs After the Treatment in Prevention Mode
[0185] During the experiment, no abnormal behaviors from treated mice are observed. the body weight of each mouse is monitored during the prevention mode. From Day 1 to Day 7, the mean body weights of all tested groups do not show significant difference as shown in
[0186] The major internal organs for histological analysis on Day 7 are also collected. Histological staining shows that the concurrent application of NP and IMQ does not cause any obvious damage to the liver, spleen and kidney, even though these organs take up comparatively more gold content than their counterparts as shown in
[0187] As expected, the alkylation of NPs significantly promoted the entry of NPs to viable epidermis (VE) (for example P=0.004, t test) and keratinocytes as shown in
[0188]
2.2.2 Topical Application of Au.sub.3@PEG-Octadecyl.sub.30% NPs Treats the IMQ-Induced Psoriatic Skin Condition in the “Treatment Mode”.
[0189] To evaluate the translational potential of Au.sub.3@PEG-octadecyl.sub.30% NPs, their abilities to treat psoriasis are determined by topically applying daily doses of NPs for 4 days onto mice with pre-established psoriasis obtained from 6 days of IMQ application as shown in
[0190] It is noted that symptoms of psoriasis dissipate naturally when IMQ cream is discontinued. Applying the IMQ cream is stopped from Day 7 onwards for two reasons. (1) long-term (>8 days) use of IMQ cream may cause significant body weight loss (>20%) and higher mortality of mice; (2) separating the application of IMQ cream and AuNPs excludes the possibility that AuNPs inhibit IMQ from activating the inflammatory cascade, allowing for testing the true therapeutic potential of the NPs.
[0191] Compared to PBS control, four days of Au.sub.3@PEG-octadecyl.sub.30% NPs or BC treatment resulted in lower PASI scores, smaller scales and shorter induration as shown in
[0192] However, with BC treatment, thinning of the dermis is observed, which likely cause the wrinkles seen in
[0193] Irrespective of the treatment, the animals maintain a stable body weight from Day 7 to Day 11 as shown in
[0194] On day 11, the spleen to body weight percentage is 0.729% in PBS control, and 0.66% in the octadecyl.sub.30%-PEG-Au.sub.3 NPs treatment group. Daivobet® ointment reduces the percentage to 0.395%, which is below the healthy group's value of 0.437%, indicating possible adverse systemic effects of the corticosteroid drug. The concentrations of skin cytokine IL-17, IL-12/23 p40, and IL-1β are also reduced to near-normal level upon treatment with both octadecyl.sub.30%-PEG-Au.sub.3 NPs and Daivobet® ointment.
2.2.3 Topical Application of Au.sub.3@PEG-Octadecyl.sub.30% NPs Do not Cause Long-Term Toxicity
[0195] The long-term toxicity of Au.sub.3@PEG-octadecyl.sub.30% NPs is evaluated by topically applying them to psoriatic mice and sacrificing the animals after 4 weeks as shown in
[0196] On Day 38, blood markers of ALT, AST and BUN remain in the normal range, indicating the normal liver and kidney function as shown in
[0197] The anti-psoriatic potential of Au.sub.3@PEG-octadecyl.sub.30% NPs in the “prevention mode” is interrogate by concurrently applying IMQ and NPs for 6 days. Remarkably, it reveals that Au.sub.3@PEG-octadecyl.sub.30% NPs, without drug loading, exhibit anti-psoriatic inflammation efficacy by inhibiting the development of psoriasis phenotype and significantly reducing epidermis thickness, proliferative keratinocytes, and CD3.sup.+ T cell infiltration, and psoriasis-related cytokines. Notably, Au.sub.3@PEG-methoxy NPs and Au.sub.3@T12 NPs also exhibit anti-inflammatory effects, although not as effective as these of the Au.sub.3@PEG-octadecyl.sub.30% NPs, while free PEG-octadecyl.sub.30% mixture, free PEG strands, or PBS do not show obvious effect in inhibiting psoriasis.
[0198] Accordingly, the data indicate that Au.sub.3@PEG-octadecyl.sub.30% NPs and Au.sub.3@PEG-methoxy NPs inhibit the development of IMQ-induced psoriasis and inflammation, and surface alkylation of NPs leads to more effective inhibition. These data reinforce our conclusion that AuNPs are self-therapeutic for anti-psoriatic inflammation.
[0199] Furthermore, a “treatment mode” is adopted by establishing psoriasis in a mouse IMQ model for 6 days before topically applying 4 doses of Au.sub.3@PEG-octadecyl.sub.30% NPs. During the treatment period, both Au.sub.3@PEG-octadecyl.sub.30% NPs and standard steroid and vitamin D analog therapy show similar levels of anti-psoriatic efficacy when compared to the PBS treated mice. At the end of treatment, Au.sub.3@PEG-octadecyl.sub.30% NPs exhibit an anti-psoriasis efficacy similar to that of standard steroid and vitamin D analog therapy, but with significantly reduced side effects such as skin thinning and hair loss of the ointment.
[0200] Moreover, no NPs are found in major organs and no cutaneous or systemic toxicity is observed in the animals four weeks post-application.
Embodiment Three: Mechanism for the Anti-Psoriasis Efficacy of Alkyl-Terminated Gold Nanoparticles
3.1.2 Reported Anti-Inflammation and Antiangiogenic Mechanism of Gold NPs
[0201] The mechanisms of anti-inflammatory of gold salts involve inhibition of the release of lysosomal enzymes of phagocytic cells.sup.46, modulation of some prostaglandins.sup.47, and inhibition the proliferation of synovial cells as well as collagen synthesis.sup.48. Similar to gold salts, the AuNPs are reported to interfere with the transmission of inflammatory signaling and acted as immuno-suppressant.sup.49.
3.1.1 RNA Sequencing (RNA-Seq) Technology
[0202] Transcriptomes are essential for interpreting the functional elements of the genome and understanding development and disease. The introduction of high-throughput next-generation sequencing (NGS) technologies revolutionized transcriptomics.sup.50. RNA sequencing (RNA-Seq) technique uses NGS to reveal the presence and quantity of RNA in a biological sample. Of particular interest is the discovery of differentially expressed genes across different conditions. Since RNA-seq first appeared in literature in 2008.sup.51, the number of publications containing RNA-Seq data has increased exponentially over the past decade owing to the decreasing costs. A typical RNA-Seq experiment consists of isolating RNA, converting it to complementary DNA (cDNA), preparing the sequencing library, and sequencing it on an NGS platform.
[0203] To gain insights into the anti-psoriatic efficacy of Au.sub.3@PEG-octadecyl.sub.30% NPs, RNA-Seq is performed on skin samples of mice that receive treatments of “IMQ only”, “IMQ+Au.sub.3@PEG-methoxy NPs”, or “IMQ+Au.sub.3@PEG-octadecyl.sub.30% NPs” in “prevention mode” by BGISEQ platform.
3.2 Materials and Methods
[0204] 3.2.1 Whole-Transcriptome Analysis with Total RNA-Seq
[0205] After conducting efficacy studies in the “prevention mode”, about 60 mg of skin biopsies is harvested from each IMQ-induced psoriatic mouse that are distributed in three treatment groups (n=3 for each group), including (i) IMQ only without NP treatment, (ii) IMQ with topical application of Au.sub.3@PEG-methoxy NPs, and (iii) IMQ with topical application of Au.sub.3@PEG-octadecyl.sub.30% NPs. The harvested skin is snap frozen in liquid nitrogen, stored at −80° C., and later sent to Beijing Genomics Institute (BGI) via courier mail for RNA extraction, RNA library construction, and bioinformatic analysis. The mRNA is isolated from total RNA using oligo (dT) magnetic beads following the manufacturer's instructions for cDNA library construction. Double stranded cDNA is sequenced using the DNBseq platform. At least 20 million clean reads per sample on the DNBseq platform are generated for data analysis.
3.2.2 RNA-Seq Data Analysis
[0206] Differential expressed gene (DEG) detection, gene ontology (GO) analysis of DEG, and other analysis based on gene expression are performed by BGI. GO terms and DEGs with corrected p values (Q values) smaller than 0.05 are considered significantly enriched. Gene analysis is performed using the Dr. Tom analysis platform (BGI). For heatmap analysis, the mean normalized FPKM (Fragments Per Kilobase Million) of each group is normalized against the mean normalized FPKM of the IMQ only control group, with the normalized, relative expression levels expressed as log 2. For scatter plots, the normalized FPKM of each sample from different groups is shown in log 2 (FPKM+1) scale.
3.2.3 Quantitative Reverse-Transcription Polymerase Chain Reaction (qRT-PCR).
[0207] After conducting efficacy studies in the “prevention mode”, about 60 mg of skin biopsies is harvested from each IMQ-induced psoriatic mouse that are distributed in three treatment groups (n=3 for each group), including (i) IMQ only without treatment, (ii) IMQ with topical application of free PEG strands, and (iii) IMQ with topical application of a 7:3 molar mixture of free PEG to PEG-octadecyl strands (“PEG-octadecyl.sub.30%”). The harvested skin is snap frozen in liquid nitrogen and stored at −80° C.
[0208] RNA is isolated using the Trizol reagent (Thermo Fisher Scientific) and reverse-transcribed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) to generate cDNA. qRT-PCR is performed on StepOnePlus™ Real-Time PCR System using TB SYBR Green Premix Ex Taq kit (Takara) following the manufacturer's instructions. Gene expression is quantified using pre-designed primers purchased from Shanghai Rui Mian Bio Tech (see sequences below).
TABLE-US-00004 Forward sequence (5′ Reverse sequence Gene to 3′) (5′ to 3′) Mouse CAGACTACCTCAACCGTTCCAC TCCAGCTTTCCCTCCGCATTG I117a (SEQ ID NO: 1) A (SEQ ID NO: 2) Mouse AACCAGGGCATTTCTGTCCCAC GGCATTGATGCAGCCTGAGTG I117f (SEQ ID NO: 3) T (SEQ ID NO: 4) Mouse TTGAACTGGCGTTGGAAGCACG CCACCTGTGAGTTCTTCAAAG I112b (SEQ ID NO: 5) GC (SEQ ID NO: 6) Mouse TGGACCTTCCAGGATGAGGACA GTTCATCTCGGAGCCTGTAGT I11b (SEQ ID NO: 7) G (SEQ ID NO: 8) Mouse GGTGCCTATGTCTCAGCCTCTT GCCATAGAACTGATGAGAGGG Tnf (SEQ ID NO: 9) AG (SEQ ID NO: 10) Mouse CATCACTGCCACCCAGAAGACT ATGCCAGTGAGCTTCCCGTTC GAPDH G (SEQ ID NO: 11) AG (SEQ ID NO: 12)
3.3 Results
3.3.1 Overview of the RNA-Seq Results
[0209] The RNAs of 9 skin samples from 3 treatment groups (n=3 for each group, “IMQ only”, “IMQ+Au.sub.3@PEG-methoxy NPs”, or “IMQ+Au.sub.3@PEG-octadecyl.sub.30% NPs”) are sequenced by BGISEQ platform. The average mapping ratio with reference genome is 95.54%, the average mapping ratio with gene is 77.65%; and 18200 genes are identified. In order to reflect the correlation of gene expression between samples, the Pearson correlation coefficients of all gene expressions between every two samples are calculated and shown in the heatmap as shown in
[0210] Next, the Venn diagram and Volcano map are used to show the differential expressed genes (DEGs) in each pairwise comparison. There are 2428 DEGs detected in Pair 1 (“IMQ only” vs “IMQ+Au.sub.3@PEG-octadecyl.sub.30% NPs”), with 1374 downregulated genes and 1054 upregulated genes in the “IMQ+Au.sub.3@PEG-octadecyl.sub.30% NPs” group. In Pair 2 (“IMQ only” vs “IMQ+Au.sub.3@PEG-methoxy NPs”), there are 343 DEGs detected, with 208 downregulated genes and 135 upregulated genes in “IMQ+Au.sub.3@PEG-methoxy NPs” group. While there are only 25 DEGs in Pair 3 (“IMQ+Au.sub.3@PEG-methoxy NPs” vs “IMQ+Au.sub.3@PEG-octadecyl.sub.30% NPs”) comparison.
[0211] The data indicate that application of Au.sub.3@PEG-octadecyl.sub.30% NPs modulates more genes when compared to Au.sub.3@PEG-methoxy NPs, although the number of DEGs between the two NP groups is limited.
3.3.2 Au.sub.3@PEG-octadecyl.sub.30% NPs Significantly Suppresses Proinflammatory Genes in Psoriatic Skin
[0212] Gene ontology (GO) analysis demonstrates that “IMQ+Au.sub.3@PEG-octadecyl.sub.30% NPs” group shows significant upregulation of keratinocyte differentiation as well as downregulation of epidermis development and inflammatory processes when compared to the “IMQ only” group of
[0213] In terms of the enriched differentially expressed genes (DEGs) obtained by benchmarking the “IMQ+Au.sub.3@PEG-octadecyl.sub.30% NPs” group against the “IMQ only” group (IMQ only vs. IMQ+Au.sub.3@PEG-octadecyl.sub.30% NPs), Au.sub.3@PEG-octadecyl.sub.30% NPs lead to significant suppression of three classes of genes that are linked to psoriasis as shown in
[0214] The inhibited genes include (i) cytokines that are released during psoriasis pathogenesis (for example, 65-fold lower expression for Il12b, 11-fold for IL17f, and 6-fold for Il1b which directly validates our observed ELISA data in
[0215]
TABLE-US-00005 TABLE 4 Enriched biological processes found in the pairwise comparison “IMQ only” vs “IMQ + Au.sub.3@PEG-octadecyl.sub.30% NPs” (Q value < 0.05). GO_ biological process Q value ribosome biogenesis 5.04E−13 sterol biosynthetic process 9.74E−08 rRNA processing 4.36E−07 cholesterol biosynthetic process 1.83E−06 lipid metabolic process 4.70E−06 cell cycle## 4.94E−05 steroid metabolic process 4.94E−05 cholesterol metabolic process 7.56E−05 oxidation-reduction process 9.21E−05 cell division## 1.81E−04 epidermis development## 3.25E−04 steroid biosynthetic process 7.10E−04 Myelination 7.10E−04 immune response## 8.01E−04 response to toxic substance 0.0019 keratmocyte differentiation## 0.0019 proteasomal ubiquitin-independent protein catabolic process 0.0028 negative regulation of cell proliferation## 0.0029 positive regulation of establishment of protein localization to 0.0044 telomere regulation of catalytic activity 0.0053 cellular response to hypoxia 0.0061 mitotic cell cycle 0.0064 positive regulation of neuron projection development 0.0067 glutamine metabolic process 0.0083 isoprenoid biosynthetic process 0.0083 proteasomal protein catabolic process 0.0083 mitotic spindle assembly 0.0149 skeletal muscle tissue regeneration 0.0162 positive regulation of protein serine/threonine kinase activity 0.0184 inflammatory response## 0.0230 cytoskeleton organization 0.0230 Aging 0.0230 skeletal muscle fiber adaptation 0.0234 glutathione metabolic process 0.0240 negative regulation of interferon-gamma production 0.0269 positive regulation of gene expression 0.0276 peptide cross-linking## 0.0349 positive regulation of cell migration## 0.0371 glycogen metabolic process 0.0403 fatty acid metabolic process 0.0403 response to vitamin E 0.0403 Note: The lines indicated by “##” are the enriched biological processes shown in FIG. 51A that are directly related to the anti-psoriasis efficacy of the Au.sub.3@PEG-octadecyl.sub.30% NPs.
TABLE-US-00006 TABLE 5 Enriched biological processes (top 40) found in the pairwise comparison “IMQ only” vs “IMQ + Au.sub.3@PEG-methoxy NPs” (Q value < 0.05). GO_ biological process Q value sterol biosynthetic process 1.82E−13 lipid metabolic process 3.17E−13 cholesterol biosynthetic process 3.09E−11 steroid biosynthetic process 2.95E−10 isoprenoid biosynthetic process 3.68E−08 cholesterol metabolic process 5.50E−08 steroid metabolic process 7.22E−08 response to organic cyclic compound 0.0076 lipid biosynthetic process 0.0116 response to virus 0.0116 keratinocyte differentiation 0.0116 immune response 0.0139 regulation of cell proliferation 0.0146 keratinization 0.0159 response to toxic substance 0.0178 positive regulation of fat cell differentiation 0.0178 muscle contraction 0.0197 ergosterol biosynthetic process 0.0212 regulation of midbrain dopaminergic neuron differentiation 0.0212 negative regulation of myoblast proliferation 0.0212 skeletal muscle atrophy 0.0228 cell chemotaxis 0.0228 fatty acid metabolic process 0.0250 negative regulation of insulin secretion 0.0288 epidermal growth factor receptor signaling pathway 0.0346 xylulose metabolic process 0.0413 isopentenyl diphosphate biosynthetic process 0.0413 isopentenyl diphosphate biosynthetic process, mevalonate 0.0413 pathway skeletal muscle fiber development 0.0413 dimethylallyl diphosphate biosynthetic process 0.0413 negative regulation of planar cell polarity pathway 0.0413 involved in axis elongation fatty acid biosynthetic process 0.0422 smoothened signaling pathway 0.0447 response to fatty acid 0.0447 positive regulation of canonical Wnt signaling pathway 0.0447 negative regulation of lipid biosynthetic process 0.0470
[0216] Further, psoriasis is induced to mice via IMQ and concurrently applied free PEG or free PEG-octadecyl.sub.30% strands for 6 consecutive days, followed by harvesting the treated skin on Day 7. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis reveals no significant difference in the expression levels of key genes in psoriasis pathology (including Il17a, Il17f, Il12b, Il1b and Tnf) in both treatment groups when compared to the IMQ control as shown in
TABLE-US-00007 TABLE 6 Enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathways in the three pairwise comparison groups (gene level, Q < 0.05). Pairwise comparison Q groups KEGG Pathway value “IMQ only” vs “IMQ + Ribosome biogenesis in eukaryotes 3.99E−07 Au.sub.3@PEG-octadecyl.sub.30% Proteasome 4.49E−07 NPs” Steroid biosynthesis 0.0024 Spliceosome 0.0132 Calcium signaling pathway 0.0132 Biosynthesis of antibiotics 0.0138 RNA transport 0.0138 AMPK signaling pathway 0.0151 Methane metabolism 0.0171 Terpenoid backbone biosynthesis 0.0241 Aminoacyl-tRNA biosynthesis 0.0241 Cytokine-cytokine receptor 0.0361 interaction Primary bile acid biosynthesis 0.0390 Glutathione metabolism 0.0396 Biosynthesis of secondary 0.0399 metabolites IL-17 signaling pathway## 0.2079 “IMQ only” vs “IMQ + Steroid biosynthesis 3.27E−08 Au.sub.3@PEG-methoxy Terpenoid backbone biosynthesis 3.27E−08 NPs” Biosynthesis of secondary 4.72E−06 metabolites Biosynthesis of antibiotics 7.42E−06 Metabolic pathways 0.0068 Sesquiterpenoid and triterpenoid 0.0088 biosynthesis “IMQ + Au.sub.3@PEG- IL-17 signaling pathway 0.0084 methoxy NPs” vs “IMQ + Au.sub.3@PEG- octadecyl.sub.30% NPs” Note: The genes indicated by “##” are also shown in FIG. 54.
TABLE-US-00008 TABLE 7 Enriched biological processes (top 40) found in the pairwise comparison “IMQ + Au.sub.3@PEG-methoxy NPs” vs “IMQ + Au.sub.3@PEG-octadecyl.sub.30% NPs” (Q < 0.05). Q GO_ biological process value membrane raft polarization 0.0338 glandular epithelial cell differentiation 0.0338 negative regulation of gliogenesis 0.0338 peptide cross-linking 0.0338 collagen catabolic process 0.0338 posttranslational protein targeting to membrane, translocation 0.0338 myelination 0.0338 detection of mechanical stimulus involved in sensory perception 0.0338 of touch regulation of neutrophil mediated killing of gram-negative 0.0338 bacterium oxygen metabolic process 0.0338 defense response to other organism 0.0338 protein insertion into plasma membrane 0.0338 cell aggregation 0.0338 positive regulation of pancreatic trypsinogen secretion 0.0338 response to methamphetamine hydrochloride 0.0338 positive regulation of cell proliferation in midbrain 0.0338 cellular response to glial cell derived neurotrophic factor 0.0338 ovarian cumulus expansion 0.0359 response to symbiotic bacterium 0.0359 response to hormone 0.0359 negative regulation by host of viral exo-alpha-sialidase activity 0.0359 negative regulation by host of viral glycoprotein metabolic 0.0359 process negative regulation of exo-alpha-sialidase activity 0.0359 negative regulation of glycoprotein metabolic process 0.0359 central nervous system development 0.0362 epidermis development 0.0362 protein oxidation 0.0362 transformation of host cell by virus 0.0362 keratinocyte differentiation 0.0362 fatty acid beta-oxidation using acyl-CoA oxidase 0.0362 T-helper 2 cell cytokine production 0.0362 cartilage development 0.0362 negative regulation of cation channel activity 0.0362 skeletal system development 0.0391 protein localization to paranode region of axon 0.0391 cell communication by electrical coupling 0.0391 neuronal signal transduction 0.0391 regulation of chemokine production 0.0391 mitotic cell cycle phase transition 0.0391 male germ-line stem cell asymmetric division 0.0391 positive regulation of NK T cell differentiation 0.0391
TABLE-US-00009 TABLE 8 List of representative DEGs in the three pairwise comparisons that are related to psoriasis-related genes. “IMQ + Au.sub.3@PEG- “IMQ only” vs “IMQ only” vs methoxy NPs” vs “IMQ + Au.sub.3@PEG- “IMQ + Au.sub.3@PEG- “IMQ + Au.sub.3@PEG- Gene octadecyl.sub.30% NPs” methoxy NPs” octadecyl.sub.30% NPs” Category Gene ID Symbol Log.sub.2 ## Q value Log.sub.2 Q value Log.sub.2 Q value i. Cytokine 12985 Csf3 −6.18 0.0001 −3.14 0.0126 −3.02 0.9757 16160 Il12b −5.90 0.0013 −3.71 0.1108 −2.18 1.0000 50929 Il22 −4.86 0.0182 −1.44 0.5938 −3.39 1.0000 257630 Il17f −3.66 0.0000 −2.16 0.1154 −1.49 1.0000 16171 Il17a −3.57 0.0000 −1.73 0.3281 −1.84 1.0000 329244 Il19 −3.22 0.0023 −2.66 0.0000 −0.54 1.0000 83430 Il23a −2.98 0.0003 −1.45 0.1222 −1.53 1.0000 16176 Il1b −2.84 0.0004 −1.36 0.4208 −1.48 1.0000 16175 Il1a −2.39 0.0014 −2.67 0.0015 0.29 1.0000 54448 Il1f6 −2.08 0.0074 −1.76 0.0400 −0.31 1.0000 77125 Il33 −1.96 0.0000 −0.24 0.8963 −1.71 0.1408 53603 Tslp −1.53 0.0421 −0.98 0.4045 −0.54 1.0000 16181 Il1rn −1.40 0.0028 −1.10 0.0114 −0.29 1.0000 21926 Tnf −1.37 0.0041 −0.95 0.2306 −0.41 1.0000 215257 Il1f9 −1.02 0.0477 −0.71 0.2197 −0.31 1.0000 ii. hemokine 20311 Cxcl5 −5.22 0.0290 1.65 0.8068 −6.85 0.0133 330122 Cxcl3 −5.07 0.0010 −2.86 0.1778 −2.20 1.0000 20310 Cxcl2 −4.89 0.0113 −2.73 0.3847 −2.16 0.9324 20302 Ccl3 −3.80 0.0002 −2.25 0.1959 −1.54 1.0000 20303 Ccl4 −3.05 0.0003 −1.86 0.3134 −1.19 1.0000 14825 Cxcl1 −2.68 0.0000 −1.91 0.0145 −0.76 1.0000 20297 Ccl20 −1.40 0.0000 −2.91 0.0092 1.51 0.9622 iii. AMPs 27358 Defb3 −4.73 0.0143 −1.81 0.1623 −2.91 0.9463 56519 Defb4 −4.62 0.0051 −0.81 0.7821 −3.79 0.6442 381493 S100a7a −4.14 0.0000 −1.20 0.6179 −2.93 0.3007 20201 S100a8 −2.56 0.0092 −0.95 0.0979 −1.61 0.8439 244332 Def14 −2.33 0.0016 −1.14 0.2706 −1.18 0.2181 67860 S100a16 −1.15 0.0005 −0.57 0.1773 −0.57 0.9926 16687 Krt6a −3.58 0.0000 −1.39 0.1561 −2.18 0.0804 16688 Krt6b −3.26 0.0000 −1.19 0.2394 −2.06 0.1334 16666 Krt16 −2.37 0.0000 −1.30 0.0345 −1.06 0.5152 16664 Krt14 −1.28 0.0001 −0.39 0.6814 −0.88 0.6417 14283 Fosl1 −3.41 0.0001 −2.73 0.0323 −0.67 1.0000 21802 Tgfa −1.28 0.0194 −1.30 0.0091 0.02 1.0000 230738 Zc3h12a −1.20 0.0134 −1.40 0.0094 0.21 1.0000 329502 Pla2g4e −2.08 0.0001 −1.74 0.0000 −0.33 1.0000 78390 Pla2g4d −2.58 0.0006 −1.02 0.3111 −1.55 0.4074 211429 Pla2g4b −2.29 0.0008 −1.96 0.0099 −0.33 1.0000 17394 Mmp8 −2.91 0.0377 2.43 0.5623 −5.33 0.5122 17386 Mmp13 −1.68 0.0049 1.55 0.2444 −3.21 0.0170 17395 Mmp9 −1.08 0.0007 2.81 0.2130 −3.88 0.0308 319191 Hist1h2ai −5.98 0.0044 −0.31 0.9235 −5.66 0.1016 319152 Hist1h3h −5.45 0.0102 −0.61 0.8071 −4.83 0.4815 Note: i. cytokine, ii. Chemokine, iii. antimicrobial peptides (AMPs), and iv. Others [e.g., keratins (Krt) and matrix metalloproteinases (Mmp)]. The log.sub.2 values of the representative genes in the pairwise comparasion of “IMQ only” vs “IMQ + Au.sub.3@PEG-octadecyl.sub.30% NPs” column, indicated by “##”, are shown in FIGS. 50A-50B. These genes are selected since they are downregulated genes downstream of the IL-17 siganlling pathways as shown in FIG. 54.
indicates data missing or illegible when filed
TABLE-US-00010 TABLE 9 Raw data for the significantly regulated genes shown in Table 8. Expression (FPKM), n = 3 Gene “IMQ + Au.sub.3@PEG- “IMQ + Au.sub.3@PEG- Gene ID Symbol “IMQ only” octadecyl.sub.30% NPs” methoxy NPs” 32985 Csf3 0.57 2.21 2.44 0 0 0.09 0.14 0.37 0.13 16160 ## Il12b 0.21 0.68 0.41 0.02 0 0 0 0 0.12 50929 Il22 0.99 0.44 0.33 0 0 0.06 0 0.48 0.23 257630 Il17f 7.45 3.18 1.63 0.48 0.27 0.33 0.85 1.9 0.27 ## 16171 Il17a 4.24 2.57 1.69 0.54 0.22 0.05 0.05 2.07 0.85 ## 329244 Il19 8.98 6.2 6.16 2.03 0.13 0.38 0.74 2.27 0.92 83430 Il23a 1.98 2.14 1.57 0.09 0.18 0.56 1.09 0.44 0.82 16176 ## Il1b 7.89 28.09 37.11 1.76 6.53 4.17 1.96 25.65 7.39 16175 Il1a 8.87 37.46 31.86 9.12 3.91 4.83 4.67 3.09 6.65 54448 Il1f6 125.47 434.14 478.29 112.56 47.09 133.57 114.81 113.19 135 77125 Il33 56.25 29.99 31.2 15.18 10.6 8.62 52.73 39.69 16.44 53603 Tslp 100.05 150.12 119.02 68.21 33.38 61.62 68.58 68.75 60.54 16181 Il1rn 176.06 264.85 290.7 148.91 91.84 180.96 159.46 158.43 201.24 21926 ## Tnf 4.73 2.79 2.09 1.41 1.28 1.55 2.32 1.72 1.44 215257 Il1f9 6.7 3.91 5.3 3.79 1.63 0.9 4.69 1.07 3.1 20311 Cxcl5 3.47 0 0.46 0.07 0.04 0 0.26 12.57 0.55 330122 Cxcl3 0.66 11.6 10.89 0.13 0 0.7 0.06 3.07 0.87 20310 Cxcl2 0.68 29.68 35 0.23 0.53 1.9 0.41 9.72 2.26 20302 Ccl3 2.79 23.63 25.14 0.66 2.59 1.35 0.58 8.9 3.95 20303 Ccl4 3.03 5.66 8.99 0.4 1.54 0.62 0.2 4.58 1.01 14825 Cxcl1 9.17 12.25 10.92 1.48 2.17 2.32 1.26 5.35 3.53 20297 Ccl20 15.93 13.39 10.86 4.66 5.68 7.21 2.35 0.24 3.4 ## 27358 Defb3 844.94 2509.79 3382.65 36.63 3.48 258.67 176.13 1391.94 772.69 56519 Defb4 1.35 4.91 6.85 0 0 0.64 1.41 7.05 0.62 381493 S100a7a 3.18 21.27 26.28 0.53 0.92 0.44 0.43 17.04 9.07 ## 20201 S100a8 6446.06 8851.08 11443.7 1194.98 283.67 3757.88 5346.27 6187.3 4543.96 244332 Defb14 28.05 88.09 123.88 22.12 11.76 19.43 30.97 49.26 44.29 ## 67860 S100a16 654.18 641.75 687.94 398.1 251.79 388.12 561.93 429.34 525.99 16687 Krt6a 3261.82 3479.3 4377.28 472.12 232.5 375.26 1742.4 2627.34 513.87 16688 Krt6b 4291.22 4234.44 5536.2 740.5 356.26 604.56 2674.02 3644.6 726.15 16666 Krt16 6600.56 7098.97 8859.96 1824.08 1244.8 2023.13 4398.18 3938.65 2082.43 ## 16664 Krt14 6702.37 6950.78 9997.84 4252.37 3311.5 3856.63 8709.45 7556.63 4489.55 14283 Fosl1 2.7 10.39 10.42 1.55 0.4 0.71 0.58 0.65 3.08 ## 21802 Tgfa 22.42 39.99 28.78 16.28 8.58 18.95 14.32 13.59 15.04 ## 230738 Zc3h12a 130.53 176.07 117.72 58.18 50.99 105.29 78.87 57.83 44.46 ## 329502 Pla2g4e 117.85 183.35 155.54 47.84 21.08 55.83 49.99 48.83 59.84 ## 78390 Pla2g4d 7.35 19.41 22.59 2.38 1.76 5.56 5.3 13.1 11.05 211429 Pla2g4b 44.45 152.5 132.41 27.88 15.51 36.63 26.43 37.44 37.09 17394 Mmp8 0.38 1.54 2.98 0.02 0.6 0.2 0.19 32.55 0.37 17386 Mmp13 1.35 0.64 1.07 0.43 0.27 0.39 4.47 4.77 0.65 17395 Mmp9 3.75 2.24 2.32 1.43 1.5 1.55 8.73 57.14 1.56 319191 Hist1h2ai 1.36 0.9 2.87 0 0 0 1.68 1.38 1.61 319152 Hist1h3h 1.42 0.98 0.86 0 0 0 0.68 1.07 0.75 Note: The genes indicated by “##” are also shown in FIG. 51C.
[0217] Next, the DEGs (for example, Q≤0.05) related to (1) cytokines, (2) chemokines, (3) antimicrobial peptides (AMPs), and (4) other psoriasis-related genes are analysed, in the octadecyl.sub.30%-PEG-Au.sub.3 NPs treatment vs. IMQ control comparison as shown in
[0218] First, in line with the ELISA results of
[0219] Second, the chemokines are small cytokines representing a large group of small chemotactic proteins (about 8-11 kDa in size) that guide the movement of leukocytes to sites of inflammation.sup.42. In the skin, epidermal keratinocytes are able to express multiple chemokines that can attract certain leukocytes, such as T cells or dendritic cells (DCs), to migrate to the epidermis. It is recognized that CCL4 chemokine is a chemoattractant for cells bearing CCR1 and CCR5 receptors, for example, Th1 cells, immature dendritic cells, NK cells, and monocytes.sup.44. CCL20 attracts cells with CCR6 receptor—Th1 lymphocytes, dendritic cells and monocytes, while CXCL8 and CXCL2 are chemoattractants for neutrophils expressing receptors for IL-8 (CXCR1 and CXCR).
[0220] The heat map in
[0221] Third, AMPs generally are small amino acids residues (composed of 12-50 amino acids) that have positive charge and amphipathic structure.sup.45. These antimicrobial molecules are best known for their integral role in killing pathogenic microorganisms; but they can also affect inflammatory responses by acting as chemotactic agents, angiogenic factors, and regulators of cell proliferation. Various AMP, such as b-defensins, S100 proteins and cathelicidin, are highly expressed in psoriatic lesions.sup.41. The heat map in
[0222] Finally, the expression of miscellaneous genes that are related to psoriasis is further examined. Keratins (KRT) are the major structural intermediate filament proteins in keratinocytes and are expressed in a highly specific pattern at different differentiation stages of keratinocytes. Recent studies have recognized KRT6/16/17 as key early barrier alarmins and upregulation of these keratins alters proliferation, cell adhesion, migration and inflammatory features of keratinocytes, contributing to hyperproliferation and innate immune activation of keratinocytes, followed by the autoimmune activation of T cells that drives psoriasis.sup.46-47.
[0223] After treatment with octadecyl.sub.30%-PEG-Au.sub.3 NPs, the downregulation of KRT6/16/17 genes are observed in
3.3.3 Molecular Basis of Alkylation Effects
[0224] The data suggest a molecular basis that explains how alkylation enhances anti-psoriasis efficacy. The pairwise comparison of “IMQ+Au.sub.3@PEG-methoxy NP” vs. “IMQ+Au.sub.3@PEG-octadecyl.sub.30% NP” reveals significant inhibition of genes related to psoriasis (for example, 15-fold lower expression for Mmp9 and 9-fold for Mmp13; as shown in
[0225] RN A-seq is used to profile the anti-psoriatic efficacy mechanism of alkyl-terminated AuNPs. RNA-seq results reveal that Au.sub.3@PEG-octadecyl.sub.30% NPs concurrently with induction of psoriasis downregulated genes that are linked to epidermal hyperproliferation and inflammation. These genes are not only significantly enriched in cytokine-cytokine receptor interaction signaling pathway, but also in the downstream of the IL-17 signaling pathway and the downstream of the TNF signaling pathway. The results also demonstrate that Au.sub.3@PEG-methoxy NPs downregulated similar types of genes or biological processes that are suppressed by Au.sub.3@PEG-octadecyl.sub.30% NPs, but less effective. These data directly validate the efficacy observed and suggest that the alkylated NPs inhibit psoriasis by blocking the communication between different inflammatory cells. In addition, the upregulation of membrane-raft polarization in Au.sub.3@PEG-octadecyl.sub.30% NPs treated skin explains the enhanced delivery of Au.sub.3@PEG-octadecyl.sub.30% NPs to keratinocytes that is superior to that of the Au.sub.3@PEG-methoxy NPs. Moreover, the qRT-PCR data suggest limited influence of the PEG strands or alkyl chains on gene expression, indicating the changes in the expression of psoriasis marker genes following treatment with Au.sub.3@PEG-octadecyl.sub.30% NPs primarily stem from the Au core.
[0226] The sub-15 nm Au.sub.3@PEG-octadecyl.sub.30% NPs are self-therapeutic agents for treating psoriasis. It has been proven that NP size and alkyl loading are critical parameters for topical delivery of NPs to the epidermal cells of healthy and psoriatic skin. The sub-15 nm size of Au.sub.3@PEG-octadecyl.sub.30% NPs allows penetration of the SC and accumulation in the epidermis without the aid of excipients, and an optimal loading of octadecyl groups (30 mol %) maintains the colloidal stability of Au cores in skin while boosting their uptake by epidermal cells.
[0227] The results are significant because they demonstrate that surface engineering of NPs with functional groups (through alkylation in this case) can overcome a major delivery hurdle to epidermal keratinocytes for treating psoriasis. Moreover, the therapeutic component of Au.sub.3@PEG-octadecyl.sub.30% NPs is the Au core, even though Au.sub.3@PEG-octadecyl.sub.30% NPs, devoid of known chemical and biological anti-psoriatic drugs, inhibit psoriasis by inhibiting genes that are enriched in the downstream of IL-17 signaling pathway and linked to epidermis hyperproliferation and inflammation. Optimal alkylation of the Au cores yields improved efficacy and more significantly inhibited inflammatory genes by promoting the entry of Au cores to epidermal cells.
[0228] It is demonstrated that Au.sub.3@PEG-octadecyl.sub.30% NPs treat psoriasis as effectively as a commercial ointment that contains steroid- and vitamin D analog, but without long-term retention in major internal organs or inducing long-term toxicity.
[0229] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.
[0230] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto.
REFERENCES
[0231] 1. Mehrmal, S., Uppal, P., Nedley, N., Giesey, R. L. & Delost, G. R. The global, regional, and national burden of psoriasis in 195 countries and territories, 1990 to 2017: A systematic analysis from the Global Burden of Disease Study 2017. J. Am. Acad. Dermatol, 84, 46-52 (2021). [0232] 2. Griffiths, C. E. & Barker, J. N. Pathogenesis and clinical features of psoriasis. Lancet 370, 263-271 (2007). [0233] 3. Boehncke, W. H. & Schön, M. P. Psoriasis. Lancet 386, 983-994 (2015). [0234] 4. Pariser, D. M. et al. National Psoriasis Foundation clinical consensus on disease severity. Arch. Dermatol. 143, 239-242 (2007). [0235] 5. Lowes, M. A., Bowcock, A. M. & Krueger, J. G. Pathogenesis and therapy of psoriasis. Nature 445, 866-873 (2007), [0236] 6. Auerbach, R. Methotrexate in psoriasis: Revised guidelines. J. Am. Acad. Dermatol. 19, 145-156 (1988). [0237] 7. Warren, E. W. & Khanderia, U. Use of retinoids in the treatment of psoriasis. Clin. Pharm, 8, 344-351 (1989), [0238] 8. Rosmarin, D. M., Lebwohl, M., Elewski, B. E. & Gottlieb, A. B. Cyclosporine and psoriasis: 2008 National Psoriasis Foundation* Consensus Conference. J. Am. Acad. Dermatol. 62, 838-853 (2010). [0239] 9. Lebwohl, M. et al. From the Medical Board of the National Psoriasis Foundation: Monitoring and vaccinations in patients treated with biologics for psoriasis. J. Am. Acad. Dermatol. 58, 94-105 (2008). [0240] 10. Fernandez-Ruiz, M. & Aguado, J. M. Risk of infection associated with anti-TNT-α therapy. Expert Rev. Anti. Infect. Ther. 16, 939-956 (2018). [0241] 11. Castela, E. et al. Topical corticosteroids in plaque psoriasis: A systematic review of efficacy and treatment modalities. J. Eur. Acad. Dermatology Venereol. 26, 36-46 (2012). [0242] 12. Fereig, S. A., El-Zaafarany, G. M., Arafa, M. G. & Abdel-Mottaleb, M. M. A. Tackling the various classes of nano-therapeutics employed in topical therapy of psoriasis. Drug Deliv. 27, 662-680 (2020). [0243] 13, Arora, R., Katiyar, S. S., Kushwah, V. & Jain, S. Solid lipid nanoparticles and nanostructured lipid carrier-based nanotherapeutics in treatment of psoriasis: a comparative study, Expert Opin. Drug Deliv. 14, 1.65-177 (2017). [0244] 14. Avasatthi, V. et al. A novel nanogel formulation of methotrexate for topical treatment of psoriasis: optimization, in vitro and in vivo evaluation. Pharm. Dev. Technol. 21, 554-562 (2016). [0245] 15. Ferreira, M. et al. Methotrexate loaded lipid nanoparticles for topical management of skin-related diseases: Design, characterization and skin permeation potential. Int. J. Pharm. 512, 14-21 (2016). [0246] 16, Sapino, S., Oliaro-Bosso, S., Zonari, D., Zanotti, A. & Ugazio, E. Mesoporous silica nanoparticles as a promising skin delivery system for methotrexate. Int. J. Pharm. 530, 239-248 (2017). [0247] 17. Kohler, N., Sun, C., Wang, J. & Zhang, M. Methotrexate-modified superparamagnetic nanoparticles and their intracellular uptake into human cancer cells. Langmuir 21, 8858-8864 (2005). [0248] 18. Wong, L. S., Tymms, K. E. & Buckley, N. A. Potential for methotrexate exposure through contamination during parenteral use as an immunosuppressant, Intern. Med. J. 39, 379-383 (2009). [0249] 19, Bessar, H. et al. Functionalized gold nanoparticles for topical delivery of methotrexate for the possible treatment of psoriasis. Colloids Surfaces B Biointerfaces 141, 141-147 (2016), [0250] 20. Pinto, M. F. et al. A new topical formulation for psoriasis: Development of methotrexate-loaded nanostructured lipid carriers, Int. J. Pharm. 477, 519-526 (2014), [0251] 21. Fratoddi, I. et al. Effects of topical methotrexate loaded gold nanoparticle in cutaneous inflammatory mouse model. Nanomedicine Nanotechnology, Biol. Med. 17, 276-286 (2019), [0252] 22. Ozcan, A. et al. Nanoparticle-Coupled Topical Methotrexate Can Normalize Immune Responses and Induce Tissue Remodeling in Psoriasis. J. Invest. Dermatol. 140, 1003-1014.e8 (2020). [0253] 23. Ferreira, M. et al. Topical co-delivery of methotrexate and etanercept using lipid nanoparticles: A targeted approach for psoriasis management. Colloids Surfaces B Biointerfaces 159, 23-29 (2017). [0254] 24. Viegas, J. S. R. et al. Nanostructured lipid carrier co-delivering tacrolimus and TNF-α siRNA as an innovate approach to psoriasis. Drug Deliv. Transl. Res. 10, 646-660 (2020). [0255] 25. Zheng, D. et al. Topical delivery of siRNA-based spherical nucleic acid nanoparticle conjugates for gene regulation. Proc. Natl. Acad. Sci. U.S.A. 109, 11975-11980 (2012). [0256] 26. Lewandowski, K. T. et al. Topically Delivered Tumor Necrosis Factor-α-Targeted Gene Regulation for Psoriasis. J. Invest. Dermatol. 137, 2027-2030 (2017). [0257] 27. Nemati, H. et al. Using siRNA-based spherical nucleic acid nanoparticle conjugates for gene regulation in psoriasis. J. Control. Release 268, 259-268 (2017). [0258] 28. Liu, H. et al. Targeting the IL-17 Receptor Using Liposomal Spherical Nucleic Acids as Topical Therapy for Psoriasis. J. Invest. Dermatol. 140, 435-444.e4 (2020). [0259] 29, Korkmaz, E. & Falo, L. D. Spherical Nucleic Acids as Emerging Topical Therapeutics: A Focus on Psoriasis. J. Invest. Dermatol. 140, 278-281 (2020). [0260] 30. Kim, J. Y. et al. Nanoparticle-Assisted Transcutaneous Delivery of a Signal Transducer and Activator of Transcription 3-Inhibiting Peptide Ameliorates Psoriasis-like Skin Inflammation. ACS Nano 12, 6904-6916 (2018). [0261] 31. Liang, H. et al. Topical nanoparticles interfering with the DNA-LL37 complex to alleviate psoriatic inflammation in mice and monkeys. Sci. Adv. 6, 1-15 (2020). [0262] 32. Hornos Carneiro, M. F. &. Barbosa, F. Gold nanoparticles: A critical review of therapeutic applications and toxicological aspects. J. Toxicol. Environ. Heal.—Part B Crit. Rev. 19, 129-148 (2016). [0263] 33. Arvizo, R. R. et al. Inhibition of tumor growth and metastasis by a self-therapeutic nanoparticle. Proc. Natl. Acad. Sci. U.S.A. 110, 6700-6705 (2013). [0264] 34. Saha, S. et al. Gold Nanoparticle Reprograms Pancreatic Tumor Microenvironment and Inhibits Tumor Growth. ACS Nano 10, 10636-10651 (2016). [0265] 35, Zhang, Y. et al. Gold nanoparticles inhibit activation of cancer-associated fibroblasts by disrupting communication from tumor and microenvironmental cells. Bioact. Mater. 6, 326-332 (2021), [0266] 36. Ho, L. W. C. et al. Effect of Alkylation on the Cellular Uptake of Polyethylene Glycol-Coated Gold Nanoparticles. ACS Nano 11, 6085-6101 (2017). [0267] 37. Flo, L. W. C., Yin, B., Dai, G. & Choi, C. H. J, Effect. of Surface Modification with Hydrocarbyl Groups on the Exocytosis of Nanoparticles. Biochemistry (2020) doi: 10, 1021/acs.biochem.0c00631. [0268] 38. Lowes, M. A., Suárez-Fariñas, M. & Krueger, J. G. Immunology of psoriasis. Annu. Rev. Immunol. 32, 227-255 (2014). [0269] 39. van der Fits, L. et al. Imiquimod-Induced Psoriasis-Like Skin Inflammation in Mice Is Mediated via the IL-23/IL-17 Axis. J. Immunol. 182, 5836-5845 (2009). [0270] 40, Sonavane, G. et al. In vitro permeation of gold nanoparticles through rat skin and rat intestine: Effect of particle size. Colloids Surfaces B Biointerfaces 65, 1-10 (2008). [0271] 41. Ogawa, E., Sato, Y., Minagawa, A. & Okuyama, R. Pathogenesis of psoriasis and development of treatment. J. Dermatol. 45, 264-272 (2018). [0272] 42. Sokolowska-Wojdylo, M., Nedoszytko, B., Ruckemann-Dziurdzińska, K., Roszkiewicz, J. & Nowicki, R. J. Chemokines and cytokines network in the pathogenesis of the inflammatory skin diseases: atopic dermatitis, psoriasis and skin mastocytosis. (2014) doi:10.5114/pdia.2014.40920. [0273] 43. Lee, C. H. & Hwang, S. T. Y. Pathophysiology of chemokines and chemokine receptors in dermatological science: A focus on psoriasis and cutaneous T-cell lymphoma. Dermatologica Sin. 30, 128-135 (2012). [0274] 44. Méhul, B. et al. Noninvasive proteome analysis of psoriatic stratum corneum reflects pathophysiological pathways and is useful for drug profiling. Br. J. Dermatol. 177, 470-488 (2017). [0275] 45. Jones, R. Antimicrobial peptides in the pathogenesis of psoriasis Shin. Bone 23, 1-7 (2014). [0276] 46. Zhang, X., Yin, M. & Zhang, L. J. Keratin 6, 16 and 17-Critical Barrier Alarmin Molecules in Skin Wounds and Psoriasis. Cells 8, 1-14 (2019). [0277] 47, Lessard, J. C. et al. Keratin 16 regulates innate immunity in response to epidermal barrier breach. Proc. Natl. Acad. Sci. U.S.A. 110, 19537-19542 (2013). [0278] 48. Benhadou, F. et al. Epidermal autonomous VEGFA/Flt1/Ntp1 functions mediate psoriasis-like disease. Sci. Adv. 6, (2020). [0279] 49. Ruiz-Romeu, E. et al. MCPIP1 RNase Is Aberrantly Distributed in Psoriatic Epidermis and Rapidly Induced by IL-17A. J. Invest. Dermatol. 136, 1599-1607 (2016). [0280] 50. Rioux, et al. The tissue-engineered human psoriatic skin substitute: A valuable in vitro model to identify genes with altered expression in lesional psoriasis. Int. J. Mol. Sci. 19, (2018). [0281] 51. Mezentsev, A., Nikolaev, A. & Bruskin, S. Matrix metalloproteinases and their role in psoriasis. Gene 540, 1-10 (2014). [0282] 52. R. Brown, K., Andrew Lyon, L., P. Fox, A., D. Reiss, B. & Natan, M. Hydroxylamine Seeding of Colloidal Au Nanoparticles. 3. Controlled Formation of Conductive Au Films. Chem. Mater. 12, 314-323 (2000). [0283] 53. Mühlpfordt, H. The preparation of colloidal gold particles using tannic acid as an additional reducing agent. Experientia 38, 1127-1128 (1982). [0284] 54. Frens, G. Controlled nucleation for the regulation of the particle size in monodisperse gold suspension. Nature 241, 20-22 (1973). [0285] 55. Haiss, W., Thanh, N. I. K., Aveyard, J. & Fernig, D. G. Determination of size and concentration of gold nanoparticles from UV-Vis spectra. Anal. Chem. 79, 4215-4221 (2007). [0286] 56. Chou, L. Y. T. & Chan, W. C. W. Fluorescence-Tagged Gold Nanoparticles for Rapidly Characterizing the Size-Dependent Biodistribution in Tumor Models. Adv. Healthc. Mater. 1, 714-721 (2012). [0287] 57. Dunnett, C. W. Multiple Comparisons between Several Treatments and a Specified Treatment, in Linear Statistical Inference (eds. Caliński, T. & Klonecki, W.) 39-47 (Springer New York, 1985). [0288] 58. Giljohann, D. A. et al. Gold nanoparticles for biology and medicine. Angew. Chemie—Int. Ed. 49, 3280-3294 (2010). [0289] 59. Daniel, M.-C. & Astruc, D. Gold Nanoparticles: Assembly, Supramolecular Chemistry, Quantum-Size-Related Properties, and Applications toward Biology, Catalysis, and Nanotechnology. Chem. Rev, 104, 293-346, (2004). [0290] 60. Enustun, B. V & Turkevich, J. Coagulation of Colloidal Gold. J. Am. Chem. Soc. 85, 3317-3328 (1963). [0291] 61. Polte, J. et al. Mechanism of Gold Nanoparticle Formation in the Classical Citrate Synthesis Method Derived from Coupled In Situ XANES and SAXS Evaluation. J. Am. Chem. Soc. 132, 1296-1301 (2010). [0292] 62. Brown, K. R., Walter, D. G. &. Natal), M. J. Seeding of Colloidal Au Nanoparticle Solutions. 2. Improved Control of Particle Size and Shape. Chem. Mater. 12, 306-313 (2000). [0293] 63. Worthen, A. J., Tran, V., Cornell, K. A., Truskett, T. M. & Johnston, K. P. Steric stabilization of nanoparticles with grafted low molecular weight ligands in highly concentrated brines including divalent ions. Soft Matter 12, 2025-2039 (2016). [0294] 64. Yamashita, S. Heat-induced antigen retrieval: Mechanisms and application to histochemistry. Prog. Histochem. Cytochem. 41, 141-200 (2007), [0295] 65. Gilleron, J. et al. Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape. Nat. Biotechnol. 31, 638-646 (2013). [0296] 66. McGrath, J. A. & Uitto, J. Anatomy and organization of human skin. Rook's Textbook of Dermatology 1-53 (2010) doi:https://doi.org/10.1002/9781444317633. ch3 [0297] 67. Larese Filon, F., Mauro, M., Adami, G., Bovenzi, M. & Crosera, M. Nanoparticles skin absorption: New aspects for a safety profile evaluation. Regul. Toxicol. Pharmacol. 72, 310-322 (2015). [0298] 68. Liu, Y. et al. Dopamine Receptor-Mediated Binding and Cellular Uptake of Polydopamine-Coated Nanoparticles, ACS Nano 15, 13871-13890 (2021), [0299] 69. Bos, J. D. & Meinardi, M. M. H. M. The 500 Dalton rule for the skin penetration of chemical compounds and drugs. Exp. Dermatol. 9, 165-169 (2000). [0300] 70. Vogt, A. et al. Nanocarriers for drug delivery into and through the skin Do existing technologies match clinical challenges? J. Control. Release 242, 3-15 (2016). [0301] 71. Hornos Carneiro, M. F. & Barbosa, F. Gold nanoparticles: A critical review of therapeutic applications and toxicological aspects. J. Toxicol. Environ. Heal.—Part B Crit. Rev. 19, 129-148 (2016). [0302] 72. Zhang, Y. et al. Gold nanoparticles inhibit activation of cancer-associated fibroblasts by disrupting communication from tumor and microenvironmental cells. Bioact. Mater. 6, 326-332 (2021). [0303] 73. Crisan, D. et al. Topical silver and gold nanoparticles complexed with Cornus mas suppress inflammation in human psoriasis plaques by inhibiting NT-κB activity. Exp. Dermatol. 27, 1166-1169 (2018). [0304] 74. Yang, H. et al. Mechanism for the Cellular Uptake of Targeted Gold Nanorods of Defined Aspect Ratios. Small 12, 5178-5189 (2016), [0305] 75. Chen, Z. et al. Specific Delivery of Oligonucleotides to the Cell Nucleus via Gentle Compression and Attachment of Polythymidine. ACS App. Mater. Interfaces 11, 27624-27640 (2019). [0306] 76. Hurst, S. J., Lytton-Jean, A. K. R. & Mirkin, C. A. Maximizing DNA loading on a range of gold nanoparticle sizes. Anal. Chem. 78, 8313-8318 (2006). [0307] 77. Dunnett, C. W. Multiple comparisons between several treatments and a specified treatment, in Linear Statistical Interference (eds. Caliński, T. & Klonecki, W.) 39-47 (Springer New York, 1985). [0308] 78. Rijzewijk, J. J., Van Erp, P. E. & Bauer, F. W. Two binding sites for Ki67 related to quiescent and cycling cells in human epidermis. Acta Derm. Venereol. 69, 512--515 (1989). [0309] 79. Veale, D. J., Barnes, L., Rogers, S. &. FitzGerald, O. Immunohistochemical markers for arthritis in psoriasis. Ann. Rheum. Dis. 53, 450-454 (1994). [0310] 80. Lowes, M. A., Bowcock, A. M. & Krueger, J. G. Pathogenesis and therapy of psoriasis. Nature 445, 866-873 (2007). [0311] 81. Vasseur, P. et al. Liver fibrosis is associated with cutaneous inflammation in the imiquimod-induced murine model of psoriasiform dermatitis. Br. J. Dermatol 179, 101-109 (2018). [0312] 82. Liang, H. et al. Topical nanoparticles interfering with the DNA-LL37 complex to alleviate psoriatic inflammation in mice and monkeys. Sci. Adv. 6, 1-15 (2020). [0313] 83. Kragballe, K. et al. A 52-week randomized safety study of a calcipotriol/betamethasone dipropionate two-compound product (Dovobet®/Daivobet®/Tacionex®) in the treatment of psoriasis vulgaris. Br. J. Dermatol. 154, 1155-1160 (2006). [0314] 84. Camisa, C. & Garofola, C. 45—Topical Corticosteroids. in (ed. Wolverton, S. E. B. T.-C. D. D. T. (Fourth E.) 511-527.e6 (Elsevier. 2021). doi:https://doi.org/10.1016/B978-0-323-61211-1.00045-0. [0315] 85, Stenn, K. S., Paus, R., Dutton, T. & Sarba, B. Glucocorticoid Effect on Hair Growth Initiation: A Reconsideration. Skin Pharmacol. Physiol. 6, 125-134 (1993). [0316] 86. Higby, G. J. Gold in medicine—A review of its use in the west before 1900. Gold Bull. 15, 130-140 (1982). [0317] 87, Champion, G. D., Graham, G. G. & Ziegler, J. B. The gold complexes. Baillieres. Clin. Rheumatol, 4, 491-534 (1990). [0318] 88. Kean, W. F. & Kean, I. R. L. Clinical pharmacology of gold. Inflammopharmacology 16, 112-125 (2008). [0319] 89. Mukherjee, P. et al. Antiangiogenic properties of gold nanoparticles. Clin. Cancer Res. 11, 3530-3534 (2005). [0320] 90. Dimartino, M. J. R. Walz, D. T. Inhibition of lysosomal enzyme release from rat leukocytes by auranofin. A new chrysotherapeutic agent. Inflammation 2, 131-142 (1977). [0321] 91. Stone, K. J., Mather, S. J. & Gibson, P. P. Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone. Prostaglandins 10, 241-251 (1975). [0322] 92. Goldberg, R. L., Parrott, D. P., Kaplan, S. R. & Fuller, G. C. Effect of gold sodium thiomalate on proliferation of human rheumatoid synovial cells and on collagen synthesis in tissue culture. Biochem. Pharmacol. 29, 869-876 (1980), [0323] 93. Sumbayev, V. V et al. Gold Nanoparticles Downregulate Interleukin-1β-Induced Pro Inflammatory Responses. Small 9, 472-477 (2013). [0324] 94. Koch, C. M. et al. A beginner's guide to analysis of RNA sequencing data. Am. J. Respir. Cell Mol. Biol. 59, 145-157 (2018). [0325] 95. Nagalakshmi, U. et al. The Transcriptional Landscape of the Yeast Genome Defined by RNA Sequencing. Science (80-.). 320, 1344 LP-1349 (2008). [0326] 96. Schuck, S. & Simons, K. Polarized sorting in epithelial cells: raft clustering and the biogenesis of the apical membrane. J. Cell Sci. 117, 5955-5964 (2004). [0327] 97. Ho, L. W. C., Liu, Y., Han, R., Bai, Q. & Choi, C. H. J. Nano-Cell Interactions of Non-Cationic Bionanomaterials. Acc. Chem. Res. 52, 1519-1530 (2019).