Formulation‐based approaches for dermal delivery of vaccines and therapeutic nucleic acids: Recent advances and future perspectives

Abstract A growing variety of biological macromolecules are in development for use as active ingredients in topical therapies and vaccines. Dermal delivery of biomacromolecules offers several advantages compared to other delivery methods, including improved targetability, reduced systemic toxicity, and decreased degradation of drugs. However, this route of delivery is hampered by the barrier function of the skin. Recently, a large body of research has been directed toward improving the delivery of macromolecules to the skin, ranging from nucleic acids (NAs) to antigens, using noninvasive means. In this review, we discuss the latest formulation‐based efforts to deliver antigens and NAs for vaccination and treatment of skin diseases. We provide a perspective of their advantages, limitations, and potential for clinical translation. The delivery platforms discussed in this review may provide formulation scientists and clinicians with a better vision of the alternatives for dermal delivery of biomacromolecules, which may facilitate the development of new patient‐friendly prophylactic and therapeutic medicines.


| INTRODUCTION
There is an increasing clinical demand to use biological macromolecules in the clinic for various applications. The global sales of biologics accounted for over 70% of the worldwide revenue from the 10 topselling pharmaceutical drugs. 1 This is of particular importance for managing severe diseases like inflammatory conditions, cancer, and autoimmune diseases as well as controlling infections and dispensing vaccines. Macromolecular therapeutics, including hormones, peptides, cytokines, antibodies, and nucleic acids (NAs), provide higher specificity and potency compared to small molecule drugs. 1,2 For example, the sales of tumor necrosis factor (TNF)-α inhibitors reached $40 billion in 2019 due to increased application in dermatology and the expansion of indications. 3 However, the successful delivery of biomacromolecules has been challenging due to their complex structure and high molecular weight (between 300 and 1,000,000 Da) in addition to stability issues encountered during manufacturing, storage, and administration. 1,4 Moreover, a short in vivo half-life, rapid clearance after injection and eventual degradation due to exposure to proteases and peptidases in the body are additional limitations. 5,6 As a result, this necessitates repeated injections of high doses to maintain therapeutic concentrations, resulting in adverse effects, and reduced patient compliance. 7,8 Consequently, alternative routes to systemic administration of biomacromolecules are appealing to overcome such limitations and improve patient compliance. While the dermal and transdermal route has been extensively studied for the systemic delivery of hormones such as calcitonin 9,10 and insulin, [11][12][13][14] the dermal delivery of macromolecules such as NAs is of particular importance for the treatment of various skin diseases 15 as well as for prophylactic purposes in vaccines. 16 The main biological obstacle for efficient dermal delivery of biomacromolecules is the barrier function of the skin, mainly the stratum corneum (SC). The SC consists of flattened and tightly packed corneocytes embedded in a highly lipophilic matrix composed of ceramides, cholesterol, and fatty acids ( Figure 1). This layer is followed by the viable epidermis, which is composed of viable keratinocytes (KCs) and Langerhans cells (LCs). Underneath the epidermis is the dermis, which contains dendritic cells (DCs), lymphatic as well as blood vessels, nerve fibers, collagen and elastic fibers, which gives structural support to the epidermis (Figure 1). [17][18][19] The barrier properties of the SC limit the penetration of molecules that are less than 500 Da and that have moderate lipophilicity (log P = 1-3), 20 making the dermal delivery of macromolecules such as proteins, antigens, antibodies, cytokines, and NA an exceedingly difficult task.
Physical, or active, methods of penetrating the skin have been studied extensively. This includes iontophoresis, 21 electroporation, 22 microneedles, 10,23 microjets, 24 and laser ablation. 25,26 However, despite their efficacy, these techniques suffer from certain limitations including challenges in use over large skin areas. Another important aspect to be considered is to guarantee cargo protection against degradation and to enhance the cellular internalization, when needed, both of which are difficult to control by physical methods. Simpler methods that are passive and noninvasive can facilitate dermal penetration while overcoming the limitations of physical methods. Passive dermal delivery can leverage a variety of tools, including lipid and polymer-based nanocarriers, peptides, hyaluronic acid (HA)-derivatives, inorganic nanoparticles, and ionic liquids, among others, for enhanced delivery of macromolecules.
In this review, we focus on the most recent formulation-based noninvasive passive delivery strategies over the last 5 years for dermal delivery of biomacromolecules, particularly antigens and NAs with applications in transcutaneous vaccination and treatment of inflammatory skin disorders such as psoriasis, atopic dermatitis, cutaneous cancers (e.g., squamous cell carcinoma, melanoma), tissue damage (e.g., burns, wounds), and fibrotic skin conditions.

| Merits of skin as immunization organ
While significant effort is directed toward the development of new vaccines, the most common method for vaccine administration remains to be by subcutaneous or intramuscular injections using hypodermic  27 ). Moreover, hazardous medical waste from used needles can be an additional source of infection. 28 Thus, there is a need for simpler, safer and more effective routes for vaccination.
Noninvasive transcutaneous immunization (TCI) offers a promising alternative to subcutaneous or intramuscular vaccine delivery owing to the large surface area of the skin and its connection to the rest of the body via a network of blood vessels and drain lymph nodes (LNs). 16,29 The epidermis consists mainly of KCs and LCs. KCs are the main immune effector cell type in skin. 30 They secrete cytokines, chemokines, and antimicrobial peptides; additionally, KCs express toll-like receptors (TLRs) that allow the immune system to recognize certain pathogens. 31 Dermal antigen presenting cells (APCs), including LCs in the epidermis and DCs in the dermis also play an important role in the initiation of immune responses (Figure 1(c)). LCs capture antigens in the tissue environment and present them using major histocompatibility complex molecules.
Activated LCs then migrate to present the antigens to CD8+ (cytotoxic) and CD4+ (helper) T cells in the draining LNs. Dermal DCs also play a role in activating the adaptive immune response to induce long lasting protection against those pathogens. 32,33 Moreover, fibroblasts, which are the major cell component in the dermis, produce cytokines like interleukin (IL)-6 and transforming growth factor (TGF)-β to participate in cutaneous immunity. 34 Accordingly, the skin serves as a highly immunologically active organ that can facilitate a more robust immune response than other tissues that have a limited population of APCs. 35 In addition to vaccination against infectious diseases, noninvasive TCI is a promising approach for other conditions including allergies, 36 autoimmune diseases, 37 and cancer. 38 The most common food allergy in children is an allergy to cow's milk proteins (CMA). Persistence of CMA is a serious concern due to the risks of accompanying atopic disorders, asthma, anaphylaxis, and other allergen-related chronic or acute symptoms. 39 Immunotherapy is considered the only curative treatment method for such allergies. 40,41 However, conventional oral immunotherapy for CMA requires hospitalization due to the risk of severe allergic reactions, including anaphylaxis. Therefore, a simpler and safer approach such as epicutaneous immunotherapy (ECIT) is required. However, the skin barrier function restricts the penetration of vaccine components such as antigens and NAs. Here we focus on the most recent passive approaches (particularly in the last 5 years) adopted for the noninvasive TCI, and their applications for cancer immunotherapy, protection against infectious diseases and allergy management.

| Antigen patches
Viaskin ® is an epicutaneous delivery system where the powdered antigen is electrosprayed on the surface of the patch. Under occlusive conditions, the antigen becomes solubilized the by natural transepidermal water loss due to emerging perspiration and is passively delivered to the SC. It has been extensively tested as a method for allergen desensitization of sensitized mice in preclinical studies [42][43][44] and clinical trials for peanut allergy. 45,46 Tordesillas et al. 47 showed that topical application of a model antigen with a Viaskin ® patch resulted in acquisition of the antigen by epidermal LCs, after which it was transported to the LN, where it was presented to naïve T cells and primed LAP + Foxp3 − regulatory T cells (Tregs). In this case, generation of immune tolerance was dependent on uptake through the hair follicle.
A recent placebo-controlled double-blind clinical trial used Viaskin ® epidermal occlusive patches loaded with lyophilized food antigens for ECIT. This system showed inductions of tolerance to peanut allergies and CMA. 36,48 However, the efficiency of antigen and adjuvant delivery was low.

| Hydrophilic gel patches
In a recent approach to enhance cytotoxic T-lymphocytes (CTL) production, Kamei et al. 49 combined a hydrophilic gel patch comprising crosslinked HiPAS™ acrylate medical adhesives, octyldodecyl lactate, glycerin, and sodium HA as a transcutaneous delivery device with mXCL1-V21C/A59C as an adjuvant. The transcutaneous delivery of ovalbumin (OVA) as a model antigen and mXCL1-V21C/A59C by the hydrophilic gel resulted in strong induction of OVA-specific CTLs and inhibited the growth of OVA-expressing tumors more efficiently than the intradermal injection of OVA with mXCL1-V21C/A59C.

| Solid-in-oil systems
In solid-in-oil (S/O) systems, the hydrophilic proteins (e.g., vaccine components, allergens) are dispersed in an oil-vehicle with the assistance of nonionic surfactants. They are prepared by freeze-drying water-in-oil emulsions containing therapeutic cargo followed by dispersion of the surfactant-medicine complexes into an oil vehicle that has affinity to the SC. 50 This system is attractive as a nanocarrier for peptides such as antigens and vaccine components owing to the high encapsulation efficacy for hydrophilic molecules and capacity for coloading immunomodulatory agents like adjuvants. [51][52][53] Recently, Kitaoka et al. 54 used a S/O nanodispersion system to deliver both the hydrophilic allergen molecules (β-lactoglobulin) and the lyophilic adjuvant (R-848) through the epidermis. The authors applied isopropyl myristate as an oil vehicle in the S/O nanodispersion and sucrose laurate L-195 as a surfactant. 55 This system enhanced the penetration of β-lactoglobulin through pig skin more than fivefold compared to the antigen solution in PBS. The system was then applied as a patch onto intact ears of mice with a model whey allergy. The level of total immunoglobulin E (IgE) was lower, and the levels of β-lactoglobulin-specific IgG subclasses were higher compared to similar model mice treated with β-lactoglobulin in a PBS solution. The extent of ear swelling was lower in mice treated with the S/O nanodispersion, and the cytokines secreted by splenocytes indicated the skewing of the immune reaction toward Th1-type immunity.
Transcutaneous immunotherapy using S/O nanodispersions has also been implemented for the treatment of Japanese cedar pollinosis, which represents a major health concern in Japan. It is a type I allergic disease treated by subcutaneous or sublingual administration of whole antigens from the pollen extract. Kong and coworkers implemented S/O nanodispersions loaded with vaccine T cell epitope peptides derived from pollen allergen coated with hydrophobic surfactants for TCI in a pollinosis mouse model. This model showed suppression in serum antibody IgE and cytokine production as well as alleviated allergic symptoms compared to mice that received subcutaneous injections. 50 In a further advancement, the same group conjugated the pollen extract to galactomannan (PE-GM) to mask IgE-binding epitopes in the PE and incorporated it in a S/O nanodispersion ( Figure 2). This system efficiently delivered the PE-GM through skin and improved uptake by DCs. Topical application in a pollinosis mouse model resulted in reduced antibody secretion and alteration of the ratio of type 1 T helper (Th1)/ type 2 T helper (Th2) cells, achieving a comparable therapeutic effect to subcutaneous injections, demonstrating the potential to alleviate Japanese cedar pollinosis. 56 Recently, transcutaneous immunotherapy against cancer with a S/O system was demonstrated. This system successfully delivered OVA, a model antigen, through the skin and induce OVA-specific antibodies. Mice vaccinated with OVA-S/O were challenged with E.
G7-OVA thymoma cells, which express OVA. A significant inhibition of tumor growth was observed in the vaccinated mice. 58 In a further advancement, Wakabayashi et al. 57 introduced a transcutaneous vaccine against melanoma by incorporating tyrosinerelated protein 2 peptide, (K-TRP-2), as a peptide antigen against melanoma in a S/O nanodispersion coloaded with the adjuvant R-848.
The S/O showed enhanced skin permeability of the peptide. A significant inhibition of melanoma growth was observed in mice vaccinated with this system in addition to the suppression of lung metastasis, highlighting the potential of the S/O nanodispersion as a transcutaneous antigen carrier for cancer vaccines. This technology is simple enough that self-medication could be possible, making this an attractive system for clinical translation.

| Hyaluronic acid conjugates
HA is a linear polysaccharide present in the extracellular matrix of the skin. It is known to promote KC proliferation as well as elasticity regeneration, and it is used in dermatologic clinics as dermal fillers. 59 HA receptors are highly expressed on skin cells, as epidermal KCs and dermal fibroblasts contribute to antigen recognition by producing immune mediators and presenting antigens to local DCs. 60 HA is a hydrophilic molecule that also has a lipophilic patch domain, giving it an amphiphilic nature that allows it to diffuse through the SC. It is reported to induce disordering of lipid organization within the SC and cause structural changes in keratin. 61 HA has been widely implemented as a delivery agent of microscale particles for intranasal delivery of influenza vaccines. 62 It also acts as a transdermal nanocarrier of macromolecules, such as human growth hormone 63 as well as small molecules 64  In the skin, HA is capable of releasing cargo by degrading into small fragments. The low molecular weight fragments are recognized in the body as damage-associated molecular pattern molecules, which can potentiate an immune response against released antigens by activating Toll-like receptors 4 (TLR4) and TLR2 and by stimulating the secretion of different cytokines. 65  HA-conjugates can be sterilized by filtration and stored in lyophilized powder forms vaccines to be reconstituted before use. The dissolved HA vaccine can be easily applied to the skin as lotion or skin toner or easily incorporated in skin patches facilitating self-administration, which may of particular use in countries or regions with limited access to healthcare.

| Natural polymeric nanocapsules
Polymeric nanocapsules (NCs) are one of the nanoscale carriers that have been widely employed for vaccination. Having a size similar to pathogens, NCs can be efficiently recognized by the immune system, leading to a potent immune response. 30,67 They can be formulated using a variety of materials, including polypeptides, polyaminoacids, polysaccharides, proteins, and others that can be rationally designed for site-specific delivery and controlled release of cargo. 68 Additionally, their core-shell structure permits the encapsulation of adjuvants in the hydrophobic core and incorporation of antigens or hydrophilic molecules in the polymeric shell to be recognized by the immune system. 69 Moreover, NCs are reported to facilitate transdermal delivery of a variety of molecules. The type of the polymeric shell in NCs can influence complement activation and immune system interactions as well as their in vivo performance. 70 Chitosan (CS) is a natural polymer that is known to enhance skin penetration besides its effect as an adjuvant. 71 Bussio et al. 72 developed NCs for transcutaneous antigen delivery comprising a polymeric corona made of CS and oily core of vitamin E for its immune adjuvant properties. 73 The positively charged NCs were prepared using a solvent displacement technique and were then incubated with OVA as a model antigen to guide their electrostatic assembly. The association efficiency of OVA was high (75%), maintaining the antigen integrity as evaluated by western blot. The antigen association in the NC shell promoted interaction with the immune system, which was demonstrated by complement activation while not affecting cell viability of macrophages.
Ex vivo studies using porcine skin showed that CS-NCs enhanced the penetration of OVA by 15 times compared to OVA in solution.
This effect was primarily attributed to their nano-size (100 nm) that allows for a higher penetration, as the antigen diffuses through different routes, including transcellular and intercellular pathways as well as through hair follicles. 74 Penetration was also aided by the positive superficial charge of CS-NCs and their ability to open tight junctions. 75 In another study, HA-NCs were prepared with an oily core of α-tocopherol stabilized by benzalkonium chloride and Lipocol ® HCO-40 (LP-HCO). 76 Although the electrostatic interaction was successful in associating OVA with CS NCs and antigens such as influenza antigen, 77 this approach was not applicable to HA-NCs. Changing the pH of the protein to at least 2 units under its isoelectric point (4.5) resulted in aggregation. However, incubating the antigen with HA-NCs without changing protein pH (6.9) resulted in 67% OVA association while maintaining the physicochemical properties of the HA-NCs and preserving the structure and the integrity of the OVA antigen.
HA-NCs produced immune complement activation over a range of concentrations, a property similar to vaccine adjuvants like aluminum salts. Studying their penetration across pig skin revealed that the association of OVA to HA-NCs resulted in 22 times higher and 33 times higher penetration and retention compared to OVA in solution, respectively. The authors proposed that the possible mechanisms by which these glycosaminoglycan-coated NCs overcame the SC to deliver OVA could be mediated by HA receptors widely expressed on KCs and fibroblasts as well as the hydration effect that forms paths for transport of OVA across the SC to aid in antigen retention within the hydrated epidermal layers. 78 Moreover, the hydrophobic domains of HA, with its eight CH groups, formed a complex with phospholipids and disturbed the SC, enhancing the skin permeability and facilitating OVA absorption across the skin. 79

| Lipid-based vesicles
Phospholipid vesicles, including liposomes, transfersomes, and ethosomes, have emerged as popular nanocarriers for dermal delivery of encapsulated vaccines owning to their physiochemical and structural resemblance to biological membranes. 80 They have the advantage of scalable and cost-effective production as well as not requiring major consideration for safety issue during pre-clinical or clinical trials. 81 Liposomes Since induction of protective T cell immunity by cancer vaccines requires both tumor-associated antigens and adjuvants to be deliv- being nontoxic to LCs. In this case, the liposomes demonstrate superiority to antibody-antigen conjugates, as they allow the coformulation of antigens and adjuvants. The liposome-targeted delivery to LCs allows the use of lower adjuvant doses, reducing adverse effects while not compromising the CTL immunity. This immune cell targeted delivery approach is of particular importance for the treatment of LC histiocytosis, which is one of the most common pediatric cancers and is characterized by the formation of lesions of the skin, lungs bone marrow, and other organs due to the abnormal proliferation of lan-gerin+ myeloid progenitor cells. 83 In a subsequent study, the uptake and intracellular routing of liposomes was studied in model cell lines by confocal and live cell imaging as well as by flow cytometry. The liposomes were made from a mixture of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)polyethylene glycol (PEG), and they were made to encapsulate protein antigens. The liposomes were internalized into early endosomal compartments and accumulated in late endosomes and lysosomes of primary human LCs followed by a release of the protein antigens. These data further support the applicability of the targeted liposomal particles for protein vaccine applications. 84 Transfersomes Transfersomes are elastic liposomes containing edge activators (e.g., surfactants) to form ultra-deformable vesicles. Their ability to squeeze through the intercellular regions of the intact SC gives them greater potential for dermal antigen transport to APCs compared to liposomes. 85 Hepatitis B surface antigen (HBsAg) plasmid DNAcationic complex deformable liposomes were topically applied onto BALB/c mice, and immunity against the antigen was evaluated by measuring serum anti-HBsAg titers and various cytokines level

Archaeosomes
Archaeosomes are modified liposomes prepared with the polar lipids found in Archaea and display superior chemical and colloidal stability. 93 The polar lipids composition is composed of sn2,3 ether-linked saturated archaeolipids, imparting resistance to the bilayer against chemical, physical and enzymatic attacks that destroy ordinary phospholipids bilayers.
Phagocytic cells can uptake archaeosomes up to 50-fold greater than conventional liposomes, making them an attractive vaccine carrier. 94 Archaeosomes contains polar lipids, which cause pronounced capture by phagocytic and immature APCs via receptor-specialized uptake, resulting in strong CD4+ and CD8+ CTL responses to the encapsulated antigens. 95,96 Ultra-deformable archaeosomes (UDA) are able to cross the intact SC and reach the epidermis.
Higa et al. 97 solubilized the total antigens from Leishmania braziliensis promastigotes with sodium cholate (dsLp) and formulated them within UDA and ultra-deformable liposomes (UDL). In vitro, the UDA was extensively taken up by macrophages and induced rapid cytokine secretion. Topical application on BALB/c mice twice per week on consecutive days for 7 weeks showed that, despite the immunostimulatory effects of dsLp on macrophages in vitro, it raised no measurable in vivo response unless associated to UDL or UDA. The UDA elicited the highest systemic response, comparable to alum. Although both UDL and UDA acted as penetration enhancers, only UDA succeeded as a topical adjuvant due to its high uptake by APCs. Based on these findings, choosing materials with potent immunostimulatory properties is an important consideration when designing a system for passive dermal immunization.

| Virus-like particles
Virus-like particles (VLPs) are inert, empty capsids of viruses that retain the virus structure without DNA/RNA from the virus itself.
VLPs can be engineered to display antigens on their surfaces. 98 An example is pathogen mimetic reconstituted VLP vaccine developed against the hepatitis B virus (HBV) antigen. 99 Reconstituted hepatitis B surface antigen vesicles (HBsAg-REVs) integrated with monophosphoryl lipid A were prepared using a delipidation-reconstitution method. The natural HBsAg vesicles spontaneously self-assembled to form immunogenic spherical VLPs that promoted uptake by competent APCs and targeted LN-residing DCs. 100 The humoral and cellular immune responses elicited by HBsAg-REVs via transcutaneous administration were comparable to the marketed intramuscular hepatitis B vaccine formulation. It was reported that the magnitude of immune response increased with the period of antigen contact with skin.
In another study, Runcan et al. 101 investigated the penetration and cellular uptake of VLPs, composed of the HIV-1 precursor protein Pr55gag, applied ex vivo to human skin. The authors compared VLP administration on human skin pre-treated with cyanoacrylate tape stripping (CSSS) to administer by skin pricking and intradermal injection (invasive). CSSS and pricking treatments allowed the penetration of VLPs in the viable skin layers. VLPs were similarly taken up by APCs harvested from culture media of skin explants treated with CSSS and invasive methods. CSSS pre-treatment resulted in significantly increased levels of IL-1α in cell culture media compared to untreated and pricked skin. This provided evidence that dermal application with CSSS (mild barrier disruption) is for effective cellular uptake of VLPs that provides stimulatory signals allowing the activation of APCs and uptake of antigenic material (Figure 3). Recently, topical delivery of tetanus toxoid vaccine to mice using STAR particles generated immune responses that were similar to intramuscular vaccine injection. Moreover, application of STAR particles to the skin of human participants indicated they were well-tolerated and effective in creating skin pores. Star particles could widen the range of compounds that can be topically applied for a variety of skin diseases. 102

| Ionic liquids
Ionic liquids (ILs) have gained significant attention for topical and transdermal delivery due to their potential benefits, including a broad safety profile and their ability to tune the physiological properties of active pharmaceutical ingredients. 12,103,104 Moreover, ILs in vaccine dosage forms can act as stabilizer, solubilizer, targeted delivery inducer, and permeation enhancer. 105 Tahar et al. 106 109,110 and skin damage 111,112 as well as monogenetic skin disorders such as pachyonychia congenita 113 due to the presence of well-defined molecular targets that can be silenced to impart therapeutic benefits. 15,114 The first skin disease to undergo clinical studies using small interfering RNA (siRNA) was pachyonychia congenita, which is an autosomal dominant genodermatosis caused by mutations in keratin. The intradermal injection of siRNA targeting the disease-relevant mutations showed promising regression of the disease (NCT00716014). 113 However, frequent intralesional administration was associated with notable pain that F I G U R E 4 STAR particles. (a) Stainless steel mSTAR (m:metal) particles containing six arms are shown next to table salt for scale; (b, c) mSTAR particles are shown on a fingertip (b, arrow) and on a flat substrate at higher magnification (c). (d, f) Representative micrographs of gentian violetstained porcine skin following treatment with Aloe vera gel (d), abrasive gel (e), and four-arm STAR particles. (g-r) Representative histological images of skin sections after delivery of fluorescent molecules to porcine skin treated with mSTAR particles ex vivo, as imaged by fluorescence microscopy. The skin was cryosectioned after exposure to sulforhodamine B (SRB) or 4 kDa FITC-Dextran (FITC-Dex) for 1 h (g, h, m, n), 6 h (i, j, o, p), or 24 h (k, l, q, r) following treatment with Aloe vera gel (g, i, k, m, o, q) or 5.4 wt% six-arm mSTAR particles in gel (h, j, l, n, p, r). (s) Skin permeability after treatment with Aloe vera gel with or without mSTAR particles and exposed to FITC-Dex in two different MW in aqueous solution. The "a" mark indicates that Dex was delivered below the detection limit. Reprinted (adapted) with permission from 102 Copyright 2020, Nature Publishing Group hindered its translation efficacy. Consequentially, there is an increasing interest for passive delivery methods that are less painful. Topical administration of NAs is advantageous over systemic administration due to the accessibility of the disease site, avoiding off-target effects and enzymatic degradation of the NAs. 115 However, NAs are negatively charged, hydrophilic, large and susceptible to degradation, making their delivery to the skin nontrivial. To address these challenges, a variety of passive delivery systems have been developed in the past few years. In the following sections, we describe these systems and discuss their potential to manage a variety of dermatological disorders.

| Inflammatory skin diseases
Atopic dermatitis (AD) is an inflammatory disease that affects 10-20% of children 116 and 1-3% of adults worldwide. 117,118 It is associated with itching, severe skin redness, lesions, and papules that significantly have a negative impact on life quality. 119 Corticosteroids are most commonly prescribed for AD topical treatment; however, their prolonged use has been accompanied by numerous side effects that necessitate shifting to novel therapeutic strategies. 120 AD is generally associated with the upregulation of inflammatory cytokine transcription factor nuclear factor kappa B (NF-κB), leading to chronic skin inflammation. NF-κB is overproduced in macrophages and DCs, and it regulates the expression of different inflammatory cytokines as IL-6 and TNF-α. 108,121,122 Accordingly, the knockdown of NF-κB with antisense oligodeoxynucleotides showed promising therapeutic outcomes in mouse models of AD. 123 RelA (p65) is one of the two subunits of NF-κB along with p50, and it is typically associated with NF-κB transcriptional activation, which is related to allergy induction. The suppression of RelA by RelA small interfering RNAs (siRNAs) is reported to alleviate the disease symptoms in mice. 124 Moreover, pathogenesis of AD has been associated with higher levels of some cytokines as IL-4, IL-5, IL-13 118 and knockdown of these cytokines with topical NA therapies has shown promise in mice. 125,126 Psoriasis is another inflammatory skin condition characterized by itchy and scaly patches where several genes that encode for proinflammatory cytokines are upregulated in the plaque area. 127 It is a multifaceted condition involving multiple interactions between the immune system and the KCs. One of the more effective ways to treat psoriatic skin lesions is RNA interference using siRNAs. Once entered into the cytoplasm of the cell, siRNA binds to a specific mRNA and stops further translation of that mRNA transcript. 128  Generally, the skin barrier function of the SC is disrupted in psoriasis and AD due to the altered function of ceramide and the sebaceous gland. Accordingly, the major barrier of siRNA delivery to ADaffected skin are the cell-cell tight junctions (TJs) that control the paracellular pathway in the granular layer of skin. 131 Hence, an appropriate NA carrier should be able to break through TJs and deliver the siRNA to its target in therapeutically relevant concentrations while preserving its stability.

| Peptides
Peptides that promote the transport of NAs into the skin and elicit a therapeutic response have been implemented as carriers for siRNA and gene delivery. TD-1 is a short synthetic peptide that enabled the successful delivery of anti-glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) siRNA through non-follicular rat skin. 132 Hsu and Mitragotri developed a skin-penetrating and cell-entering peptide (SPACE) that was used to deliver IL-10 and GAPDH siRNA to both mouse and human skin. 133  Another LNC, consisting of a glycerol stearate and oleic acid core coated with polyethylenimine (PEI), was reported to deliver a combination of tacrolimus and siRNA against TNF-α through the skin. This system generated a synergistic effect by reducing the severity of psoriatic plaques in an imiquimod-induced psoriasis mouse model compared to treatment with tacrolimus alone or TNF-α siRNA-loaded nanocarriers alone. 141

| Liposome-peptide hybrids
The combination of liposomes and functional peptides has been also studied. The incorporation of SPACE peptides in cationic ethosomes was shown to enhance dermal siRNA delivery, resulting in effective gene silencing in vivo. This was achieved either by conjugating the SPACE peptide to the siRNA, conjugating it to the surface of the ethosomal particle or incorporating it directly into the formulation. 142 In another recent study, liposomes encapsulating siRelA were pre-

| Lipid-polymer hybrids
Desai et al. designed a biodegradable, cyclic cationic head lipid-polymer hybrid NC (CyLiPn) composed of poly(lactic-co-glycolic acid) (PLGA) and cationic amphiphiles. 122 CyLiPn was formed in an aqueous solution by self-assembly with a negatively charged hydrophobic PLGA core encapsulating capsaicin and a positively charged cationic lipid shell incorporating siRNA against TNF-α. This system delivered siRNA as deep as 360 μm into skin and showed a significant downregulation of inflammatory genes (i.e., TNF-α, NF-κB, IL-17, IL-23, and Ki-67) in a mouse psoriatic model compared to drugs alone, which was comparable to Topgraf ® .

| Ionic liquids
ILs have emerged as a versatile class of materials for dermal and transdermal delivery of a variety of macromolecules including hormones like insulin, 13 protein molecules 12 and NAs. 144

| Dendrimers
Polyamidoamine (PAMAM) dendrimers possess an amine-terminated surface that can be easily functionalized, providing a positive charge for interaction with siRNA. 152 Pandi et al. 153

| Skin fibrosis
Skin fibrosis such as hypertrophic scars and keloids affect millions of people worldwide. Fibrotic tissue is like a scar showing a bumpy, irregular and thick surface due to the excessive accumulation of protein under the skin. [156][157][158] A major cause of fibrotic diseases is the uncontrolled overexpression of connective tissue growth factor (CTGF).
Inhibition of CTGF expression with siRNA is an attractive strategy to modulate the fibrotic mechanism, which could inhibit or reverse the process of fibrosis. 159,160

| Mesoporous silica nanoparticles
Recently, Kang et al. 161 investigated the anti-fibrotic activity of siRNA against CTGF using mesoporous silica nanoparticles (MSNs). MSNs are attractive carriers due to their high surface area and large pore sizes. 162,163 Moreover, the ease of surface modification makes them a suitable option for siRNA loading. 162

| Wound healing and scar therapy
Chronic wound healing remains a serious problem worldwide, causing a significant public health burden. 164 This process is characterized by three stages: inflammation, proliferation, and remodeling. Aberrant wound healing can occur when any of these processes do not occur normally. 165 The development of drug delivery strategies to the wound site is complicated by common features associated with aberrant wound healing, including poor vascularization and hypoxia, infection, oxidative stress, excess proteases leading to therapeutic degradation, and frequent mechanical disturbances during body movement. 166,167 These challenges necessitate the development of new topical wound healing therapies with improved efficacy.
Therapeutic strategies for wound healing include small molecule drugs, growth factors, stem cells, fibroblasts and RNA interference (RNAi) therapies such as siRNA and microRNA (miRNA). 167 Among these, RNAi therapies provide an attractive option over as they are more targeted toward abnormal cells, providing a safer option to correct aberrant wound healing. 168 These therapeutic drugs can often target genetic defects that lead to chronic wounds, for exaMPLE, certain genes overexpressed in diabetic patients. 169 However, many RNAi therapies rely on scaffold-based strategies, which are often delivered subcutaneously, an invasive method compared to passive topical delivery strategies. 170

| Inorganic nanoparticles
Inorganic nanoparticles, especially gold nanoparticles, are a popular nanocarrier for NA delivery due to their low toxicity and immunogenicity combined with ease of surface functionalization. 166 For goldnanoparticle formulations, tightly packed siRNA is conjugated to the surfaces of the nanoparticles, resulting in SNA constructs. They display desirable properties such as nuclease resistance and the ability to enter cells via scavenger-mediated endocytosis. 176

| Polymeric nanoparticles
Polymeric nanoparticles offer tunability combined with biocompatibility and protection of cargo degradation. Their charge and size can be optimized for use as oligonucleotide vehicles. 178 PEI is a commonly used cationic polymer for siRNA delivery; however, associated cytotoxicity and non-biodegradability limits its use. 178,179 Co-polymerization of PEI with other polymers can mediate this issue, a strategy recently used by Cho and coworkers. 112 The authors developed a degradable poly(sorbitol-co-PEI), a copolymer of sorbitol diacrylate with PEI, to deliver siRNA targeting CTGF in a cutaneous murine wound healing model. The delivery of siRNA using this system showed a significant reduction in scar contraction post-healing with almost no contraction observed in some mice. Besides PEI, cyclodextrins are a class of natural oligosaccharides that are used for oligonucleotide delivery. 178 They are generally nontoxic and non-immunogenic, hence conjugation with PEI has been explored to improve biocompatibility. Li et al. 180

| Layer-by-layer assemblies
Hammond et al. 182,183 used layer-by-layer (LbL) approaches to assemble ultrathin polymer films for localized and controlled delivery of siRNA to chronic wounds to accelerate wound healing in vivo. In their reports, a commercially available woven nylon bandage was applied directly into healing wounds for the delivery of siRNA.
LbL assemblies have also been used for scar therapy. The process of wound healing often results in thick, collagen-enriched tissue called scar tissue, which can negatively impact quality of life. 156,157 Cutaneous scars from serious traumatic injury can cause long-lasting complications due to scar contraction and poor tissue remodeling reducing the range of motion and joint mobility and subsequently impairing function. 158,184 Castleberry et al. 111 applied the LbL technology to anti-scar therapy. The authors implemented the nanolayered platform to deliver anti-CTGF siRNA and investigated its potential to improve scar outcomes in a third-degree burn-induced scar model in rats.
CTGF is a therapeutic target within wounds to ameliorate fibrosis without impairing normal wound healing. They described an ultrathin polymer coating that can be uniformly assembled onto a commercially available suture using LbL assembly (  186 Interestingly, delivery of fibroblasts alone did not significantly accelerate wound closure; however, co-delivery with growth factors resulted in significantly faster healing. While nonbiodegradable scaffolds may be useful in situations where healing rates are slow, long-term studies are needed to evaluate their safety. In a novel study, genetically modified Lactobacilli were delivered topically to a wound site, resulting in accelerated wound healing mediated by C-X-C motif chemokine 12 (CXCL12) production from the bacteria. 187 This strategy provides an alternative to local protein delivery, which is limited by protein degradation. It is also safer than using genetically modified cells, such as mesenchymal stem cells, as probiotic bacteria are naturally present on skin.

| Skin cancer
Cutaneous malignancies such as melanoma and nonmelanoma skin cancers represent a significant burden on global health. 163   They can be screened for their affinity to certain receptors on the cell surface and subsequently used for targeted delivery. 23,[192][193][194][195] For example, Gan et al. 193 used a screening process to identify an epidermal growth factor (EGF) receptor (EGFr)-targeting peptide to target A431 squamous cell carcinoma. Ruan et al. 196 combined SPACE with EGF to create a fusion protein that could specifically target melanoma cells that overexpress EGFr. The authors used this fusion protein carrier to deliver c-Myc siRNA to a B16F10 mouse melanoma model in vivo, which significantly reduced tumor growth compared to naked siRNA. Notably, the effect the topically admin-

| Mesoporous silica nanoparticles
The effectiveness of MSNs for transdermal siRNA delivery to cutaneous cancer was recently demonstrated by Lio et al. 198

| CRITICAL ANALYSIS AND OUTLOOK
Noninvasive dermal delivery systems allow simple application to different areas of the body in a patient-friendly way. Transdermal patches are an ideal drug delivery platform that have been widely used in the clinic for the delivery of hormones and small-molecule drugs; however, their use for vaccines and NA delivery is limited by poor permeation of biomacromolecules, limiting the amount of the cargo that can be delivered to the skin. 35,199 The efficiency of this technology can be improved when coupled with other tailored approaches that have shown success in dermal delivery. Such delivery systems can also be formulated in semi-solid dosage forms as creams or gels for convenient topical application.
Novel transport-promoting peptides can be identified via highthroughput screening approaches or computational approaches as molecular docking to aid the design of peptides of improved stability.
Also, using a combination of peptides with different functions can allow for synergistic effects by promoting skin and cellular penetration simultaneously. 142,194 Formulation approaches can be leveraged to formulate lipid or polymer-based nanocarriers with excipients that possess multiple functionalities such as enhancing skin permeation, facilitating cellular uptake and enhancing the stability of the biological molecules either during storage or after administration until reaching the target cells. In some cases, such lipids can inhibit the gene silencing process. 200 Recently, a zwitterionic lipid (cephalin) was found to be more effective than DOTAP in delivering saRNA to human skin explants. 200  Bioconjugation chemistry can be harnessed to design and optimize stimuli-responsive conjugates that enable skin penetration and cargo release upon exposure to the target microenvironment. 2 Further modification with targeting moieties can enable delivery to specific cell subtypes within the skin.
ILs are a relatively recently studied drug delivery system whose physicochemical properties can be tuned to enhance the transport and stability of biological molecules. They are designer materials that can be tailored to attain the desired concentration and adequate viscosity desired for topical application without the need for thickening agents. They can also be formulated with cation and anion counterparts that possess a desirable pharmacological effect, acting as bioactive delivery system to augment the efficacy of the loaded bioactive cargo. They offer a promising tool for noninvasive dermal delivery of antigens and NAs. Recently, the use of ILs has moved to into clinical trials. Ko and coworkers reported the clinical translation of CAGE for the treatment of rosacea, which is an inflammatory skin disease. 205    Apart from intradermal injection, other current clinical trials are based on physical disruption methods (mainly microneedles and microjets) with exception for a few transdermal patches that are used for the dermal delivery of some peptide therapeutics. Helix BioPharma developed a cream containing IFNα-2b encapsulated in liposomes that was used in Phase II human trials for treating low-grade squamous intraepithelial lesions. 212 Only one recent Phase II clinical trial was performed for the dermal delivery of an anti-TNF antibody using a hydrogel formulation (NCT01936337). However, despite the wide research on using nanocarriers for NA delivery to the skin, most of the studies are still in an early preclinical phase. More effort should be directed to explore new transporter mechanisms and to identify synergistic combinations to overcome the skin barrier with minimal invasiveness or irritation. These efforts should be extended to deliver antibodies given their potential role in treating a variety of skin conditions.
It is worth pointing out that combining noninvasive active delivery techniques with the discussed formulation approaches can synergistically promote dermal delivery and achieve better therapeutic outcomes. While not discussed in detail in this review, iontophoresis and sonophoresis have shown promising outcomes in this regard. For example, iontophoresis has been employed to deliver cetuximab, an anti-EGFR monoclonal antibody, to the deep layers of skin. 21 Combining iontophoresis with cetuximab-conjugated liposomes showed promising outcomes in a squamous cell carcinoma mouse model. 213 This technique has also been used to deliver the anti-TNF-α drug etanercept for topical psoriasis. 214 The combined use of PAMAM dendrimers as carriers for antisense oligonucleotides (ASOs) and iontophoresis caused more than a twofold reduction in tumor volume in a skin cancer model compared to iontophoresis alone or the passive delivery of a ASO-dendrimer complex. 190 The combined use of iontophoresis with charged liposomes has also been reported to enhance the TCI using OVA and silver nanoparticles. 215 While sonophoresis has been reported to improve the delivery of a variety of macromolecules, 216 few reports exists on its combined use with other carriers. Low frequency ultrasound has been implemented to enhance dermal penetration of quaternized starch and miR-197 (a microRNA targeting subunits of IL-17 and IL-22 receptors) complexes. This showed a significant reduction in psoriatic levels when tested in a human skin xenograft model in mice. 107 Sonophoresis also enhanced the permeation of PAMAM and low molecular weight peptide dendrimers, while increased the dermal retention of high molecular weight peptide dendrimers. 217 Given the ability of dendrimers to form complexes with siRNA, sonophoresis may be useful to further enhance their dermal delivery.
Iontophoretic patches and sonophoretic devices can be used for self-administration. However, use of such combinations should be carefully considered (e.g., the intensity of current or frequency of the ultrasound) to ensure that delivery is directed to target layers of skin while avoiding further penetration into systemic circulation. The combinatorial use of active techniques with rationally designed formulations may help the performance of therapeutic moieties at lower doses, while using physical methods at lower strengths to ensure safety and potentially enable a take-home therapy.

| REMAINING CHALLENGES
Despite the recent efforts to advance noninvasive dermal delivery of vaccines and topical NA therapies (summarized in Table 1), there still exist some limitations to overcome before these systems can become clinically viable.
The use of peptides and dendrimers may be constrained to some NAs with a certain size as SiRNA and oligonucleotides as opposed to DNA. Lipid nanoparticles and cationic polymers, despite their efficiency for cytosolic delivery, still suffer from some toxicity and instability issues. Chemical conjugations, despite being tunable to provide cellular targetability and controlled release rates, suffer from timeconsuming and labor-intensive processes.
The stability of the biological macromolecules during manufacturing and storage is of critical importance. Care should be taken during the manufacturing process to avoid excessive agitation or shear, as well as drastic changes in pH, ionic strength, or temperature, as those factors can affect the molecular structure and result in a loss of biological activity. 218,219 When designing lipid or polymer-based nanocarriers, the formulation parameters such as the choice of excipients and the loading method should be optimized based on the individual properties of the macromolecule and its possible interaction with any of the formulation ingredients. What works with a particular cargo might adversely affect the properties and transport of others.
Another important aspect is the translatability of the experimental results obtained with animal models to humans. With exception to studies performed on human skin explants, the T A B L E 1 A summary of formulation-based strategies for dermal delivery of antigens and nucleic acids majority of the studies have shown success in small animal models.
One should take into consideration the biological and structural differences before extrapolating findings to humans since the human skin is thicker, has fewer hair follicles and is less permeable than rodent skin.

| CONCLUSIONS
Looking at the clinical landscape for vaccines and cutaneous disorders reveals the critical need for noninvasive approaches to deliver biological macromolecules. Recent efforts have been made to overcome the barrier function of the skin by implementing a variety of smart delivery systems. To further advance this field, we need a better understanding and practice of how to deliver various macromolecules safely and efficiently to target cells. Translational value of preclinical work can be increased by using human-relevant models such as human skin explants, tissue-engineered skin models, and human-based tissues grafted onto mice. Addressing these aspects and combining them with a deeper understanding of cell biology can lead to the improved design of bioengineered materials to navigate the skin and improve prophylactic and therapeutic outcomes.