Monovalent Antibody-Conjugated Lipid-Polymer Nanohybrids for Active Targeting to Desmoglein 3 of Keratinocytes to Attenuate Psor

1 Monovalent antibody-conjugated lipid-polymer nanohybrids for active 2 targeting to desmoglein 3 of keratinocytes to attenuate psoriasiform 3 inflammation 4 5 Zih-Chan Lina, Tsong-Long Hwangb,c,d,e,f, Tse-Hung Huangd,g,h,i, Kohei Taharaj, Jiří 6 Trousilk, Jia-You Fangb,c,d,f,* 7 a Graduate Institute of Biomedical Sciences, Chang Gung University, Kweishan, 8 Taoyuan, Taiwan 9 b Graduate Institute of Natural Products, Chang Gung University, Kweishan, Taoyuan, 10 Taiwan 11 c Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang 12 Gung University, Kweishan, Taoyuan, Taiwan 13 d Research Center for Food and Cosmetic Safety and Research Center for Chinese 14 Herbal Medicine, Chang Gung University of Science and Technology, Kweishan, 15 Taoyuan, Taiwan 16 e Department of Chemical Engineering, Ming Chi University of Technology, New 17 Taipei City, Taiwan 18 f Department of Anesthesiology, Chang Gung Memorial Hospital, Kweishan, Taoyuan, 19 Taiwan 20 g Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital, 21 Keelung, Taiwan 22 h School of Traditional Chinese Medicine, Chang Gung University, Kweishan, 23 Taoyuan, Taiwan 24 i School of Nursing, National Taipei University of Nursing and Health Sciences, 25 Taipei, Taiwan 26 j Laboratory of Pharmaceutical Engineering, Gifu Pharmaceutical University, Gifu, 27 Japan 28 k Institute of Macromolecular Chemistry, Czech Academy of Sciences, Prague, Czech 29 Republic 30 31 Correspondence: Jia-You Fang, Pharmaceutics Laboratory, Graduate Institute of 32 Natural Products, Chang Gung University, 259 Wen-Hwa 1st Road, Kweishan, 33 Taoyuan 333, Taiwan, Tel: +886-3-2118800, Fax:+886-3-2118236 E-mail: 34 [email protected]. 35 1 1 Abstract 2 To improve the treatment of psoriasiform inflammation, we developed actively 3 targeted nanocarriers loaded with the phosphodiesterase 4 inhibitor AN2728. 4 Methods: Phospholipid–poly(lactic-co-glycolic acid) nanohybrids were prepared and 5 conjugated with monovalent anti-desmoglein 3 antibody to bind keratinocytes. 6 Results: The actively targeted nanohybrids were 229 nm in mean size with a nearly 7 neutral surface charge. Flow cytometry and confocal microscopy showed a 9-fold 8 increase in keratinocyte uptake of targeted nanohybrids relative to non-targeted 9 nanoparticles. The nanoparticles localized mainly in lysosomes after internalization. 10 AN2728-loaded antibody-conjugated nanocarriers inhibited cytokine/chemokine 11 overexpression in activated keratinocytes without affecting cell viability. The targeted 12 nanohybrids also suppressed neutrophil migration by reducing CXCL1 and CXCL2 13 release from keratinocytes. Following subcutaneous administration in mice, the 14 nanohybrids distributed to the epidermis and hair follicles. In a psoriasis-like skin 15 mouse model, the actively targeted nanoparticles were superior to free drug and 16 non-targeted nanoparticles in mitigating skin inflammation. Intervention with the 17 targeted nanosystem reduced the epidermal thickness of the psoriasiform lesion from 18 191 to 42 μm, decreased the Psoriasis Area Severity Index by 74%, restored barrier 19 function, and returned chemokine levels to baseline. 20 Conclusions: Our developed nanosystem was safe and demonstrated efficient 21 targeting properties for the treatment of cutaneous inflammation. 22 23 Keywords: desmoglein 3; keratinocyte; psoriasis; lipid-polymer nanohybrid; active 24 targeting; monovalent antibody 25 2 1 Introduction 2 Psoriasis is an autoimmune skin inflammation involving interactions between 3 keratinocytes and immune cells. Psoriasis is characterized by keratinocyte 4 proliferation and immune cell accumulation in epidermis/dermis [1]. The clinical 5 observation includes erythematous papules with white multilayered scales and 6 thickened acanthotic epidermis. While the global prevalence of psoriasis is about 7 2‒3%, its incidence seems to be increasing [2]. Keratinocytes demonstrate a critical 8 capacity to initiate psoriatic inflammation by activating the onset of the pathogenic 9 event and sustaining the prolonged phase [3]. Long-term therapies incompletely 10 resolve psoriasis because of their inefficiency after prolonged application and side 11 effects [4]. Therefore, new treatment strategies are needed to improve therapy. 12 Loading antipsoriatic drugs into nanocarriers is one approach to improve their 13 therapeutic efficiency. The nanoparticles protect the drugs from degradation and 14 provide controlled and targeted delivery, leading to improved therapy, reduced dose, 15 and minimized adverse effects [5]. Active targeting of nanoparticles to target cells is 16 possible by furnishing the nanoparticle surface with specific ligands for receptors on 17 the cell membrane. Antibody-based targeting is promising for active targeting due to 18 its high specificity [6]. Increasing applications of monoclonal antibodies in the 19 targeted treatment of psoriasis also encourages the potential of antibody-conjugated 20 nanocarriers. Desmoglein 3 (Dsg3) is a desmosomal glycoprotein that provides 21 calcium-dependent adhesive integrity among keratinocytes [7]. Anti-Dsg monoclonal 22 antibody has been proven to specifically target keratinocytes [8, 9]. Since Dsg3 is 23 overexpressed in keratinocytes, it is an appropriate target for mitigating psoriatic 24 inflammation with minimum effects on normal tissue. 25 There are very few actively targeted antibody-conjugated nanosystems for treating 26 cutaneous inflammation. To improve psoriasis management, we developed Dsg3 3 1 antibody-conjugated lipid-polymer hybrid nanoparticles encapsulating AN2728. The 2 model drug AN2728 (crisaborole) is a phosphodiesterase 4 (PDE4) inhibitor approved 3 by the USFDA for atopic dermatitis treatment [10] and is also successful in relieving 4 psoriasis [11]. PDE4 inhibitors limit the breakdown of cyclic adenosine 5 monophosphate (cAMP) to decrease the levels of proinflammatory cytokines and 6 chemokines [12]. Apremilast is an oral PDE4 inhibitor that was approved for psoriasis 7 treatment in 2014 [13]. PDE4 inhibitors attenuate inflammation by directly 8 modulating the function of keratinocytes [14]. Lipid-polymer nanohybrids were 9 selected as the AN2728 nanocarrier because these hybrid materials combine the 10 advantages of lipid-based and polymer-based nanosystems, including excellent 11 storage stability, easy fabrication, high biocompatibility, facile cell uptake, and high 12 loading capacity of lipophilic drugs [15]. Polyethylene glycol (PEG)ylated 13 phospholipid and poly(lactic-co-glycolic acid) (PLGA) were used to fabricate the 14 nanocarriers because of their biodegradability and approval by the USFDA [15]. To 15 effectively target the nanoparticles to keratinocytes, we used a monovalent anti-Dsg3 16 antibody fragment with free thiol moieties. Full antibody ligands have a number of 17 disadvantages for nanoparticle conjugation that lead to off-target effects [16]. 18 Foremost, the orientation of the antibody on the nanoparticle surface is usually 19 random, which reduces its selective binding ability [17]. Further, the large size of 20 whole antibodies causes steric hindrance during receptor targeting. Full antibody 21 ligands are also immunogenic and have poor stability. In comparison, antibody 22 fragments, including fragment antigen-binding (Fab) region, single-chain variable 23 fragment (scFv), and monovalent fragment, retain specific antigen binding and 24 specificity during nanoparticle conjugation [18]. Of these fragment types, monovalent 25 fragments involve less complicated preparations. The monovalent antibody used in 26 this study is a half-antibody fragment that has better stability than the full-length 4 1 antibody [19]. We evaluated the effects of our nanosystem on psoriasis mitigation in 2 imiquimod (IMQ)-stimulated keratinocytes and an IMQ-induced psoriasiform lesion 3 mouse model. 4 Methods 5 Preparation of Dsg3 antibody-conjugated nanoparticles 6 PLGA (50 mg) and AN2728 (2 mg) or rhodamine 800 (0.2 mg) in dichloromethane 7 were injected into 8 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol) 9 -2000] (DSPE-PEG-maleimide, 1 mg), soybean phosphatidylcholine (SPC, 1 mg), 10 and polyvinyl alcohol emulsifier then immediately rigorously emulsified using a 11 high-power sonicator. This resulted in the formation of PLGA nanoparticles 12 encapsulating AN2728 or rhodamine 800. Dsg3 antibody (50 μL, 1 mg/mL, 13 Invitrogen) was reduced to a monovalent fragment by incubating the antibody in 7.5 14 mM 2-mercaptoethylamine (2ME) in HEPES buffer at 37 °C for 1 h [20]. The 15 nanoparticles were incubated with the monovalent antibody for 4 h at room 16 temperature to fabricate Dsg3 antibody-conjugated nanoparticles (DPNPs). Four types 17 of nanoparticles were fabricated with the following compositions from core to outer 18 surface: (i) PLGA matrix, (ii) SPC-decorated surface, (iii) DSPE-PEG-maleimide 19 cross-linker intercalated in the SPC surface, and (iv) anti-Dsg3 antibody conjugated to 20 DSPE-PEG-maleimide (DPNPs). The proposed structure of DPNPs loaded with 21 AN2728 is illustrated in Figure 1A. The nanoparticles (iii) without antibody 22 conjugation were named PNPs. We removed the solvent by evaporation on a magnetic 23 stirrer for 24 h and purified AN2728- or rhodamine 800-loaded nanoparticles by 24 ultracentrifugation (12,000 ×g) for 30 min followed by washing thrice and 25 resuspending in phosphate-buffered saline (PBS). The nanodispersions were 26 centrifuged at 48,000 ×g and

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