Hydrogel-forming microneedles with programmed mesophase transitions for controlled drug delivery

Abstract

The present invention provides an array of microneedles composed of tri-block amphiphiles that can be programmed to undergo in situ sequential mesophase transitions for controlled drug release.

Description

[0001] HYDROGEL-FORMING MICRONEEDLES WITH PROGRAMMED MESOPHASE TRANSITIONS FOR CONTROLLED DRUG DELIVERY

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a drug delivery system in the form of solid microneedles that undergo a first mesophase transition to swollen hydrogel needles upon contact with the biological fluids, followed by a second phase transition to hydrogel microparticles, thereby enabling the controlled release of a drug incorporated therein. The hydrogel microparticles further undergo enzymatic degradation and transform into soluble hydrophilic polymers enabling substantially complete clearance of the delivery system once the drug has been released.

[0004] BACKGROUND OF THE INVENTION

[0005] There is a critical demand for advanced drug delivery systems to offer an efficient drug localization at targeted sites and to circumvent the complications associated with drug delivery routes including reduced drug stability stemming from rapid metabolism of certain drugs, nonspecific biodistribution, and lack of patient adherence. Microneedles (MNs), which consist of an array of needles with heights ranging from 50 to 1500 pm, show promising prospects in transdermal drug delivery. MNs based delivery systems are able to penetrate the skin barrier without stimulating nerve fibres, allowing a minimally-invasive alternative to parenteral drug administration. MNs provide an efficient approach for drug penetration and an improved bioavailability, as they create micron-sized pores, allowing the transfer of drugs and macromolecules through the distinct layers of the skin. Hence, the use of MNs holds the potential of broadening the spectrum of the drugs that can be delivered by conventional transdermal dosage forms, which are limited to drugs with molecular masses of less than 500 Da, having high partition coefficient and high potency. Additionally, the utilization of MNs for drug delivery offers a strategic advantage by circumventing challenges associated with oral drug delivery within the gastrointestinal (GI) tract such as first-pass metabolism, enzymatic degradation, and GI irritation.

[0006] Microneedles are classified into five different categories according to their design and drug delivery mechanism, namely solid, hollow, coated, dissolving, and hydrogel-forming microneedles (Al-Japairai et al., Inter J Pharma 2020, 587, 119673). Among these different types, hydrogel-forming (HF) MNs are an emerging type in which the hydrogel is formed in situ as a result of the uptake of interstitial fluids. This process leads to swelling and an increase in the dimensions of the resultant microneedles, while maintaining physical and chemical structure integrity. These hydrogel-forming (HF) MNs eventually allow for improved drug delivery into the skin (Turner et al., Macromol Biosc 2021, 21(2), 2000307). HF MNs are mainly fabricated from crosslinked polymers where altering the degree of crosslinking could be used to control the drug release and provide long-term and continuous drug administration for improved therapeutic outcomes (Tekko et al., Int J Pharm 2020, 586, 119580; Anjani et al., Eur J Pharm Biopharm 2021, 158, 294-312; Migdadi et al., J Cont Rel 2018, 285, 142-151; and Pan et al., Inter J Pharma 2022, 617, 121612).

[0007] Amphiphilic block copolymers have been utilized as building blocks in the development of nanocarriers due to their ability to self-assemble in aqueous solution, forming polymeric assemblies that can encapsulate an active ingredient inside the hydrophobic core and shield it from the biological environment (Torchilin at al., Adv Drug Deliv Rev 2011, 63 (3), 131-135). Moreover, due to the enhanced permeability and retention (EPR) effect, these assemblies are able to accumulate at the target site, such as inflamed or cancerous tissue, depending on their structural complexity and size (Mura et al., Nat Mater 2013, 12(11), 991— 1003; Zhao et al., Cancer Biol. Med. 2021, 18(2), 319-335). Furthermore, stimuli-responsive moieties can be incorporated to facilitate their disassembly and achieve targeted delivery.

[0008] Recently, stimuli-responsive polymeric MNs have attracted significant attention in the field of drug delivery. These MNs can release their payloads in response to endogenous or exogenous stimuli such as pH, reactive oxygen species, light, temperature, enzymes, and mechanical forces. Such MNs can be used to deliver drugs in a more controllable manner, improve the therapeutic efficacy, reduce potential side effects, and provide a precise dosing (Yang et al., Chem Eng J 2021, 426, 130561; Makvandi et al., Mater Today 2021, 47, 206- 222; and Liu et al., Pharma 2023, 15(5), 1407).

[0009] In the past decade, modular pathways were developed for the synthesis of polymeric amphiphiles based on dendrons with enzymatically cleavable end-groups. Using the high molecular precision, which emerges from the hydrophobic dendritic blocks, enzymatically degradable polymeric amphiphiles of various compositions, architectures and hydrophilic to hydrophobic ratios were synthesized and their self-assembly and enzymatic degradation were carefully characterized (Harnoy et al., JACS 2014, 136(21), 7531-7534; Segal et al., JACS 2017, 139(2), 803-810; and Slor et al., Biomacromol 2021, 22(3), 1197-1210). These studies have resulted in profound understanding of the structural parameters that govern the interaction of polymeric assemblies with enzymes. Based on these studies, the design and synthesis of triblock based amphiphiles that were used to fabricate hydrogel forming micro-fibers by electrospinning (Edelstein-Pardo et al., Chem. Mater. 2022, 34(14), 6367-6377), and enzymatically degradable hydrogels (Rathee et al., ACS Macro Lett. 2023, 12 814-820; and Rathee et al., Biomacromol. 2024, 25(6), 3607-3619) were recently demonstrated.

[0010] WO 2023 / 002476 describes a stimuli-induced delivery system comprising selfassembled amphiphilic tri-block copolymers in the form of micelles. The delivery system enables the release of a cargo from within the micelles following a transition to di-block copolymers micelles and subsequent activation by a protein to induce disassembly of the micelles.

[0011] WO 2024 / 052920 describes controlled-release delivery systems composed of a formulation containing a mixture of tri- and di-block copolymers that can be programmed to undergo sequential mesophase transitions in response to multiple stimuli by tuning the molecular composition of the formulation.

[0012] WO 2024 / 243887 describes a microneedle drug administration apparatus which comprises substrates, microneedle arrays, and a liquid-absorbing swelling material which is placed between the substrates to form a sandwich structure. The apparatus uses a mild water absorption and swelling mechanism and the natural peristaltic behavior of the gastrointestinal tract to pierce the intestinal wall using the microneedles to complete drug delivery.

[0013] CN 118477057 describes a near-infrared response temperature-sensitive drug-loaded microneedle patch comprising a bonding layer and a plurality of microneedles arranged on the bonding layer in an array mode, and drug hydrogel is loaded in the microneedles; wherein the microneedle comprises polyethylene glycol and soluble polysaccharide; the drug hydrogel comprises a temperature-sensitive material, a polymer gel material and a drug.

[0014] CN 118021707 describes a soluble microneedle transdermal drug delivery system comprising a substrate and a needle body, the nano capsule-like body is loaded by a biocompatible degradable polymer component.

[0015] CN 117159457 describes a preparation method of a temperature-sensitive phasechange medicine-carrying microgel-microneedle array capable of controlling medicine administration, which is characterized by a soap-free emulsion polymerization method. WO 2023 / 164306 describes microneedles for drug delivery that include a wax or other meltable material and methods of making and using such microneedles.

[0016] There is an unmet need for drug delivery systems to enable the controlled drug release at the target site of action with improved efficacy and reduced side effects.

[0017] SUMMARY OF THE INVENTION

[0018] The present invention provides a drug delivery system comprising a microneedle array that is designed to undergo multiple phase transitions following administration to release the drug incorporated therein in a controlled manner.

[0019] Disclosed herein are programable microneedles that are capable of in situ transformation into enzymatically degradable drug eluting hydrogels upon contact and absorption of biological fluids. The microneedles therefore afford the controlled drug release at the site of action, following which, full degradation by target enzymes into soluble hydrophilic polymers is achieved. The programmable mesophase transitions can be applied to afford local and effective treatment by increasing the residence time of the drug at the target site, enabling improved penetration, and sustainable delivery for an extended period of time, while minimizing side effects.

[0020] According to a first aspect, there is provided a delivery system comprising an active pharmaceutical ingredient incorporated within a plurality of solid microneedles comprising triblock amphiphile comprising two hydrophobic segments each comprising at least one stimulus-responsive cleavable site and a hydrophilic segment therebetween, wherein upon contacting a biological fluid, the solid microneedles undergo a first and a second phase transition thereby releasing the active pharmaceutical ingredient incorporated therein in a controlled manner, wherein the first phase transition comprises a transition from solid microneedles to hydrogel microneedles and the second phase transition comprises a transition from hydrogel microneedles to hydrogel microparticles, and wherein upon exposure of the hydrogel microparticles to a stimulus, the stimulus-responsive cleavable sites undergo cleavage thereby disassembling the hydrogel to dissolved polymers and fragments thereof.

[0021] According to certain embodiments, the plurality of microneedles have widths of about 50 pm to about 500 pm and heights of about 200 pm to about 1000 pm, including each value within the specified ranges. In other embodiments, the microneedles are extending from an approximately planar base substantially perpendicular to the microneedles. In some embodiments, the base comprises a polymer selected from a polysaccharide, cellulose derivatives, poly(acrylates and methacrylates), PEG derivatives, gelatin, silk fibroin, hyaluronic acid, and dextrin. Each possibility represents a separate embodiment. In further embodiments, the base comprises a polymer comprising alginic acid or derivatives thereof. In some embodiments, the base comprises a polymer selected from sodium, potassium and calcium alginate. In additional embodiments, the plurality of microneedles are disposed on a patch substrate. In one embodiment, the tip portion of the microneedles and the bottom portion of the microneedles are different.

[0022] According to various embodiments, the hydrophobic segments comprise a lipid. In one embodiment, the hydrophobic segments comprise a fatty acid. In another embodiment, the hydrophobic segments comprise a steroid. In yet another embodiment, the hydrophobic segments comprise cholesterol.

[0023] According to some embodiments, the hydrophobic segments comprise hydrophobic dendrons. In various embodiments, the hydrophobic dendrons comprise between 0 to 5 generations. In other embodiments, the hydrophobic dendrons comprise between 0 to 3 generations.

[0024] In certain embodiments, each generation of the hydrophobic dendron comprises a linear or branched C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C1-C20 alkylene, C2-C20 alkenylene, C2-C20 alkynylene or arylene moiety which is substituted at each end with a group selected from the group consisting of -O-, -S-, -NH-, -C(=O)-, -C(=O)-O-, -O-C(=O)-O-, -C(=O)-NH-, -NH-C(=O)-NH-, -NH-C(=O)-O-, -S(=O)-, -S(=O)-O-, -PO(=O)-O-, and any combination thereof. Each possibility represents a separate embodiment. In further embodiments, each generation of the hydrophobic dendron is terminated with cleavable fatty acids or steroidal end- groups.

[0025] In various embodiments, each generation of the dendron is derived from a compound having the following structure HX-Z-XH or HX-Z-CO2H, wherein X is independently at each occurrence NH, S or O, and Z is selected from C1-C10 alkylene, C2-C10 alkenylene, C2-C10 alkynylene, and arylene. Each possibility represents a separate embodiment. In other embodiments, each generation of the dendron is derived from a compound selected from the group consisting of HX-CH2-CH2-XH, HX-(CH2)I-3-CO2H and HX-CH2-CH(XH)-CH2-XH wherein X is independently at each occurrence NH, S or O. Each possibility represents a separate embodiment. In particular embodiments, each generation of the dendron is derived from a compound selected from the group consisting of HS-CH2-CH2-OH, HS-(CH2)I-3-CO2H and HS-CH2-CH(OH)-CH2-OH. Each possibility represents a separate embodiment.

[0026] In further embodiments, where the hydrophobic segments comprise hydrophobic dendrons, the stimulus cleavable sites are present at one or more of the terminal repeating units (i.e., terminal generations) of the hydrophobic dendrons, and / or in intermediary generations of the dendrons. Each possibility represents a separate embodiment. In additional embodiments, the stimulus-responsive cleavable site comprises a cleavable bond selected from the group consisting of a disulfide, a diselenide, an ester (including a boronate ester and a phosphate ester), an amide, an amidine, an imine, a carbamate, a carbonate, an acetal, a urea, a thiourea, a trithionate, a sulfate, a sulfamate, a phosphate, a phosphoamide, a hydrazone, an ether, a silyl ether, an oxyme, a boronic acid, a boronic ester, a nitro, and an azo. Each possibility represents a separate embodiment. Preferably, the stimulus-responsive cleavable site comprises an ester, an amide, and / or a urea. According to particular embodiments, the stimulus is an enzyme. According to other particular embodiments, the stimulus cleavable sites are selected from an ester, an amide, and a urea, and the stimulus is an enzyme selected from an esterase / lipase, an amidase, and a urease, respectively.

[0027] According to some embodiments, the hydrophilic segment comprises polyethylene glycol (PEG), poly aery lie acid, poly (hydroxy ethyl acrylate), poly(oligo-ethylene glycol acrylate), polyacrylamide, polymethyl oxazoline, poly ethyl oxazoline, polyvinylpyrrolidone (PVP), polysarcosine, polypeptide, polypeptoid, hydrophilic polymethacrylate, polyamine, hydrophilic nylon, polyvinyl alcohol, hydrophilic protein, or polycarbohydrate. Each possibility represents a separate embodiment. In one embodiment, the hydrophilic segment comprises polyethylene glycol (PEG). In particular embodiments, the hydrophilic segment has a molecular weight of about 0.5 to about 100 kDa, including each value within the specified range. In other particular embodiments, the hydrophilic segment has a molecular weight of about 0.5 to about 50 kDa, including each value within the specified range. In yet other particular embodiments, the hydrophilic segment has a molecular weight of about 0.5 to about 25 kDa, including each value within the specified range.

[0028] According to certain embodiments, the hydrophilic segment is linked to each hydrophobic segment by a group selected from the group consisting of -Z-X-, -X '-Z-X2-, and -Z^X^Z^X2-, wherein Z, Z1, and Z2are each independently selected from C1-C20 alkyl, C2- C20 alkenyl, C2-C20 alkynyl, C1-C10 alkylene, C2-C10 alkenylene, C2-C10 alkynylene, and arylene; X, X1, and X2are each independently selected from -O-; -S-; -NH-; -C(=O)-; -C(=O)- O-; -O-C(=O)-O-; -C(=O)-NH-; -NH-C(=O)-NH-; -NH-C(=O)-O-; -S(=O)-; -S(=O)-O-; - PO(=O)-O-; triazolylene, and any combination thereof. Each possibility represents a separate embodiment.

[0029] According to some embodiments, the microneedles further comprise an excipient. In particular embodiments, the excipient comprises polyethylene glycol (PEG). In other embodiments, the PEG has a molecular weight of about 10 to about 200 kDa, including each value within the specified range. In yet other embodiments, the PEG has a molecular weight of about 20 to about 150 kDa, including each value within the specified range. In various embodiments, the PEG has a molecular weight of about 20 to about 100 kDa, including each value within the specified range. In certain embodiments, the PEG has a molecular weight of about 25 to about 100 kDa, including each value within the specified range. In additional embodiments, the PEG has a molecular weight of about 30 to about 100 kDa, including each value within the specified range.

[0030] According to further embodiments, the active pharmaceutical ingredient is a steroid selected from the group consisting of dexamethasone, aldosterone, beclomethasone, betamethasone, budesonide, cloprednol, cortisone, cortivazol, deoxycortone, desonide, desoximetasone, difluorocortolone, fluclorolone, flumethasone, flunisolide, fluocinolone, fluocinonide, fluocortin butyl, fluorocortisone, fluorocortolone, fluorometholone, flurandrenolone, fluticasone, halcinonide, hydrocortisone, icomethasone, meprednisone, methylprednisolone, paramethasone, prednisolone, prednisone, tixocortol and triamcinolone; an antibiotic selected from the group consisting of ampicillin, dapsone, chloramphenicol, neomycin, cefaclor, cefadroxil, cephalexin, cephradine, erythromycin, clindamycin, lincomycin, amoxicillin, ampicillin, bacampicillin, carbenicillin, dicloxacillin, cyclacillin, picloxacillin, hetacillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, ticarcillin, rifampin, tetracycline, fusidic acid, lincomicyn, novobiocine, and spectinomycin; an antiinflammatory agent selected from the group consisting of betamethasone, prednisolone, piroxicam, aspirin, flurbiprofen, diflunisal, ibuprofen, fenoprofen, fenamate, ketoprofen, nabumetone, naproxen, diclofenac, indomethacin, sulindac, tolmetin, etodolac, ketorolac, oxaprozin, celecoxib, meclofenamate, mefenamic acid, oxyphenbutazone, phenylbutazone, salicylates, and phyto sphingosine; an anti-fungal agent selected from the group consisting of clotrimazole, miconazole, ketoconazole, nystatin, amphotericin B, and pimaricin; or respective pharmaceutically acceptable salts or derivatives thereof. Each possibility represents a separate embodiment.

[0031] According to some embodiments, the microneedles are characterized by a drug-loading capacity in the range of about 3% to about 50%, including each value within the specified range. According to other embodiments, the microneedles are characterized by a drug-loading capacity in the range of about 5% to about 30%, including each value within the specified range. According to yet other embodiments, the microneedles are characterized by a drugloading capacity in the range of about 5% to about 20%, including each value within the specified range.

[0032] According to several embodiments, the delivery system is suitable for administration via a route selected from transdermal, nasal, and vaginal. Each possibility represents a separate embodiment. In currently preferred embodiments, the delivery system is suitable for transdermal administration through the skin of a subject. In additional embodiments, there is provided a method of transdermally administering an active pharmaceutical ingredient to a subject in need thereof, the method comprising applying the delivery system disclosed herein to the skin of a subject.

[0033] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0034] BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 depicts a schematic illustration of the process of fabrication of Tri-C9 MNs with an alginate base, (i) Casting of the polymeric solution into PDMS mold to form the tips; (ii) Subjecting the template to a vacuum for 10 min; (iii) Casting the baseplate-forming solution; (iv) Drying for 24 hours; and (v) Demolding of the MNs array.

[0036] Figure 2 depicts HPLC chromatogram of dexamethasone at a wavelength of 241.5 nm.

[0037] Figures 3A-3C depict the fabrication of MNs from Tri-block C9 amphiphile into a PDMS mold. (3A) Fabrication from an ethanol solution; (3B) Fabrication from chloroform; and (3C) The empty PDMS mold. Scale bar - 1mm. Figure 4 depicts a microscope image showing a MN patch made of 5% w / v Tri-C9 / PVP (1:1). Scale bar - 1mm.

[0038] Figures 5A-5C depict microscope images of MN patches. (5A) A patch containing 3% (w / v) Tri-C9 amphiphile; (5B) A patch containing 5% (w / v) Tri-C9 amphiphile; and (5C) A patch containing 10% (w / v) Tri-C9 amphiphile. Scale bar - 1mm.

[0039] Figures 6A-6F depict microscope images of Tri-C9 MN patches incorporated with 3% w / v PLGA in different compositions before and after application of manual force. (6A) MNs containing 3% w / v Tri-C9 and PLGA at a 1:1 ratio (top) and MNs containing tips composed of 3% w / v PLGA and bottoms composed of 3%w / v Tri-C9 (bottom) with a sodium alginate base before application of manual force; (6B) A microscope image of needles of 3%w / v Tri-C9 and PLGA (1:1) after insertion into the parafilm layer; (6C) The first layer of parafilm into which the MNs composed of 3% w / v Tri-C9 and PLGA (1:1) were inserted (at 93%); (6D) The second layer of parafilm into which the MNs composed of 3% w / v Tri-C9 and PLGA (1:1) were inserted (at 60%); (6E) A microscope image of needles composed of tips of 3%w / v PLGA and bottoms of 3% w / v Tri-C9 after insertion into the parafilm layer; and (6F) The first layer of parafilm into which the MNs composed of tips of 3% w / v PLGA and bottoms of 3% w / v Tri- C9 were inserted.

[0040] Figures 7A-7C depict microscope images of a Tri-C9 MN patch incorporating 10% w / w microcrystalline cellulose (MCC) before and after application of manual force. (7 A) Micrograph of the patch before application of manual force; (7B) A microscope image of needles after insertion into the parafilm layer; and (7C) A representative image of the first layer of parafilm into which the MNs were inserted.

[0041] Figures 8A-8I depict microscope images of 3% w / v Tri-C9 MN with or without different concentrations of PEG 3.4 kDa excipient before and after application of manual force. (8A) MNs containing 3%w / v Tri-C9 before application of manual force; (8B) MNs containing 3% w / v Tri-C9 after application of manual force; (8C) A representative image of the first layer of parafilm into which MNs containing 3%w / v Tri-C9 were inserted; (8D) MNs containing Tri-C9 MN incorporated with 5% w / w (with respect to the Tri-C9 polymer) PEG 3.4 kDa before application of manual force; (8E) MNs containing Tri-C9 MN and 5% w / w PEG 3.4 kDa after application of manual force; (8F) A representative image of the first layer of parafilm into which MNs containing Tri-C9 MN and 5% w / w PEG 3.4 kDa were inserted; (8G) MNs containing Tri-C9 MN incorporated with 10% w / w (with respect to the Tri-C9 amphiphile) PEG 3.4 kDa before application of manual force; (8H) MNs containing Tri-C9 MN and 10% w / w PEG 3.4 kDa after application of manual force; and (81) A representative image of the first layer of parafilm into which MNs containing Tri-C9 MN and 10% w / w PEG 3.4 kDa were inserted.

[0042] Figures 9A-9D depict representative images of the MN patches with 10% w / w PEG / Tri-C9. (9A) 3.4 kDa PEG; (9B) 10 kDa PEG; (9C) 35 kDa PEG; and (9D) 100 kDa PEG.

[0043] Figures 10A-10J depict the insertion test in the parafilm skin simulant model. (10A) Percentage of holes created in the first two parafilm layers by the MNs; (10B) Percentage of MN height reduction post insertion to parafilm layers, results are expressed as means ± s.d., n=10; (10C) A micrograph of MNs composed of Tri-C9 MN and PEG 3.4 kDa after insertion into parafilm layer; (10D) A micrograph of MNs composed of Tri-C9 MN and PEG 10 kDa after insertion into parafilm layer; (10E) A micrograph of MNs composed of Tri-C9 MN and PEG 35 kDa after insertion into parafilm layer; (10F) A micrograph of MNs composed of Tri- C9 MN and PEG 100 kDa after insertion into parafilm layer; (10G) A microscope image showing the holes observed on the first layer of a paraffin film by MNs composed of Tri-C9 MN and PEG 3.4 kDa; (10H) A microscope image showing the holes observed on the first layer of a paraffin film by MNs composed of Tri-C9 MN and PEG 10 kDa; (101) A microscope image showing the holes observed on the first layer of a paraffin film by MNs composed of Tri-C9 MN and PEG 35 kDa; and (10J) A microscope image showing the holes observed on the first layer of a paraffin film by MNs composed of Tri-C9 MN and PEG 100 kDa.

[0044] Figures 11A-11I depict the evaluation of mechanical strength against static force. (11A) A microscope image showing the morphological changes of 35 kDa / Tri-C9 MNs after placement of 0 g; (11B) A microscope image showing the morphological changes of 35 kDa / Tri-C9 MNs after placement of 50 g; (11C) A microscope image showing the morphological changes of 35 kDa / Tri-C9 MNs after placement of 500 g; (11D) A microscope image showing the morphological changes of 35 kDa / Tri-C9 MNs after placement of 1000 g; (HE) A microscope image showing the morphological changes of 100 kDa / Tri-C9 MNs after placement of 0 g; (11F) A microscope image showing the morphological changes of 100 kDa / Tri-C9 MNs after placement of 50 g; (11G) A microscope image showing the morphological changes of 100 kDa / Tri-C9 MNs after placement of 500 g; (11H) A microscope image showing the morphological changes of 100 kDa / Tri-C9 MNs after placement of 1000 g; and (111) Percentage of MN height reduction after compression, results are expressed as means ± s.d., n=10.

[0045] Figure 12 depicts DSC curves for neat Tri-C9 (solid line), neat 35kDa PEG (dashed line) and Tri-C9 with 10% w / w 35kDa PEG (dotted line).

[0046] Figures 13A-13F depict SEM images of MNs and fragments. (13A) Blank Tri-C9 MNs with 35 kDa PEG; (13B) DEX-loaded Tri-C9 MNs; (13C) Blank Tri-C9 MNs with 100 kDa PEG; (13D) DEX-loaded Tri-C9 MNs with 100 kDa PEG; (13E) A fractured tip and arrow pointing to the area analysed by EDS; and (13F) The baseplate separated from the needles and analysed individually. Scale bar - 100 pm.

[0047] Figures 14A-14B depict representative images of DEX-loaded MNs following the parafilm insertion test. (14A) Tri-C9 MNs with 35 kDa PEG; and (14B) Tri-C9 MNs with 100 kDa PEG.

[0048] Figures 15A-15D depict digital microscopic view of MN tips upon exposure to PBS solution (pH=7.4) for different time durations. (15A) t= 0; (15B) t=14 seconds; (15C) t= 26 seconds; and (15D) Images of the aqueous medium containing the obtained hydrogel after 15 minutes and 8 days.

[0049] Figure 16 depicts the in vitro release of dexamethasone from Tri-C9 MNs (pH 7.4, 37°C). The values are mean ± s.d. of three experiments.

[0050] Figure 17 depicts HPLC overlay after dissolving the gel formed at the end of the release experiment.

[0051] Figures 18A-18D depict skin insertion test of DEX-loaded Tri-C9 MNs (35kDa PEG). (18A) A schematic representation of the compression tester set-up for the determination of insertion force of MNs using ex vivo chicken skin; (18B) Representative force-displacement curves of the MNs pressed against chicken skin. The point of insertion exhibits small plateau marked by a circle; (18C) Top view of chicken skin after MN insertion; and (18D) Top view of chicken skin after staining by trypan blue.

[0052] Figures 19A-19C depict microscope images of CTZ-loaded MNs. (19A) Tri-C6 MNs; (19B) Tri-C9 MNs; and (19C) Tri-C12 MNs. Scale bar - 500 pm.

[0053] Figures 20A-20I depict the insertion capabilities of CTZ-loaded MNs composed of Tri-block amphiphiles with different lengths of end groups. (20A) A microscope image of Tri- C6 MNs before mechanical force; (20B) A representative image of the first layer of parafilm into which Tri-C6 MNs were inserted; (20C) A microscope image of Tri-C6 MNs after insertion into the parafilm layer; (20D) A microscope image of Tri-C9 MNs before mechanical force; (20E) A representative image of the first layer of parafilm into which Tri-C9 MNs were inserted; (20F) A microscope image of Tri-C9 MNs after insertion into the parafilm layer; (20G) A microscope image of Tri-C12 MNs before mechanical force; (20H) A representative image of the first layer of parafilm into which Tri-C12 MNs were inserted; and (201) A microscope image of Tri-C12 MNs after insertion into the parafilm layer.

[0054] Figures 21A-21H depict the insertion capabilities of CTZ-loaded MNs composed of combinations between tri-block amphiphiles. (21A) A schematic representation of a MNs array composed of Tri-C6 and Tri-C9 at a 1:1 ratio, clotrimazole and PEG 35kDa on a sodium alginate base; (2 IB) A microscope image of Tri-C6 and Tri-C9 at a 1:1 ratio, clotrimazole and PEG 35kDa MNs array before application of mechanical force; (21C) A representative image of the first layer of parafilm into which Tri-C6 and Tri-C9 at a 1:1 ratio, clotrimazole and PEG 35kDa MNs were inserted; (21D) A microscope image of needles of Tri-C6 and Tri-C9 at a 1:1 ratio, clotrimazole and PEG 35kDa MNs after insertion into the parafilm layer; (2 IE) A schematic representation of a MNs array composed of a tip of Tri-C12 and bottom of Tri-C6 loaded with clotrimazole on a sodium alginate base; (21F) A microscope image of MNs composed of a tip of Tri-C12 and bottom of Tri-C6 loaded with clotrimazole before application of mechanical force; (21G) A representative image of the first layer of parafilm into which needles composed of a tip of Tri-C12 and bottom of Tri-C6 loaded with clotrimazole were inserted; and (21H) A microscope image of needles composed of a tip of Tri-C12 and bottom of Tri-C6 loaded with clotrimazole after insertion into the parafilm layer.

[0055] Figures 22A-22H depict the insertion capabilities of AmB-loaded MNs composed of different formulations of Tri-C9 amphiphile. (22A) A schematic representation of a MNs array composed of needles of Tri-C9, amphotericin B and PEG 35kDa on a sodium alginate base; (22B) A microscope image of Tri-C9, amphotericin B, and PEG 35kDa MNs array before application of mechanical force; (22C) A representative image of the first layer of parafilm into which Tri-C9, amphotericin B, and PEG 35kDa MNs were inserted; (22D) A microscope image of needles of Tri-C9, amphotericin B, and PEG 35kDa MNs after insertion into the parafilm layer; (22E) A schematic representation of a MNs array composed of a tip of amphotericin B and bottom of Tri-C9 and PEG 35kDa on a sodium alginate base; (22F) A microscope image of MNs composed of a tip of amphotericin B and bottom of Tri-C9 and PEG 35kDa before application of mechanical force; (22G) A representative image of the first layer of parafilm into which needles composed of a tip of amphotericin B and bottom of Tri-C9 and PEG 35kDa were inserted; and (22H) A microscope image of needles composed of a tip of amphotericin B and bottom of Tri-C9 and PEG 35kDa after insertion into the parafilm layer.

[0056] Figures 23A-23D depict microscope images of AmB loaded MN patches prepared from ethanol or methanol viscous solutions. (23A) Tri-C6, AmB, and PEG 35kDa prepared using ethanol; (23B) Tri-C6, AmB, and PEG 35kDa prepared using methanol; (23C) Tri-C9, AmB, and PEG 35kDa prepared using ethanol; and (23D) Tri-C9, AmB, and PEG 35kDa prepared using methanol.

[0057] Figures 24A-24F depict microscope images of AmB loaded MN patches prepared from ethanol viscous solutions using different excipient concentrations. (24A) Tri-C6, AmB, and 10% PEG 35kDa; (24B) Tri-C6, AmB, and 20% PEG 35kDa; (24C) Tri-C9, AmB, and 10% PEG 35kDa; (24D) Tri-C9, AmB, and 20% PEG 35kDa; (24E) Tri-C12, AmB, and 10% PEG 35kDa; and (24F) Tri-C12, AmB, and 20% PEG 35kDa.

[0058] Figures 25A-25F depict the insertion capabilities of AmB-loaded MNs composed of Tri-C6 and Tri-C9 with 10%w / w PEG 35kDa as an excipient. (25A) A microscope image of Tri-C6-based MNs array before application of mechanical force; (25B) A representative image of the first layer of parafilm into which Tri-C6-based MNs array was inserted; (25C) A microscope image of Tri-C6-based MNs array after insertion into the parafilm layer; (25D) A microscope image of Tri-C9-based MNs array before application of mechanical force; (25E) A representative image of the first layer of parafilm into which Tri-C9-based MNs array was inserted; and (25F) A microscope image of Tri-C9-based MNs array after insertion into the parafilm layer.

[0059] Figures 26A-26I depict the insertion capabilities of AmB-loaded MNs composed of Tri-C6, Tri-C9, and Tri-C12 with 20%w / w PEG 35kDa as an excipient. (26A) A microscope image of Tri-C6-based MNs array before application of mechanical force; (26B) A representative image of the first layer of parafilm into which Tri-C6-based MNs array was inserted; (26C) A microscope image of Tri-C6-based MNs array after insertion into the parafilm layer; (26D) A microscope image of Tri-C9-based MNs array before application of mechanical force; (26E) A representative image of the first layer of parafilm into which Tri- C9-based MNs array was inserted; (26F) A microscope image of Tri-C9-based MNs array after insertion into the parafilm layer; (26G) A microscope image of Tri-C 12-based MNs array before application of mechanical force; (26H) A representative image of the first layer of parafilm into which Tri-C12-based MNs array was inserted; and (261) A microscope image of Tri-C12-based MNs array after insertion into the parafilm layer.

[0060] Figure 27 depicts a schematic illustration showing a two-step process of fabrication of AmB-loaded MNs composed of Tri-C6, Tri-C9, or Tri-C12 with 10%w / w PEG 35kDa as an excipient.

[0061] Figures 28A-28C depict microscope images of AmB loaded Tri-C6 MN patches prepared from a solution of methanol and ethanol at a 1:1 v / v ratio in the two-step fabrication process.

[0062] Figures 29A-29C depict microscope images of AmB loaded Tri-C9 MN patches prepared from a solution of methanol and ethanol at a 1:1 v / v ratio in the two-step fabrication process.

[0063] Figures 30A-30C depict microscope images of AmB loaded Tri-C12 MN patches prepared from a solution of methanol and ethanol at a 1:1 v / v ratio in the two-step fabrication process.

[0064] Figures 31A-31I depict the insertion capabilities of AmB-loaded MNs composed of Tri-C6, Tri-C9, or Tri-C12 with 10%w / w PEG 35kDa as an excipient prepared from a solution of methanol and ethanol at a 1:1 v / v ratio in the two-step fabrication process. (31 A) A microscope image of Tri-C6-based MNs array before application of mechanical force; (3 IB) A representative image of the first layer of parafilm into which Tri-C6-based MNs array was inserted; (31C) A microscope image of Tri-C6-based MNs array after insertion into the parafilm layer; (3 ID) A microscope image of Tri-C9-based MNs array before application of mechanical force; (3 IE) A representative image of the first layer of parafilm into which Tri- C9-based MNs array was inserted; (31F) A microscope image of Tri-C9-based MNs array after insertion into the parafilm layer; (31G) A microscope image of Tri-C12-based MNs array before application of mechanical force; (31H) A representative image of the first layer of parafilm into which Tri-C12-based MNs array was inserted; and (311) A microscope image of Tri-C12-based MNs array after insertion into the parafilm layer. DETAILED DESCRIPTION OF THE INVENTION

[0065] The present invention provides an array of microneedles and use thereof in local drug delivery. In particular, the microneedles array is composed of a tri-block amphiphile that is designed to transform into hydrogel based MNs, followed by their transition into hydrogel microparticles, thereby acting as a reservoir (depot) of a drug incorporated therein. Accordingly, sustained release of the drug at a target site is afforded. Following drug release, the hydrogel undergoes complete degradation and clearance upon exposure to a stimulus, for example an enzyme.

[0066] The present invention is based, in part, on the unexpected finding of a microneedles array composed of tri-block amphiphiles that can be programmed to undergo sequential mesophase transitions and afford the controlled release of a drug incorporated therein. In particular, upon application to the skin of a subject in need thereof and imbibition of interstitial fluids, the solid microneedles transform into hydrogel microneedles followed by subsequent transition into microgel particles. Thereafter, complete degradation and clearance is afforded by target enzymes at the site of action. The multiple mesophase transitions afford improved control over drug release rates, enhanced penetration, and short application times. In addition, by tailoring the end-groups, decomposition and clearance by e.g., enzymatic degradation, can be achieved.

[0067] The programmable microneedles-based drug delivery system of the present invention can be applied to provide local and more effective treatment than hitherto used transdermal drug delivery systems by increasing the residence time and concentration of the drug at the target site while minimizing side effects.

[0068] The present invention provides a drug delivery system comprising a microneedles array. The term “microneedles array” as used herein refers a plurality of microneedles, for example between 2 and 1,000 needles having sizes in the micrometer range. For example, the microneedles have widths of about 50 pm to about 500 pm and heights of about 200 pm to about 1000 pm, including each value within the specified ranges. Typical sizes include, but are not limited to, microneedles having widths of about 50 pm to about 400 pm, about 50 pm to about 300 pm, about 50 pm to about 200 pm, or about 50 pm to about 100 pm; and heights of about 200 pm to about 900 pm, about 200 pm to about 800 pm, about 200 pm to about 700 pm, or about 200 pm to about 600 pm, including each value within the specified ranges. In some embodiments, the plurality of microneedles has a variety of shapes that are capable of piercing the stratum corneum. For example, the microneedles may have a square pyramidal shape, a triangular pyramidal shape, a stepped pyramidal shape, a conical shape, a microblade shape, or the shape of a hypodermic needle. Each possibility represents a separate embodiment. The microneedles in the array are typically aligned in an ordered fashion.

[0069] According to certain embodiments, the microneedles array is extending from an approximately planar base substantially perpendicular to the microneedles. In some embodiments, the base comprises a polymer having carboxylic acid side-groups and salts thereof. In other embodiments, the base comprises a hydrophilic polymer that forms a readily pourable solution in water, at a concentration between 0.5% and 20% by weight, including each value within the specified range. Without being bound by any theory, the main advantages of utilizing a hydrophilic polymer in the base stem from facilitating the adhesion of the microneedles array to the tissue, and the washing off after application to shorten the application time and improve patient’s compliance.

[0070] The base of the array may be composed of polymers such as, but not limited to, polysaccharides, cellulose derivatives (e.g., carboxymethyl cellulose), poly(acrylates and methacrylates), PEG derivatives, polyvinyl pyrrolidone, polyvinyl alcohol, polylactic-glycolic acid, gelatin, dextrin, collagen, chitosan, silk fibroin, hyaluronic acid and derivatives thereof. Each possibility represents a separate embodiment. Currently preferred is the use of alginic acid and derivatives thereof including alginate salts as the base material. Suitable alginate salts include, but are not limited to, sodium alginate, potassium alginate, and calcium alginate. Each possibility represents a separate embodiment. In some aspects and embodiments, the microneedles array is disposed on a patch substrate (i.e., a backing layer) which typically further comprises an adhesive material.

[0071] According to various aspects and embodiments, the microneedles are composed of a tri-block amphiphile having two hydrophobic termini and a central hydrophilic segment located in between. The hydrophobic termini may be composed of a lipid, a fatty acid, a steroid and the like. Each possibility represents a separate embodiment. In some embodiments, the hydrophobic termini comprise cholesterol.

[0072] In certain aspects and embodiments, the hydrophobic termini comprise a hydrophobic dendron. A “dendron” as used herein is a hyper-branched monodisperse organic molecule defined by a tree-like or generational structure. In general, dendrons possess three distinguishing architectural features: a linker moiety; an interior area containing generations with radial connectivity to the linker moiety; and a surface region (peripheral region) of terminal moieties. According to certain embodiments, each generation of the hydrophobic dendron comprises a linear or branched C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C1-C20 alkylene, C2-C20 alkenylene, C2-C20 alkynylene or arylene moiety which is substituted at each end with a group selected from the group consisting of -O-, -S-, -NH-, -C(=O)-, -C(=O)-O-, - O-C(=O)-O-, -C(=O)-NH-, -NH-C(=O)-NH-, -NH-C(=O)-O-, -S(=O)-, -S(=O)-O-, -PO(=O)- O-, and any combination thereof. Each possibility represents a separate embodiment.

[0073] In some aspects and embodiments, each generation is derived from a compound having a structure represented by the following structure HX-Z-XH or HX-Z-CO2H, wherein X is independently at each occurrence NH, S or O, and Z is selected from C1-C10 alkylene, C2-C10 alkenylene, C2-C10 alkynylene, and arylene. Each possibility represents a separate embodiment.

[0074] According to other aspects and embodiments, each generation of the dendron is derived from a compound selected from the group consisting of HX-CH2-CH2-XH, HX-(CH2)I-3-CO2H and HX-CH2-CH(XH)-CH2-XH, wherein X is independently at each occurrence NH, S or O. Each possibility represents a separate embodiment. For example, each generation of the dendron may be derived from a compound selected from the group consisting of HS-CH2-CH2- OH, HS-(CH2)I.3-CO2H and HS-CH2-CH(OH)-CH2-OH. Each possibility represents a separate embodiment.

[0075] The hydrophobic dendron of the present invention typically comprises between 0 to 5 generations, for example between 0 to 3 generations, including each integer within the specified ranges. In one embodiment, the hydrophobic dendron is a generation 0 (GO) dendron. In another embodiment, the hydrophobic dendron is a generation 1 (Gl) dendron. In yet another embodiment, the hydrophobic dendron is a generation 2 (G2) dendron. In other embodiments, the hydrophobic dendron is a generation 3 (G3) dendron.

[0076] In various embodiments, the dendron comprises a repeating unit selected from the group consisting of: wherein X1is independently, at each occurrence, selected from the group consisting of O, S and NH; and m is an integer from 1 to 15, including each integer within the specified range.

[0077] According to the principles of the present invention, the hydrophobic termini comprise at least one stimulus-responsive cleavable site, for example an enzymatically cleavable site. Enzymatically cleavable sites typically include a functional group such as, but not limited to, a disulfide, a diselenide, an ester (including a boronate ester and a phosphate ester), an amide, an amidine, an imine, a carbamate, a carbonate, an acetal, a urea, a thiourea, a trithionate, a sulfate, a sulfamate, a phosphate, a phosphoamide, a hydrazone, an ether, a silyl ether, an oxyme, a boronic acid, a boronic ester, a nitro, and an azo. Each possibility represents a separate embodiment. Functional groups that can be cleaved by enzymes include, for example -O-C(O)-R’, -C(O)-OR’ -NH-C(O)-R’ or -C(O)-NHR’, wherein R’ is C1-C12 alkyl or an aryl. Each possibility represents a separate embodiment.

[0078] It will be appreciated to one skilled in the art that an amide bond is enzymatically cleavable by an amidase. Suitable amidases that can cleave an amide bond include, but are not limited to, aryl-acylamidase, aminoacylase, alkylamidase, and phthalyl amidase. Each possibility represents a separate embodiment. Where an ester bond is present in the hydrophobic termini, it can be cleaved by an esterase or a lipase. Suitable esterases that can cleave an ester bond include, but are not limited to, carboxylesterase, arylesterase, and acetylesterase. Each possibility represents a separate embodiment. Where a urea bond is present in the hydrophobic termini, it can be cleaved by a urease.

[0079] Where hydrophobic termini comprise hydrophobic dendrons, the enzymatically cleavable site may be present at one or more of the terminal repeating units (i.e., terminal generations) of the hydrophobic dendron, and / or in intermediary generations of the dendron. The enzymatically cleavable hydrophobic end group may be present only at the terminal repeating units of the hydrophobic dendron (i.e., the enzymatically cleavable hydrophobic end group is not present in intermediary generations of the dendron) or it may be present only at the intermediary generations of the dendron (i.e., the enzymatically cleavable hydrophobic end group is not present in the terminal repeating units of the hydrophobic dendron). Each possibility represents a separate embodiment. In some embodiments, each generation of the hydrophobic dendron is terminated with cleavable fatty acids or steroidal end-groups.

[0080] It is to be understood that the hydrophobic termini of the tri-block amphiphile may be the same or different, with each possibility representing a separate embodiment of the present invention.

[0081] The term “alkyl” used herein alone or as part of another group denotes a saturated aliphatic hydrocarbon, including straight-chain and branched-chain alkyl groups. In one embodiment, the alkyl group has 1-20 carbons designated here as C1-C20 alkyl. In another embodiment, the alkyl group has 1-10 carbons designated here as C1-C10 alkyl. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec -butyl, t-butyl, and the like.

[0082] The term “alkylene” used herein alone or as part of another group denotes a bivalent radical which is bonded at two positions connecting together two separate additional groups (e.g., CH2). Examples of alkylene groups include, but are not limited to -(CH2)-, -(CFh -, - (CH2)3-, -(CH2)4-, etc.

[0083] The term “alkenyl” used herein alone or as part of another group denotes an aliphatic hydrocarbon containing at least one double bond, including straight-chain and branched-chain alkenyl groups. In one embodiment, the alkenyl group has 2-20 carbons designated here as C2-C20 alkenyl. In another embodiment, the alkenyl group has 2-10 carbons designated here as C2-C10 alkenyl.

[0084] The term “alkenylene” used herein alone or as part of another group denotes a bivalent radical containing at least one double bond, which is bonded at two positions connecting together two separate additional groups (e.g., -CH=CH-).

[0085] The term “alkynyl” used herein alone or as part of another group denotes an aliphatic hydrocarbon containing at least one triple bond, including straight-chain and branched-chain alkynyl groups. In one embodiment, the alkynyl group has 2-20 carbons designated here as C2-C20 alkynyl. In another embodiment, the alkynyl group has 2-10 carbons designated here as C2-C10 alkynyl.

[0086] The term “alkynylene” used herein alone or as part of another group denotes a bivalent radical containing at least one triple bond, which is bonded at two positions connecting together two separate additional groups (e.g., -C= C-). The term “aryl” used herein alone or as part of another groups denotes an aromatic ring system containing from 5-14 ring carbon atoms. The aryl ring can be a monocyclic, bicyclic, tricyclic and the like. Non-limiting examples of aryl groups are phenyl, naphthyl including 1 -naphthyl and 2-naphthyl, and the like. In some embodiments, aryl includes heteroaryl.

[0087] The term “arylene” denotes a bivalent radical of aryl, which is bonded at two positions connecting together two separate additional groups (e.g., -Cetk-).

[0088] Each of the alkyl, alkylene, alkenyl, alkenylene, alkynyl, alkynylene, aryl, and arylene can be substituted by one or more substituents.

[0089] Suitable substituents include, but are not limited to, alkyl, aryl, halide, -OR, -COR, - COOR, OCOR, -SR, -CSR, -COSR, SCOR, -CSSR, SCSR, -NRR’, -CNRR’, -CONRR’ and -NR’ COR, wherein each R and R’ is alkyl, alkenyl, alkynyl, aryl or H, wherein each R and R’ is optionally further substituted as described herein. Each possibility represents a separate embodiment.

[0090] Exemplary substituents for the alkyl, alkylene, alkenyl, alkenylene, alkynyl, alkynylene, aryl, and arylene include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, halogen, haloalkyl, hydroxy, alkoxy, carbonyl, carboxyl, ester, amido, alkylamido, dialkylamido, nitro, cyano, amino, alkylamino, dialkylamino, thio, and thioalkyl. Each possibility represents a separate embodiment.

[0091] According to the principles of the present invention, the tri-block amphiphile has a hydrophilic central unit located between the two hydrophobic termini.

[0092] In some aspects and embodiments, the hydrophilic central unit comprises a hydrophilic polymer such as, but not limited to, polyethylene glycol (PEG), polyacrylic acid, poly (hydroxy ethyl acrylate), poly (oligo-ethylene glycol acrylate), polyacrylamide, polymethyl oxazoline, polyethyl oxazoline, polysarcosine, polypeptide, polypeptoid, hydrophilic polymethacrylate, poly amine, hydrophilic nylon, polyvinylpyrrolidone (PVP), polyvinyl alcohol, hydrophilic protein, and polycarbohydrate. Each possibility represents a separate embodiment. In particular aspects and embodiments, the hydrophilic polymers include, but are not limited to, polyethylene glycol (PEG), polyacrylic acid, poly(2-hydroxyethyl acrylate), and poly(oligo-ethylene glycol acrylate). Each possibility represents a separate embodiment. A currently preferred hydrophilic polymer comprises PEG, for example PEG having at least 10 repeating units of ethylene glycol monomers. Typically, the hydrophilic polymer has a molecular weight of about 0.5 to about 100 kDa, including each value within the specified range. Exemplary molecular weights of the hydrophilic polymer within the scope of the present invention include, but are not limited to, from about 0.5 to about 75 kDa, from about 0.5 to about 50 kDa, or from about 0.5 to about 25 kDa, including each value within the specified ranges. In one embodiment, the molecular weight of the hydrophilic polymer is from about 0.5 to about 10 kDa, including each value within the specified range. In another embodiment, the molecular weight of the hydrophilic polymer is from about 5 to about 15 kDa, including each value within the specified range. In some embodiments, the hydrophilic polymer does not contain hyaluronic acid or derivatives thereof. In other embodiments, the hydrophilic polymer does not contain a peptide or a polypeptide.

[0093] The hydrophilic polymer is chemically bound to the hydrophobic termini by chemical bonds which include, but are not limited to, -Z-X-, -X'-Z-X2-, and -Z^X^Z^X2-, wherein Z, Z1, and Z2are each independently selected from C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C1-C10 alkylene, C2-C10 alkenylene, C2-C10 alkynylene, and arylene; X, X1, and X2are each independently selected from -O-; -S-; -NH-; -C(=O)-; -C(=O)-O-; -O-C(=O)-O-; -C(=O)-NH- ; -NH-C(=O)-NH-; -NH-C(=O)-O-; -S(=O)-; -S(=O)-O-; -PO(=O)-O-; triazolylene, and any combination thereof. Each possibility represents a separate embodiment. For the sake of clarity, the optional groups that chemically bond the hydrophilic polymer to the hydrophobic termini are depicted in one direction. However, it is to be understood by the person having ordinary skill in the art that the relevant embodiments are not limited to a specific direction, for example, the group -X'-Z-X2- may refer to either (hydrophobic terminus)-X1-Z-X2-(hydrophilic polymer) or to (hydrophobic terminus)-X2-Z-X1-(hydrophilic polymer). Furthermore, groups that are described with direction are intended to cover both directions, e.g., -C(=O)-NH- refer also to -NH-C(=O)-.

[0094] Currently preferred linkages between the hydrophilic polymer and the hydrophobic termini are -X'-Z-X2-, wherein Z is C2 alkenylene and X1and X2are each -C(=O)-NH-. In some embodiments, the linkages between the hydrophilic polymer and the two hydrophobic termini are the same.

[0095] According to some aspects and embodiments, the tri-block amphiphile comprises a hydrophilic middle segment which is a PEG and hydrophobic termini which are dendrons containing esterase / amidase / urease cleavable alkyls, fatty acids, or steroidal end-groups. Each possibility represents a separate embodiment. According to other aspects and embodiments, the esterase / amidase / urease cleavable alkyls include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and the like. Each possibility represents a separate embodiment.

[0096] According to some embodiments, the tri-block amphiphile is represented by the structure of Formula (I):

[0097] A^-B-L^A2

[0098] (I)

[0099] According to some embodiments, each one of A1and A2is a hydrophobic terminus as described herein.

[0100] According to some embodiments, each one of L1and L2is a linkage moiety between the hydrophobic termini and the hydrophilic polymer as described herein.

[0101] According to some embodiments, B is a hydrophilic polymer as described herein.

[0102] According to some embodiments, A1is the same as A2.

[0103] According to some embodiments, A1is selected from the group consisting of: alkyl, alkenyl, alkynyl and aryl, each of which is optionally substituted as defined herein. According to some embodiments, A1comprises at least 10 carbon atoms. According to some embodiments, A1comprises at least 20 carbon atoms. According to some embodiments, A1comprises at least 30 carbon atoms. According to some embodiments, A1comprises at least 40 carbon atoms. According to some embodiments, A1comprises at least 50 carbon atoms.

[0104] According to some embodiments, A1is an aryl group. According to some embodiments, A1is a substituted aryl. According to some embodiments, A1is a disubstituted aryl. According to some embodiments, A1has the structure -A^R^. It is to be understood that each R1may be different or both R1groups are the same.

[0105] According to some embodiments, Ar1is naphthyl or phenyl. According to some embodiments, Ar1is phenyl. According to some embodiments, A1has the structure -Ceth-R^. According to some embodiments, both R1are positioned meta.

[0106] According to some embodiments, each R1comprises at least 5 carbon atoms. According to some embodiments, each R1comprises at least 10 carbon atoms. According to some embodiments, each R1comprises at least 15 carbon atoms. According to some embodiments, each R1comprises at least 20 carbon atoms.

[0107] According to some embodiments, each R1has the formula -Y1-(CH2)n-CHW1- (CH2)mW1. It is to be understood that each W1may be different or both W1groups are the same.

[0108] According to some embodiments, Y1is O, S or NH. According to some embodiments, Y1is O.

[0109] According to some embodiments, n is 1, 2, 3, 4 or 5. According to some embodiments, n is 1.

[0110] According to some embodiments, m is 1, 2, 3, 4 or 5. According to some embodiments, m is 1.

[0111] According to some embodiments, W1is an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl or an optionally substituted aryl. According to some embodiments, W1is an optionally substituted alkyl. According to some embodiments, W1is an optionally substituted alkyl represented by the formula -E1-(CH2)j-G1.

[0112] According to some embodiments, E1is S, O or NH. According to some embodiments, E1is S.

[0113] According to some embodiments, j is 1, 2, 3, 4 or 5. According to some embodiments, j is 2.

[0114] According to some embodiments, G1is -C00M1, -0C0-M1, M1, -COM1, -NHM1, - NM*2, -CNHM1or -CNMJ2. According to some embodiments, G1is -C00-M1or -0C0-M1. According to some embodiments, G1is -C00-M1. According to some embodiments, G1is -0C0-M1.

[0115] According to some embodiments, M1is an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl or an optionally substituted aryl. According to some embodiments, M1comprises at least 4 carbon atoms. According to some embodiments, M1is an optionally substituted alkyl. According to some embodiments, M1is an unsubstituted alkyl. According to some embodiments, M1is C2-20 alkyl. According to some embodiments, M1is a C5-15 alkyl. According to some embodiments, M1is a C9 alkyl. According to some embodiments, M1is a linear alkyl. According to some embodiments, M1is -(CH2)8CH3. According to some embodiments, A2is selected from the group consisting of: alkyl, alkenyl, alkynyl and aryl, each of which is optionally substituted as defined herein. According to some embodiments, A2comprises at least 10 carbon atoms. According to some embodiments, A2comprises at least 20 carbon atoms. According to some embodiments, A2comprises at least 30 carbon atoms. According to some embodiments, A2comprises at least 40 carbon atoms. According to some embodiments, A2comprises at least 50 carbon atoms.

[0116] According to some embodiments, A2is an aryl group. According to some embodiments, A2is a substituted aryl. According to some embodiments, A2is a disubstituted aryl. According to some embodiments, A2has the structure -Ar2R22. It is to be understood that each R2may be different or both R2groups are the same.

[0117] According to some embodiments, Ar2is naphthyl or phenyl. According to some embodiments, Ar2is phenyl. According to some embodiments, A2has the structure -CeH3-R22. According to some embodiments, both R2are positioned meta.

[0118] According to some embodiments, each R2comprises at least 5 carbon atoms. According to some embodiments, each R2comprises at least 10 carbon atoms. According to some embodiments, each R2comprises at least 15 carbon atoms. According to some embodiments, each R2comprises at least 20 carbon atoms.

[0119] According to some embodiments, each R2has the formula -Y2-(CH2)n-CHW2- (CH2)mW2. It is to be understood that each W2may be different or both W2groups are the same.

[0120] According to some embodiments, Y2is O, S or NH. According to some embodiments, Y2is O.

[0121] According to some embodiments, n is 1, 2, 3, 4 or 5. According to some embodiments, n is 1.

[0122] According to some embodiments, m is 1, 2, 3, 4 or 5. According to some embodiments, m is 1.

[0123] According to some embodiments, W2is an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl or an optionally substituted aryl. According to some embodiments, W2is an optionally substituted alkyl. According to some embodiments, W2is an optionally substituted alkyl represented by the formula -E2-(CH2)j-G2. According to some embodiments, E2is S, O or NH. According to some embodiments,

[0124] E2is S.

[0125] According to some embodiments, j is 1, 2, 3, 4 or 5. According to some embodiments, j is 2.

[0126] According to some embodiments, G2is -C00M2, -0C0-M2, M2, -COM2, -NHM2, - NM22, -CNHM2or -CNM22. According to some embodiments, G2is -C00-M2.

[0127] According to some embodiments, M2is an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl or an optionally substituted aryl. According to some embodiments, M2comprises at least 4 carbon atoms. According to some embodiments, M2is an optionally substituted alkyl. According to some embodiments, M2is an unsubstituted alkyl. According to some embodiments, M2is C2-20 alkyl. According to some embodiments, M2is a C5-15 alkyl. According to some embodiments, M2is a C9 alkyl. According to some embodiments, M2is a linear alkyl. According to some embodiments, M2is -(CH2)8CH3.

[0128] According to some embodiments, L1is represented by the formula -V1-(CH2)i-Q1-.

[0129] According to some embodiments, each one of V1and Q1is selected from the group consisting of: -CONH-, -S-, -O-, -COO-, -OCO-, -CO-, -NHCO-, -NH-, -COS-, and -SCO-. According to some embodiments, each one of V1and Q1is selected from the group consisting of: -CONH- and -S-. According to some embodiments, each one of V1is -CONH-. According to some embodiments, each one of Q1is -S-.

[0130] According to some embodiments, i is 1, 2, 3, 4 or 5. According to some embodiments, i is 2.

[0131] According to some embodiments, L2is represented by the formula -V2-(CH2)i-Q2-.

[0132] According to some embodiments, each one of V2and Q2is selected from the group consisting of: -CONH-, -S-, -O-, -COO-, -OCO-, -CO-, -NHCO-, -NH-, -COS-, and -SCO-. According to some embodiments, each one of V2and Q2is selected from the group consisting of: -CONH- and -S-. According to some embodiments, each one of V2is -CONH-. According to some embodiments, each one of Q2is -S-.

[0133] According to some embodiments, i is 1, 2, 3, 4 or 5. According to some embodiments, i is 2. According to some embodiments, B is polyethylene glycol, as described herein. According to some embodiments, B is polyethylene glycol having molecular weight of about lOkDa.

[0134] According to some embodiments, the tri-block amphiphile is depicted by the structure of formula (II):

[0135] (ID, wherein R is a C2-20 alkyl and n is an integer from 2 to 3,000, including each value within the specified range. The tri-block amphiphiles can be synthesized as is known in the art. A non-limiting synthesis of the tri-block amphiphiles is depicted in Scheme 1.

[0136] Scheme 1:

[0137] According to some embodiments, the tri-block amphiphile is depicted by the structure of formula (III): wherein R' is a C2-20 alkyl and n is an integer from 2 to 3,000, including each value within the specified range.

[0138] An additional non-limiting synthesis route of the tri-block amphiphiles is depicted in Scheme 2. 1 Scheme 2:

[0139] R=propanol / hexanol i nonsol i endecanol i dodecanol

[0140] According to some aspects and embodiments, the microneedles further comprise an excipient. Suitable excipients within the scope of the present invention include, but are not limited to, polyethylene glycol (PEG), hydroxyalkyl methylcellulose, hydroxyalkyl cellulose, microcrystalline cellulose, cellulose esters, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acids, polybutyric acid, poly lactide (PLA), polyglycolide (PGA), poly lactide-co- glycolide (PLGA), poly ethylene oxide - poly propylene oxide - poly ethylene oxide, or a mixture or combination thereof. Each possibility represents a separate embodiment. In certain aspects and embodiments, the weight ratio between the tri-block amphiphile and the excipient is within a range of about 100:1 and about 1:100, including all iterations of ratios within the specified range. Suitable ratios between the tri-block amphiphile and the excipient include, but are not limited to, 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100. Each possibility represents a separate embodiment.

[0141] Currently preferred is the use of polyethylene glycol (PEG) as an excipient. PEG has beneficial physical and / or chemical properties such as water-solubility, non-toxic, odorless, lubricating, non-volatile, and non-intrusive which are particularly suitable for pharmaceutical utility. Typically, the PEG excipient encompassed by the present invention has a molecular weight of about 10 to about 200 kDa, including each value within the specified range. Exemplary molecular weight include, but are not limited to, about 20 to about 150 kDa, about 20 to about 130 kDa, about 20 to about 100 kDa, about 25 to about 150 kDa, about 25 to about 130 kDa, about 25 to about 100 kDa, about 30 to about 150 kDa, about 30 to about 130 kDa, and about 30 to about 100 kDa, including each value within the specified ranges.

[0142] Encompassed by the present invention is the incorporation of an active pharmaceutical ingredient into the microneedles. The active pharmaceutical ingredient may be blended together with the tri-block amphiphile and optional excipient or covalently linked to the triblock amphiphile. In some embodiments, the active pharmaceutical ingredient is dissolved in the tri-block amphiphile and optional excipient medium prior to casting the microneedles such that no drug particles are visible in the microneedles array.

[0143] Suitable active pharmaceutical ingredients within the scope of the present invention include, but are not limited to, analgesic agents, anesthetic agents, antiarthritic agents, antibacterial agents, antifungal agents, anticancer agents, anti-infective agents, antiinflammatory agents, muscle relaxants, peptide drugs, protein drugs, steroids, vasodilators, vaccines, and mixtures thereof. Each possibility represents a separate embodiment.

[0144] For example, a steroid such as, but not limited to, dexamethasone, aldosterone, beclomethasone, betamethasone, budesonide, cloprednol, cortisone, cortivazol, deoxycortone, desonide, desoximetasone, difluorocortolone, fluclorolone, flumethasone, flunisolide, fluocinolone, fluocinonide, fluocortin butyl, fluorocortisone, fluorocortolone, fluorometholone, flurandrenolone, fluticasone, halcinonide, hydrocortisone, icomethasone, meprednisone, methylprednisolone, paramethasone, prednisolone, prednisone, tixocortol and triamcinolone, may be incorporated into the microneedles. Each possibility represents a separate embodiment. Another example includes the incorporation of an antibiotic such as, but not limited to, ampicillin, dapsone, chloramphenicol, neomycin, cefaclor, cefadroxil, cephalexin, cephradine, erythromycin, clindamycin, lincomycin, amoxicillin, ampicillin, bacampicillin, carbenicillin, dicloxacillin, cyclacillin, picloxacillin, hetacillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, ticarcillin, rifampin, tetracycline, fusidic acid, lincomicyn, novobiocine, and spectinomycin into the microneedles. Each possibility represents a separate embodiment. An additional example includes the incorporation of an antiinflammatory agent such as, but not limited to, betamethasone, prednisolone, piroxicam, aspirin, flurbiprofen, diflunisal, ibuprofen, fenoprofen, fenamate, ketoprofen, nabumetone, naproxen, diclofenac, indomethacin, sulindac, tolmetin, etodolac, ketorolac, oxaprozin, celecoxib, meclofenamate, mefenamic acid, oxyphenbutazone, phenylbutazone, salicylates, and phyto sphingosine into the microneedles. Each possibility represents a separate embodiment. A further example is the incorporation of an anti-fungal agent such as, but not limited to clotrimazole, miconazole, ketoconazole, nystatin, amphotericin B, and pimaricin into the microneedles. Each possibility represents a separate embodiment. It is to be understood that any pharmaceutically acceptable salt or derivative of these agents is included within the scope of the present invention.

[0145] The active pharmaceutical ingredient is typically incorporated into the microneedles with a drug-loading capacity of about 3% to about 50%, including each value within the specified range. Suitable drug-loading capacities include, but are not limited to, about 3% to about 45%, about 3% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, or about 5% to about 20%, including each value within the specified ranges.

[0146] The microneedles of the drug-delivery system disclosed herein may be homogenous such that the tri-block amphiphile, optional excipient, and active pharmaceutical ingredient are homogeneously distributed within the needles. Alternatively, non-homogenous microneedles may be used, for example microneedles having a tip portion which is different than the bottom portion. Thus, in some embodiments, the tip portion comprises the tri-block amphiphile, optional excipient, and active pharmaceutical ingredient and the bottom portion comprises the tri-block amphiphile and optional excipient without an active pharmaceutical ingredient. In other embodiments, the tip portion comprises the tri-block amphiphile and optional excipient without an active pharmaceutical ingredient and the bottom portion comprises the tri-block amphiphile, optional excipient, and active pharmaceutical ingredient. In further embodiments, the tip and bottom portions of the microneedles each comprise a different tri-block amphiphile and / or a different optional excipient and ratios thereof. In some embodiments, the microneedles are not hollow or porous. In other embodiments, the microneedles do not include a coating, such as a drug coating layer.

[0147] The microneedles comprising the tri-block amphiphiles can be prepared as is known in the art, for example using a vacuum-deposition micro-molding method by injecting the polymeric solution onto a micro-mold surface, followed by the application of a vacuum and drying. In particular, the amphiphiles with(out) an excipient are typically dissolved in an organic solvent (e.g., chloroform) and the solution is cast into a mold followed by the application of a vacuum to form the microneedles. Optionally, casting can be performed in two or more stages whereby the polymeric solution is divided into two or more portions that can be the same or different, each portion is cast into a mold followed by the application of a vacuum. In order to form the base, an aqueous solution comprising the base material is added on top of the inverted microneedles followed by drying and de-molding.

[0148] The microneedles may be administered via a route selected from transdermal, nasal, and vaginal. Each possibility represents a separate embodiment. Currently preferred is the transdermal administration through the skin of a subject by pressing the microneedle array or patch against the skin surface. In some embodiments, the administration is performed with the help of a designated apparatus. The present invention also provides a method of transdermally administering an active pharmaceutical ingredient to a subject in need thereof, the method comprises applying the delivery system disclosed herein to the skin of a subject. In some embodiments, the application is performed with the help of a designated apparatus.

[0149] According to the principles of the present invention, the delivery system of the present invention is designed to undergo at least two mesophase transitions due to absorption of interstitial fluids to thereby release the active pharmaceutical ingredient incorporated therein in a controlled manner. The first mesophase transition involves the transition from solid microneedles to hydrogel microneedles. The term “hydrogel” as used herein refers to a three- dimensional hydrated assembly of polymeric chains following imbibition of the interstitial fluids of the body. The second phase transition involves the transition from hydrogel microneedles to hydrogel microparticles which act as a sustained release depot formulation. The term “sustained release depot” as used herein refers to a pharmaceutical formulation which provides prolonged, long or extended release of a therapeutically effective amount of an active pharmaceutical ingredient to the local sites of action in a subject. This term may further refer to a pharmaceutical formulation which provides prolonged, long or extended exposure to (pharmacokinetics) and duration of action of (pharmacodynamics) a therapeutically effective amount of an active pharmaceutical ingredient in a subject. The subject may be a mammal, preferably a human.

[0150] Without being bound by a particular theory it is believed that the release of an active pharmaceutical ingredient from the depot formulation can occur by either one of two different mechanisms. The first mechanism includes the release by diffusion through aqueous filled channels generated in the hydrogel. The second mechanism includes the release of the biologically active agent due to degradation of the tri-block amphiphile. By altering the properties of the amphiphile, for example the ratio of the hydrophilic middle portion to hydrophobic termini, the release profile can be controlled. According to various aspects and embodiments, the release of an active pharmaceutical ingredient by the system disclosed herein is performed in a continuous manner without pulse release. The release profile can be a zero order release profile, a first order release profile, a second order release profile, a third order release profile, or any pseudo orders known. Each possibility represents a separate embodiment.

[0151] Following the release of the active ingredient and upon exposure of the hydrogel microparticles to a stimulus, the stimulus-responsive cleavable sites undergo cleavage thereby disassembling the hydrogel to dissolved polymers and fragments thereof. The stimulus may be a chemically-induced stimulus or a physically-induced stimulus, with each possibility representing a separate embodiment. Physically induced stimuli include, but are not limited to, a change in at least one of temperature, pH, light (UV light, visible light or near infrared light), or electric field. Each possibility represents a separate embodiment. Chemically induced stimuli include, but are not limited to, contacting with an agent such as a redox agent, a transport protein or an activating enzyme. Each possibility represents a separate embodiment. As used herein, the term “contacting” refers to bringing in contact with the tri-block amphiphiles of the present invention. Contacting can be accomplished for example in cells or tissue cultures, or living organisms. Each possibility represents a separate embodiment. In one embodiment, the present invention encompasses contacting the delivery system of the present invention with an agent within a human subject. In other embodiments, the term “contacting” may be performed ex-vivo on a surface, on a device, in cell / tissue culture dish, in food and water. Each possibility represents a separate embodiment. In currently preferred embodiments, the agent that induces cleavage of the stimulus- responsive cleavable sites is an activating enzyme. Suitable activating enzymes within the scope of the present invention include, but are not limited to, amidases, esterases, lipases, and ureases, as detailed hereinabove. Advantageously, the tri-block amphiphiles of the present invention are biodegradable such that following the cleavage of the stimulus-responsive cleavable sites, degradation of the polymer occurs. The term “biodegradable” as used herein refers to a component which erodes or degrades at its surfaces over time due, at least in part, to contact with substances found in the surrounding tissue fluids, or by cellular action. In some embodiments, the microneedles array is composed entirely of organic material that is fully biodegradable and can be completely eliminated from the body following drug release. In certain embodiments, the microneedles array does not include metals or other electrically conductive materials.

[0152] The delivery system of the present invention can further be provided in the form of a kit comprising the microneedle array and written instructions for use. In some embodiments, the kit may further comprise a compartment comprising an agent (e.g., an enzyme) capable of cleaving the stimulus-responsive cleavable sites. When the enzyme is in a lyophilized form, the kit may optionally contain a sterile and physiologically acceptable reconstitution medium such as water, saline, buffered saline, and the like. Each possibility represents a separate embodiment. According to some embodiments, the kit may further comprise an applicator to facilitate the administration / application of the microneedles array to the subject.

[0153] The term “about” as used herein refers to ±10% of a specified value. Throughout the description and claims, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, mean “including but not limited to”, and are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0154] As used herein, the singular forms “a”, “an”, and “the” include plural forms unless the context clearly dictates otherwise. Thus, for example, reference to “a cleavable site” also includes a plurality of cleavable sites, which may be the same or different with each possibility representing a separate embodiment. As used herein, the term “and” or the term “or” include “and / or” unless the context clearly dictates otherwise.

[0155] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES

[0156] Materials

[0157] Dexamethasone (DEX; Alfa Aesar), sodium alginate (Fisher Chemical) and Trypan blue powder (Thermo Scientific) were purchased from Holland-Moran Inc. Israel. Silicone MPatch microneedle templates were purchased from Micropoint Technologies Pte Ltd. (Pioneer Junction, Singapore) and were pyramidal in shape with dimensions of 10 x 10 needle array, 200 pm base, 500 pm height, and 500 pm pitch. Organic solvents and PEG with average MWs of 3.4, 10, 35 and 100 kDa were purchased from Sigma Aldrich. Polyvinyl pyrrolidone (PVP) K-30 (MW 40 kDa) was purchased from BASF. Microcrystalline cellulose was supplied by FMC BioPolymer (Avicel®, Philadelphia, PA, USA). PLGA-Purasorb PDLG 5010 (50:50) was obtained from Corbion Purac (Gorinchem). Phosphate buffer saline (PBS, pH 7.4) was purchased from Hyclone Laboratories. GeBaFlex tubes with a molecular weight cutoff (MWCO) of 8 kDa, porcine liver esterase (PLE), and bovine serum albumin (BSA, Probumin®) were purchased from Merck.

[0158] Methods

[0159] Tri-C9 MNs fabrication

[0160] Arrays of MNs were prepared based on Dawud et al. (Pharmaceutics 2023, 15(2), 526) using a modified vacuum-deposition casting method. Silicone MN molds were used to fabricate MNs arrays of 10x10 pyramid- shaped microneedles, with dimensions of 500 pm needle height, 200 pm base diameter, and interspacing of 500 pm. MNs composed of tri-block amphiphile (Tri-C9) composed of hydrophobic dendrons containing esterase cleavable aliphatic end- groups was fabricated (Scheme 1), and the parameters affecting their characteristics were studied as depicted in Table 1. Briefly, the amphiphile was first dissolved in ethanol or chloroform to examine the effect of the organic solvent on film formation. Next, aqueous solution of sodium alginate was added to form a base layer and enhance the amphiphile's filmforming ability. The mechanical properties of the obtained MNs were then assessed. To achieve the desired mechanical properties, different excipients were introduced into the formulation. The formulation containing PEG excipient was thereafter evaluated with different concentrations and molecular weights of the excipient.

[0161] The following structure depicts tri-block polymeric amphiphile composed of a central hydrophilic polyethylene glycol (PEG) block (10 kDa PEG) conjugated with two hydrophobic dendritic branching units functionalized with ester-based nonyl end-groups (Tri-C9) which can be cleaved by skin esterases:

[0162] Table 1.

[0163] PEG-containing MNs arrays fabrication

[0164] Fabrication of the MNs arrays was performed as illustrated in Figure 1. In particular, a solution of 3% w / v of tri-block amphiphile, and 10% w / w (calculated according to the tri-block amphiphile) PEG with different molecular weights in chloroform, was cast into the mold and degassed in a vacuum for 10 minutes to form the MN tips. Then, an aqueous solution of 4% w / v sodium alginate was added to the mold and allowed to dry overnight in a desiccator. After drying, the microneedle patches were detached from the mold and kept for further investigation. MN patches were characterized in terms of mechanical-strength, insertion capability, and surface morphology. Insertion capabilities of MNs and mechanical properties testing

[0165] Insertion test was performed using eight stacked layers of parafilm (-140 pm each) as a skin simulant to investigate the penetration ability of the MNs arrays, as well as the insertion depth (Dawud et al., Pharma 2023, 15 (2), 526; Larraneta et al., Inter J Pharma 2014, 472 (1- 2), 65-73; and Abu-Much et al., Biomater Sci 2022). This skin simulant model was developed and validated by Larraneta et al. (Inter J Pharma 2014, 472 (1-2), 65-73), showing that the insertion profiles obtained are consistent with the insertion depths obtained with optical coherence tomography (OCT), and the force that was used in this test achieved insertion profiles equivalent to those obtained using neonatal pig skin. The obtained MNs were inserted manually into the parafilm layers, held for 30 seconds, and then removed. The number of holes created in each layer and the height of the MNs' after insertion were evaluated using a stereomicroscope (Olympus-SZ61, Tokyo, Japan). To further evaluate the mechanical strength of the MNs, they were positioned in an upright manner, then underwent compression with forces ranging from 50 to 1000 g for 5 min. Subsequently, morphological and dimensional changes of the needles were evaluated (Weimer et al., Pharma 2021, 13 (10), 1601).

[0166] Fabrication and characterization of DEX-loaded MNs

[0167] DEX-loaded MNs were prepared in a similar manner to that described above, by adding 1 mg of dexamethasone to the tip solution (final concentration 1% w / v). MNs arrays were characterized by evaluating their penetration efficiency, surface morphology and drug content. To determine the encapsulation efficiency (EE) and drug loading, DEX-loaded MNs arrays were immersed in distilled water with continuous stirring to disintegrate the MNs patch completely. Subsequently, water was evaporated using a rotary evaporator. Then, 5 mL of acetonitrile was added to the resulting residue, and after serial dilutions, dexamethasone was quantified using HPLC. All measurements were recorded on a Waters Alliance e2695 separations module equipped with a Waters 2998 photodiode array detector. Chromatographic separation was obtained using XBridge® Protein BEH, C4, 3.5 pm, 150x4.6 mm column and a mobile phase consisted of water and acetonitrile (5:95) with a flow rate of 1 mL / min. The samples analysis time was 15 min and the column temperature was maintained at 37°C with an injection volume of 30 pL. Dexamethasone was detected at 241.5 nm (Figure 2) according to Xiong et al. (J Pharma and Biomed Ana 2009, 49 (3), 646-654), and Song et al. (J Liq Chroma Rel Tech 2004, 27 (14), 2293-2306).

[0168] Morphological characterization of the MNs upon the incorporation of the drug was performed using a Quanta 200 FEG environmental scanning electron microscope (SEM) in high vacuum (WD ~10 cm, 3-20 kV). Before imaging, a thin layer of palladium (Pd) was deposited onto the needle arrays to ensure better visualization. In addition, to elucidate the polymer disposition within the lead MNs formulation, an elemental analysis was performed using energy dispersive spectroscopy (EDS) on a separated single tip and baseplate of the MNs array. Swelling studies of hydrogel-forming MN in PBS solution

[0169] In order to better visualize the swelling of MNs, curcumin (1 mg) was incorporated into Tri-C9 MNs patch with 35kDa PEG using the same preparation procedure as above. The tips of the MNs were placed on supporting parafilm platform to ensure exposure of the MNs tips, where the tri-block amphiphile is mainly located, to the aqueous media. PBS solution (pH 7.4) was then added, and the transition stages of the tips were visualized during the test using HAYEAR 4K UHD microscope camera.

[0170] In-vitro drug release studies

[0171] The in-vitro release of dexamethasone from the hydrogel-forming MNs arrays was determined using PBS (pH 7.4) solution containing 1 mg / mL bovine serum albumin (BSA), which exhibits an affinity to hydrophobic entities (Slor et al., ACS Polym. Au 2022, 2 (5), 3 SO- 386; Sulkowska et al., J Mol Struc 2003, 651-653, 133-140; and Al-Husseini et al., Lang 2019, 35 (52), 17054-17060). Porcine liver esterase (PLE) was used as a model for esterase activity. Briefly, Tri-C9 MNs with 10% w / w 35 kDa were placed in GeBaFlex tubes (8 MWCO kDa) containing 0, 15 pM or 45 p M of PLE enzyme. The tube was then immersed in 40 mL of the release medium (PBS containing BSA). At predefined points, 0.5 mL of the release medium was sampled and replaced with 0.5 mL of fresh pre-warmed release medium to maintain sink conditions. The samples were analysed using HPLC with UV-detection at 241.5 nm. At the end of the experiment, acetonitrile was added to the samples inside the dialysis tube to fully dissolve them and the obtained solutions were injected to HPLC with UV-detection at 293 nm to analyse the degree of degradation of the Tri-C9.

[0172] Ex-vivo skin insertion test of DEX-loaded MNs

[0173] DEX-loaded Tri-C9 MNs (35 kDa PEG) were evaluated for their penetration ability into chicken skin. A tension / compression tester consisting of motorized vertical test stand equipped with a digital force gauge (PCE-VTS 50-DFG N 500) was employed (PCE Instruments UK Ltd, Manchester, United Kingdom). In this test, the MNs samples were glued to the moving probe of the machine and were pressed against fixed chicken skin at a rate of 1 mm / min, until the desired normal force of 20 N was achieved (in average 200 mN per needle), followed by 30 second dwell time under the loaded state, followed by withdrawal of the upper holder that holds the MNs samples in the opposite normal direction. Continuous force and displacement measurements were recorded to identify the point of needle insertion. The curves of force versus displacement were generated for each test and the average insertion force was determined from 5 independent measurements. After MNs array removal, the skin area, where the MN were inserted, was inspected using HAYEAR 4K UHD microscope camera. To better visualize the insertion area, the skin was stained with 0.4% w / v trypan blue aqueous solution (left for 5 minutes, after which the excess of trypan blue was rinsed with ethanol and wiped away; Yu et al., Inter J Bio Macromol 2021, 191, 783-791).

[0174] Example 1: Preparation of Tri-C9 MNs

[0175] To demonstrate the fabrication of MNs from the dendritic tri-block amphiphile, ethanol and chloroform were first used to cast Tri-C9 amphiphile. However, aggregates were formed instead of a homogeneous MNs array (Figures 3A-3C). Without being bound by any theory or mechanism of action, this is attributed to the swift solvent evaporation rate and the fragility of the film, wherein the polymer chains face limited opportunities to entangle, hindering the formation of a stable film layer on the MNs mold (Ramos -Hernandez et al., Coatings 2023, 13 (2), 425). Consequently, polyvinyl pyrrolidone (PVP) was incorporated into the amphiphile solution in ethanol, due to its film- strengthening ability (Steward et al., Advan Coll Interf Sci 2000, 86 (3), 195-267; and Brady et al. In Developing Solid Oral Dosage Forms; Elsevier, 2017; pp 181-223). Upon drying, the MNs array was detachable, yet the resultant MNs showed poorly defined shape, non-homogeneous spreading of the polymer, and incomplete filling of the MNs mold cavities (Figure 4). Considering these findings, the baseplate was prepared using an aqueous solution containing sodium alginate to allow easy de-molding and a more definite structure. Without being bound by any theory or mechanism of action, it is contemplated that although Tri-C9 amphiphile does not dissolve in water, it can swell into a hydrogel mesophase, therefore, the addition of sodium alginate aqueous solution further facilitates the penetration of the polymer into the needle cavities of the mold. Furthermore, with the evaporation of water and increase in the relative concentration of the polymers, the sodium alginate chains can interpenetrate the Tri-C9 network to form a continuous matrix with increased stability, while the organic -based polymeric solution exhibited negligible viscosity, which led to poor filmforming ability (Felton, Inter J Pharma 2013, 457 (2), 423-427).

[0176] Next, Tri-C9 solutions at concentrations of 3%, 5%, and 10% (w / v) in chloroform were cast into the MNs molds to form the needles, and sodium alginate aqueous solution was added to form the baseplate. Increasing the amphiphile concentration led to a decrease in the capability of MNs formation (Figures 5A-5C). High concentrations (5% and 10%) of Tri-C9 amphiphile resulted in aggregates deposited in the mold cavities that prevent the sodium alginate solution from being homogenously spread over the template. Therefore, the concentration of 3% w / v Tri-C9 was chosen for further investigation.

[0177] To obtain an insight into the mechanical strength of the MNs, they were inserted manually into the skin-simulant parafilm model (Larraneta et al., Inter J Pharma 2014, 472(1- 2), 65-73). Significant bending of the needles was observed, indicating poor mechanical properties. Therefore, several excipients were examined with the aim of increasing the MNs’ mechanical strength. Initially, poly (lactic co-glycolic acid) (PLGA), a common polymer with good mechanical properties (Abdel-Haq et al., Inter J Pharma 2021, 601, 120578; Lu et al., Pharma 2023, 16 (3), 454; and Jahshan et al., ACS Appl Bio Mater 2021, 4 (5), 4131-4139), was incorporated into the MNs formulation, by either combining the Tri-C9 amphiphile with PLGA at the same ratio and casting into the MNs mold or by forming tri-layer MNs by casting the PLGA into the tips followed by casting the Tri-C9 amphiphile solution and then the baseplate as detailed above. While PLGA improved the insertion capability of the MNs in parafilm, a phase separation between the two polymers was noticed suggesting incompatibility between them (Figures 6A-6F). Then, microcrystalline cellulose (MCC), a widely used compression excipient serving as a binder in pharmaceutical dosage forms, was examined but the obtained MNs still showed inadequate mechanical properties (Figures 7A-7C). MCC did not integrate effectively into the MNs array, presumably due to its reduced affinity to chloroform, in comparison to Tri-C9 amphiphile, leading to decreased compatibility between the two. Additionally, MCC has strong molecular interactions with water, leading to the diffusion of MCC into the film surface upon addition of the aqueous sodium alginate layer.

[0178] Lastly, PEG with a molecular weight of 3.4 kDa was used to increase the engagement of the polymer chains. Two concentrations of PEG were used, 5% and 10% w / w PEG / Tri-C9. The incorporation of PEG significantly improved the MNs arrays in terms of structural integrity, uniformity, and mechanical strength, as confirmed by the insertion test using parafilm layers, providing vital indication for meeting the mechanical requirements of MNs (Figures 8A-8I).

[0179] Example 2: Fabrication and characterization of Tri-C9 MNs with PEG

[0180] Based on the obtained findings, PEG has been selected as the leading excipient and was integrated into the MNs at a concentration of 10% w / w PEG / Tri-C9. Next, MNs preparations with varying molecular weights of PEG (3.4 kDa, 10 kDa, 35 kDa, and 100 kDa) were investigated. The fabricated MNs patches consisted of an array of 10 x 10 pyramidal needles with a base width and height of 200 |im and 500 |im, respectively. Tri-C9 amphiphile and PEG were mainly concentrated at the tips of the needles while the baseplate consisted of sodium alginate, as depicted in Figure 1. Tri-C9 MNs made with 3.4 kDa and 10 kDa PEG showed bent-shaped needles and incomplete tips, while MNs formed with 35 kDa and 100 kDa PEG demonstrated well-defined sharp tips and uniform distribution on the substrate, confirming that the molecular weight of PEG affects both the physical appearance and mechanical properties of Tri-C9 MNs (Figures 9A-9D). Without being bound by any theory or mechanism of action, it is contemplated that higher molecular weight PEG polymers contribute to increased stiffness, while intermediate molecular weight PEGs have a plasticizing effect on the MNs patches thereby adversely affecting their structural properties and flexibility (Ortega-Toro et al., Food Hydrocolloids 2016, 56, 9-19; and Faradilla et al., Carbohyd Poly 2019, 205, 330-339).

[0181] Example 3: Insertion capabilities of MNs in a skin-simulant model and mechanical properties testing

[0182] The mechanical performance and insertion testing of MNs with PEG having different molecular weights were evaluated as described by Larraneta et al. (Inter J Pharma 2014, 472(1- 2), 65-73) whereby parafilm was used as a skin simulant. 8 layers of parafilm were assembled to create a film with an approximate thickness of 1 mm and the MNs were inserted by manual pressure to imitate the practical use in clinical settings.

[0183] Tri-C9 MNs with 35 kDa and 100 kDa PEG exhibited complete perforation of the first parafilm layer and a number of holes were created in the second layer, whereas Tri-C9 MNs with 3.4 kDa and 10 kDa PEG displayed lesser penetration ability accompanied with higher height reduction percentage of the needles, indicating poorer mechanical performance of the formulations with intermediate molecular weight PEGs (Figures 10A-10J). In this regard, Tri- C9 MNs with 35 kDa PEG created well-defined square-shaped pores in the parafilm and the needles were slightly compressed rather than bent, demonstrating their mechanical adequacy and uniformity (Figure 10E). On the other hand, the remaining MNs formulations were significantly bent (Figures 10C, 10D, and 10F).

[0184] To further investigate the mechanical strength of the lead MNs formulations, with 35 kDa and 100 kDa PEG, the resistance of MNs to increasing static forces was measured by placing different weights on top of the MNs arrays for 5 min. As shown in Figures 11 A- 111, the MNs underwent deformation as compared to the original form, in which the sharp tips of MNs presented increased bending following application of 50 g (-4.9 mN / needle) to 1000 g (~98 mN / needle) weights on the MNs. However, the MNs remained intact and did not break, indicating their good mechanical strength and potential competency for transdermal drug delivery (Yu et al., Mater Sci Eng: C 2017, 80, 187-196; and Shan et al., J Nanobio technol 2022, 20 (1), 238).

[0185] Taken together, incorporating PEG with high molecular weights into the MN formulations led to an improvement in the mechanical properties due to the increased polymeric chain density at the MNs tips (Dawud et al., Pharma 2023, 15 (2), 526; and Kim, et al., Euro J Pharmac Biopharma 2016, 105, 148-155). Furthermore, the results shown in Figure 12 illustrate the increase in the melting point of the polymer, whereby a higher melting point indicates more intertwined polymer chains and stronger intermolecular interactions. Based on the skin-simulant model results and observed mechanical properties, Tri-C9 with PEG 35 kDa and 100 kDa were selected as lead formulations for further investigation.

[0186] Example 4: Preparation and characterization of DEX-loaded MNs

[0187] The anti-inflammatory drug, dexamethasone (DEX), was used as a lipophilic model drug and was successfully incorporated into the needle tips of Tri-C9 MNs with 35 kDa and 100 kDa PEG. Both formulations displayed comparable encapsulation efficiencies with insignificant difference (t-test, p>0.05), and drug-loading content of ca. 8.5 wt % (Table 2). The pyramidal morphology of the MNs arrays was confirmed using scanning electron microscopy (SEM).

[0188] Table 2.

[0189] Figures 13A-13D show drug-loaded Tri-C9 MNs containing 35kDa and 100 kDa PEG, compared to their blank counterparts. All formulations had intact structures with quadrangular pyramidal shapes indicating that the MNs can be fabricated from the amphiphilic tri-block polymer and successfully load the drug. To better describe the composition of the MN tips and baseplate, an elemental analysis was performed using energy dispersive spectrometry (EDS). Since carbon and oxygen are indicatives of both Tri-C9 amphiphile and sodium alginate, there is a higher carbon to oxygen ratio in the Tri-C9 amphiphile. In addition, sodium is characteristic of the sodium alginate baseplate, sulfur is derived only from the Tri-C9 amphiphile, and the drug is the only source of fluorine. The distributions of carbon, oxygen, fluor, sodium and sulfur were therefore determined in order to gain further insight into the arrangement of the constituent materials. Table 3 summarises the quantitative contents of the constituent materials. Analysis of the tip of a single needle, showed a significantly higher carbon to oxygen ratio in comparison with the baseplate (Figures 13E-13F), which was found to contain higher oxygen to carbon ratio. In addition, the tip part was also found to contain higher amount of sulfur than sodium. Conversely, when analysing the baseplate, the amount of sodium surpassed that of sulfur. Without being bound by any theory, it is suggested that the polymer is predominantly positioned in the upper part of the MNs, while sodium alginate is primarily located at the baseplate of the microneedles patch. Additionally, a fluorine atom signal was observed only in the tip analysis, and was absent in the baseplate, confirming the successful encapsulation of the drug within the polymer in the tips of the MNs.

[0190] Table 3.

[0191] The drug-loaded MN patches were evaluated in terms of their insertion ability by conducting the parafilm insertion test. Both DEX-loaded Tri-C9 MNs demonstrated more than 95% penetration in the first parafilm layer, equivalent to ca. 140 pm insertion depth, indicating their ability to deliver the drug into the epidermis layer by penetrating the stratum corneum. The MNs height reduction percentage was calculated after the insertion test for each formulation, in which DEX-loaded MNs with 35 kDa PEG exhibited a height reduction of 23% ± 8, whereas DEX-loaded MNs with 100 kDa PEG showed a height reduction of 29% ± 6. Although the MNs displayed greater deformation than in the absence of the drug (Figures 10A- 10J), no evidence of breakage or fractures was observed (Figures 14A-14B). It can be concluded that both the molecular weight of the PEG excipients as well as the drug loading could influence the mechanical properties of Tri-C9 MNs whereby loading with drug molecules resulted in the weakening of the mechanical strength. Based on the aforementioned findings, Tri-C9 with 35 kDa PEG was chosen for further investigations.

[0192] Example 5: MNs arrays behaviour upon exposure to an aqueous medium

[0193] To assess the ability of the MNs to imbibe the interstitial fluids of the body and undergo mesophase transitions, the swelling capacity and dynamic transition stages in phosphate - buffered saline were examined. Upon introducing into PBS, the wetting process of the MNs involved two main stages, which included dissolving and swelling phases that occurred simultaneously. The first stage included rapid dissolution of the hydrophilic PEG excipient and the sodium alginate as a result of interaction with water molecules, while in the second stage, the amphiphilic tri-block polymer absorbed water and swelled into a hydrogel, due to the existence of the hydrophobic dendrons. As depicted in Figures 15A-15D, the MNs patch swelled rapidly and subsequently a third stage of disintegration of the formed hydrogel was observed within 30 seconds. At this stage, the swollen hydrogel needles started to disintegrate and formed microgel particles, which gradually precipitated at the bottom of the container. The hydrogel particles that were formed remained stable even after 1 week.

[0194] Example 6: In vitro drug release from MNs tips in PBS

[0195] The release of dexamethasone from MNs composed of Tri-C9 and 35kDa PEG was then examined. DEX-loaded MNs were placed into a dialysis tube (MWCO 8kDa) with an aqueous buffer (PBS, pH 7.4) in the presence or absence of an esterase (PLE) to evaluate the release profile of the hydrophobic dexamethasone cargo during the several mesophase transitions of the Tri-C9 amphiphiles. Both conditions (with / without esterase) exhibited controlled release with rather similar release rates in the first 48 hours. As presented in Figure 16, in the absence of PLE, approximately 90% of the drug was released within 6 days, while in the presence of the enzyme, the formulation exhibited a slightly decelerated release rate, resulting in a release of approximately 80% within the same time frame.

[0196] The data acquired from the drug release study were analysed using zero-order, first- order, and Korsmeyer-Peppas release kinetics models in order to shed light on the release mechanism. Table 4 presents the correlation coefficient (R2) values for these kinetic models, showing that the first-order release and Korsmeyer-Peppas model provided the best fit for the data.

[0197] Korsmeyer-Peppas kinetic model describes the drug transport mechanism by fitting the first drug release data (below 60% release) and calculating the value of the release exponent (n). Using DEX-loaded MNs, the release exponents under different PLE concentrations ranged from 0.77 to 0.84, indicating non-Fickian or anomalous transport. Without being bound by any theory or mechanism of action, this suggests that the mechanism of dexamethasone release is mainly governed by swelling and diffusion, where the slow rearrangement of polymeric chains and the diffusion of the drug from the hydrogel, simultaneously cause the time-dependent anomalous effects (Abu Ammar et al., J Cont Rel 2018, 272, 54-61; and Siepmann et al., Adv Drug Del Rev 2001, 48 (2-3), 139-157). The atypical geometry of the MNs could potentially also contribute to the drug release behaviour as well as certain physicochemical processes that are not considered by the mathematical model applied.

[0198] Table 4.

[0199] Notably, although the Tri-C9 amphiphiles contain aliphatic end-groups linked by ester bonds to the dendritic branches, no significant difference in the release kinetics in the presence or absence of the enzyme were observed. Without being bound by any theory or mechanism of action, this may be explained by the slow enzymatic degradation of hydrogels composed of triblock amphiphiles as compared to similar architectures that are characterized by a lower degree of hydrophobicity (Rathee et al., ACS Macro Lett. 2023, 12 (6), 814-820). As the release of dexamethasone was faster than the expected enzymatic degradation, similar release rates regardless of the presence of the enzyme and its concentration are contemplated. Moreover, as dexamethasone is a moderately lipophilic drug (logP 1.83) it can readily diffuse to the release medium, so that the potential contribution of the hydrolysis of the amphiphiles by the enzyme becomes even more limited. Nevertheless, as eventual degradation of the amphiphiles can be a critical requirement to allow their clearance after releasing their cargo, the degree of the degradation of the amphiphiles at the end of the release experiment was analysed. The solutions inside the dialysis tubes were diluted with acetonitrile to allow complete dissolution of the polymer residues, and then analysed by HPLC. The chromatograms (Figure 17) clearly showed full degradation of the amphiphiles into hydrophilic polymers for the samples that were incubated with the enzyme, while the samples without the enzyme were found to show only partially degraded tri-block amphiphiles (due to spontaneous hydrolysis of the esters). When considering both the release rates and the HPLC analysis with respect to the degree of degradation of the amphiphiles, the release of dexamethasone from the formulation can be mainly attributed to the successful entrapment within the amphiphilic polymer matrix. This underlines the feasibility of the designed tri-block amphiphiles-based MNs to afford the controlled and prolonged drug release. Such sustained release behaviour could increase the patient’ s adherence to the treatment by reducing the need for frequent administrations as well as minimizing the side effects often associated with higher doses.

[0200] Example 7: Ex vivo skin insertion test

[0201] To further elucidate the mechanical properties and insertion capability of DEX-loaded MNs, the MNs were subjected to penetration testing using ex vivo chicken skin (Figure 18A). Skin penetration is characterized as a series of sequential small penetrations where the MNs gradually tear the skin, while the force increases until it reaches a plateau followed by more rapid increase, indicating successful skin piercing (Economidou et al., Micromachines 2021, 12(2), 117; Zhang et al., Acta Pharma Sinica B 2018, 8(3), 449-457, Lim et al., Inter J Pharma 2021, 600, 120475). Figure 18B shows the force-displacement curve, in which the force was normalized by the number of needles (force per a single needle) in each MNs array. At the insertion point, an abrupt change in slope in the form of a small plateau is evident, and then while MNs being further inserted to the skin, the resisting force increases again due to the friction between the needles and the skin tissues, as well as the compression of the skin-MN system (Ranamukhaarachchi et al., Biomed Microdevices 2019, 21(4), 100). The DEX-loaded MNs required a mean insertion force of 101 ± 1 mN per needle (n=5). The insertion was confirmed by visualizing the skin after the compression test. The MNs demonstrated full penetration into the skin as shown in Figure 18C. To improve the visibility of the puncture sites, the skin was stained with trypan blue solution and blue pinholes were observed on it, indicating that the MNs had sufficient mechanical strength to successfully penetrate the skin (Figure 18D). Example 8: MNs composed of Tri-block amphiphiles with different lengths of end group

[0202] To evaluate the effect of the lengths of the hydrophobic end groups on the penetration of the MNs, three amphiphiles designated Tri-C6, Tri-C9 and Tri-C12 as depicted in the following structure were synthesized. where R=C5Hn (Tri-C6), C8HI7(Tri-C9), or C11H21 (Tri-C12).

[0203] The amphiphiles were blended with an antifungal medication, clotrimazole (CTZ), and PEG 35kDa as an excipient to result in formulations containing 3% w / v of the amphiphile, 1% w / v CTZ, and 10% w / w relative to the amphiphile of PEG 35kDa. Figures 19A-19C show images of the MNs arrays evidencing that all three amphiphiles were successful in forming a complete array of needles.

[0204] CTZ-loaded MNs patches were characterized for their insertion abilities. Tri-C6 and Tri-C9 based MNs patches showed full penetration into the first parafilm layer (Figures 20A, 20B, 20D, and 20E), whereas Tri-C12 based MNs exhibited poorer insertion performance, in which the number of pores created were 75 (Figures 20G and 20H). The MNs height post insertion further demonstrated that the Tri-C6 and Tri-C9 MNs had sufficient strength to pierce the parafilm layer with minimal change in the height of the needles (Figures 20C and 20F) as compared to Tri-C12 MNs (Figure 201).

[0205] In order to demonstrate the ability to fine-tune the hydrophobicity of the resulted polymer-blend, the MNs were cast in two different manners. In the first manner, 3% w / v of Tri-C6 and Tri-C9 at a 1:1 ratio was blended with 10% w / w PEG 35kDa and 1% w / v clotrimazole and cast into MNs (Figure 21A). In the second manner, the tips of the MNs were composed only of Tri-C12 and the bottom of the MNs were composed of Tri-C6 loaded with CTZ (Figure 21E). Figures 21B-21D and 21F-21H show that both formulations demonstrated acceptable insertion capabilities highlighting the feasibility to cast different MNs arrays by altering the composition of the polymeric solution.

[0206] Another antifungal drug, amphotericin B (AmB), was incorporated into Tri-C9-based MNs. Two different formulations were developed differing in the solvent and the fabrication steps. The first formulation, Fl-Org (Figure 22A), was composed of Tri-C9 amphiphile at a concentration of 3% w / v, PEG 35kDa at a concentration of 10% w / w of the Tri-C9, and 0.5% w / v AmB dissolved in chloroform. The formulation was prepared by casting the organic amphiphile solution into the MNs mold followed by application of vacuum. In the second formulation, F2-Aq (Figure 22E), the drug was dissolved and cast separately in an aqueous medium with the addition of a sodium bicarbonate buffer at pH 11.0. The tri-block amphiphile and PEG were dissolved in ethanol and cast onto the tip-forming layer. Finally, sodium alginate 4% w / v was used as a back-layer for both formulations.

[0207] Figures 22B and 22F show the MNs patches formed depicting sharp microneedles in both formulations. The needles were characterized by a yellow color stemming from the successful entrapment of AmB. When the MNs were tested against parafilm layer, both showed full penetration into the first parafilm layer, as evidenced by well-defined square-shaped holes created at each layer (Figures 22C and 22G). The morphology of the MNs after the insertion was determined. It was found that the Fl-Org formulation exhibited a height reduction of 25.0+11.0 (n=28; Figure 22D) while the F2-Aq formulation exhibited a height reduction of 29.5+15.0 (n=30; Figure 22H).

[0208] Another approach was adopted to formulate AmB -loaded MNs considering the ability of the dendritic amphiphiles to form a gel network when in contact with water, due to hydrophobic interaction between end-groups of the amphiphile, and the good solubility of AmB at acidic environment (pH 2.0). AmB and each of Tri-C6 and Tri-C9 were dissolved together with PEG 35kDa in acidic water (pH 2.0) and mixed with either methanol or ethanol at a ratio of 1:1 (v / v). The concentration of PEG 35 kDa in both formulations was 10% w / w with respect to the Tri-Cnamphiphile. Each formulation showed a different behaviour depending on the hydrophobicity of the polymer and the nature of the organic solvent. In particular, both Tri-C6 and Tri-C9 based formulations resulted in a viscous solution when dissolved in ethanol. When methanol was used as the solvent, the resulting solutions were even more viscous than those prepared in ethanol. In view of the viscous nature of the solutions, centrifugation was employed using a centrifugal force to enabled efficient filling of the mold cavities. As shown in Figures 23A-23D, Tri-C6 and Tri-C9 based MNs patches were successfully formed either using ethanol or methanol as the organic solvent.

[0209] In order to test the effect of increased concentration of the PEG excipient, formulations containing 10% w / w and 20% w / w PEG 35 kDa with respect to the Tri-Cnamphiphile were prepared from ethanol solutions. Figures 24A-24F show that good MNs arrays were obtained in both concentrations of excipient.

[0210] To examine the penetration ability of the formed MNs, both formulation, Tri-C6 and Tri-C9 MNs loaded with AmB were inserted into eight stacked parafilm sheets. As shown in Figures 25A-25F, in both cases, approximately 100 pores were created in the first layer with minimal morphological changes observed in the needles after insertion. However, bending of needles was more noticeable for Tri-C9 MNs.

[0211] To further examine the effect of PEG concentration on the mechanical properties of the MN, the insertion test was performed on the MNs with an increased amount of PEG. For Tri- C6 MNs, increasing the PEG concentration improved the insertion performance of the needles, as 100 pores with more precise and defined shapes were created without significant change in the height of the needles after the insertion (Figures 26A-26C). In contrast, increasing the amount of PEG adversely affected Tri-C9 MNs (Figures 26D-26F). The pores created had less defined shapes, demonstrating poor insertion ability (Figure 26E). This was further confirmed by examining the needles after insertion, where broken segments were observed (Figure 26F). Similar results were obtained when testing the Tri-C12 MNs (Figures 26G-26I). Overall, increasing the amount of PEG contributed to the physical appearance of the needles as well as their insertion performance.

[0212] Another approach was adopted to fabricate AmB-loaded MNs using a two-step fabrication process as illustrated in Figure 27. In particular, 100 pL of a solution containing 0.5 % (w / v) AmB, 3% tri-block TriC6,9,12 amphiphile (w / v), and 10% (w / w of amphiphile) PEG 35kDa in EtOH:MeOH (1:1 v / v) was cast into a MNs mold followed by application of vacuum for 10 minutes. The casting and vacuum application were repeated to enable higher drug loading. The sodium alginate (SA, 4% w / v) base was then applied followed by drying and demolding. Figures 28A-28C, 29A-29C, and 3OA-3OC show the morphology of the TriC6,9,12 MNs arrays that were obtained. To examine the penetration ability of the formed MNs arrays, the needles were inserted into eight stacked parafilm sheets as a skin model. The results are shown in Figures 31A-31I and are summarized in Table 5.

[0213] Table 5.

[0214] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

CLAIMS1. A delivery system comprising an active pharmaceutical ingredient incorporated within a plurality of solid microneedles comprising tri-block amphiphile comprising two hydrophobic segments each comprising at least one stimulus-responsive cleavable site and a hydrophilic segment therebetween, wherein upon contacting a biological fluid, the solid microneedles undergo a first and a second phase transition thereby releasing the active pharmaceutical ingredient incorporated therein in a controlled manner, wherein the first phase transition comprises a transition from solid microneedles to hydrogel microneedles and the second phase transition comprises a transition from hydrogel microneedles to hydrogel microparticles, and wherein upon exposure of the hydrogel microparticles to a stimulus, the stimulus-responsive cleavable sites undergo cleavage thereby disassembling the hydrogel to dissolved polymers and fragments thereof.

2. The delivery system according to claim 1, wherein the microneedles are extending from an approximately planar base substantially perpendicular to the microneedles.

3. The delivery system according to claim 2, wherein the base comprises a polymer selected from a polysaccharide, cellulose derivatives, poly(meth)acrylates, PEG derivatives, gelatin, silk fibroin, hyaluronic acid, and dextrin.

4. The delivery system according to claim 2, wherein the base comprises a polymer comprising alginic acid or derivatives thereof.

5. The delivery system according to claim 2, wherein the base comprises a polymer selected from sodium, potassium and calcium alginate.

6. The delivery system according to any one of claims 1 to 5, wherein the plurality of microneedles have widths of about 50 pm to about 500 pm and heights of about 200 pm to about 1000 pm.

7. The delivery system according to any one of claims 1 to 6, wherein the hydrophobic segments comprise a lipid, a fatty acid, or a steroid.

8. The delivery system according to any one of claims 1 to 6, wherein the hydrophobic segments comprise hydrophobic dendrons.

9. The delivery system according to claim 8, wherein the hydrophobic dendrons comprise between 0 to 5 generations.

10. The delivery system according to claim 9, wherein each generation of the hydrophobic dendron comprises a linear or branched C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl,C1-C20 alkylene, C2-C20 alkenylene, C2-C20 alkynylene or arylene moiety which is substituted at each end with a group selected from the group consisting of -O-, -S-, - NH-, -C(=O)-, -C(=O)-O-, -O-C(=O)-O-, -C(=O)-NH-, -NH-C(=O)-NH-, -NH-C(=O)- O-, -S(=O)-, -S(=O)-O-, -PO(=O)-O-, and any combination thereof.

11. The delivery system according to any one of claims 1 to 10, wherein the stimulus- responsive cleavable site comprises a cleavable bond selected from the group consisting of a disulfide, a diselenide, an ester, an amide, an amidine, an imine, a carbamate, a carbonate, an acetal, a urea, a thiourea, a trithionate, a sulfate, a sulfamate, a phosphate, a phosphoamide, a hydrazone, an ether, a silyl ether, an oxyme, a boronic acid, a boronic ester, a nitro, and an azo.

12. The delivery system according to claim 11, wherein the stimulus-responsive cleavable site comprises a cleavable bond selected from the group consisting of an ester, an amide and a urea.

13. The delivery system according to any one of claims 1 to 12, wherein the stimulus is an enzyme.

14. The delivery system according to claim 13, wherein the enzyme is selected from an esterase, a lipase, an amidase, and a urease.

15. The delivery system according to any one of claims 1 to 14, wherein the hydrophilic segment comprises polyethylene glycol (PEG), polyacrylic acid, poly(hydroxyethyl acrylate), poly(oligo-ethylene glycol acrylate), polyacrylamide, polymethyl oxazoline, polyethyl oxazoline, polyvinylpyrrolidone (PVP), polysarcosine, polypeptide, polypeptoid, hydrophilic polymethacrylate, polyamine, hydrophilic nylon, polyvinyl alcohol, hydrophilic protein, or polycarbohydrate.

16. The delivery system according to claim 15, wherein the hydrophilic segment comprises polyethylene glycol (PEG).

17. The delivery system according to claim 15 or 16, wherein the hydrophilic segment has a molecular weight of about 0.5 to about 100 kDa.

18. The delivery system according to any one of claims 1 to 17, wherein the tri-block amphiphile is depicted by the structure of formula (II):wherein R is a C2-20 alkyl and n is an integer from 2 to 3,000.

19. The delivery system according to any one of claims 1 to 17, wherein the tri-block amphiphile is depicted by the structure of formula (III):wherein R' is a C2-20 alkyl and n is an integer from 2 to 3,000.

20. The delivery system according to any one of claims 1 to 19, wherein the active pharmaceutical ingredient is a steroid selected from the group consisting of dexamethasone, aldosterone, beclomethasone, betamethasone, budesonide, cloprednol, cortisone, cortivazol, deoxycortone, desonide, desoximetasone, difluorocortolone, fluclorolone, flumethasone, flunisolide, fluocinolone, fluocinonide, fluocortin butyl,fluorocortisone, fluorocortolone, fluorometholone, flurandrenolone, fluticasone, halcinonide, hydrocortisone, icomethasone, meprednisone, methylprednisolone, paramethasone, prednisolone, prednisone, tixocortol and triamcinolone; an antibiotic selected from the group consisting of ampicillin, dapsone, chloramphenicol, neomycin, cefaclor, cefadroxil, cephalexin, cephradine, erythromycin, clindamycin, lincomycin, amoxicillin, ampicillin, bacampicillin, carbenicillin, dicloxacillin, cyclacillin, picloxacillin, hetacillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, ticarcillin, rifampin, tetracycline, fusidic acid, lincomicyn, novobiocine, and spectinomycin; an anti-inflammatory agent selected from the group consisting of betamethasone, prednisolone, piroxicam, aspirin, flurbiprofen, diflunisal, ibuprofen, fenoprofen, fenamate, ketoprofen, nabumetone, naproxen, diclofenac, indomethacin, sulindac, tolmetin, etodolac, ketorolac, oxaprozin, celecoxib, meclofenamate, mefenamic acid, oxyphenbutazone, phenylbutazone, salicylates, and phyto sphingosine; an anti-fungal agent selected from the group consisting of clotrimazole, miconazole, ketoconazole, nystatin, amphotericin B, and pimaricin; or respective pharmaceutically acceptable salts or derivatives thereof.

21. The delivery system according to any one of claims 1 to 20, wherein the microneedles further comprise an excipient.

22. The delivery system according to claim 21, wherein the excipient comprises polyethylene glycol (PEG).

23. The delivery system according to claim 22, wherein the PEG has a molecular weight of about 10 to about 200 kDa.

24. The delivery system according to any one of claims 1 to 23, which is suitable for administration via a route selected from transdermal, nasal, and vaginal.

25. The delivery system according to claim 24, which is suitable for transdermal administration through the skin of a subject.

26. A method of transdermally administering an active pharmaceutical ingredient to a subject in need thereof, the method comprising applying the delivery system according to any one of claims 1 to 25 to the skin of a subject.

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