Polypeptide-corticosteroid conjugates as a topical treatment approach to psoriasis

Journal Pre-proof Polypeptide-corticosteroid conjugates as a topical treatment approach to psoriasis

Irene Dolz-Pérez, Marwa A. Sallam, Esther Masiá, Daniel Morelló-Bolumar, M. Dolores Pérez del Caz, Patrick Graff, Doaa Abdelmonsif, Sarah Hedtrich, Vicent J. Nebot, María J. Vicent PII:

S0168-3659(19)30735-7

DOI:

https://doi.org/10.1016/j.jconrel.2019.12.016

Reference:

COREL 10059

To appear in:

Journal of Controlled Release

Received date:

27 September 2019

Revised date:

18 November 2019

Accepted date:

11 December 2019

Please cite this article as: I. Dolz-Pérez, M.A. Sallam, E. Masiá, et al., Polypeptidecorticosteroid conjugates as a topical treatment approach to psoriasis, Journal of Controlled Release (2019), https://doi.org/10.1016/j.jconrel.2019.12.016

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© 2019 Published by Elsevier.

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Polypeptide-Corticosteroid Conjugates as a

Topical

Treatment Approach to Psoriasis Irene Dolz-Pérez1, Marwa A. Sallam2,3, Esther Masiá1,4, Daniel MorellóBolumar5, M. Dolores Pérez del Caz6, Patrick Graff7, Doaa Abdelmonsif8, Sarah Hedtrich7,9, Vicent J. Nebot5*, María J. Vicent1,4* 1

Polymer Therapeutics Laboratory, Centro de Investigación Príncipe Felipe, Av. Eduardo Primo

2

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Yúfera 3, Valencia 46012, Spain. Department of Industrial Pharmacy, Faculty of Pharmacy, Alexandria University, El Sultan Hussein

3

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St Azarita, Egypt.

John A. Paulson school of engineering and applied sciences, Harvard University, 52 Oxford St,

Screening Platform, Centro de Investigación Príncipe Felipe, Av. Eduardo Primo Yúfera 3, Valencia

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4

e-

Cambridge, MA 02138, USA.

46012, Spain.

Polypeptide Therapeutic Solutions S.L. C/ Benjamin Franklin 19 (Paterna), Valencia 46980, Spain

6

Servicio de cirugía plástica, Hospital Universitario y Politécnico La Fe, Av. de Fernando Abril

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5

7

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Martorell 106, Valencia 46026, Spain

Institute of Pharmacy (Pharmacology and Toxicology), Freie Universität Berlin, Königin-Luise Str.

2+4, Berlin 14195, Germany. 8

Department of Medical Biochemistry, Faculty of Medicine, Alexandria University, El Sultan

Hussein St Azarita, Egypt 9

Faculty of Pharmaceutical Sciences, University of British Columbia, 2405 Wesbrook Mall,

Vancouver, BC, Canada

* Address Correspondence to [email protected] and [email protected] Keywords Polymer therapeutics, polymer-drug conjugates, skin, topical delivery, psoriasis, antiinflammatory activity 1

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Abstract Topical treatment of mild-to- moderate psoriasis with corticosteroids suffers from challenges that include reduced drug bioavailability at the desired site of action. The retention of therapeutics within the epidermis can safely treat skin inflammation, scaling, and erythema associated with psoriasis while avoiding possible side effects associated with systemic treatments. We successfully synthesized and characterized a pH-responsive biodegradable poly- L-glutamic acid (PGA)- fluocinolone acetonide (FLUO) conjugate that allows the controlled release of the FLUO to reduce skin inflammation. Additionally, the application of

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a hyaluronic acid (HA)-poly-L-glutamate cross polymer (HA-CP) vehicle boosted skin permeation. During in vitro and ex vivo analyses, we discovered that PGA-FLUO inhibited pro-inflammatory cytokine release, suggesting that polypeptidic conjugation fails to affect the

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anti- inflammatory activity of FLUO. Additionally, ex vivo human skin permeation studies using confocal microscopy revealed the presence of PGA-FLUO within the epidermis, but a

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minimal presence in the dermis, thereby reducing the likelihood of FLUO entering the

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systemic circulation. Finally, we demonstrated that PGA-FLUO applied within HA-CP effectively reduced psoriasis-associated phenotypes in an in vivo mouse model of human psoriasis while also lowering levels of pro- inflammatory cytokines in tissue and serum.

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Overall, our experimental results demonstrate that PGA-FLUO within an HA-CP penetration

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enhancer represents an effective topical treatment for psoriasis.

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1. Introduction The skin is the largest and most physiologically complex organ of the human body, creating a barrier between the host and the external environment [1]. Skin barrier integrity is essential for the protection of the whole body; any dysfunction can enhance the risk of developing infective and inflammatory disorders [2], such as psoriasis [3]. Psoriasis is a common inflammatory chronic disease mediated by the immune system with a predominantly cutaneous involvement [4]. There are several clinical phenotypes of psoriasis that are classified based on the characteristics of the disease, including patient age at

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disease onset, degree of skin involvement, morphologic pattern, and anatomical location [5]. Scaly skin, erythematous plaques, and inflammatory cell infiltration characterize plaque

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psoriasis, the most prevalent type of psoriasis [6, 7]. In recent years, the immune response involved in the onset of the disease has been extensively investigated, uncovering the relative

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importance of the Interleukin (IL)-23/IL-17 axis [7, 8]. The current development of antipsoriatic therapeutics, mainly IL-17 and IL-23 inhibitors, focuses on systemic treatments that

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target the immune system [8]. However, topical treatment with corticosteroids remains one of the most widely employed treatments for approximately 80% of patients affected with mild to psoriasis

[9].

Corticosteroids

immunosuppressive,

and

vasoconstrictive

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moderate

induce anti- inflammatory,

antiproliferative,

effects through binding to

intracellular

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[10].

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corticosteroid receptors and the modulation of pro-inflammatory cytokine gene transcription

However, many topical corticosteroids that are currently employed or under assessment in clinical trials still require improvements to their physico-chemical properties to promote skin penetration (e.g., solubility in water) and suffer from local cutaneous (e.g., skin atrophy, acne) and systemic side effects (e.g., osteoporosis, diabetes, weight gain) that limit their clinical use [11]. These effects mainly correlate with the high doses required and the elevated frequency of applications and have been noted for both high and reduced potency corticosteroids. Therefore, any advanced therapeutic approaches that allow a reduction in dose and the avoidance of systemic circulation of corticosteroids could significantly enhance treatment efficacy and patient compliance. The application of nanomedicines as advanced therapeutics in the field of topical and transdermal delivery of active compounds [12-16] and immunotherapy [17] is currently in 3

Journal Pre-proof exponential growth. Liposomes have been the most used carriers for skin delivery, although their large sizes and heterogeneous size distribution limit their efficiency when acting as skin permeation enhancers [18, 19]. The application of controlled polymerization techniques and the tailoring of critical nanocarrier parameters, such as size and Z-potential, can significantly enhance the penetration of active substances into the skin, as has been demonstrated for polypeptide-based

nanostructures

[20, 21]. Furthermore,

the

mode of the drug

encapsulation/entrapment [22] or polypeptide carrier-conjugation [23, 24] has a significant influence on the site of drug release and the kinetic profile of drug release. Although currently underexplored for skin delivery, a rationally-designed bioresponsive linker can

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optimize the therapeutic output of topical treatment by enhancing efficiency and reducing side effects [25, 26]. Finally, the vehicle used for the administration of a given therapeutic may also have an essential synergistic effect; formulations with an optimal interaction

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between the vehicle and the therapeutic agent can enhance penetration and hydration of the

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skin [27].

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We aimed to potentiate the therapeutic capacity of the topical corticosteroid fluocinolone acetonide (FLUO) as a local cutaneous treatment of psoriasis by enhancing its skin permeation and controlling drug release within the epidermis. We proposed the

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conjugation of FLUO to a biodegradable poly-L-glutamic acid (PGA) carrier via a pH-

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responsive ester linker, and we now report the synthesis, physico-chemical characterization, and in vitro, ex vivo, and in vivo evaluation of PGA-FLUO for the topical controlled delivery

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of FLUO. We discovered that this strategy enhanced FLUO skin penetration and provided more significant inhibition of local skin inflammation, and established that the formulation of PGA-FLUO within a hyaluronic acid-poly-L-glutamate cross polymer vehicle (HA-CP; Yalic© [28], previously described as a penetration enhancer [29]) increased the permeation and residence of PGA-FLUO in deeper skin layers. Overall, our results suggest that our new polypeptide-conjugated corticosteroid approach to the topical treatment of psoriasis can provide for enhanced patient outcomes.

2. Materials and Methods The materials and physico-chemical characterization methods employed to fully characterize PGA-FLUO can be found in Supplementary Information (SI).

4

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2.1. Synthesis of the Poly-L-Glutamate-Fluocinolone acetonide Conjugate FLUO conjugation to PGA was performed according to a previously described procedure [30]. A detailed synthetic procedure for the generation of PGA-FLUO can be found in the SI. 2.2. Cellular Uptake in Keratinocytes Cellular trafficking and uptake of PGA-FLUO were assessed in HaCaT cells (human

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immortalized non-tumorigenic keratinocyte cell line) supplied by CLS Cell Lines Service (Eppelheim, Germany) using Oregon Green (OG)- labeled polymers (PGA-FLUO-OG).

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Briefly, HaCaT cells were seeded in 6-well plates at a density of ~120,000 cells/cm2 (1 mL cell suspension per well) and allowed to adhere for 24 h. Then, 15 µL of OG- labeled

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polymers were added at different time points (from 0 to 27 h) during cell incubation. OG fluorescence was analyzed by flow cytometry and live-cell confocal microscopy (see SI for

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more details).

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2.3. Visualization of Dermal Penetration of PGA-FLUO

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Breast skin samples were obtained from healthy women undergoing plastic surgery with informed consent (kindly donated from Hospital La Fe, Valencia, Spain). Immediately

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after excision, the subcutaneous fatty tissue was removed using a scalpel. The skin was cut into 4 x 4 cm pieces, wrapped in aluminum foil, and stored at -20ºC until their use. Penetration studies were performed using Franz diffusion cells (Logan Instruments Corp., USA). Skin samples were fixed between the donor and the receptor chamber so that the stratum corneum was placed upwards. The receptor chamber was filled with 8 mL of PBS 0.01 M pH 7.4 and mixed with a magnetic stirring bar at 37ºC. Then, 100 µL of a solution of 10 mg/mL of PGA-FLUO-OG in water or the HA-CP vehicle (1%) labeled with Cyanine5.5 (HA-CP-Cy5.5) were applied topically onto the skin surface. Aliquots (2 mL) were taken from the receptor chamber at 8 and 24 h of study and immediately refilled with fresh solution. After 24 h, skin samples were gently rinsed twice with PBS-BSA 0.1% (PBS supplemented with bovine serum albumin) and kept in 4% paraformaldehyde (PFA) for 24 h at room temperature. Then, samples were washed with 30% sucrose in PBS solution for 24 h at 4ºC. After that, skin samples were washed twice with PBS and preserved in a 5

Journal Pre-proof cryopreservation solution (40% PB 0.1 M, 30% ethylene glycol and 30% glycerol) at 4ºC until use. Finally, the samples were placed into optimum cutting temperature (OCT) inclusion medium, and slides of 5 µm were generated via cryostat (version CM1850 UV, Leica, Germany). Samples were analyzed by confocal microscopy (see SI for further details). 2.4. In Vitro Anti-Inflammatory Activity The ability of FLUO (0.49 ng/mL), PGA-FLUO (0.49 ng/mL of FLUO equivalents) and PGA to reduce the levels of pro- inflammatory cytokines released to the culture media were evaluated following stimulation of macrophages (Raw264.7 cell line) with 5 ng/mL of

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bacterial lipopolysaccharide (LPS) from E. coli. Briefly, cells were seeded in 96-well plates at a concentration of 6,000 cells/well, and after 24 h of stimulation with LPS the treatments

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were added for 72 h. In vitro cell viability of macrophages was assessed using the MTS assay following a previously described protocol (see SI for detailed information). For the

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quantification of pro- inflammatory cytokines, the plates were centrifuged 5 min, 22°C, 4000 rpm, and 90 μL of supernatant were collected and frozen at -80ºC until use. Pro- inflammatory

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cytokines levels were measured by LUMINEX multiplex immunoassay (Affymetrix m Th1/2/9/17/22/Treg 17plex, eBioscience, Spain). This study was performed using LUMINEX

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inhibition of each cytokine.

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200 equipment (LUMINEX Corporation, USA) and results expressed as a percentage of

2.5. Tissue Viability and Quantification of Pro-Inflammatory Cytokines after

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Treatment with PGA-FLUO in an Inflammatory Ex Vivo Human Skin Model Tissue viability assay was performed in an ex vivo human skin model [31]. After inflammatory insult via combined treatment with bacterial LPS (15 µg/mL) and EGF (2.5 ng/mL) for 24 h (see SI for more details), 3 µL of 10 mg/mL of PGA, FLUO, PGA-FLUO applied in sterile water and HA-CP vehicle (1%), and HA-CP (1%) were topically applied. MTT assay was used to evaluate tissue viability after 24 h of treatment (see SI for detailed information). IL-6 concentrations in the culture media were measured by ELISA assay (Invitrogen, Spain). Cytokines concentrations were determined according to standard solutions. 2.6. Skin Equivalent Construction and Skin Irritation Testing

6

Journal Pre-proof Human skin equivalents were prepared from primary human keratinocytes and fibroblasts from juvenile foreskin following circumcision from three different donors (EA: 1/081/13, written consent was obtained) as previously described [32, 33]. Negative control (solvent control), untreated control, and positive control (sodium dodecyl sulfate (SDS) 1%) skin equivalents were studied to ensure the adequate development of the model. PGA, FLUO, PGA-FLUO applied in sterile water and HA-CP vehicle (1%), and HA-CP (1%) at 5 mg/mL (35 μg/cm2 ) were topically applied on days 11 and 13 of skin equivalent cultivation. On day 14, tissue viability was evaluated by MTT assay (see SI for detailed information).

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To develop an inflammatory phenotype in the skin equivalents emulating characteristics of psoriatic skin in vitro, such as hyperproliferation [34], 20 ng/mL of recombinant TNF-α (eBioscience, Hatfield, UK) were added to the culture media on day 10

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and 12. Treatments (2 μg/cm2 ) were applied topically at days 11 and 13 to test any reduction in the release of the pro-inflammatory cytokine IL-6 to the culture media. At day 14, culture

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media was collected for quantification of IL-6 levels using an ELISA assay (Invitrogen,

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Spain). Cytokines concentrations were determined according to standard solutions. 2.7. Establishment of Psoriatic Like Plaque Model in Animals

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Animal experiments were performed following the European Communities Council

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Directive (86/609/ECC) guidelines and by the Spanish Royal Decree 1201/2005. 6-week-old male BALB/c mice were purchased from Envigo Laboratories Inc. (Spain, EU). One day

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before the first imiquimod (IMQ) application, an approximately 2 x 3 cm square of the back of the mice was shaved. Psoriasis- like symptoms were induced by the daily application of 62.5 mg of commercially available IMQ cream (5%) (Imunocare; Industrial Farmacéutica Cantabria, Spain) on the shaved back and the right ear for seven consecutive days, translating in a daily dose of 3.125 mg of the active compound. This dose was empirically determined to cause optimal and reproducible skin inflammation in mice [35-37]. 2.8. Evaluation of the Anti-Inflammatory Activity of PGA-FLUO in an In Vivo Psoriatic Model Seven days after the induction of psoriasis- like disease via the topical application of IMQ, mice were split into representative groups. Healthy and untreated mice were used as controls (negative and positive control, respectively). Previous studies were conducted to optimize the optimal concentration of FLUO, demonstrating that the concentration of 0.15 7

Journal Pre-proof wt% of FLUO equivalents had the maximum therapeutic potential (see SI for further details). Therefore, conjugate-based treatments with 0.15% of FLUO equivalents were dissolved in the HA-CP vehicle (1%) and a commercial base cream (Cold cream®, Pharmacy Boix, Spain). Free FLUO was dissolved with 0.5 mL of Hydrolite® (Guinama S.L., Spain) and incorporated in the base cream. HA-CP vehicle (1%) and the base cream were applied as controls, following the same methodology. Treatments were immediately applied topically in the back and the right ear during five consecutive days. Treatments safety was evaluated by tracking body weight every two days compared to positive and negative controls (see SI for more details). Ear thickness was measured with a digital caliper before every treatment every

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two days. The anti-psoriatic potential of the formulations was evaluated using an objective scoring system based on the clinical Psoriasis Area and Severity Index (PASI) score. Erythema, scaling, and thickening were assigned independently on a scale from 0 to 4: 0,

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none; 1, slight; 2, moderate; 3, marked; 4, very marked. After five days of treatment, mice

e-

were sacrificed using a CO2 atmosphere, and spleen, dorsal skin, ears, and blood were extracted for further analysis. Spleen weight was measured immediately after sacrificed using

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a balance.

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2.9. Histology

Hematoxylin- Eosin (H&E) staining was performed on both ears and the dorsal skin.

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The tissues were washed with fresh PBS and fixed in 4% of PFA for 24 h. PFA was

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eliminated by successive washing with PBS, and a common dehydration and paraffin inclusion procedure was carried out, providing blocks that were sliced into 5 μm sections. The staining was performed as required (Dako Autostainer 48, US), and the slides were finally assembled with Eukitt. The slides were observed under the microscope, scanned with a Panoramic 250 Flash III slide scanner and processed with CaseViewer software (both from 3DHISTECH Ltd, Budapest, Hungary). 2.10. Pro-Inflammatory Interleukin Levels in Serum and Tissue Pro-inflammatory interleukin levels were measured both in serum and in tissue. Fresh serum was isolated from the blood through centrifugation (4000 rpm, 10 min, 4ºC) and stored at -80o C until use. To performed protein extraction from frozen tissue from the dorsal skin, samples were mixed with PBS pH 7.4 containing a protease and phosphatase inhibitor cocktail and then were digested by Ultra Turrax Scatter at 10000 rpm for 20 min. After 8

Journal Pre-proof digestion, samples were centrifuged at 4000 rpm for 20 min at 4°C, and supernatants were collected and stored at -80o C until use. IL-23 and INF-γ protein expression in skin lysates and serum were quantified by LUMINEX multiplex immunoassay (ProcartaPlexT M multiplex immunoassay), according to the manufacturer’s instruction (Invitrogen, Spain). 2.11. Statistical Analysis Data from in vitro studies are reported as the mean ± SEM of at least three independent experiments. In vivo experimental results are expressed as mean ± SEM with at least n = 7 samples per group. Statistical analysis was performed using one-way ANOVA test and

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Bonferroni’s and Tukey’s post hoc tests; comparisons with p<0.05 were considered statistically significant with a 95% confidence interval.

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3. Results and Discussion

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3.1. Synthesis and Characterization of the Poly-L-Glutamate-Fluocinolone acetonide

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Conjugate

We aimed to conjugate the poorly water-soluble corticosteroid FLUO to a water-

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soluble PGA carrier via an ester bond, aiming to improve FLUO bioavailability after topical administration in the skin while also providing a modulated release rate of FLUO in a

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specific skin layer. We chose PGA based on its successful application in the synthesis of polymer-drug conjugates in terms of cargo loading capacity through the multifunctional

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carboxylic acid backbone [23], elevated biodegradability of the polyaminoacid backbone under physiologically relevant conditions [38], and the ability of PGA-based nanomedicines to bypass biological barriers with a non-toxic and safe biodistribution profile [39-42]. Furthermore, PGA is composed of repeating units of glutamic acid, an essential amino acid that can provide the skin with the necessary nutrition for proper cellular function, and hence, enhances skin hydration, thereby exhibiting a soothing effect on damaged skin [43]. We prepared PGA-FLUO following well-established synthetic procedures [30, 44], attaching FLUO to the polypeptidic backbone via an ester linker (Figure 1A) through an esterification reaction using EDC, a carbodiimide coupling reagent, and DMAP as a catalyst. We employed a PGA backbone with a molecular weight of 15 kDa, as determined by sizeexclusion chromatography (SEC) (100 units of glutamic acid, see Figure S1 and Figure S2) and the drug molar content of ~8 mol%, as determined via 1 H NMR (Figure S3). We 9

Journal Pre-proof optimized the synthetic methodology to improve reproducibility and scalability (up to multigram scale), yielding the desired products with high purity. We characterized the synthesized conjugate through a battery of techniques to ensure identity, purity, total drug loading (TDL), and free drug (FD) content. The synthetic strategy yielded the desired conjugate with high purity as revealed via NMR spectroscopy (Figure 1B). After the conjugation of FLUO to PGA, we observed a broadening of the bands retaining characteristic peaks of the parent drug derivative at 6.5 - 7.5 ppm. Indeed, we proved the integrity of the acetonide moiety within the conjugate via NMR spectroscopy as

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shown by the prevalence of the associated peaks at 0.5 and 1.5 ppm, respectively (Figure 1B and Figure S4). As shown in Figure 1C, UV-Vis spectra of PGA-FLUO shows the preservation of the characteristic maximum absorbance of the parent drug (239 nm).

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However, the signals of PGA and FLUO overlap at 239 nm, so we quantified the total drug loading at 260 nm. UV-Vis analysis (Figure S5) allowed us to determine a total drug loading

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(TDL) by weight of 16-24 wt%, which agreed with the estimated value obtained from NMR

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analysis (6.5 to 8.9 mol%) for the different batches.

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Journal Pre-proof Figure 1. General synthetic route for the synthesis of the poly-L-glutamate (PGA)fluocinolone acetonide conjugate and physico-chemical characterization. (A) i) EDC, DMAP, DMF, 0ºC, N2 (see SI for details). (B) Chemical identity and purity demonstrated by 1

H NMR spectroscopy. (C) UV-Vis quantification of FLUO content within the polypeptide-

drug conjugate at 260 nm in a solution of MilliQ H2 O/ACN (60:40) and (D) SEC in aqueous media containing 0.1 M NaNO 3 and 0.005% (w/w) azide as additive (refractive index and UV channel) of PGA and PGA-FLUO. SEC-RI-MALS analysis allowed Mw determination for PGA (Mw=15.24 KDa and Đ=1.18) and PGA-FLUO (Mw=18.91KDa and Đ=1.38)

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(Figure S2) suggesting a FLUO content of 19.4 wt%. Homogeneous conjugation of the drug to the PGA backbone was evidenced by SEC chromatograms obtained from parental PGA and PGA-FLUO conjugate. Refractive index

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detection for both PGA and PGA-FLUO correlated well (Figure 1D, solid lines, note the peak at 17 min comes from the Na+ counter ion from carboxylate side chains in the PGA

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backbone), and UV-Vis detection at the absorbance peak of FLUO (260 nm) (Figure 1D,

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dashed lines) verified homogeneous FLUO distribution throughout PGA backbone. Table 1 summarizes the most relevant parameters for the physico-chemical characterization of the polymer-drug conjugate from three different batches and scales, demonstrating batch to batch

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reproducibility together with the robustness of the synthetic methodology followed (Figure

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S6). Additionally, we found a free drug (FD) content of 0.10-0.12 wt% of the total drug loading in all the conjugates as determined by reversed phase high-performance liquid

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chromatography (RP-HPLC) (Figure S7). The results obtained demonstrated a free drug content below 2 wt% (relative to total drug loading) after exhaustive purification of the conjugate, thereby providing evidence for the purity and high drug loading. We employed Batch 3 for the subsequent complete physico-chemical and biological evaluation. Table 1. Physico-chemical Characterization of Poly-L-glutamate conjugates.

Conjugate PGA-FLUO (batch 1) PGA-FLUO (batch 2)

a

Yield

Conj.

TDL

(%)

Eff. (%)

(wt%)

b

TDL

(wt%)

(mol%)

Amount

Linker

200 mg

Ester

49.80

41.90

16.23

0.12

200 mg

Ester

34.32

52.90

20.50

0.11

11

c

FD

Rh

d

Z-pot

e

(nm)

(mV)

6.57

93.27

-44.7

8.53

91.32

-53.7

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1g

Ester

59.48

99.80

23.80

0.10

8.87

88.82

-49.5

a

Total drug loading (TDL) determined by UV-Vis. bFree drug (FD) determined by RP-HPLC and calculated as percentage of the total drug loading. cTDL determined by 1 H NM R. d Hydrodynamic radius (1 mg/ mL, intensity) by DLS. eZ-potential measured via DLS in 1mM KCl. Conjugation efficiency (Conj. Eff. (%)) expressed as percentage of conjugated molar drug content compared to the targeted molar drug content.

3.2. Self-assembling Behavior of PGA-FLUO We studied the behavior of PGA-FLUO in an aqueous environment as the conjugation of the corticoid motifs into the hydrophilic PGA backbone may result in the generation of an

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amphiphilic system with appropriate hydrophobic-hydrophilic balance enabling the selfassembly into larger nanometer-sized objects [45]. At the molecular level, self-assembly of

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PGA-FLUO proceeded primarily via the hydrophobic effect, as evidenced by the pyrene assay performed to determine the critical aggregation concentration (CAC). The presence of

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hydrophobic pockets within the assemblies allowed the determination of the CAC via pyrene probe (Figure 2A) resulting in a low value of 0.06 mg/mL, in close agreement with other

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anionic polymer amphiphiles reported in the literature [46]. We then employed circular dichroism (CD) to determine the conformation of the conjugates as a function of PGA-FLUO

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concentration and at different pHs that mimic the different environments to which the

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conjugates will be exposed after topical administration. At pH 7.4, PGA-FLUO took on a predominantly random coil conformation, as

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revealed by the absolute minimum ellipticity value at 200 nm. We also recorded a significant redshift evolution of the random coil negative band below and above the CAC threshold value together with the appearance of a cotton effect in the region of 270-300 nm (Figure 2B). A bisignated band is attributed to the ketones within the FLUO molecules [47]. The increase in the cotton band intensity is most likely related to the increasing concentration of the FLUO content rather than a supramolecular organization of the inner core of the assembled conjugates as derived by the flat shape of the CD spectra when representing the molar ellipticity and hence correcting the concentration effect (Figure S8). At pH 5.5, in contrast, we found a partial alpha- helix characteristic conformation that evolved when increasing the concentration above the CAC. Under this condition, the random coil to alpha-helix transition remains incomplete according to the pKa values reported in literature (only roughly 50% of side chains are protonated at pH 5.5) [48] and the fact that we 12

Journal Pre-proof failed to observe precipitation or cloudiness at pH 5.5, as can be observed at pH 3 (Figure S9) [49]. We also observed a FLUO induced cotton band that evolved analogously to the experiments undertaken at pH 7.4. Moreover, transmission electron microscopy (TEM) micrographs confirmed the spherical aspect of the self- assembled carriers with geometrical sizes in the range of 50-100 nm with no particular evidence for core-shell morphologies (Figure 2C). The conjugates exhibited a hydrodynamic radius (Rh ) of 40-100 nm in aqueous solutions at different pH and ionic strengths, and Z-potential measurements resulted in a highly negative value of -50 mV, confirming the assembly of multiple PGA-FLUO molecules within the globular nanoassembly (Figure 2D and Figure S10 and S11). The compaction of

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the assembled conjugates upon increasing ionic strength reinforces the critical character of hydrophobic interactions. These interactions can drive the self-assembly process exposing the polyglutamate anionic backbone to the surface and hence experiencing a screening of anionic

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Pr

e-

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repulsions upon increasing ionic strength and leading to the recorded decrease in Rh [50].

Figure 2. Self-assembly behavior of PGA-FLUO and drug release kinetics. (A) Pyrene fluorescence assay of PGA-FLUO demonstrates conjugate self-assembly from 0.06 mg/mL.

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Journal Pre-proof (B) Secondary structure of PGA-FLUO by circular dichroism at pH 7.4 and 5.5, showing a transition from random coil to alpha- helix. (C) TEM micrographs of PGA-FLUO bearing self-assembly triggering of a sample prepared in ddH2 O at 2 mg/mL. (D) Hydrodynamic radius by DLS in different solvents (ddH2 O, PB 10mM, and PBS pH 7.4) with a constant size of ~100 nm. (E) Illustration of the self-assembly behavior of the PGA-FLUO conjugate. (F) Drug release profiles by RP-HPLC at different pHs (5.5 and 7.4) and under proteolytic conditions (cathepsin B) of PGA-FLUO alone and within the HA-CP vehicle. Experiments were conducted in triplicate (mean ± SEM).

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In summary, conjugation of the highly hydrophobic corticoid FLUO to PGA results in an amphiphilic PGA-FLUO conjugate that self-assembles into spherical nanosized objects driven mainly by hydrophobic interactions of the FLUO motifs (Figure 2E). Although we

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found no clear evidence for a core-shell structure by TEM, spectroscopic analyses suggest an

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unordered arrangement of FLUO within an inner hydrophobic core. At the outer layer of the constructs, CD analysis and Z-potential values indicate a PGA backbone composition with a

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high anionic carboxylate form at pH 7.4 in a relatively constrained random coiled conformation, probably due to electrostatic repulsions, which might transition to partial

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alpha-helix conformation at pH 5.5 without altering the solubility of the system.

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into the Skin

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3.3. FLUO Release Kinetics after Conjugation Drives Sustained and Controlled Release

We subsequently studied drug release kinetics under hydrolytic and proteolytic conditions owing to the pH-labile nature of the ester bond and cathepsin B-mediated degradation of the polypeptidic backbone. We incubated samples of PGA-FLUO at 37ºC at different pHs including 5.5 (lysosome) and 7.4 (blood) for up to 72 h. Of note, the acetonide groups at FLUO are expected to be much less labile than the ester bonds under the applied hydrolytic conditions [51]; this is evidenced from the absence of chromatographic peak changes of FLUO in our RP-HPLC experiments. Additionally, we performed drug release kinetics in the presence of HA-CP given the application of this vehicle to administer the conjugate into the skin. Overall, the data collected suggests a small but sustained FLUO release in all cases (Figure 2F and Figure S12). The results revealed that drug release kinetics from the conjugate at 72 h is two- fold faster at pH 7.4 (5 wt% of free FLUO) than at pH 5.5 (2.4 wt% of free FLUO).

14

Journal Pre-proof Although these results might seem counterintuitive, we must consider the conformational and spatial arrangement of the drug within the polymer-drug conjugate and the resulting assembled nanocarrier. Although acids or bases chemically catalyze ester-bond hydrolysis [52-54], these results could be explained in terms of PGA backbone response to pH. At pH 5.5, protonation of PGA side chains represents roughly 50% molar of the overall carboxylates as derived from the pKa of linear PGA reported in the literature [48]. These effects induce a partial random coil to alpha- helix conformation transition as shown above, although the system remains fully soluble according to turbidimetric data. At pH 7.4, 100% molar side chains in PGA are carboxylates, yielding a full random coil conformation.

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Whether the observed change in release kinetics derives from conformational aspects (e.g., rigidity, compactness, accessibility to PGA-FLUO bonds) or auxiliary effect of vicinal carboxylates, the result observed represents an inverse release profile to that expected. We

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hope to explore the rationale behind this phenomenon in future studies.

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We also studied drug release in the presence of cathepsin B (5 U), a lysosomal

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cysteine protease that significantly contributes to the proteolytic conditions faced by the polymer-drug conjugates following cell uptake. We found the release of FLUO to be significantly higher following the addition of cathepsin B when compared to hydrolytic

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conditions, with 20 wt% drug release within 72 h (Figure 2F). This significant increase in

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drug release can be ascribed to the digestion of the PGA backbone, which could enhance the

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accessibility of the PGA-FLUO bond and, therefore, elevated hydrolytic cleavage [55]. Additionally, the incorporation of PGA-FLUO in HA-CP caused a marked decrease in the release of FLUO under hydrolytic conditions, obtaining half of the free FLUO released at pH 7.4 (~2.5 wt% of free FLUO) and pH 5.5 (~1.2 wt% of free FLUO). However, more importantly, we observed a marked decrease in FLUO release under proteolytic conditions, obtaining ~2.5-3 wt% of free FLUO in comparison with the 20 wt% drug release without the HA-CP vehicle. 3.4. Compatibility of PGA-FLUO with the HA-CP Vehicle As PGA-FLUO will be delivered topically using an HA-CP vehicle that can enhance permeation to the epidermis [28, 29], we studied the compatibility of the intended formulations. The incorporation of PGA-FLUO at a working concentration of 10 mg/mL within 1% of HA-CP vehicle established the complete solubility and stability of the conjugate 15

Journal Pre-proof in the vehicle. Furthermore, the vehicle displayed no effect over nanosized PGA-FLUO in terms of homogenous globular shape and an expected size of about 80-100 nm, as evidenced in TEM micrographs (Figure S13). As shown, the incorporation of PGA-FLUO into the HACP vehicle caused a marked decrease in the release of FLUO under hydrolytic or proteolytic conditions (Figure 2F). The increase in the viscosity caused sequestration of the PGA conjugate within the cross-polymer vehicle, thereby contributing to the delayed release of FLUO compared to aqueous dispersion (Figure S14). We observed a more evident decrease in drug release under proteolytic conditions, where the reduction in the release kinetics most likely relates to the lower mobility of enzyme and substrate. Indeed, we discovered a

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significant decrease of cathepsin B activity in the presence of the HA-CP vehicle (Figure S15). Overall, these results suggest a protective effect of the HA-CP vehicle on PGA-FLUO.

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3.5. In Vitro Skin Compatibility and Cell Trafficking Studies of the PGA-FLUO

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Cell viability assays performed on keratinocytes (HaCaT cells) and human fibroblasts after 72 h of treatment with increasing doses of FLUO or PGA-FLUO at the same drug

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equivalents revealed the absence of cell toxicity up to the concentrations tested, demonstrating the safety of the PGA-FLUO conjugation strategy for dermal applications

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(Figure 3A and 3B). Additionally, we evaluated cell viability of Raw264.7 murine macrophages in response to FLUO after 72 h of treatment (Figure S16), allowing us to select

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line.

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0.49 ng/mL as FLUO concentration for any subsequent pharmacological study in this cell

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Figure 3. In vitro evaluation of PGA-FLUO. Cell viability assays in HaCaT keratinocytes (A) and human fibroblasts (B) after 72 h of treatment with FLUO or PGA-FLUO (n=3). (C) Uptake study by flow cytometry of PGA-FLUO fluorescently labeled with OG (PGA-FLUO-

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OG) in HaCaT keratinocytes. Results are represented as the average of positive cells (%) ±

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SEM. ***p<0.001, **p<0.01, *p<0.05 (n=3). (D) Confocal images from the uptake of PGAFLUO-OG at 24 h post-treatment in HaCaT keratinocytes, in which co- localization with

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Lysotracker red was observed. Red fluorescence corresponds to Lysotracker red; green fluorescence corresponds to OG; blue fluorescence corresponds to DAPI; and yellow fluorescence marks co-localization between Lysotracker red and OG.

We also performed flow cytometry (Figure 3C) and fluorescence confocal microscopy (Figure 3D) at non-toxic concentrations in keratinocytes as our target cell to understand the cell internalization of an OG- labeled PGA-FLUO (PGA-FLUO-OG). As expected for this type of macromolecular systems [56, 57], we observed energy-dependent endocytic uptake, reaching 30% positive cells at 24 h post-treatment (Figure 3C). In agreement with these results, we obtained confocal images to identify the subcellular localization of PGA-FLUO-OG within keratinocytes after 24 h of uptake, demonstrating a clear co-localization with the lysosomal marker Lysotracker red (Figure 3D). These findings

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Journal Pre-proof indicate the suitability of our polypeptidic nanocarrier for lysosomotropic drug delivery [58], where the combination of acidic pH and presence of proteolytic enzymes (cathepsin B) promote the sustained release of FLUO from PGA-FLUO conjugate (also see Figure 2F), thereby allowing binding to the intracellular glucocorticoid receptor, and the subsequent downstream effects [59]. 3.6. FLUO Conjugation and the Application of the HA-CP Vehicle Enhances Skin Permeation To visualize skin penetration of PGA-FLUO and explore the role of the HA-CP

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vehicle [29], we performed a qualitative study on human skin for 24 h comparing the permeation of 10 mg/mL of PGA-FLUO-OG applied in water (Figure S17) and embedded in

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Cy5.5-labeled HA-CP vehicle (1%) (HA-CP-Cy5.5) (Figure S18). After fixation and tissue processing, the confocal microscopy images acquired to identify skin penetration revealed a

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significant accumulation of the PGA-FLUO-OG within the stratum corneum, and a lower accumulation in the epidermis (Figure 4A); however, PGA-FLUO-OG in HA-CP-Cy5.5

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displayed enhanced penetration through the epidermal layer (Figure 4B). Moreover, aliquots extracted from the receptor chamber did not contain OG and the Cy5.5 signals compared to a

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negative control with MilliQ water, indicating that neither the conjugate nor the vehicle can cross the whole skin and reach the systemic circulation, ensuring the safety of the treatment

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(Figure 4C). The signals in the spectra arises from the biological material (proteins 200-280

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nm and hemoglobin traces at 400 nm). We quantified OG intensity in various skin layers by ImageJ software after the permeation studies with/without the HA-CP-Cy5.5 vehicle; this revealed that the presence of the HA-CP-Cy5.5 vehicle significantly enhanced the epidermal accumulation of PGAFLUO-OG, proven by the higher fluorescence intensity detected both in the stratum corneum and the viable epidermis (Figure 4D). Furthermore, we extracted FLUO from the whole skin after 24 h of application of PGA-FLUO (10 mg/mL) in water or embedded in the HA-CP vehicle (1%) and analyzed the samples by RP-HPLC to quantify the amount of FLUO into the skin (Figure S19). We normalized the values obtained by RP-HPLC using the amount of FLUO released from the conjugate with/without the HA-CP vehicle (obtained from drug release kinetics studies), demonstrating that the amount of FLUO extracted after 24 h was almost two- fold higher when we applied the conjugate in the HA-CP (Figure 4E). We also demonstrated by LC-MS measurements in separated skin layers that the amount of FLUO 18

Journal Pre-proof extracted from the skin mainly derived from the epidermal layer, as we found a higher concentration of FLUO in the epidermis when compared to the dermis (Figure S20A) with non-detectable FLUO signals observed in the receptor compartment (Figure S20B). These findings prove the ability of the HA-CP vehicle to promote skin permeation and conjugate accumulation in the epidermal layer, most likely via improved vehicle-mediated hydration in both the stratum corneum and epidermal layers [60]. This then allows for the sustained release of the drug in the target epidermal layer while reducing the need for repeated

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applications to the skin.

Figure 4. Evaluation of skin permeation of PGA-FLUO-OG and FLUO quantification in the skin. (A) Confocal images after permeation studies of PGA-FLUO-OG formulated in water. (B) Confocal images after permeation studies of PGA-FLUO-OG in HA-CP-Cy5.5 (1%). (C) UV-Vis measurements of the aliquots extracted from the receptor chamber after 8 19

Journal Pre-proof (left) and 24 h (right) of permeation studies compared to negative control (MilliQ water) and OG spectra. (D) Quantification of OG intensity by ImageJ software in the stratum corneum (SC) and the viable epidermis (n=5) after permeation studies. (E) Quantification of FLUO extracted from the whole skin (see SI for details) analyzed by RP-HPLC after the permeation studies of PGA-FLUO with and without the HA-CP vehicle (n=3). Asterisks indicate statistically significant differences after ANOVA analyses followed Bonferroni’s post hoc tests, mean ± SEM. In all cases, we considered differences to be significant when p***<0.001; p**<0.01; p*<0.05.

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3.7. PGA-FLUO Reduces the Release of Pro-inflammatory Cytokines In vitro studies: We tested the anti- inflammatory activity of FLUO derivatives in

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Raw264.7 macrophages (at a non-toxic concentration (Figure 5A)) using a LUMINEX multiplex immunoassay by assessing any reduction in pro- inflammatory cytokines release to

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the culture media from macrophages treated with 5 ng/mL of bacterial (E. coli) LPS. The results obtained suggested that FLUO not only retains its anti- inflammatory activity

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following incorporation into the polypeptidic backbone, but also that the conjugation to PGA promotes the inhibitory effect of FLUO on the release of pro- inflammatory cytokines IL-1b

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and INF-γ (Figure 5B).

Figure 5. In vitro PGA-FLUO evaluation in macrophages. (A) Cell viability assay in Raw264.7 macrophages after 72 h of treatment with FLUO, PGA-FLUO, and vehicles at 0.49 ng/mL of FLUO equivalents. (B) Reduction of pro- inflammatory cytokine release after treatment for 72 h in macrophages stimulated with bacterial LPS (n=3). Asterisks indicate statistically significant differences after ANOVA analyses followed Tukey’s post hoc tests

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Journal Pre-proof mean ± SEM. In all cases, we considered differences to be significant when p***<0.0001; p**<0.01; p*<0.05. Ex vivo studies: We explored two ex vivo systems to corroborate the data obtained in the cell studies: (i) an ex vivo human skin model (Figure 6A) and (ii) a skin equivalent model (Figure 6B). Ex vivo models represent valuable research tools for investigations into different skin diseases (such as psoriasis or atopic dermatitis) and are considered more relevant than in vitro research involving cell lines. We employed two ex vivo models to obtain complementary information regarding the safety and the anti- inflammatory activity of PGA-

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FLUO. Firstly, in an ex vivo human skin model (Figure 6C), analysis of tissue viability after

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treatment with 3 µL of a solution of 10 mg/mL of PGA, FLUO, PGA-FLUO applied in water and the HA-CP vehicle (1%), and HA-CP (1%) for 24 h revealed that all treatments allowed

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the maintenance of tissue viability. Additionally, in the skin equivalents model, the application of two doses of 5 mg/mL of the same treatments on day 11 and 13 also displayed

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excellent biocompatibility as indicated by the absence of significant cytotoxic effects (Figure 6D and Figure S21).

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Additionally, we developed inflammatory models to assess the anti- inflammatory

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potential of the FLUO derivatives at the same concentration evaluated in the viability assays. We studied IL-6 secretion by ELISA assay in an optimized inflammatory ex vivo human skin

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model after inflammatory insult via the combined treatment with bacterial LPS (15 µg/mL) and EGF (2.5 ng/mL) for 24 h in the culture media (Figure S22-24). Treatment for 24 h revealed that the reduction in IL-6 release following FLUO treatment was similar to PGAFLUO treatment (both applied in water or HA-CP vehicle). All treatments reduced IL-6 levels by around 20% compared to the corresponding untreated control (Figure 6E). To ratify this data, we also studied IL-6 secretion in the second inflammatory skin equivalents model induced by the application of 20 ng/mL of recombinant TNF-α in the culture media on days 10 and 12 of culture [32, 34, 61, 62]. Of note, this model provides a more permeable stratum corneum than the ex vivo human skin model, given the reconstitution of this skin model from cells. On day 11 and 13, we topically applied the same treatments at 5 mg/mL and evaluated the release of IL-6 by ELISA assay. We discovered a significant reduction in IL-6 secretion (almost 50%) in the case of PGA-FLUO treatment applied in 21

Journal Pre-proof water or the HA-CP vehicle when compared to the free drug, most probably due to the lower amount of model variance when compared to the higher variation observed among the human

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skin samples (Figure 6F).

Figure 6. Evaluation of tissue viability and anti-inflammatory activity of FLUO derivatives in inflammatory skin models. (A) Establishment of the inflammatory ex vivo human skin model. (B) Establishment of the inflammatory skin equivalents model. (C) Relative tissue viability compared to the control in an ex vivo human skin model after 24 h of treatment (10 mg/mL of PGA, FLUO, PGA-FLUO in water and HA-CP vehicle (1%), and HA-CP (1%)) by MTT assay. (D) Relative tissue viability compared to the control in a skin equivalents model after two doses of treatment (5 mg/mL of PGA, FLUO, PGA-FLUO in water and HA-CP vehicle (1%), and HA-CP (1%)) by MTT assay. (E) IL-6 levels in an

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Journal Pre-proof inflammatory ex vivo human skin model after the induction with the combination of bacterial LPS (15 µg/mL) and EGF (2.5 ng/mL), following treatment (10 mg/mL of PGA, FLUO, PGA-FLUO in water and HA-CP vehicle (1%), and HA-CP (1%)) for 24 h. Values expressed as relative inhibition of IL-6 (%) compared to the control. (F) IL-6 levels in an inflammatory skin equivalents model stimulated with TNF-α (20 ng/mL), following two doses of treatment (5 mg/mL of PGA, FLUO, PGA-FLUO in water and HA-CP vehicle (1%), and HA-CP (1%)). Values are expressed as relative inhibition of IL-6 (%) compared to the control. Asterisks indicate statistically significant differences after ANOVA analyses followed

significant when p***<0.001; p**<0.01; p*<0.05.

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Bonferroni’s post hoc tests, mean ± SEM. In all cases, we considered differences to be

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3.8. PGA-FLUO in HA-CP Reduces Imiquimod-induced Skin Inflammation In Vivo Several reports have demonstrated that IMQ can activate immune cells via a toll- like

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receptor to induce psoriasis- likes inflammation [63]. Therefore, we established an in vivo model based on topical IMQ treatment to evaluate the anti-psoriatic activity of PGA-FLUO.

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Firstly, we optimized the FLUO equivalent concentration for this experimental setup, which we established at 0.15 wt% dose (Figure S25-32). We used this concentration to compare the

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anti-psoriatic activity of FLUO applied in a commercially-available cream used for current corticosteroids (due to its hydrophobicity and poor water solubility) with PGA-FLUO in the

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same cream and also applied in the HA-CP vehicle due to its high solubility in aqueous

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formulations. We applied treatments to the back and the right ear of IMQ-treated 6-week-old male BALB/c mice for five days. We also applied the cream and the HA-CP alone as controls. Treatment safety was evaluated by tracking body weight every two days and comparing results to healthy animals. Encouragingly, we discovered that treatments failed to significantly alter body weight (Figure S33). Histological examination of the mouse ear and back sections revealed a marked difference in the extent of epidermal thickening and elongation of epidermal ridges between the treated groups. PGA-FLUO treatment resulted in a more significant diminishment of ear thickness and epidermal ridges and, remarkably, the choice of the vehicle employed for the administration also influenced overall effects, with the group treated with PGA-FLUO in HA-CP providing similar results to those observed in healthy control animals. Overall, PGA-FLUO in HA-CP exhibited significant differences compared to all other groups, presenting minimal epidermal thickening, an intact stratum corneum, and histological features compared to the negative control (Figure 7). Importantly,

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Journal Pre-proof following analysis of the control vehicles groups, we discovered that the group treated with the HA-CP vehicle presented a decrease in the epidermis thickness, whereas treatment with the commercially available cream failed to elicit any anti-psoriatic effect. This finding suggests that the probable increase in skin hydration via HA-CP treatment can lead to an improvement in the symptoms (dryness, scaling) produced by IMQ application. Therefore, the use of this HA-CP vehicle offers a synergistic effect with the conjugated corticosteroids triggering an enhanced cutaneous anti-psoriatic effect. Signs of epidermal thickening due to inflammation were not only evident in the back but also by microscopical examination of the

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ear sections (Figure 7).

Figure 7. Histological analysis of mouse tissues after five days of anti-psoriatic treatment in an IMQ-induced skin inflammation model. The images demonstrate a marked reduction of the epidermal thickening in the group treated with PGA-FLUO compared to free FLUO. The group treated with PGA-FLUO in HA-CP prompts the maximum reduction of both back and ear thickness. Original magnification displayed in each image was 10x. One representative picture is shown for each treatment regimen. We also evaluated erythema, scaling, and thickness of the dorsal skin by PASI scoring after five days of treatment (Figure 8A). While an examination of the back every two days revealed that the group treated with FLUO displayed a reduction in all the parameters, this

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Journal Pre-proof treatment did not lead to the restoration of values observed in healthy control mice (negative control). Again, PGA-FLUO treatment prompted an enhanced effect when applied in cream and a more marked effect when administered in the HA-CP vehicle, presenting significant morphological tissue recovery (similar to the negative control, a value of 0 in the PASI score at the end of the experiment in the three measured parameters). The group treated with the HA-CP vehicle also exhibited a reduction in PASI scores, probably due to its ability to hydrate the skin and diminish IMQ- induced skin dryness. We measured ear thickness every two days with a caliper during treatment discovering that all treated groups showed a decrease compared to the positive control group, with a maximum reduction (20%) of

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thickness observed in the group treated with PGA-FLUO in HA-CP, ratifying the dorsal results (Figure 8B).

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Several studies have reported that IMQ can produce a significant spleen enlargement with a weight increase attributed to the release of inflammatory cytokines [64] (Figure 8C).

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In our IMQ-induced model, we established an average spleen weight in the positive control

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of 250 mg compared to 100 mg in the negative control. All treated groups displayed a marked decreased in the spleen weight (FLUO 125 mg and PGA-FLUO applied in cream 100 mg), which was maximal for the group treated with PGA-FLUO in HA-CP. We found no

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significant difference in the spleen weights between the negative control and the group

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treated with PGA-FLUO in HA-CP, confirming the ability of the vehicle embedded

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conjugate to reach the epidermal layer of the skin and reduce inflammation. 3.9. PGA-FLUO Reduces Pro-inflammatory Cytokines Levels in Serum and Tissue TNF-α, IL-17, IL-23, and INF-γ act as significant mediators of psoriasis, and they mark the onset of IMQ-induced psoriasis [64-66]. In our psoriatic mice model, we discovered the significantly higher expression of INF-γ and IL-23 for the positive control compared to the negative control in tissue, and INF-γ in serum, which confirmed adequate in vivo psoriatic model development. In tissue, the levels of INF-γ were reduced by 31% and 51% in the groups treated with PGA-FLUO in cream and PGA-FLUO in HA-CP, respectively, compared to 6% for the free FLUO treated group. Furthermore, IL-23 levels were reduced by 21% and 34% for PGA-FLUO in cream and PGA-FLUO in HA-CP, respectively, as compared to free FLUO (24%) (Figure 8D). We observed a similar reduction in values and differences among groups following the evaluation of INF-γ levels in serum, establishing that PGA-FLUO in HA-CP displayed the highest anti- inflammatory capacity (Figure 8E). Of note, the group 25

Journal Pre-proof treated with the HA-CP vehicle displayed a reduction in INF-γ levels in tissue and serum by 28% and 27%, respectively. The possible explanation can be ascribed to the intrinsic nature of HA-CP, as it results from crosslinking of HA with amino acids, which protect its enzymatic degradation by hyaluronidases, and consequently allows a prolonged moisturizing

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effect that could trigger its impact in reducing inflammation [29].

Figure 8. Reduction of skin inflammation, ear thickness, splenomegaly, and proinflammatory cytokine levels in an in vivo psoriatic mice model after anti-psoriatic treatments. The activity of FLUO and PGA-FLUO applied in cream, PGA-FLUO in HACP, and vehicles was evaluated after five days of topical treatment. (A) The severity of skin inflammation in the dorsal skin was measured by PASI scoring (erythema, scaling and thickness) every two days during treatments using a scale from 0 to 4. (B) Relative reduction of ear thickness after the treatments compared to the positive control. (C) Spleen weight (mg) determination after five days of treatment. Relative reduction compared to positive control of pro-inflammatory cytokines related to psoriasis in tissue (D) and serum (E) (see SI for more information) measured by LUMINEX assay. The results were compared to positive control. 26

Journal Pre-proof Asterisks indicate statistically significant differences after ANOVA analyses followed Bonferroni’s post hoc tests, mean ± SEM. In all cases, we considered differences to be significant when p***<0.001; p**<0.01; p*<0.05.

In summary, the topical application of PGA-FLUO within an HA-CP vehicle ameliorates IMQ-induced psoriasis, returning indicators of disease to basal levels. The results suggest that the synergism observed derives from the conjugation of the corticosteroid to a polypeptidic carrier combined with the application of a highly hydrating vehicle as a

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permeation enhancer for topical administration. The application of FLUO as a water-soluble PGA conjugate resulted in higher drug concentration available at the site of application,

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while the inclusion of PGA-FLUO in HA-CP results in higher uptake by dermal cells and improved skin permeation, leading a deeper penetration of PGA-FLUO into the viable layers

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of the skin. Furthermore, the incorporation of PGA-FLUO in HA-CP might increase skin hydration, an additional factor required for treatment success, while HA-CP may also protect

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the conjugate from enzymatic degradation, thereby allowing it to reach the target epidermal layer. Our results indicate that the combination of a polymer-corticosteroid conjugate and an

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HA-CP vehicle provides a synergistic effect that may make corticosteroid treatments more

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4. Conclusions

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efficient and safer for the treatment of psoriasis.

Herein, we have demonstrated that the polypeptide conjugation of a corticosteroid used for the topical treatment of psoriasis (PGA-FLUO) provides a significant improvement in the pharmacological activity due to greater bioavailability in the require skin layer. Polypeptide conjugation may contribute to improve the penetration and drug exposure, thereby increasing the amount of corticosteroid in the viable epidermis, and therefore, increasing the effectiveness of the treatment. An adequate drug release kinetic profile represents a crucial parameter for the achievement of a suitable drug concentration in the epidermis, and therefore, polymer-drug linker design represents an essential feature. However, the mode of conjugate administration also plays a vital role, as the application of the polypeptide-conjugated corticosteroid within a polymeric vehicle (HA-CP) protected the conjugate, resulting in the slower and sustained release of the drug in the epidermis and allowing for sustained residence time. All these parameters are important as in human 27

Journal Pre-proof psoriasis, inflammation in the epidermis leads to uncontrolled keratinocyte proliferation and erythema in the affected area. PGA conjugation of FLUO allowed enhanced targeting to the viable epidermis while the use of the HA-CP vehicle further promoted this targeting effect by enhancing skin penetration. Cleavage of the polymer-drug linker under lysosomal conditions after being endocytosed by keratinocytes allows FLUO release. Overall, this provides a means to target the anti- inflammatory activity of FLUO to the required skin layer and avoid possible systemic side effects. In conclusion, the synergistic combination of PGA-FLUO and HA-CP provides an

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elevated reduction in inflammation and, therefore, represents a possible palliative treatment of inflammatory skin diseases. Our strategy opens a new horizon in the field of localized dermal delivery that can be extended to the conjugation of other therapeutic moieties for other aggressive

skin diseases,

such as cancer.

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implemented

The

use of

polymer/polypeptide therapeutic-based formulations for various skin diseases could

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revolutionize current therapeutic strategies of dermatology, overcoming old challenges, and

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offering new perspectives for the treatment and prevention of dermatological conditions.

Acknowledgements:

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The authors would like to thank Dr. Stuart P. Atkinson for English revision. This work has been supported by Spanish Ministry of Science, Innovation and Universities (RTC-2017-

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6465-1 and D.M-B, Grant Industrial PhD DI-14-06926), Generalitat Valenciana (I.D-P, Grant

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VALi+d ACIF-2016-021 and BEFPI/2018/055) and Science development and technology fund - Egypt (STDF grant no. 12362). Part of the equipment employed in this work has been funded by Generalitat Valenciana and co- financed with FEDER funds (PO FEDER of Comunitat Valenciana 2014–2020).

Author Contributions: - Conception and design: V.J.N, M.J.V. - Synthesis and characterization of the polymer-drug conjugates: I.D-P. - Ex vivo and In vivo experiments optimization and development: I.D-P., E.M., D.M-B., MS, DA, PG. - Analysis and interpretation of data: I.D-P., E.M., V.J.N, M.J.V. - Writing, review, and/or revision of the manuscript: I.D-P., S.H., M.S, V.J.N., M.J.V. - Study supervision: V.J.N, M.J.V.

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Conflict of Interest D.M-B., V.J.N., and M.J.V. declare that a patent with reference WO2019020344 (A1) has been filed related to the present work.

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Table 1. Physico-chemical Characterization of Poly-L-glutamate conjugates.

Amount

2)

mg

PGA-FLUO (batch

1g

3)

49.8

(wt%)

(wt%)

(mol%)

(nm)

(mV)

41.90

16.23

0.12

6.57

93.27

-44.7

52.90

20.50

0.11

8.53

91.32

-53.7

99.80

23.80

0.10

8.87

88.82

-49.5

0 Ester

34.3 2

Ester

59.4

oo f

200

Ester

Z-pote

pr

PGA-FLUO (batch

(%)

Rhd

8

e-

mg

(%)

Linker

TDLc

Pr

1)

Eff.

FDb

al

200

d

TDLa

rn

PGA-FLUO (batch

Conj.

Jo u

Conjugate

Yiel

33

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rn

al

Pr

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pr

oo f

Highlights  Rationally designed polypeptide-based conjugates for topical delivery.  Increased anti-inflammatory activity after corticosteroid conjugation, importance of bioresponsive polymer-drug linkers.  Improved skin penetration using a hyaluronic acid-polyglutamic acid cross-polymer as vehicle.  Synergistic combination of drug conjugation and penetration enhancer provides an elevated reduction in inflammation including an anti-psoriatic activity in vivo and, therefore, represents a possible palliative treatment of inflammatory skin diseases.

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