Nucleic Acid Delivery Into Skin for the Treatment of Skin Disease: Proofs-Of-Concept, Potential Impact, and Remaining Challenges

15 March 2005

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2386 East Heritage Way, Suite B, Salt Lake City, Utah 84109 USA Phone +1-877-628-7300 • Email—[email protected] www.pachyonychia.org COREL-07857; No of Pages 12 Journal of Controlled Release xxx (2015) xxx–xxx

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Nucleic acid delivery into skin for the treatment of skin disease: Proofs-of-concept, potential impact, and remaining challenges

Michael Zakrewsky, Sunny Kumar, Samir Mitragotri ⁎

Center for Bioengineering and Department of Chemical Engineering, University of California Santa Barbara, Santa Barbara, CA 93106, USA article info abstract

Article history: Nucleic acids (NAs) hold signiﬁcant potential for the treatment of several diseases. Topical delivery of NAs for the Received 10 June 2015 treatment of skin diseases is especially advantageous since it bypasses the challenges associated with systemic Received in revised form 7 September 2015 administration which suffers from enzymatic degradation, systemic toxicity and lack of targeting to skin. Howev- Accepted 9 September 2015 er, the skin's protective barrier function limits the delivery of NAs into skin after topical application. Here, we Available online xxxx highlight strategies for enhancing delivery of NAs into skin, and provide evidence that translation of topical NA

1. Introduction applied drugs. Nevertheless, significant efforts have been expended over the years to overcome the skin barrier. This review highlights strat- Nucleic acids (NAs) hold great potential for the treatment of various egies to effectively deliver NAs into the skin. Focus is placed on their ap- diseases, and there has been a significant amount of both academic plications for the treatment of dermatological diseases. Further, we as well as commercial interest in a variety of NA-based therapeutics provide evidence of the transformative impact of topical NA therapies including genes, antisense oligodeoxynucleotides (ODNs), siRNA, on the treatment of skin diseases. The efforts here highlight significant aptamers, and CpG oligonucleotides [1–4]. However, translation of advances in topical delivery of NAs over the last decade, and aim to these platforms to the clinic has been significantly limited by challenges guide future technologies and their translation into the clinic. associated with delivering NAs to the diseased site. Enzymatic degradation in the blood, rapid clearance from systemic circulation, poor bio- 2. The skin barrier availability at the target site, and immunological response have yet to be adequately addressed for the implementation of systemically admin- The outermost layer of skin, the SC, is primarily responsible for istered NA therapies [1,4]. its barrier function. The SC is a thin layer only 10–20 μmthickthat Delivery of NAs through the skin offers a potential solution to these is made up of corneocytes. Corneocytes are anucleate cells heavily issues, especially for the treatment of dermatological diseases (Table 1). enriched with intracellular keratin filaments. Corneocytes are held to- The skin is the largest organ of the body, and can provide a pain free and gether in a “brick and mortar” structure by a lipid matrix composed of compliant interface for drug delivery [5,6]. Topical delivery of NAs offers ceramides, free fatty acids, and cholesterol. Materials traversing the several advantages over alternative delivery routes including avoidance skin barrier must, therefore, diffuse through the tortuous lipid channels, of major degradative pathways in the GI tract, avoidance of enzymatic and/or traverse transcellularly through corneocytes, or enter the skin degradation and clearance from the bloodstream, sustained and con- through hair follicles or sweat ducts (Fig. 1). Transport within the lipid trolled delivery, reduction in systemic toxicity, the ability to easily bilayers, however, is the most common mode of passage through the observe and treat sites of adverse reactions, and improved patient com- skin. This results in the exclusion of most foreign materials, and more pliance [6]. Concurrently, it provides a means to directly target the dis- specifically, renders passage of large, hydrophilic molecules (N500 Da eased sites for the treatment of dermatological diseases e.g. skin cancer, and Log Po/w b 1.5) such as NAs (typically NN10,000 Da, Log Po/w b 0) psoriasis, and atopic dermatitis. to virtually negligible levels without some form of enhancement Topical delivery of NAs, however, is quite challenging. The challenge strategy. originates from the outermost layer of the skin – the stratum corneum The layer underlying the SC, the epidermis, can also serve as (SC) – which serves as a formidable barrier to the entry of topically another transport barrier [7]. The epidermis is the first viable tissue layer of the skin where the pathology of several dermatological disor- – μ ⁎ Corresponding author. ders resides. The epidermis is 50 100 m thick and is composed primar- E-mail address: [email protected] (S. Mitragotri). ily of keratinocytes. As keratinocytes migrate upward from deeper

Table 1 overwhelming. Needle-phobia is a serious concern for a large number Advantages of NA topical delivery for the treatment of skin disease. of children as well as adults leading to significant patient non- Advantages of NA topical delivery compliance [9]. Moreover, intradermal injections are limited only to the site of application, and injection into multiple sites during a single • Targeted delivery into skin • Large surface area for drug application administration is challenging. To avoid many of these drawbacks, • Easily observe and excise sites of adverse reaction microneedle arrays have been developed. Microneedle arrays comprise • Easily monitor disease progress and adjust treatment needles that are only 100–700 μminlength(Fig. 2). When placed on the • Needle-free application skin, their sharp tips allow easy insertion into the stratum corneum, • Sustained and controlled delivery • Avoidance of GI tract while the short length ensures adequate penetration into the skin • Limited to negligible systemic toxicity without disrupting nerves in deeper skin tissue. Microneedles have • Limited to negligible clearance from diseased tissue been used extensively for the delivery of NAs. Mikszta et al. used • User-friendly application microneedle arrays to deliver plasmid DNA encoding a hepatitis B surface antigen for immunization [10], and they showed extensive immunological response in mice. Antibody titers following application of portions of the epidermis to the SC they gradually begin to keratinize microneedle arrays were significantly higher and less variable than and secrete lipids that eventually form the SC bilayers. This process con- when delivered using either intradermal or intramuscular injection. tinues as keratinocytes terminally differentiate into corneocytes and Chabri et al. [11] used microneedles to deliver cationic lipid-DNA com- serves to rejuvenate the SC from underneath while the outermost plexes (~100 nm diameter) into the skin. Ding et al. [12] demonstrated layer of the SC sloughs away. Within the epidermis, keratinocytes are successful immunization of mice with co-administration of diphtheria held together by cell–cell tight junctions. In the epidermis, claudin-1, toxoid and CpG oligonucleotide delivered by microneedle array, and claudin-4, occludin, and zonula occludens-1 are responsible for inhib- Gonzalez-Gonzalez et al. [13] demonstrated effective delivery of anti- iting paracellular transport [8]. This makes transport of NAs, other luciferase siRNA and gene silencing in luciferase expressing transgenic large macromolecules, and drug carriers such as nanoparticles and mice. NA–lipid complexes difficult both vertically, deeper into the skin, as well as laterally from the site of administration to peripheral areas of 3.1.2. Microporation the skin. For effective treatment, both the SC as well as epidermal trans- Microporation is another technique that employs physical dis- port barriers must be overcome to deliver NAs to all areas of the disease. ruption of the SC for delivery of large therapeutics or therapeutic carriers. An array of resistive elements can be placed on the skin. An 3. Methods of transport enhancement electric current pulsed through the array results in localized ablation of corneocytes in contact with the array [14]. Alternatively, Over the years, a large number of strategies have been devised for erbium:yttrium–aluminum–garnet (Er: YAG) laser arrays can be used perturbing the SC to enhance the delivery of drugs into and through for localized ablation of the SC and epidermis [15]. Similar to micro- the skin. These strategies can be generally categorized into three main needle arrays, microporation has gained considerable interest over the groups: physical, active, and passive methods. The advantages and dis- last decade. For example, Lee et al. [16] used laser microporation to de- advantages of each class of perturbation methods are summarized in liver antisense oligonucleotide as well as plasmid DNA into the skin. De- Table 2. Their use for the delivery of NAs is described below. livery of antisense oligonucleotide was enhanced 3–30 fold compared to intact skin in vitro. In addition, expression of GFP in nude mice was en- 3.1. NA delivery using physical methods hanced 160 fold after application of GFP plasmid DNA. The amount of enhancementcorrelatedwithboththelaserfluency as well as the size 3.1.1. Microneedles of oligonucleotide. The same group also showed enhanced delivery of Intradermal injections are the simplest and most direct method siRNA [17]. siRNA delivery into skin was enhanced 3.5 fold and localized for delivering NAs into the skin. Here, the barrier properties of the SC mainly in the dermis. Hessenberger et al. [18] used laser microporation are overcome completely by injecting NAs directly into the viable to deliver CpG oligonucleotides into the skin and successfully protected tissue layers of the skin. Intradermal injections are typically used for against immune response to grass pollen in mice. They used the Precise evaluating efficacy of NAs or other cutaneous therapeutics, or as the Laser Epidermal System (Pantec Biosolutions) which creates well- positive control for evaluating dermal delivery technologies, however, defined arrays of micropores in the skin, and allows precise control the downsides of intradermal injection for treating skin disease are over the number, density, and depth of the micropores giving the user

Fig. 1. Transport pathways into the skin. A: Intercellular pathway through lipid bilayers. B: Transcellular pathway through keratin-rich corneocytes. C: Shunt pathway through hair follicles and sweat ducts.

Table 2 Advantages and disadvantages of physical, active, and passive topical NA delivery methods.

Class Methods Advantages Limitations

Physical Microneedles; microporation • High enhancement • Cost • No therapeutic limitations • Limited application area • Easily incorporate large depot • Slow return of skin barrier function • Delivery not majorly affected by diseased state of skin • Potentially irritating • No direct cell internalization enhancement Active Electroporation; iontophoresis; • Skin penetration and cell internalization • Cost sonophoresis • Controlled delivery • Limited application area • Easily incorporate large depot • Localized penetration zones • Repeated application at the same site • Transient perturbation of skin Passive Polymeric nanoparticles; liposomes; • Low cost • Nanoparticles, liposomes may localize in hair follicles peptides; dendrimers • Application to large skin surface • Peptides, dendrimers may be limited by size of NA • Can protect therapeutic from degradation • Lower enhancement • Multi-functional (SC perturbation, epidermal tight junction • Delivery can vary depending on state of SC and epidermis perturbation, cell membrane perturbation) • Targeting of speciﬁc cell-types

unprecedented tuneability over the amount and localization of thera- of SC microstructure creating pores in the skin [14].Ontheotherhand, peutic delivered [19]. electroporation is the application of short duration (b0.5 s) and high intensity (b100 V) electric pulses to the skin [20] which result in transient 3.2. NA delivery using active methods permeabilization of the lipid bilayers in the skin and concurrently permeabilize cell membranes of epidermal keratinocytes. Electropora- 3.2.1. Electroporation tion is also expected to create aqueous pores through the skin. However, Electroporation can be used to permeabilize the skin and enhance electroporation, unlike microporation, acts through non-thermal mech- passive diffusion of drug. The mechanism of electroporation is quite dif- anisms as lipid rearrangement, reduced skin resistance, and enhanced ferent from that of electrically-induced microporation. Electrically- transdermal transport are observed in the absence of a significant tem- induced microporation utilizes electric fields to induce thermal ablation perature rise in the pulse medium [20]. Using electroporation, Regnier et al. [21] showed enhanced permeability of the stratum corneum to a phosphorothioate antisense ODN. The permeability enhancement lasted up to 1 hr post-electroporation in rat skin in vitro. Specifically, transport was enhanced N 4-fold into the SC and N 3-fold into viable skin tissue when oligonucleotide solution was applied immediately following electroporation. Further, Zhang et al. [22] demonstrated effective gene transfection and expression in the epidermis of human skin.

3.2.2. Iontophoresis Iontophoresis can be used to drive transport of charged drugs like NAs. Applying a continuous low intensity (b10 V) electric field at a constant current [20] has been used extensively to deliver a wide range of charged therapeutics including calcitonin, luteinizing hormone- releasing hormone, and dexamethasone [5]. In contrast to electroporation which acts primarily on the skin structure, iontophoresis is not believed to cause major changes to the skin. Minor structural effects can be observed and may partially contribute to delivery enhancement through sweat ducts and hair follicles [20,23]. Nevertheless, iontophoresis is believed to primarily act on the drug itself, driving transport of a charged molecule by means of an applied electric field. Using iontophoresis, Kigasawa et al. [24] demonstrated successful delivery of anti- IL-10 siRNA. Specifically, using an atopic dermatitis model in rats they showed a 73% reduction in IL-10 mRNA levels after treatment with anti-IL-10 siRNA and iontophoresis. The same group later extended the technique for vaccination as well as treatment of melanoma tumors [25].Kigasawaet al. reported enhanced delivery of CpG oligonucleotides into the epidermis and dermis using iontophoresis compared to that by free diffusion. Further, for the treatment of melanoma tumors in hairless mice, CpG oligonucleotides delivered using iontophoresis resulted in tumor reduction comparable to subcutaneous injection. Abu Hashim et al. also demonstrated the ability to deliver NF-κB decoy ODN into skin using iontophoresis for atopic dermatitis treatment [26].While passive diffusion of FITC-NF-κB decoy ODN was negligible for all con- centrations tested, ~100 pmol/cm2 was delivered after 6 hr of iontopho- Fig. 2. Microneedle arrays offer controlled length and sharp tips for easy insertion through resis using 0.5 mA current density. In addition, the authors observed a the SC and into the epidermis. (a) Scanning electron micrograph of a 20 × 20 silicon fl microneedle array. (b) Scanning electron micrograph of a microneedle tip. Figure linear dependence between the ux of ODN into the skin with both cur- reproduced with permission from Henry, et al. [104]. rent density and concentration of drug. When the technique was

Fig. 3. Sonophoresis enhances delivery of NA into skin through localized perturbation zones. (a) Top view of skin exposed to sulforhodamine B and ultrasound. (b and c) FITC-NA delivery into skin after ultrasound treatment. (b) Cross-section of skin corresponding to a localized perturbation zone. (c) Cross-section of skin corresponding to a non-localized perturbation zone. Figure modiﬁed with permission from Tezel et al. [28].

Fig. 4. The anatomy of spherical nucleic acid nanostructures. An inorganic core is densely functionalized with oligonucleotides containing three segments: a recognition sequence, a spacer segment, and a chemical-attachment group. Additionally, other functional groups such as dye molecules, quenchers, modiﬁed bases, and drugs can be attached along any segment of the oligonucleotide. Figure reproduced with permission from Cutler et al. [37].

Fig. 5. Experimental design to screen for skin-penetrating peptides using phage-display. The same strategy could be used to screen for aptamers. cell-penetrating peptide to enhance intracellular delivery, and AT1002 resulted in significantly enhanced apoptosis and reduction in tumor was used as a tight-junction modulator to enhance permeation into size in mice. the epidermis and dermis. Tat peptide has also been shown to enhance macromolecule transport across the SC [47,48]. The system was later 4. Potential impact applied to treat atopic dermatitis in mice [49]. Application of anti-RelA siRNA complexed with Tat peptide and co-incubated with AT1002 led The burden of skin disease is alarming and growing fast [53]. 1 out to reductions in ear thickness, clinical skin severity, topical cytokine of 3 individuals is estimated to have a skin disease at any given time, levels, and serum IgE production. Yi et al. [50] successfully delivered resulting in direct healthcare costs over $30 billion annually in the US anti-microphthalmia-associated transcription factor (anti-MITF) siRNA alone [54]. Symptoms from the ~3000 different skin diseases range conjugated to TD-1 R8 peptide (ACSSSPSKHCGRRRRRRRR) for the treat- from itching, redness, and irritation to physical disfigurement, or ment of patients with melisma. Treatment with a cream formulation death. Furthermore, skin diseases manifest externally leading to severe containing TD-1 R8 peptide co-administered with anti-MITF siRNA re- and even debilitating emotional distress and social prejudice [54]. sulted in reductions in tyrosinase, tyrosinase-related protein 1, and Dermatological drugs aimed at treating skin disease reap an estimated melanocortin 1 receptor leading to measurable inhibition of melanin $24.4 billion in annual revenue [55]. In addition, many other skin con- production and melanocyte apoptosis. In fact, after 4 weeks application ditions, not considered disease, are of major concern. These include cos- patients demonstrated a significant lightening of facial hypermelanosis metic conditions like wrinkling, cellulite, discoloration, and skin pliancy. lesions and almost completely restored dark lesions to normal skin color Growing demand for more effective treatments of cosmetic conditions after 12 weeks. is evidenced by the emergence and growth of the cosmeceutical market which was estimated to be ~$35 billion globally in 2013 [56].TopicalNA therapies have exciting potential to reduce this burden and be transfor- 3.3.5. Dendrimers mative in the way we deal with and treat skin disease (Table 3). Exam- Although less studied than peptides for delivering NAs into skin, ples are discussed in detail below. dedrimers are similarly advantageous due to their diversity, ease of Targeting orphan disease can help speed translation by allowing synthesis, and functional group density. Bielinska et al. [51] demonstrat- fast-tracked regulatory processing from orphan drug designation. ed the use of dendrimers for topical gene delivery. Dendrimer com- There are several orphan skin diseases with recognized NA targets. plexes resulted in measurable gene expression of chloramphenicol Hidradenitis suppurativa (Hurley disease) is an orphan disease that af- transacetylase that was ~7-fold higher than with naked plasmid. In fects over 100,000 people in the US [57], most commonly women [58]. addition, Venuganti et al. [52] used a similar dendrimer in combination It is a chronic and debilitating disease characterized by painful abscess- with iontophoresis to deliver antisense oligonucleotide. Treatment with es, redness, and scarring, leading to significant physical and psycholog- antisense oligonucleotide targeting the anti-apoptotic protein, Bcl-2, ical impairment and reduced quality of life [59]. Currently, the most

Table 3 Topical NA delivery can have a large impact in the clinic due to the large number of skin diseases with NA therapeutic targets responsible for their etiology and pathogenesis.

Disease/Condition People affected Causative molecule Role in pathogenesis Up/downregulation Ref. and NA target for for therapy therapy

Hurley disease 100,000 in US TNFα, IL-17, IL-23 Promotes inflammation through activation of vascular Down [61,62] endothelial cells and immune cells Epidermolysis 500,000 globally IL-1β, IL-1β receptor Mutant K14 leads to upregulation of IL-1β, which then Down [67] bullosa results in a positive feedback signaling cascade that ends up further activating mutant K14 expression Mutant K5, K14 Mutations result in keratin filament aggregation in Down (mutant) [68,69] keratinocytes leading to dysfunction Collagen VII Mutation results in loss of anchoring-fibril formation Up (wild-type) [70] Pachyonychia congenita 5000–10,000 globally Mutant K6a, K6b, K17 Mutations disrupt keratin filament formation resulting Down (mutant) [72] in skin cell fragility Netherton syndrome 1/50,000 globally Serine protease Mutation in serine protease inhibitor Kazal type 5 leads Down [79] to serine protease hyperactivity and excessive protein degradation in skin Psoriasis 4.5 million adults in US TNFα, IL-17, IL-23 Same as above Down [64] IL-4 IL-4 producing T helper cell population is repressed Up [84] β defensin-2 Highly upregulated in psoriatic lesions Down [34] Atopic dermatitis 10%–20% of children; RelA Important member of the NF-κBinflammatory pathway Down [49] 1%–3% of adults IL-4, -5, -10, -13 Overexpressed in AD lesions Down [85] Th2-type immune Th2-type dominated immunological response Down [88] response implicated in AD pathogenesis Cellulite 80%–90% post-adolescent ACE ACE has been linked to higher production of angiotensin Down [89] women II, a vasoconstrictive peptide, along with lower production of bradykinin, a vasodilatative peptide Collagenase, lipase, Enzyme hyperactivity leads to breakdown of subcutaneous Down [90] elastase and dermal ECM. Lipase suppression leads to build up of (collagenase, fatty tissue elastase); up (lipase) HIF-1 HIF-1 induces fibrosis and inflammation which is Down [89] implicated in the formation of cellulite Wrinkling Universal with aging Elastase Breakdown of elastic fibers leads to reduced skin Down [91] elasticity and wrinkle formation Melasma 4%–10% of all MITF Inhibition of MITF gene leads to suppression of melanin Down [50] dermatological visits production in dark facial lesions common form of treatment is surgical excision of diseased areas of the with severe EB survive past the age of 30 due to extensive wounds skin [60]. For severe forms of Hurley disease there is no FDA approved and infection. There is no FDA approved drug indication for EB. Manage- treatment. Studies into the etiology and pathogenesis of Hurley disease ment of EB consists of limiting blister formation from scratching and suggest it is a multifactorial inflammatory disease characterized by friction [66]. For more effective treatment, studies suggest IL-1β and overexpression of TNFα [61] as well as other inflammatory cytokines its receptor may be a valuable target. Indeed, overexpression of IL-1β like IL-17 and IL-23 [62]. Treatment of Hurley disease with anti-TNFα has been detected in patients with EB and treatment of EB in mice monoclonal antibodies or other biologics has been shown to be effective with anti-IL-1β or IL-1β receptor antagonist resulted in improvement [60,63].TNFα promotes inflammation through recruitment of vascular of symptoms [67]. Further, TWi Biotechnology Inc. was recently granted endothelial cells and immune cells. Further, it is overly expressed at orphan drug designation for its anti-IL-1β and anti-IL-1β receptor small sites of inflammation while repression generally results in alleviation molecule drug AC-201 [65]. EB can also be targeted at the genetic level. of inflammation [64]. While there is no FDA approved drug indication One subtype of EB (epidermolysis bullosa simplex), is predominantly for treating moderate to severe Hurley disease, several anti-TNFα bio- caused by dominant-negative mutations in the genes encoding logics are already on the market including etanercept and adalimumab. keratin-5 and keratin-14 (K5 and K14). siRNA therapies that specifically In fact, the anti-TNFα monoclonal antibody, Humira (adalimumab, knockdown mutant keratin without affecting wild-type keratins have Abbvie Inc.), was recently granted orphan drug designation by the shown promise [68,69]. A second subtype of EB (dystrophic epider- FDA to start clinical trials on moderate to severe Hurley disease patients molysis bullosa) is caused by mutations in collagen VII and gene deliv- [65]. On the other hand, these drugs can only be administered by injec- ery of wild-type collagen VII has shown promise as a therapeutic in tion or infusion and systemic side effects limit their long-term use. phase I/II clinical trials [70]. Alarmingly, the most common side effect of systemic delivery is the de- Pachyonychia congenita (PC) is a rare genetic autosomal dominant velopment of other autoimmune diseases as a direct result of treatment skin disorder that affects 5000 to 10,000 people [71]. PC may be caused due to increased expression of IL-17 and IL-23 as well as other inflam- by mutations in either K6a, K6b, K6c, K16, or K17 [66]. The disease typ- matory cytokines [64]. Therefore, topical delivery of anti-TNFα siRNA ically manifests with calluses and blisters on the feet and palms, as well or antisense oligonucleotide is an attractive platform to limit off target as discoloration, cysts, and hyperkeratosis of the hair follicles [72]. effects, improve patient compliance, and reduce hospital-related costs. Moreover, patients typically live in constant pain. Due to the large num- Even more impactful, a cocktail of topical anti-TNFα, anti-IL-17, and ber of different mutations that can result in PC, topical application of anti-IL-23 NA therapeutics may prove synergistic at alleviating Hurley personalized NA therapies holds the most potential. In fact, treatment disease symptoms while avoiding adverse reactions. of patients with topical siRNA therapies are showing promise both in Epidermolysis bullosa (EB) is another rare skin disease with few ef- mouse models as well as in humans [73–77]. fective treatments. EB affects roughly 30,000 people in the US and Netherton syndrome is an autosomal recessive disorder estimated to ~500,000 people globally [66]. EB symptoms typically include severe affect 1/50,000 individuals [78]. Symptoms present at birth or shortly skin fragility to the point where large segments of skin can be removed after and include painful peeling and scaling of skin. More alarmingly, from simply scratching or rubbing skin [66]. Alarmingly, few patients Netherton syndrome manifests significant impairment of skin barrier

Please cite this article as: M. Zakrewsky, et al., Nucleic acid delivery into skin for the treatment of skin disease: Proofs-of-concept, potential impact, and remaining challenges, J. Control. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.09.017 8 M. Zakrewsky et al. / Journal of Controlled Release xxx (2015) xxx–xxx function leading to life-threatening dehydration and frequent systemic 80%–90% of post-adolescent women [89]. It is characterized by an “or- infections. Netherton syndrome is caused by a mutation of the serine ange peel” appearance of the skin primarily in the thighs and buttocks. protease inhibitor Kazal type 5 (SPINK5) gene leading to serine protease While physical burden is not associated with cellulite, significant emo- hyperactivity and excessive protein degradation in skin. Essentially, tional burden is typically associated with cellulite. Cellulite pathology protease hyperactivity results in excessive and premature epidermal is generally not well understood, however, a few NA targets that show desquamation [79]. Netherton syndrome may be treated by down- promise are proposed in literature. For example, decreased microcircu- regulating production of serine proteases in the skin, or alternatively, in- lation in subcutaneous adipose tissue is believed to play a role in cellu- ducing expression of wild-type serine protease inhibitor [80]. Due to the lite formation. Specifically, overexpression of angiotensin-converting significant reduction in skin barrier function, initial treatment with top- enzyme has been linked to higher production of angiotensin II, a vaso- ical NAs may not require dermal enhancement. Instead, technologies to constrictive peptide, along with lower production of bradykinin, a localize drug in the skin such as cell-targeting or skin targeting peptides vasodilatative peptide [89]. Breakdown of the extracellular matrix may be required. However, as skin integrity improves more significant (ECM) due to enzyme hyperactivity may also play an important role enhancement may be required. in cellulite [90]. Normal ECM may potentially be restored by silencing Mainstream disease would also benefit from topical NA therapies. enzymes like collagenase, elastase, and lipase. Finally, fibrosis and local- For example, psoriasis is estimated to affect nearly 4.5 million adults ized inflammation may play an important role in cellulite formation in the US alone [81]. Children are also commonly affected by the disease. [89].Specifically, hypoxic inducible factor-1 (HIF-1) mutations that It is a chronic skin disease that presents as severe lesions, redness, and minimize its ability to induce fibrosis and inflammation have been itching and poses a significant burden on patients' quality of life. shown to be associated with individuals who do not typically have cel- About 20%–25% of patients are estimated to be dissatisfied with their lulite, or have reduced severity of cellulite, while individuals with wild- current treatment [81]. Of even more concern, for patients suffering type HIF-1 were shown to have a higher probability of more severe pre- from the most severe form of psoriasis, erythrodermic psoriasis, there sentation of cellulite. is no standard treatment regimen [82]. Due to the multifactorial nature Skin wrinkling is another cosmetic condition that generates signifi- of psoriasis, NA therapies may be advantageous because NA cocktails cant emotional burden. Wrinkling affects everyone as they age, howev- targeting a number of different targets can be formulated and delivered er, treatment is most typically sought by post-adolescent females. leading to down/up regulation as needed. Psoriasis is one of the more Unfortunately, few effective treatments exist for the reduction of wrin- exhaustively studied forms of inflammatory skin disease and numerous kles. Currently, the most common treatment is intradermal injections of therapeutic targets have been proposed; a subset is described here. Sim- botulinum toxin A (Botox). Botox, however, is extremely toxic and re- ilar to Hurley disease, TNFα is perhaps the most established drug target sults in temporary muscular paralysis at the site of injection. Further, for treating severe forms of psoriasis [64]. Topical delivery of anti-TNFα Botox must be administered by a trained physician limiting its use and siRNA as well as a siRNA cocktail including anti-TNFα and anti-STAT3 increasing its cost. Alternatively, topical NA therapies may be beneficial was shown to be effective at alleviating psoriasis-like symptoms in as both a treatment and prevention strategy. For example, elastase up- mice [33,83]. Activated T helper cells have also been shown to play a regulation has been proposed as an important factor for ultraviolet major role in the manifestation of psoriasis, and selective skewing irradiation induced wrinkle formation [91]. Inhibition of elastase with from Th1 phenotype to the IL-4 producing Th2 phenotype can alleviate N-phenethylphosphonyl-L-leucyl-L-tryptophane resulted in reduced psoriasis symptoms [84]. Further, selective differentiation to the Th2 formation of wrinkles in mice exposed to daily doses of ultraviolet irra- phenotype can be simply induced by subcutaneous injection of IL-4 diation. Therefore, knockdown of elastase with topically applied NAs [84]. As an alternative to injection, psoriasis may be alleviated through may be a viable option for the treatment and prevention of skin wrin- topical delivery of plasmid DNA to induce expression of IL-4 in the kling, as well as provide patients with a safer and cheaper alternative skin. Proof-of-concept has been demonstrated in a K14-VEGF transgenic to Botox injections. mouse model of psoriasis [35]. Melasma is a hyperpigmentation disorder that manifests as darken- Atopic dermatitis (AD) affects 10%–20% of children and 1%–3% of ing or browning of the skin, typically on the face [92]. Melasma dispro- adults worldwide [85]. AD typically manifests with severe redness and portionately affects women and disproportionately affects ethnicities itching, skin lesions, and papules resulting in significant impact on qual- with darker skin color, however overall, melasma is generally estimated ity of life. Similar to psoriasis, the inflammatory nature of the disease to account for 4%–10% of all dermatological-related doctor visits [93]. makes topical NA treatment appealing. Although the exact cause of AD Moreover, melasma is commonly reported to induce significant psycho- is not known, upregulation of inflammatory pathways such as the NF- logical burden, social impairment, and reduced quality of life [92,93]. κBinflammatory pathway are typically observed, and inhibition of The most common treatments are skin lightening agents like tyrosinase these pathways have shown promise. For example, delivery of anti- inhibitors and spot removing agents like retinoic acid, kojic acid, and RelA, an important member of the NF-κBinflammatory pathway, result- azelaic acid [50]. However, these are typically either ineffective or result ed in significant improvement in disease symptoms in mice [49].Inad- in unsightly white spots when used in the clinic. Topical NA therapies dition, knockdown of NF-κB with decoy ODNs also showed therapeutic may prove beneficial for treating melasma without adverse side effects promise in mice [26]. Several cytokines are also believed to be associat- associated with current treatments. Specifically, studies have shown si- ed with the pathogenesis of AD including IL-4, -5, -10, and -13. These lencing of the microphthalmia-associated transcription factor (MITF) cytokines are typically expressed in significantly higher quantities in gene could be an effective treatment strategy. MITF encodes for tyrosi- AD lesions compared to healthy skin [85], and knockdown of these cy- nase, tyrosinase-related protein 1, and melanocortin 1 receptor which tokines and others with topical NA therapies has shown promise in are all involved in melanin synthesis. Topical delivery of anti-MITF mouse models of AD [24,36,86,87]. Further, suppressing the Th2-type siRNA has shown promise in human trials [50]. dominated immunological response typical of AD may hold therapeutic promise. Indeed, delivery of CpG oligonucleotides was shown to significantly reduce AD lesions in mice by shifting from Th2-type dominant 5. Remaining challenges immune response to a more balanced Th1/Th2 immune response [88]. Cosmetic conditions would also benefit significantly from topical NA Despite significant efforts to deliver NAs into the skin as well as iden- therapies developed as either pharmaceuticals or cosmeceuticals. How- tifying NA targets for treating skin disease, significant hurdles remain. ever, cosmeceutical development may benefit from faster translation to Three major areas posed serious challenges in the past and still remain the clinic due to reduced FDA regulations provided claims of the cosme- the bottleneck to successful translation of topical NA therapies: (1) skin ceutical are appropriately chosen. For example, cellulite manifests in delivery, (2) cellular internalization, and (3) stability of NAs. These

Please cite this article as: M. Zakrewsky, et al., Nucleic acid delivery into skin for the treatment of skin disease: Proofs-of-concept, potential impact, and remaining challenges, J. Control. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.09.017 M. Zakrewsky et al. / Journal of Controlled Release xxx (2015) xxx–xxx 9 challenges must be addressed concurrently to develop successful NA microneedle arrays and microporation devices see widespread use in topical delivery systems for use in humans. the clinic. Microneedles and other physical methods are the most effective at Perhaps the most significant drawback of both physical and active enhancing delivery into the skin, and the type and size of therapeutic methods, however, is limited application area. For diseases like Hurley is not restricted. Further, large depots can be easily incorporated for disease, EB, moderate to severe psoriasis, skin wrinkling, and many sustained release. However, significant questions remain in regards to others which can occupy a large percentage of skin surface area, physi- their effectiveness at localized and homogeneous delivery into skin tis- cal and active methods may not be practical. In these cases, passive per- sue. Specifically, physical methods inherently result in localized pene- turbation methods may be most effective. tration zones. They do not address horizontal diffusion of NA which is Passive transport enhancers can be formulated as a cream and retarded by cell–cell tight junctions in the epidermis [7,8].Active applied to all areas of the body, including the face, in a patient friendly methods such as electroporation [94] and sonophoresis [28] may also manner. Further, formulations incorporating synergistic combina- be limited by localized penetration zones (Fig. 3). The effect of size of tions of skin penetrating and cell internalizing transport enhancers penetration area on clinical outcome needs to be determined. Combina- can be easily envisaged. The main limitation of passive methods tion therapies with co-delivery of NAs and, for example, tight junction remains their low transport enhancement relative to physical and active modulators may be required for efficacy in humans. methods. Further, passive transport enhancers like peptides and Also of concern, physical methods do not enhance cellular inter- dendrimers may be limited to use with NAs of a certain size like anti- nalization nor protect NAs from degradation in the skin. NAs need sense oligonucleotides and siRNA as opposed to plasmid DNA. Effort to enter into the cytoplasm or nucleus of cells to elicit a therapeutic re- has been spent over the years to identify and understand passive trans- sponse. Physical methods alone cannot facilitate this enhancement, port enhancers. High-throughput screening for chemical enhancers and therefore, they must inherently be combined with other technolo- is well-documented [105] and has proven useful for identifying gies to enable cell internalization. For example, microneedle arrays inte- synergistic combinations of chemicals that perturb the skin with mini- grating active methods like electroporation [95–97] and sonophoresis mal irritation [106]. Along the same lines, peptides can be screened in [98] have been designed to facilitate NA delivery enhancement into a high-throughput fashion using phage-display (Fig. 5). Phage libraries cells. However, incorporating active methods for cell internalization applied to the skin are selected based on their ability to transport further adds complexity and cost. Passive methods for cell internaliza- through the skin. After only a few rounds, a library of ~109 peptide se- tion, for example, tagging NAs with cell-penetrating peptides, cell- quences can be screened. Particularly advantageous, this technique penetrating dendrimers, or cell-penetrating aptamers have the poten- can be used to identify peptides that localize in particular layers of the tial to provide affordable means of permeation enhancement. In skin e.g. epidermis or dermis. Delivery of NA therapies to specific loca- fact, several cell-penetrating peptides are currently in clinical trials tions in the skin could be beneficial to limit off-target effects. Phage which makes them an attractive option [99]. On the other hand, cell- screening is a powerful technique, and an extensive number of phage li- penetrating peptides combined with physical or active dermal penetra- braries exist. Effort should be place on identifying novel peptides with tion enhancement still neglect the stability issues of NAs. siRNA, ODN, powerful skin-penetrating ability. The same methodology could be and plasmid DNA are susceptible to enzymes in the epidermis and der- used to screen for skin-penetrating aptamers, which to the authors' mis which can lead to significant degradation. To address this concern, knowledge has not been attempted to-date, further broadening the packaging NAs in cationic lipid complexes or liposomes would both pro- space of chemically and structurally distinct passive transporters for de- tect the NA from enzymatic degradation as well as aid cell internaliza- livering NAs. tion. In addition, chemical modifications of NAs like attaching charge As new transporters are identified, we can better understand how to neutral moieties via cleavable or reducible linkers or use of a phospho- overcome the skin barrier to deliver increasingly large cargos. For exam- rothioate backbone have been shown to aid in internalization [100] and ple, the number of skin-penetrating peptides identified thus far have protect from degradation [101], respectively, without compromising afforded a better understanding of their mechanism of transport en- function. hancement [107]. Interestingly, TD-1, Tat, and SPACE peptide all seem The regulatory hurdles for medical devices and drug–device combi- to bind and interact with keratin in the corneocytes in the skin facilitat- nations must also be addressed. Physical methods are sophisticated and ing transport of cyclosporine A through the transcellular pathway. As a will be highly regulated. Fabrication, sterilization, and implementation result of this work, we posit screening of low molecular weight ligands of the device must be demonstrated to result in safe and effective ther- for affinity to keratin may help identify ligands with improved NA deliv- apy. This regulation is in addition to any oversight process for the NA ery. It is also conceivable that the mechanism of enhancement is depen- itself and further complicates translation of topical NA therapies. dent on the cargo delivered, however, similar studies with NA cargos are Along this line, a lot of effort has been spent over the years to address severely lacking. fabrication concerns. For example, methods have been devised to fabri- Concurrently, definitive mechanistic studies to elucidate modes of cate microneedle devices using steel and other strong materials [102]. transport enhancement of nanoparticles, liposomes, and spherical Further, biocompatible and biodegradable microneedles have been pro- nucleic acids are lacking and studies that have been reported do not posed [103]. Many of these methods can be scaled using the same pro- reach adequate consensus. Alvarez-Roman et al. [108] have studied cesses used for integrated circuits [104] which affords cost-effective the distribution of polystyrene particles after topical application. They manufacturing as well as the potential for outsourcing manufacturing. observed almost exclusive uptake into the hair follicles. Follicular up- Lacking, however, are studies assessing effects of sterilization and im- take was dependent on size of the particles with 20 nm particles accu- plementation protocols on patient safety. For example, all physical mulating to a larger extent than 200 nm particles. Similar localization methods result in disruption of the skin barrier. This disruption will re- in the hair follicles has been observed for liposomes [109–111] and main until re-epithelialization at the site of ablation can occur. While ultradeformable liposomes [35], as well as titanium dioxide microparti- barrier function of the skin is diminished, pathogens may potentially cles [112], micro/nanoemulsion droplets [113], lipoplexes [114],and enter the skin and cause infection. Therefore, the site of application solid lipid nanoparticles [112]. However, naked NAs have also been may need to repeatedly be sterilized, or sealed with a sterile bandage, shown to localize in hair follicles when applied without any nanocarrier for days or weeks after application of the NA therapy. Depending on [115–117] which suggests nanocarriers may only hold potential as drug the frequency of application as well as the site of application (e.g. face, depots for controlled release topical formulations of NAs. Still others hands, feet), requiring a permanent or semi-permanent sterile bandage have demonstrated dispersed distributions of nanocarriers into viable may not be tolerated by the patient. Prevalence of adverse events from epidermis and dermis. Verma et al. [118] demonstrated enhanced physical ablation of the SC and epidermis must be determined before drug delivery using liposomes and also concluded that size was the

Please cite this article as: M. Zakrewsky, et al., Nucleic acid delivery into skin for the treatment of skin disease: Proofs-of-concept, potential impact, and remaining challenges, J. Control. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.09.017 10 M. Zakrewsky et al. / Journal of Controlled Release xxx (2015) xxx–xxx most important parameter. However, it is important to note that 6. Conclusion these studies were performed with fluorescent drug and therefore do not distinguish between penetration of intact liposomes versus Topical application of NAs for the treatment of skin disease is an ad- enhancement in drug delivery through simple fluidization of lipid vantageous route for the translation of NA therapies to the clinic. Topical bilayers. In fact, Kirjavainen et al. concluded that non-fusogenic application offers many advantages over alternative methods of admin- fluorescently-tagged liposomes do not penetrate skin [119]. Instead, istration including avoidance of many challenges that are current road- only liposomes that were fusogenic with SC lipids were able to enhance blocks to systemic NA therapy implementation. Although the skin does drug delivery. Moreover, fusogenic liposomes enhanced penetration pose a significant barrier to drug delivery, especially to large hydrophilic of drug applied in free solution subsequent to application of lipo- macromolecules like NAs, extensive effort has been spent to overcome somes. These data suggest that liposomes may act through incor- this barrier. These efforts have been highlighted here, and include the poration into and fluidization of the lipid bilayers to enhance drug use of microneedles, microporation, electroporation, iontophoresis, delivery. Other factors of liposome design such as charge, amount of sonophoresis, nanoparticles, liposomes, spherical nucleic acids, pep- cholesterol, and acyl chain length did not appear to influence drug tides, and dendrimers. Translation of these technologies to the clinic is delivery, In contrast, Geusens et al. concluded liposomes can penetrate sure to have a transformative impact on the burden of both skin disease intact skin if flexible enough [120]. It is reasonable that liposomes as well as cosmetic conditions. A large number of genetic and multifac- that can penetrate via the multilamellar lipid bilayers in the SC without torial inflammatory diseases result in chronic, physical and emotional breaking apart may aid delivery of cargo into the skin. Others have also burden to patients who have few treatment options other than repeated reached similar conclusions arguing ultraflexible liposomes create dosing by injection or infusion, or invasive surgery. Similarly, cosmetic hydration-driven transport mechanisms through the skin [121].How- conditions result in significant emotional burden with limited effective ever, since studies have shown localization of ultraflexible treatment options other than invasive surgery. Future efforts should liposomes in hair follicles [35], another possibility is that flexibility focus on validating the safety of long-term physical disruption of the does not aid penetration across multilamellar SC but instead enhances skin, better understanding the mechanisms of NA enhancement into transport only across the unilamellar lining of hair follicles. If confirmed, skin via passive methods, and exhaustively screening for novel ligands this may preclude treatment of regions devoid of hair follicles like palms and synergistic combinations of enhancers to realize the full potential and foot pads. Interestingly, spherical nucleic acids, are not be expected of topical NA therapies in the clinic. to fluidize lipid bilayers, nor are they be expected deform to facilitate passive diffusion through tight intercellular lipid channels in the skin; Acknowledgment therefore, we would expect them to localize in hair follicles like the ma- jority of solid nanoparticles N 40 nm studied to date [122].Yet,spherical The authors acknowledge support from Duncan and Suzanne nucleic acids exhibit extensive penetration homogenously into epider- Mellichamp Chair and fellowship as well as from the National Institutes mal and dermal tissue [38,40]. How spherical nucleic acids penetrate of health (1R21CA191133). skin has yet to be reported but should be of immediate interest given the significant potential this platform appears to have for treating skin disease. 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