In situ gelling alginate-pectin blend particles loaded with Ac2-26: A new weapon to improve wound care armamentarium

Carbohydrate Polymers 227 (2020) 115305

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In situ gelling alginate-pectin blend particles loaded with Ac2-26: A new weapon to improve wound care armamentarium

T

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Pasquale Del Gaudioa, , Chiara Amantea,b, Roberta Civalea, Valentina Bizzarroa, Antonello Petrellaa, Giacomo Pepea, Pietro Campigliaa, Paola Russoa, Rita P. Aquinoa a b

Department of Pharmacy, University of Salerno, via Giovanni Paolo II, 132, 84084 Fisciano, SA, Italy PhD Program in Drug Discovery and Development, University of Salerno, via Giovanni Paolo II, 132, I-84084 Fisciano, SA, Italy

A R T I C LE I N FO

A B S T R A C T

Keywords: Alginate Pectin Annexin A1 derived peptide Wound dressing Controlled release Peptide improved stability

In this paper, alginate-pectin blend particles loaded with Annexin A1 derived peptide Ac2-26 as an in situ forming dressing was successfully developed for wound repair applications. High mannuronic (M) content alginate and amidated pectin blend have been used to encapsulate Ac2-26 in order to enhance stability of the peptide at room temperature and to control its release through the in situ formed gel. Ac2-26 recovery and FTIR studies suggests chemical interactions between peptide and polysaccharides blend able to improve the encapsulation efficiency of Ac2-26 into the polymer matrix and control its release, till 48 h. In vitro wound healing assay on HaCaT cells highlights the ability of Ac2-26 to significantly accelerate wound healing compared to unloaded particles, with complete closure of the wound model in 24 h. Therefore, all these results suggest that Ac2-26 loaded submicrometric in situ gelling powders could be a promising wound dressing to improve wound care armamentarium.

1. Introduction Non-healing wounds affect more than 2% of the population in Western countries. It is estimated that diabetic foot ulcers and chronic non-healing wounds are responsible, nowadays, for more than 20% of hospital beds occupation (Gomes, Teixeira, Ferraz, Prudêncio, & Gomes, 2017; Sen et al., 2009). Management of such wounds as well as other non-healing wounds require good knowledge of their complexity in terms of both stage and severity, while possible infections must be taken into consideration in order to identify the proper treatment protocol (Han & Ceilley, 2017). Stimulation of wound healing is essential in the treatment of severe wounds. The use of dressing as well as scaffolds containing active agents to both support and stimulate tissue rebuilding in diabetic, venous and pressure ulcers is becoming critical to reduce wounds healing time that may require several years, while some remain unhealed for decades (Järbrink et al., 2017). Moreover, proper wound micro-environment is also very important to stimulate the healing process and reduce microbial proliferation (Junker, Caterson, & Eriksson, 2013; Scalise et al., 2015). In the last few years, in order to reduce the threat to public health represented by non-healing wounds significant efforts have been made

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to find new active ingredients and treatments to improve the effectiveness of current therapies focusing on the development of novel devices loaded with various healing stimulating ingredients able to control the delivery of their cargo (De Cicco et al., 2016; Della Porta, Del Gaudio, De Cicco, Aquino, & Reverchon, 2013; Gomes et al., 2017; Raj, Kumar Sharma, & Malviya, 2018; Rasool, Ata, & Islam, 2019). Several natural and biocompatible polysaccharides, as alginate, are currently used as wound dressing due to their natural adhesive properties and the ability to stimulate the healing process (Ganesan, 2017; Moura, Dias, Carvalho, & de Sousa, 2013; Murakami et al., 2010). Alginate is composed by a mixture of two monomeric units, guluronic acid (G) and mannuronic acid (M) in different ratio depending on the source. In wound healing applications high M content alginates should be preferred due to its ability to induce cytokine production by human monocytes, a process very useful in chronic wound healing (Iwamoto et al., 2005; Thomas, Harding, & Moore, 2000). Moreover, its ability to gel when in contact with wound fluids can provide a barrier impermeable to fluids and microorganisms, while permeable to air (Murphy & Evans, 2012). Although alginate is one of the most used polymer in would healing formulations some of the properties showed by alginate dressing as well as gel, as transpiration and exudate absorbance are not able to match all the needs for an optimal device that

Corresponding author at: Department of Pharmacy, University of Salerno, I-84084 Fisciano, SA, Italy. E-mail address: [email protected] (P. Del Gaudio).

https://doi.org/10.1016/j.carbpol.2019.115305 Received 1 August 2019; Received in revised form 6 September 2019; Accepted 6 September 2019 Available online 09 September 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.

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2.1. In situ gelling powders preparation

should also provide a proper wound environment to promote healing process and reduce bacterial proliferation (Rowan et al., 2015). On the contrary, blends or composites of alginate with other natural polysaccharides are able to compensate most of its lacks (De Cicco et al., 2014; Naseri-Nosar & Ziora, 2018; Venkatesan, Bhatnagar, Manivasagan, Kang, & Kim, 2015) and enable the production of active ingredients controlled release platforms. Active ingredients as growth factors (GFs) and wound healing peptides (WHP) have attracted many interests based on their mechanisms of action able to promote one or more stages of the healing process (Bodnar, 2013; Mangoni, McDermott, & Zasloff, 2016). Several formulations loaded with different GFs have been designed for the locally controlled release, i.e. epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), plateletderived growth factor (PDGF-BB) (Brannon-Peppas & Blanchette, 2012; Gainza, Villullas, Pedraz, Hernandez, & Igartua, 2015; Kumari, Yadav, & Yadav, 2010; Lee, Silva, & Mooney, 2011). Unfortunately, in spite of very encouraging results in wound healing treatment their use is nowadays mainly related to cosmetic products (Aldag, Teixeira, & Leventhal, 2016). In fact, only PDGF-BB as gel based formulation has been approved for its use in diabetic ulcers treatment demonstrating various adverse effects in patient extensively treated (Hirshberg, Coleman, Marchant, & Rees, 2001; Khoshkam et al., 2015; Margolis, Crombleholme, & Herlyn, 2000). Ac2-26 is the N-terminal derived peptide of Annexin A1 (ANXA1); it can be used as peptide surrogate in view of its ability to replicate the effects of the parent protein (i.e. anti-inflammatory signalling, kinase activities in signal transduction, maintenance of cytoskeleton and extracellular matrix integrity, tissue growth, apoptosis and differentiation) while binding to cellular membranes in a Ca2+-dependent manner (Kamaly et al., 2013). The ability to improve cell migration and tissue repair is related to the engaging of FPRs (formyl peptide receptor) then acting positively on cytoskeletal machinery is able to promote repair mechanisms for skeletal muscle and may have therapeutic implications with respect to the development of ANXA1 mimetic entities (Bizzarro, Fontanella et al., 2012; Leoni et al., 2013). In a previous study (Del Gaudio et al., 2015) we demonstrated the ability of Ac2-26 to promote wound healing in a murine model when released in a controlled way by an alginate gel over 72 h in spite of Ac226 instability in physiological conditions. In the present study, we have developed a polysaccharide based composite powder based on high M content alginate and high amidated pectin loaded with Ac2-26 with the aim to obtain an in situ forming hydrogel, able to produce a proper wound microenvironment, via exudate absorption of the powder when spread on the wound. Such blend has been designed also to stabilize the peptidomimetic, while prolonging its release into the wound cavity. Investigation on gelling rate and wound fluid uptake of the powder as well as stability and release profiles of the loaded peptide have been conducted. Finally, cytotoxicity of the polysaccharide powders as well as the ability to promote wound healing has been evaluated on an ex vivo model, using HaCaT (human immortalized keratinocytes) cell line.

Particles were prepared by nanospray drying processing different aqueous feeds. Feeds were prepared using a precise volume of a peptide stock solution (0.125 mg/mL), previously prepared. Ac2-26 aliquots were added to an aqueous solution of alginate and pectin blend at different polymer ratio by means of a Gilson pipet (Pipetman Classic, Gilson, Inc., Middleton, WI, USA), under gentle magnetic stirring. Alginate-pectin solutions were previously prepared with ratios between polymers ranging from 3:1 to 1:3 by dissolving both polymers in distilled water under vigorous stirring for 1 h. Total concentration of the feeds was set between 0.25% and 1.00% w/v. Ac2-26 solution were then added to the polymers solution in order to obtain a final peptide/ polymer blend ratio ranging between 0.01 and 0.02 (w/w). Feeds were processed by Nano Spray Dryer B-90 HD (Buchi Laboratoriums-Tecnik, Flawil, Switzerland) with optimized operational conditions set as follow: inlet temperature 80 °C, outlet temperature 39 °C, feed rate 0.5 mL/min, nozzle mesh about 3 μm, frequency 110 KHz, Pressure 40 hPa. After collection the powders were analysed in term of production yields, particles morphology and size distribution and Ac2-26 content and encapsulation efficiency. Each formulation was prepared at least in triplicate. 2.2. Powders physico-chemical properties 2.2.1. Morphology and particle size distribution The morphology of all powder formulations was studied by scanning electron microscopy (SEM), using a Carl Zeiss EVO MA 10 microscope with a secondary electron detector (Carl Zeiss SMT Ltd, Cambridge, UK). Powders were dispersed on a carbon tab previously stuck to an aluminium stub (Agar Scientific, Stansted, United Kingdom) and gold coated using a LEICA EMSCD005 metallizator producing a deposition of a 200–400 Å thick gold layer. Analysis was conducted at 20 KeV. At least 30 SEM images were taken into account for each run to verify particles uniformity. Particle size distribution was evaluated by dinamic light scattering (DLS) (N5, Beckman Coulter, Miami, Florida). Each sample was diluted in dichloromethane and analyzed with a detector at 90° angle. For each batch, mean diameter and size distribution were the mean of three measures. The effectiveness of the particle dispersion was verified performing the measurement after different sonication times ranging between 5 and 30 min. Results were expressed as d50 and SD. In all time intervals, good reproducibility of results was obtained. 2.2.2. Residual water content Water content was evaluated by Thermo gravimetric analysis (TGA, TG50 - Mettler Toledo, Columbus, OH, USA) by using about 15 mg of each powder batch, conducting experiment of each formulation at least in triplicate. Results were expressed as the mean of the obtained results for each batch. 2.2.3. Powders fluid uptake ability Powders fluid uptake ability was evaluated as the amount of liquid gained after gelation of each formulation; the ratio between the weight of the gel and the weight of the dried powder producing the gel, was obtained as reported in (Aquino et al., 2013). Briefly, 15 mg of dried powder was spread over a previously weighed filter paper disc. The disc was in contact with a vertical donor compartment of a Franz cell, filled with simulated wound fluid (SWF) consisting in 50% fetal calf serum (Sigma Aldrich, Milan, Italy) and 50% maximum recovery diluent (Sigma Aldrich, Milan, Italy, composed by 0.1% (w/v) peptone, peptic digest of animal tissue, and 0.9% (w/v) sodium chloride) (Bowler et al., 2012; Del Gaudio et al., 2014). SWF was thermostated at 37 °C and refilled during the experiment in order to maintain constant fluid volume into the donor. At regular time intervals, the weight of the gel was

2. Materials and methods ANXA1 N-terminal peptide Ac2-26 (1 mM) was purchased from Tocris Bioscience (Bristol, UK). Sodium alginate from brown algae (1% viscosity 65 mPa s; medium molecular weight 180 kDa, mannuronic/ guluronic ratio 70/30) was purchased from Carlo Erba reagents (Milan, Italy). Pectin Amid CF 025 D (pectin, amidated low methoxyl grade, degree of esterification 23–28%, degree of amidation 22–25%, molecular weight 120 kDa) have been kindly donated by Herbstreith&Fox (Werder/Havel, Germany). All other chemicals and reagents were obtained from Sigma Aldrich (Milan, Italy) and used as supplied.

2

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measured after removing the filter paper from donor and compared with the weight of the dry formulation, until equilibrium was reached. All experiments were performed at least in triplicate.

2.4. Peptide release studies Ac2-26 release assays were performed by means of slide-a-lyzer mini dialysis units, 10 K molecular weight cut off (Thermo Fisher Scientific Inc., Waltham, MA, USA) with an exposed surface area of 0.33 cm2. The dialysis cup was filled with 20 mg of powder and 250 μL of simulated wound fluid to promote the formation of the gel. The studies were conducted in a 5 mL receptor compartment filled with SWF thermostated at 37 °C and magnetically stirred at 200 rpm by a tefloncoated stirring bar. 100 μL aliquots of the receptor solution were sampled at specific time points and analysed by LC–MS for the determination of Ac2-26 permeated through the dialysis membrane, as previously described (see 2.3).

2.2.4. Water evaporation from hydrogel Water vapour transmission rate (WVTR) was obtained as described by ASTM standard (ASTM Standard, 2010). Briefly, 25 mm hydrogel disc, thickness about 2.2 mm, of each formulation was mounted on the top of a plastic tube containing 20 mL of distilled water. Teflon tape was used to cover the edge of the disc in order to avoid boundary loss. The assembly was kept inside an incubator at 37° ± 0.5 °C at a relative humidity of 32 ± 0.2%. Weight loss was recorded at regular time intervals and plotted against time. Water evaporation rate from in situ formed hydrogel was obtained as loss of weight over time by using the same procedure described above and noting the weight of the hydrogel at regular time intervals. Weight loss percentage was calculated by the following equation

Weight (%) =

Wt × 100 W0

2.5. Storage stability test Stability test of powders were performed following the ICH Q1AR2 C Guideline “Stability Testing of New Drug Substances and Products” by storing the powders at 40 °C and 75% relative humidity (RH) for 1 month. At defined time points, till 1 month, samples were recovered, and Ac2-26 content was determined (see 2.3).

(1)

where W0 is the initial weight and Wt is the weight at the specific time point. All experiments were performed at least in triplicate.

2.6. Cell viability Mitochondrial respiration, an indicator of cell viability, was assessed by the mitochondrial-dependent reduction of MTT (3,(4,5-dimethylthiazol-2)2,5 difeniltetrazolium bromide) to formazan, and cell viability was assessed according to the method of Mosmann (Mosmann, 1983). HaCaT cell line (Human immortalized keratinocytes) was purchased from CLS Cell Lines Service GmbH (Germany) and was maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) following the instructions reported in (Boukamp et al., 1988). The medium was supplemented with antibiotics (10,000 U/mL penicillin and 10 mg/mL streptomycin), cells were stained at 37 °C in 5% CO2 -95% air humidified atmosphere and were serially passed at 70–80% confluence. HaCaT cells were treated with particles made by alginate, pectin and alginate-pectin blend (0.01–10 μg/mL) in triplicate for 24 h. After the indicated treatment, 25 mL MTT was added and the cells were incubated for an additional 3 h. Thereafter, cells were lysed and the dark blue crystals solubilized with 100 mL of an aqueous solution containing 50% (v:v) N,N-dimethylformamide, 20% SDS, with an adjusted pH of 4.5. The optical density of each well was measured with a microplate reader (Titertek Multiskan MCC/340, LabSystem) equipped with a 550 nm filter. One Way ANOVA, followed by Bonferroni’s post test, were used for statistical analysis of the data.

2.2.5. Fourier Transform Infrared Spectroscopy (FTIR) studies Infrared analysis was performed using a FTIR spectrophotometer (IRAffinity-1S, Shimadzu Corporation, Kyoto, Japan) equipped with a MIRacle ATR accessory with ZnSe crystal plate. The samples were directly spread over the crystal plate and analysed using 256 scans with a 1 cm−1 resolution step. Each experiment was carried out in triplicate, and results averaged. 2.3. Peptide content and encapsulation efficiency LC/MS analyses were used to evaluate the loading in Ac2-26 of the different submicrometric powder formulations. Different samples of each batch were dissolved in water under vigorous stirring, about 6 min. 1 mL of each sample was pre-purified on a Chromabond HR-X SPE cartridge (Macherey-Nagel, Düren, Germany) using an elution solution made by water/acenotrile (30:70). LC/MS analyses were performed on a LTQ XL instrument (Thermo Fisher Scientific, MA, USA) equipped by an ESI ion source and a hybrid quadrupole/linear trap analyser and coupled with an Accela 600 HPLC system. Chromatography was performed on an Aeris C18 (2.0 × 150-millimeter i.d., 3 μm) reversed-phase column, using a mobile phases: A (0.1% formic acid in water, v/v) and B (0.1% formic acid in acetonitrile, v/v), using a linear gradient increase from 25 to 70% B in 30 min (De Falco et al., 2017). The flow rate was 200 μL/min. Mass spectra were acquired in positive ion mode over the m/z range from 700 to 1600. In order to quantify Ac2-26 in each analysed batch, ions at m/z 773.37 [M +4H]4+, 1030.82 [M+3H]3+, and 1545.73 [M+2H]2+were selected as specific Ac2-26 fragmentation markers. Peptidomimetic content was obtained as the ratio between actual Ac2-26 calculated weight and powder weight

Drug

Content (%) =

Ac 2 − 26 amount Powder Weight

x

100

2.7. Wound healing assay HaCaT cell line (Human immortalized keratinocytes) was purchased from CLS Cell Lines Service GmbH (Germany) and was maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) following the instructions reported in (Boukamp et al., 1988). The medium was supplemented with antibiotics (10,000 U/mL penicillin and 10 mg/mL streptomycin), cells were stained at 37 °C in 5% CO2 -95% air humidified atmosphere and were serially passed at 70–80% confluence. HaCaT cells were then seeded in the presence of a hydrogel bed formed by alginate or pectin or alginate-pectin or alginate-pectin-Ac2-26 loaded particles in growth medium as control in a 12-well plastic plate at 5 × 105 and 10 × 105 cells, respectively, per well. After 24 h incubation, cells reached 100% confluency and a wound was produced at the centre of the monolayer by gently scraping the cells with a sterile plastic p10 pipette tip. All experimental points were further treated with mitomycin C (10 μg/mL, Sigma Aldrich) to ensure the block of mitosis. The wounded cells were then incubated at 37 °C in a humidified and equilibrated (5% v/v CO2) incubation

(2)

while encapsulation efficiency (E.E.) was calculated as the ratio of actual to theoretical peptide content.

E . E . (%) =

Ac 2 − 26calculatedamount x100 Ac 2 − 26theoreticalamount

(3)

Each analysis was performed in triplicate and the results were expressed as mean ± standard deviation. 3

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responsible for the increase in stability of the Ac2-26 when encapsulated into the polymer blend matrix. Water content of the particles was mainly related to the total feed concentration, ranging between 3.5 and 5.6% (w/w), due to the increasing in surface tension of the solution coming out from the vibrating membrane (Xu et al., 2012). FT-IR studies were conducted in order to verify the presence of such interaction in the loaded particles. As shown in Fig. 2a–b, blank pectin particles presented amide I band at 1680 cm−1 and at 1592 cm−1 the amide II band, while COO stretching was observed at 1410 cm−1; blank alginate particles presented two characteristics bands at 1613 cm−1 and 1418 belonging to the antisymmetric and symmetric stretching vibration of alginate carboxyl group. Blank alginate-pectin particles presented a shift in both carboxyl peaks and vibration bands related to amide bonds (see Fig. 2c) due to the formation of hydrogen bonding (Sinitsya, Čopı́ková, Prutyanov, Skoblya, & Machovič, 2000) during the spray drying process. In fact, in the amide I band was shifted at 1675 cm−1 while the band belonging to amide II was overlapped with the shifted antisymmetric carboxyl band shifted at 1602 cm−1; symmetric stretching vibration of carboxyl group was also shifted at1412 cm−1. As shown in Fig. 2d, Ac2-26 loaded alginate-pectin particles exhibited also a characteristic band at 1547 cm−1, probably due to a partial shifting of the alginate carboxyl groups confirming the presence of hydrogen bonding between Ac2-26 and some alginate chains. In order to evaluate stability of Ac2-26 loaded particles, quantitative LC/MS analyses were performed at different time points, measuring the amount of recovered peptide loaded into the stored formulations, till one month. Reduction of encapsulated Ac2-26 was very low for both NAP_31s and NAP_11s formulations, less than 5% after one month, regardless the amount of loaded peptide, whereas in case of NAP_13s formulations a reduction around 13% was found after one month (about 8% after 15 days). Such results suggest that when the amidated pectin is not prevailing in the polymeric blend some of the alginate carboxyl groups are still able to interact with the peptide via hydrogen

chamber of an Integrated Live Cell Workstation Leica AF-6000 LX. A 10x phase contrast objective was used to record cell movements with a frequency of acquisition of 10 min. The migration rate of individual cells was determined by measuring the wound closure from the initial time to the selected time-points (bar of distance tool, Leica ASF software). For each wound ten different positions were registered, and for each position ten different cells were randomly selected to measure the migration distances. 3. Results and discussion 3.1. Preparation and characterization of the peptide loaded particles Polysaccharides blend particles loaded with different amount of Ac2-26 were produced by Nano Spray Dryer B-90 HP processing different aqueous alginate/pectin feed solutions with total concentration of the polymers set at 0.25, 0.50% and 1.00% (w/v). Preliminary experiments allowed to set proper operating spray conditions in order to obtain powders made by submicrometric particles avoiding aggregates or particle clusters into the produced powers. Process yield ranged from 68% to 85%, depending on alginate/pectin ratio and feed total concentration, the higher the concentration the higher the yield. Feed concentration also influenced particle size, as well as, particle size distribution with smaller particles obtained when lower concentrated feeds were processed due to the decreasing in surface tension of the droplets coming out from the vibrating membrane (Del Gaudio et al., 2017; Xu, Howes, Adhikari, & Bhandari, 2012). On the contrary, the presence of the peptide cargo had no significant effect on size and morphology of the particles, as shown in Table 1 and Figure 1. Good encapsulation efficiency (E.E.) was obtained for all formulations, between 77 and 89%, depending on the relative amount of alginate into the feed. This behaviour might be ascribable to an interaction between Ac2-26 and free alginate carboxyl groups via hydrogen bonding (Del Gaudio et al., 2015). Such interaction could be also

Table 1 Composition, process yield, particle size, drug content and encapsulation efficiency (E.E.) of Ac2-26 loaded polysaccharides blend powder formulations obtained by nano spray drying. Sample code

Polymers blend concentration % (w/V)

NAP_31-25 NAP_31-25_Ac1 NAP_31-25_Ac2 NAP_11-25 NAP_11-25_Ac1 NAP_11-25_Ac2 NAP_13-25 NAP_13-25_Ac1 NAP_13-25_Ac2

0.25

NAP_31-50 NAP_31-50_Ac1 NAP_31-50_Ac2 NAP_11-50 NAP_11-50_Ac1 NAP_11-50_Ac2 NAP_13-50 NAP_13-50_Ac1 NAP_13-50_Ac2

0.50

NAP_31-100 NAP_31-100_Ac1 NAP_31-100_Ac2 NAP_11-100 NAP_11-100_Ac1 NAP_11-100_Ac2 NAP_13-100 NAP_13-100_Ac1 NAP_13-100_Ac2

1.00

a

Alginate Pectin ratio 3:1

1:1

1:3

3:1

1:1

1:3

3:1

1:1

1:3

Ac2-26 polymers ratio

Yield (%)

mean diameter (nm) ± S.D.

drug content (%) ± SD

E.E. (%) ± SD

drug content (%) ± SDa

– 0.01 0.02 – 0.01 0.02 – 0.01 0.02

73.9 73.6 71.9 72.0 70.2 71.3 69.2 67.9 68.8

± ± ± ± ± ± ± ± ±

1.8 2.8 3.2 1.7 2.2 1.9 1.9 1.7 2.4

583 569 593 611 599 583 595 582 605

± ± ± ± ± ± ± ± ±

62 71 58 72 58 92 81 84 74

n.d. 0.82 1.73 n.d. 0.72 1.62 n.d. 0.71 1.69

n.d. 86 ± 88 ± n.d. 78 ± 78 ± n.d. 79 ± 77 ±

n.d. 0.78 1.64 n.d. 0.69 1.52 n.d. 0.63 1.48

– 0.01 0.02 – 0.01 0.02 – 0.01 0.02

75.8 76.4 74.9 71.4 72.8 71.9 72.9 73.4 72.8

± ± ± ± ± ± ± ± ±

1.9 2.7 2.1 3.0 2.3 2.9 2.6 2.5 3.1

656 684 678 667 689 674 691 704 686

± ± ± ± ± ± ± ± ±

92 86 106 94 90 88 96 106 97

n.d. 0.82 1.46 n.d. 0.76 1.53 n.d. 0.72 1.73

– 0.01 0.02 – 0.01 0.02 – 0.01 0.02

84.9 85.3 85.7 82.6 81.8 83.5 80.8 82.1 81.4

± ± ± ± ± ± ± ± ±

2.1 2.2 1.9 2.4 1.8 2.3 2.2 2.6 1.8

822 769 831 814 882 834 855 876 865

± ± ± ± ± ± ± ± ±

112 98 89 106 114 91 93 104 94

n.d. 0.79 1.72 n.d. 0.73 1.54 n.d. 0.72 1.50

Values registered after 1 months in accelerated storage conditions. 4

± 0.04 ± 0.03 ± 0.04 ± 0.05 ± 0.03 ± 0.04 ± 0.04 ± 0.04 ± 0.05 ± 0.06 ± 0.04 ± 0.06 ± 0.05 ± 0.07 ± 0.04 ± 0.07 ± 0.06 ± 0.07

n.d. 84 ± 86 ± n.d. 80 ± 80 ± n.d. 77 ± 78 ± n.d. 85 ± 89 ± n.d. 80 ± 81 ± n.d. 78 ± 79 ±

5 4 4 5 3 4 5 3 2 4 5 4 5 6 4 6 5 6

n.d. 0.78 1.39 n.d. 0.73 1.45 n.d. 0.63 1.52 n.d. 0.75 1.63 n.d. 0.69 1.46 n.d. 0.63 1.33

± 0.05 ± 0.06 ± 0.05 ± 0.07 ± 0.05 ± 0.07 ± 0.06 ± 0.07 ± 0.06 ± 0.08 ± 0.03 ± 0.08 ± 0.05 ± 0.06 ± 0.05 ± 0.08 ± 0.04 ± 0.08

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Fig. 1. SEM microphotographs of alginate-pectin powders produced by nanospray drying with different feed concentrations. Blank submicroparticles: NAP_31-25 (a), NAP_31-50 (b) and NAP_31-100 (c); Ac2-26 loaded submicroparticles: NAP_31-25_Ac2 (d), NAP_31-50_Ac2 (e) and NAP_31-100_Ac2 (f).

Fig. 2. FTIR spectra of alginate/pectin Ac2-26 loaded particles in comparison with blank alginate, pectin and alginate/pectin particles, NAP_31) and pure peptide. Blank particles: amidated pectin (a), alginate particles (b), alginate/pectin particles, (c), NAP_31-50_Ac2 (d) and 0.01% (w/V) Ac2-26 solution (e).

after removal of SWF. All gels demonstrated an enhanced fluid loss within 12 h, between 53% and 34%, depending on the relative amount of pectin; the higher the pectin, the lower the loss. However, after 24 h all formulations were able to retain at least 25% of the incorporated fluid, about 40% in case of powders produced with high amount of pectin probably due to the higher pectin water affinity (Yamamoto, Saeki, & Inoshita, 2002) demonstrating the ability of the powders to remain gel even when no more fluid is supplied by wound. As for fluid uptake, Ac2-26 loading, as well as concentration of the feed, did not influence significatively either uptake or loss rate of any loaded formulations (data not shown).

bonding enabling the polysaccharides blend matrix to stabilize Ac2-26 even in harsh storage conditions for, at least, one month, whereas pure peptide must be stored at −20 °C in order to prevent its degradation (Qin et al., 2019). Fluid uptake studies were conducted in order to evaluate the ability of the powders to move to a hydrogel when spread on a wound bed, using a simulated wound fluid (SWF) to mimic in vitro the wound environment. Fig. 3 reports fluid uptake of the particles prepared with different alginate/pectin ratio. All powders exhibited fast fluid uptake moving to their maximum swelling between 15 and 20 min. As expected, particles made with higher amount of amidated pectin were able to gel faster and uptake higher amount of SWF (Capel, Nicolai, Durand, Boulenguer, & Langendorff, 2006). After maximum swelling was reached all powders exhibited an equilibrium phase with partial loss of water due to the diffusion and rearrangement of the gel elastic network (Azevedo & Kumar, 2012). In situ formed hydrogel fluid loss was evaluated in order to understand the ability of the formulations to retain water when advanced healing state would not provide high amount of fluids to the gel. Fluid loss was evaluated as the weight decrease of the different formulations

3.2. In vitro peptide release studies and wound healing tests Ac2-26 release behaviour was monitored using vertical Franz-type diffusion cells. A slide-a-lyzer mini dialysis units, 10 K molecular weight cut off was used as loading chamber for the powder and inserted into acceptor compartment. Fig. 4 reports the representative release curves of AC2-26 loaded formulations obtained with alginate/pectin ratio. All formulations exhibited burst effect within 3 h related to gel formation 5

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Fig. 3. Simulated wound fluid uptake (left) and loss (right) of blank alginate/pectin submicrometric powder formulations prepared by nanospray drying. Mean ± SD; (n = 6).

Mimura, Araujo, Greco, & Oliani, 2016). Total release of Ac2-26 was achieved between 24 and 36 h in SWF, not depending on the peptide load, while still depending on alginate pectin ratio that determined the resistance of the gel, the higher the alginate the faster the release. In fact, NAP_31-50_Acs formulations released 95% of its load in 24 h, while NAP_11-50_Acs and NAP_13-50_Acs released the 88% and 82% of the loaded peptide in 30 and 48 h, respectively. Total release of the peptide dose was never achieved due to the interaction between alginate carboxyl residues and Ac2-26 via hydrogen bonding, as reported in FTIR analyses, and partial degradation of the peptide in SWF after its release. Previous studies have been demonstrated the activity of Ac2-26 to significantly improve the healing of different wounds (Bizzarro, Belvedere, Dal Piaz, Parente, & Petrella, 2012; Leoni et al., 2015) while high M content alginate has been widely applied as dressing material and drug carrier in many formulations demonstrating its ability to improve wound healing process (Del Gaudio et al., 2013; Draget & Taylor, 2011; Jones, Grey, & Harding, 2006). One of the main problems related to the use of Ac2-26, as many other peptides, is their instability. Therefore, Ac2-26 loaded polysaccharides blend particles should significatively improve its stability over time while its association with high M content alginate might have synergistic effect in healing process. Powders are intended to be administered directly to the wound. Therefore, in vitro cytotoxic activity studies of the particles made by alginate, pectin and alginate-pectin blend, produced at same operating conditions, have been conducted in order to asses safety of the formulations. The activity of the powders in inhibiting cell proliferation

Fig. 4. Release profiles of Ac2-26 loaded alginate pectin submicrometric par), ticles produced with different polymer blend ratio: NAP_31-50Ac2 ( ), NAP_13-50_Ac2 ( ). Mean ± SD; (n = 6). NAP_11-50_Ac2 (

rate and gel texture. In fact, slower gel formation (NAP_31s formulations) resulted in the release of about 50% of their load during burst effect, whereas powders with faster gelling rate, due to the presence of higher amount of amidated pectin producing a tougher gel, NAP_11s and NAP_13s, released 42% and 37% of the loaded peptide, respectively. Such release of the peptide in the first hours after administration, followed by a prolonged release, can be very useful to boost the healing process that could benefit of the strong anti-inflammatory and promigratory effects related to high concentration of Ac2-26 (Teixeira,

Fig. 5. Cell viability assessed by MTT test on HaCaT cells after 24 h treatment with naospray dried submicrometric particles, made by (A) alginate, (B) pectin, (C) representative of alginatepectin blends. Data are represented as median ± interquartile range (n = 7). Statistically significant differences were determined by one-way ANOVA followed by Bonferroni’s multiple comparison post-test.

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Fig. 6. Results of wound healing assay on HaCaT cells. Panel A: representative bright fields of wound healing assay at 0 and 24 h after the creation of the lesion on cell monolayer. Scale Bar 100 μm; Panel B: the migration rate related to Ctrl (not treated cells), Alginate, Pectin, NAP_11-50 (blank alginate/pectin particles) and NAP_11-50_Ac2 (Ac2-26 loaded particles) of cells was determined by measuring the wound closure by individual cells from the initial time to the selected time-points (bar of distance tool, Leica ASF software). The data are representative of 3 independent experiments ± SD. * p < 0.05; ** p < 0.01.

topical application of the alginate/pectin particles. These promising results suggest that submicrometric alginate/pectin particles loaded with the peptide Ac2-26 might have potential application as dressing for wound healing processes and could represent a new weapon to improve wound care armamentarium.

was evaluated by MTT assay against human immortalized keratinocytes (HaCaT) cell line. HaCaT cells after 24 h treatment with particles of polymers alone or by alginate-pectin blend particles did not exhibit any significant reduction in in the optical density (OD) at 550 nm, except for pectin particle at 10 μg/mL concentration, demonstrating that in the range of 0.01–10 μg/mL NAP particles had no cytotoxic effect, compared to the nanopaticulate powder made by pectin that demonstrated such effect at higher concentration, as shown in Fig. 5. In vitro wound healing tests were conducted to evaluate the ability to promote the wound repair process in HaCaT cells used as model to test the efficacy of the powder formulations. Fig. 6 shows the wound reduction after 24 h. As expected, alginate particles were found to improve cell migration (Lee & Mooney, 2012; Park, Lee, An, & Lee, 2017) leading to an almost complete closure of the wound (44% higher than control) whereas the treatment with pure amidated pectin particles was comparable to the control. Blank alginate-pectin particles were less efficient than pure alginate (8%) in promote wound closure; on the contrary, the powders containing the Ac2-26, even lowest amount, were able to improve migration leading to wound complete closure after 24 h, 79% and 25% better than control and pure alginate, respectively.

Acknowledgments This work was supported by a grant from Regione Campania (I) POR CampaniaFESR 2014/2020 “Fighting Cancer resistance: Multidisciplinary integrated Platform for a technological Innovative Approach to Oncotherapies (Campania Oncotherapies)” [Project N. B61G18000470007]. References Aldag, C., Teixeira, D. N., & Leventhal, P. S. (2016). Skin rejuvenation using cosmetic products containing growth factors, cytokines, and matrikines: A review of the literature. Clinical, Cosmetic and Investigational Dermatology, 9, 411. Aquino, R. P., Auriemma, G., Mencherini, T., Russo, P., Porta, A., Adami, R., et al. (2013). Design and production of gentamicin/dextrans microparticles by supercritical assisted atomisation for the treatment of wound bacterial infections. International Journal of Pharmaceutics, 440(2), 188–194. ASTM Standard (2010). ASTM Standard E96/E96M - 10. Standard test methods for water vapour transmission of materials. Philadelphia: ASTM. Azevedo, E. P., & Kumar, V. (2012). Rheological, water uptake and controlled release properties of a novel self-gelling aldehyde functionalized chitosan. Carbohydrate Polymers, 90(2), 894–900. Bizzarro, V., Belvedere, R., Dal Piaz, F., Parente, L., & Petrella, A. (2012). Annexin A1 induces skeletal muscle cell migration acting through formyl peptide receptors. PLoS One, 7(10), e48246. Bizzarro, V., Fontanella, B., Carratu, A., Belvedere, R., Marfella, R., Parente, L., et al. (2012). Annexin A1 N-terminal derived peptide Ac2-26 stimulates fibroblast migration in high glucose conditions. PLoS One, 7(9), e45639. Bodnar, R. J. (2013). Epidermal growth factor and epidermal growth factor receptor: The yin and yang in the treatment of cutaneous wounds and cancer. Advances in Wound Care, 2(1), 24–29. Boukamp, P., Petrussevska, R. T., Breitkreutz, D., Hornung, J., Markham, A., & Fusenig, N. E. (1988). Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. Journal of Cell Biology, 106(3), 761–771. Bowler, P. G., Welsby, S., Towers, V., Booth, R., Hogarth, A., Rowlands, V., et al. (2012). Multidrug-resistant organisms, wounds and topical antimicrobial protection. Journal of Cell Biology, 9(4), 387–396. Brannon-Peppas, L., & Blanchette, J. O. (2012). Nanoparticle and targeted systems for cancer therapy. Advanced Drug Delivery Reviews, 64, 206–212. Capel, F., Nicolai, T., Durand, D., Boulenguer, P., & Langendorff, V. (2006). Calcium and acid induced gelation of (amidated) low methoxyl pectin. Food Hydrocolloids, 20(6),

4. Conclusions Nano spray drying technology has been successfully used to produce stable submicrometric powders loaded with ANXA1 derived peptide AC2-26 able to move rapidly to gel (about 10 min) when in contact with wound fluids. Ac2-26 encapsulation efficiency was very high, till 83%, even after 1 month in accelerated storage conditions demonstrating the ability of the polymeric blend to stabilize the peptide. Release in SWF of encapsulated Ac2-26 from the gel exhibited a burst effect, within 3 h, followed by a prolonged release (between 24 and 48 h, depending on alginate/pectin ratio) useful to boost the healing process via strong antiinflammatory and pro-migratory effects related to high concentration of Ac2-26. FTIR studies suggested an extensive hydrogen bonding binding between the negatively charged portion of the peptide and free alginate carboxyl groups of the polysaccharidic blend able to both stabilize and control the release of Ac2-26 from the in situ formed hydrogel matrix. In vitro wound healing tests on HaCaT cells showed an acceleration of wound closure for Ac2-26 loaded formulations compared with the 7

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