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ERROR: type should be string, got "https://doi.org/10.1007/s13346-022-01257-9\nDrug Delivery and Translational Research (2023) 13:1343–1357 https://doi.org/10.1007/s13346-022-01257-9\nDrug Delivery and Translational Research (2023) 13:1343–1357 ORIGINAL ARTICLE ORIGINAL ARTICLE Abstract This work combines natural polymers with nanoemulsions (NEs) to formulate nanocomposites as an innovative wound dress-\ning. Spray-drying has been used to produce alginate-pectin in situ gelling powders as carriers for NEs loaded with curcumin \n(CCM), a model antimicrobial drug. The influence of NEs encapsulation in polymer-based microparticles was studied in \nterms of particle size distribution, morphology, and stability after spray-drying. NEs loading did not affect the size of micro-\nparticles which was around 3.5 µm, while the shape and surface morphology analyzed using scanning electron microscope \n(SEM) changed from irregular to spherical. Nanocomposites as dried powders were able to form a gel in less than 5 min \nwhen in contact with simulated wound fluid (SWF), while the value of moisture transmission of the in situ formed hydrogels \nallowed to promote good wound transpiration. Moreover, rheologic analyses showed that in situ formed gels loaded with NEs \nappeared more elastic than blank formulations. The in situ formed gel allowed the prolonged release of CCM-loaded NEs in \nthe wound bed, reaching 100% in 24 h. Finally, powders cytocompatibility was confirmed by incubation with keratinocyte \ncells (HaCaT), proving that such nanocomposites can be considered a potential candidate for wound dressings. Keywords  Nanocomposite · Nanoemulsion · Alginate · Pectin · In situ gelling powder · Wound healing Chiara Amante1 · Valentina Andretto2 · Annalisa Rosso2 · Geraldine Augusti2 · Stefania Marzocco1 · Giovanna Lollo2 · \nPasquale Del Gaudio1 Accepted: 1 November 2022 \n© The Author(s) 2022\n/ Published online: 13 December 2022 Introduction Oleoyl polyoxyl-6 glycerides ­(Labrafil®M1944CS) was \nsupplied by Gattefossé (Saint-Priest, France). Sodium alginate \nKelton LVCR from brown algae (1% viscosity 35 mPa s; man-\nnuronic/guluronic ratio 70/30) was kindly donated by Dompè \nS.p.A (L'Aquila, Italy). Pectin Amid CF 025 D (amidated low \nmethoxyl grade, degree of esterification 23–28%, degree of ami-\ndation 22–25%, molecular weight 120 kDa) was kindly offered \nby Herbstreith & Fox (Werder/Havel, Germany). Sodium chlo-\nride (NaCl) and phosphate buffered saline (PBS) tablets (pH 7.4) \nwere acquired from VWR International (Fontenay-sous-Bois, \nFrance). Mycological peptone was purchased from Oxoid Ltd, \nBasingstoke, Hants, UK) and fetal bovine serum (FBS), quali-\nfied, heat inactivated from Gibco (Thermo Fischer Scientific, Bra-\nzil). Penicillin (10,000 U/mL) and 10 mg/mL streptomycin and \nDulbecco’s Modified Eagle’s Medium (DMEM) were acquired \nfrom Euroclone (Euroclone, Milan, Italy). 3-(4,5-dimethylthiazol-\n2-yl)-2,5-diphenyltetrazolium (MTT) and DMSO were bought \nfrom Sigma (Sigma Aldrich, Milan, Italy). Dichloromethane, \nmethanol, ethanol, acetonitrile (HPLC grade), formic acid, and \nsodium taurocholate hydrate 96% were purchased from Thermo \nFisher Scientific (Illkirch, France). Milli-Q® water was obtained \nusing a Milli-Q® Academic System from Merck-Millipore (Saint-\nQuentin-en-Yvelines, France). such as polymeric scaffold is necessary [13]. Hydrogel-based \nnanocomposites are one of the most promising agents for \nwound healing thanks to their ability to favor skin adhesion \nmaintaining a moist and cool environment [14]. Therefore, this work focused on the design of nanocompos-\nites for direct wound application to deliver lipophilic drugs. To \nthis aim alginate-pectin in situ gelling powders were embedded \nwith curcumin-loaded NEs. In a previous work, we demonstrated \nthat alginate with high mannuronic content and amidated pectin \nwith a low degree of methylation were forming powders able to \nquickly gelify when in contact with wound fluids [15]. Sodium \nalginate with a high mannuronic content (over 65%) has been \nselected for its ability to form a soft and flexible hydrophilic gel \nthrough Ca/Na ionic exchange when in contact with the wound \nexudates and because it induces cytokine production by human \nmonocytes, useful in the wound healing process [16]. To enhance \nthe in situ gel-forming rate, low methoxyl grade pectin that geli-\nfies ionotropically when in contact with bivalent cations present \nin the exudate has been associated with sodium alginate [17]. g\n[\n]\nAs a model drug for wound healing purposes, curcumin \n(CCM), 1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-\n2,5-dione, has been selected as a model hydrophobic drug. Introduction controlled release at the wound site [2–4]. The combination \nof different materials into a single formulation can enhance \nthe specific properties of the single components leading to \nunique physicochemical and biological characteristics that \nthe separate systems cannot achieve individually [5].f The recent advancements in nanotechnology have provided \nsuitable strategies to overcome most of the inconveniences \nassociated with conventional dressings, such as bacterial \ninfections, while offering cell-type specificity [1]. In par-\nticular, nanosystems have been combined with macro- and \nmicrostructures to develop nanocomposites aimed at increas-\ning the stability of the encapsulated drug and promoting Different studies have reported the preparation of nano-\ncomposites loaded with antibiotics (gentamicin and rifa-\nmycin), anti-inflammatory agents, or other biomolecules, \nto increase drug residence time in the wound sites, thus \nenhancing their activity [6, 7]. Also, composites containing \ngelatin, hyaluronic acid, and chondroitin sulfate associated \nwith asiatic acid, ZnO, and CuO nanoparticles have been \ndescribed as effective wound dressing scaffolds for second-\ndegree burn wounds [8]. Giovanna Lollo and Pasquale Del Gaudio contributed equally to this \nwork. *\t Giovanna Lollo \n\t\ngiovanna.lollo@univ-lyon1.fr\n *\t Pasquale Del Gaudio \n\t\npdelgaudio@unisa.it\n1\t\nDepartment of Pharmacy, University of Salerno, Via \nGiovanni Paolo II, 132, 84084 Fisciano, SA, Italy\n2\t\nUniversity of Lyon, Université Claude Bernard Lyon 1, \nCNRS, LAGEPP UMR 5007, 43 Bd 11 Novembre 1918, \n69622 Villeurbanne, France Giovanna Lollo and Pasquale Del Gaudio contributed equally to this \nwork. *\t Giovanna Lollo \n\t\ngiovanna.lollo@univ-lyon1.fr\n *\t Pasquale Del Gaudio \n\t\npdelgaudio@unisa.it\n1\t\nDepartment of Pharmacy, University of Salerno, Via \nGiovanni Paolo II, 132, 84084 Fisciano, SA, Italy\n2\t\nUniversity of Lyon, Université Claude Bernard Lyon 1, \nCNRS, LAGEPP UMR 5007, 43 Bd 11 Novembre 1918, \n69622 Villeurbanne, France Giovanna Lollo and Pasquale Del Gaudio contributed equally to this \nwork. Giovanna Lollo and Pasquale Del Gaudio contributed equally to this \nwork. Along with nanocomposites, nanoemulsions (NEs) com-\nposed of an oily core stabilized by a surfactant shell [4, 9] have \nbeen described for wound healing applications [10]. NEs incor-\nporating poorly water-soluble drugs in the oil droplet phase can \nbe used to deliver lipophilic compounds to the superficial stra-\ntum corneum after topical application [11, 12]. However, since \nthe viscosity of these systems is not high enough for a direct \nskin application, their encapsulation in a secondary structure (0121 3456789)\n3 Drug Delivery and Translational Research (2023) 13:1343–1357 1344 Polyoxyethylene (40) stearate ­(Myrj®52) and curcumin (CCM) \nwere acquired from Sigma-Aldrich (St. Quentin-Fallavier, \nFrance). Introduction CCM \nis a yellow phenolic pigment obtained from the rhizomes of tur-\nmeric (Curcuma longa), a tropical plant native to Southern Asia \n[18]. CCM, interfering with the inflammatory, proliferative, \nand remodeling phases and preventing oxidative damage, plays \na great role in the treatment of wounds [19]. Moreover, recent \nstudies showed that CCM possesses antibacterial activity against \na broad range of microorganisms, including common bacteria \nthat are detected in wounds: Staphylococcus aureus, Escheri-\nchia coli, and Pseudomonas aeruginosa [20]. Its bacteriostatic \nactivity is linked to the interference with cellular processes by \ntargeting DNA and proteins, cell wall and membrane damages, \nand inhibition of bacterial quorum sensing [21]. Here, nano-\ncomposites were produced by mini spray-drying, and the influ-\nence of the addition of CCM-loaded NEs to the alginate-pectin \npowders was studied. Morphological analysis and particle size \ndistribution, thermal behavior, fluid uptake ability, water vapor \ntransmission rate, and viscoelastic properties of the powders \nwere assessed. In vitro studies on the spontaneously immortal-\nized human keratinocytes and cell line derived from adult skin \n(HaCat) have been performed to study the biocompatibility of \nselected powders. Preparation of curcumin‑loaded nanoemulsions Nanoemulsions (NEs) were prepared by emulsion phase \ninversion technique coupled with high stirring energy input \nas previously described [4]. Briefly, NEs composed of an oil \ncore of medium chain triglycerides (MCT) were stabilized \nby a shell of surfactants, made of a mixture of polyoxyeth-\nylene (40) stearate ­(Myrj®52) and oleoyl polyoxyl-6 glyc-\nerides ­(Labrafil®M1944CS), hydrophilic and hydrophobic \nsurfactants, respectively. To prepare the oil phase, the MCT \noil core and surfactants were homogenized under magnetic \nstirring (750 rpm) using a thermostated bath at 80 °C. Then, \nthe aqueous phase (PBS 5 mM pH 7.4), heated up to 80 °C, \nwas added into the organic melt phase. The process of high \nstirring was then performed in two cycles of 10 min using a \nrotor–stator disperser (T25 digital Ultra-Turrax® equipped \nwith an S25N10G shaft, ­IKA®-Werke GmbH & Co. KG, \nStaufen, Germany) rotating at 11,000 rpm at 80 °C. The \nresulting colloidal system was cooled to room temperature \nunder magnetic stirring for 30 min. CCM was added to the \noil phase during NEs preparation. Nanoemulsions were pre-\npared with a lipid concentration in the final phase of 142.86 \nw/v with 1.33% (w/w) of curcumin. Materials Medium-chain triglycerides, MCT ­(Miglyol®812), was purchased \nfrom Cremer Oleo GmbH & Co. KG (Hamburg, Germany). All studies and measurements presented in this work were \nperformed in triplicate. 1 3 1345 Drug Delivery and Translational Research (2023) 13:1343–1357 Static light scattering Particle size distribution and mean diameter of powders pro-\nduced by spray-drying were evaluated by Static Light Scatter-\ning Coulter LS 13,320 (Beckman Coulter, Inc., Fullerton, CA, \nUSA). About 6 mg of AP-11, AP-11-NE0.1, and AP-11-NE0.2 \nwas diluted in 6 mL of dichloromethane (DCM) and soni-\ncated three times for 10 min. After sonication, some drops of \neach formulation were placed in DCM under constant stirring, \nusing the micro liquid module making the average of three \nmeasurements for the sample. Results, calculated by instru-\nment software using the Fraunhofer model, were expressed as \nmean diameter, and to evaluate the width of particle distribu-\ntion span, Eq. 1 was used: (1)\nSpan value = d90 −d10\nd50 (1) The spray-dried powders were recovered and kept in \nclosed vials to avoid moisture absorption. The process yield \nwas calculated as the ratio between the amount of powders \nobtained and the total amount of processed material. where d10, d50, and d90 indicate the volume diameters at \n­10th, ­50th, and ­90th percentiles, respectively. All powders were produced in triplicate. All powders were produced in triplicate. Size distribution and surface potential \nof nanoemulsions and nanocomposites Powder fluid uptake studies were conducted to evaluate the \nbehavior of the powders when in contact with simulated wound \nfluid (SWF) containing 50% of FBS and 50% diluent com-\nposed of 0.1% (w/v) peptone, a peptic digest of animal tissue, \nand 0.9% (w/v) sodium chloride [22]. For fluid uptake ability \nstudies conducted on dry powders, a Franz-type diffusion cell \nwas used in an open configuration, without the donor chamber, \nwith a total volume of 5 mL and a permeation area of 0.6 ­cm2. The size distribution and surface potential of the NEs were \nstudied using the Malvern ­Zetasizer® Nano ZS instrument \n(Malvern Instruments S.A., Worcestershire, UK). Particle \nsize and polydispersity index (PDI) were determined by \ndynamic light scattering (DLS) diluting all samples with \nMilli-Q water to ensure the correct calculation of size distri-\nbution. Analyses were carried out at 25 °C with an angle of \ndetection of 173°. The ζ–potential was calculated from the \nmean electrophoretic mobility measured for samples diluted \nin NaCl 1 mM. Measurements were performed in triplicate. The stability of NEs was followed for 28 days upon storage \nat 4 °C, measuring, at regular time points, particle size, PDI, \nand ζ–potential. p\nAbout 8 mg of dried powder weighed on a microbalance \n(MTS Mettler Toledo, USA) was spread over a previously \nweighed HVLP nitrocellulose membrane (0.45 µm, Merck \nMillipore, Darmstadt, Germany). The membrane was in con-\ntact with a Franz cell (Hanson Research, USA), filled with \nSWF thermostated at 37 °C under stirring (200 rpm). At \nscheduled time intervals, the membrane with the sample was \nweighted, to establish the amount of fluid absorbed by the dry \npowder, and the Franz cell was refilled to maintain constant \nthe volume of fluid during the entire experiment. Fluid uptake \nwas calculated as the ratio between the weight of the gel and \nthe weight of the dried powder producing the gel, using the \nfollowing equation [23]: Preparation of alginate‑pectin powders \nand nanocomposites powders 250 FEG microscope with 10-kV accelerating voltage. Before \nmicroscopy, the powders were deposited on a flat steel holder \nand coated under a vacuum by cathodic sputtering with copper \n(10-nm layer). To produce alginate-pectin (AP-11) powders and nanocom-\nposites made of alginate-pectin with CCM-loaded NEs (AP- \n11-NE), mini spray-dryer (Mini Spray Dryer Büchi B 290, \nBüchi, Rungis, France) was used. AP solution was pre-\npared by dissolving both polymers in Milli-Q under vig-\norous stirring for 1 h (500 rpm). The total concentration \nof polymers was set at 1% (w/v), and an alginate-pectin \nmass ratio of 1:1 was used. For the preparation of nano-\ncomposites, AP-11-NE0.1, and AP-11-NE0.2, two different \nconcentrations of NEs were added, to obtain a final concen-\ntration of 0.1% and 0.2% w/w, to the AP solution under a \nslight stirring (250 rpm) for 15 min before the spray-drying \nprocess. All formulations were processed with optimized \nparameters: aspirator 100%, drying airflow 560–580 L/h, \nair pressure 6 atmospheres, feed rate 3 mL/min, 120 °C \ninlet temperature, 65–68 °C outlet temperature, and nozzle \ndiameter 0.7 mm. Rheological measurements Tetrafluoroethylene was then used to cover the edge of \nthe hydrogel disks to avoid boundary loss [24]. This system \nwas kept in an incubator at 37 ± 0.5 °C with a humidity of \n32 ± 0.2%. At defined time points, weight loss was noted and \nplotted against time. The rheological properties of in situ gelled powders were \nevaluated through a MCR 302 rheometer (Anton Paar, Les \nUlis, France) fitted with a 25 mm plate-plate geometry \n(PP25 with a diameter of 24.985 mm). Each powder was \ntreated with 1 mL of SWF to form a gel in around 2 min. The \ndistance between the plates was set at 0.5 mm, and the sys-\ntem was heated at 37 °C. Amplitude sweep tests were per-\nformed setting strain amplitude in the range of 0.01–200% \nand angular frequency at ω = 10 rad/s. WVTR was calculated as the ratio between the slope of the \nplot and the area of the disk normalized respect the amount \nof AP when in contact with SWF, by the following formula: (3)\nWVTR = Slope\nA (3) Curcumin‑loaded nanoemulsions: drug loading \nand encapsulation efficiency where A is the area of the sample in ­m2. Water evaporation rate from in situ formed hydrogel was \nobtained as loss of weight over time by using the same pro-\ncedure described above. After regular intervals, the weight \nwas noted. The weight remaining was calculated by the fol-\nlowing equation: The amount of CCM loaded in the NEs was quantified by \nUHPLC (ultrahigh-performance liquid chromatography) \nequipped with a PDA detector using the method reported in \nthe literature [25, 26]. Pure CCM for the calibration curve \nand NEs were dissolved in methanol/acetonitrile (50:50). AP-\n11-NE powders were solubilized in methanol/acetonitrile and \nmaintained under a stirring for 2 h to allow the complete \ndissolution of CCM from formulations. All the samples were \nvortexed for 5 min and filtered using a nylon filter of 0.22 μm \n(Whatman GmbH, Dassel, Germany) before injection in the \nUHPLC system. The UHPLC apparatus consisted of UHPLC \nWaters Acquity Arc Quaternary Solvent Manager-R and \nWaters Acquity Arc Sample Manager FTN-R, equipped \nwith Waters Acquity UHPLC 2998 PDA Detector. CCM \nwas detected using an RPC18 column (Kinetex 5 μm C18 \n100 Å, 150 × 4.6 mm, Phenomenex, Torrance, CA, USA), set \nat 30 °C, using acetonitrile and deionized water 0.1% formic \nacid (50:50) as mobile phase at a flow rate of 1 mL/min. The injection volume was 10 μL, the detection wavelength \nwas 423 nm, and the total run time was 7 min. The chroma-\ntogram of CCM exhibited a characteristic peak at 4.7 min. The UHPLC calibration curve was linear (R2 = 0.999) in the \nconcentration range of 0.04–40 μg/mL. The method was vali-\ndated according to ICH Q2(R1) guidelines. Detection and \nquantification limits (LOD and LOQ) were 0.00120 μg/mL \nand 0.0425 μg/mL, respectively. (4)\nWeight increase = Wt\nW0 × 100 (4) Morphological analysis The morphology of the nanocomposites was analyzed through \nscanning electron microscopy (SEM) at the “Centre Tech-\nnologique des Microstructures” (CTμ) facility of the Univer-\nsity of Lyon. SEM images were obtained with a FEI Quanta (2)\nFluid uptake (%) = Ww\nWd × 100 (2) 1 3 3 Drug Delivery and Translational Research (2023) 13:1343–1357 1346 Differential scanning calorimetry where Ww is the weight of the wet formulation and Wd is the \nweight of the dry formulation. All analyses were performed \nat least in triplicate. Differential Scanning Calorimetry (DSC) ­Q200® instru-\nment by TA Instruments (New Castle, DE, USA) was used \nto determine the thermal characteristics of powders. About \n5–6 mg of each sample was placed in an aluminum pan (40 \nµL) perforated. Samples were subjected to both heating \nand cooling cycles in a range from + 20 °C to + 180 °C and \nfrom + 180 °C to − 80 °C, respectively. All DSC analyses \nwere performed setting the flow rate at 10 ℃/min and the \nnitrogen atmosphere at 50 mL/min. Water evaporation from hydrogel 5–6 mg of each sample was placed in an aluminum pan (40 \nµL) perforated. Samples were subjected to both heating \nand cooling cycles in a range from + 20 °C to + 180 °C and \nfrom + 180 °C to − 80 °C, respectively. All DSC analyses \nwere performed setting the flow rate at 10 ℃/min and the \nnitrogen atmosphere at 50 mL/min. Water vapor transmission rate (WVTR) to determine the \nmoisture permeability of the wound dressing was performed \nas described by ASTM standard (ASTM Standard, 2010). Briefly, a 25-mm hydrogel disk, formed from each powder \naccurately weight and normalized respect the amount of AP \nwhen in contact with SWF, was mounted on the top of a \nplastic tube containing 20 mL of distilled water.l In vitro test Preliminary analyses conducted on NEs aimed at maxi-\nmizing drug loading while maintaining the nanometric \nsize (around 100 nm) and stability led to the formulation of \nnanosystems with a lipid concentration in the final phase of \n142.86 w/v loaded with 1.33% (w/w) of CCM. Due to the \nlipophilic character of CCM, the drug encapsulation effi-\nciency was very high (96.46 ± 5.66%), and the drug loading \nwas 1.29 ± 0.10%. Nanoemulsions formulation, physicochemical \ncharacterization, and stability NEs loaded with CCM were prepared by emulsion phase \ninversion technique coupled with a high stirring energy pro-\ncess, as previously described [29]. CCM was chosen as a \nhydrophobic model drug for its ability to enhance the wound \nhealing process [30, 31]. MTT test The effect of the powders on the vitality of HaCaT cells was \nevaluated using a colorimetric assay with 3-(4,5-dimethylthiazol-\n2-yl)-2,5-diphenyltetrazolium bromide (MTT), as previously \nreported [28]. Briefly, HaCaT cells (5 × ­103 cells/well) were plated \non 96-well plates and allowed to adhere for 24 h at 37 °C. After \nthat, the medium was substituted with either fresh medium alone \nor one containing serial dilutions of powders (25, 50, 100 μg/\nmL) and incubated for 24, 48, and 72 h. 6-Mercaptopurine was \nused as a positive control. Then, 25 µL of MTT (5 mg/mL) was \nadded, and the cells were incubated for further 3 h. Cells were \nthen lysed, and the dark blue crystals were solubilized with 100 \nµL of a solution containing 50% (v/v) N,N-dimethylformamide, \n20% (w/v) SDS with an adjusted pH of 4.5. The absorbance of \nthe resulting solution in each well was recorded at 570 nm using \nan automated microplate reader (Titertek Multiskan MCC/340-\nDASIT, Cornaredo, Milan, Italy). Each sample was measured at \nleast in triplicate, in three different experiments. The antiprolifera-\ntive activity was calculated as % viability: 100 – [(OD treated/OD \ncontrol) × 100], where OD is the optical density. In vitro release studies of nanoemulsions from AP‑11‑NE \nnanocomposites Data are reported as mean ± standard deviation (SD) of \nat least three independent experiments, each in triplicate. Analysis of variance and Bonferroniʼs test were used for data \nanalysis to perform multiple comparisons, using GraphPad \nPrism 8 (GraphPad Software). A P value less than 0.05 was \nconsidered significant. The in vitro release of NEs from AP-11-NE nanocompos-\nites was evaluated in SWF by cumulative study, in non-sink \nconditions, analyzing the CCM-loaded NEs by UHPLC as \npreviously described. 2.5 mL of SWF were added on top of \nthe AP-11-NE0.1 and AP-11-NE0.2 placed in a glass vial of \n3 mL. 60 mg of powders of each sample were used. Vials were \nkept under a slight stirring at 37 °C. At predetermined time \npoints, 200 μL were removed and replaced with fresh fluid. Samples were dissolved in 600 μL of methanol/acetonitrile \n(50:50) vortexed for 5 min and centrifugated for 2 min at 1200 \nRCF to remove the polymers. In the supernatants, 10 µg/mL \nof CCM as an internal standard was added to allow cor-\nrect detection of the drug. The analyses were performed in \ntriplicate. Solubility of curcumin A saturated solution of CCM was prepared in SWF under \nstirring (750 rpm) at room temperature and left overnight \nto reach equilibrium. Then, the sample was centrifuged \ntwo times at 1200 RCF for 10 min, and the supernatant was \ncollected and filtrated with a 0.22-μm nylon syringe filter \n(Whatman GmbH, Dassel, Germany). 200 μL of supernatant \nwere mixed with 600 μL of methanol/acetonitrile (50:50), \nand the sample was injected into the UHPLC system for \nCCM detection. Residual water content Water content contained in the powders was determined by \nThermo Gravimetric Analysis (TGA) on NETZSCH TG \n209F1 (NETZSCH-Gerätebau, Germany) using NETZSCH Water content contained in the powders was determined by \nThermo Gravimetric Analysis (TGA) on NETZSCH TG \n209F1 (NETZSCH-Gerätebau, Germany) using NETZSCH \nProteus 6.1 software to analyze the data. About 5 mg of the \npowder was placed in ceramic crucibles and heated from 20  \n°C to 1000 °C at a heating rate of 10 °C/min, under a nitrogen \natmosphere with a nominal gas flow rate of 30 mL/min. Proteus 6.1 software to analyze the data. About 5 mg of the \npowder was placed in ceramic crucibles and heated from 20  \n°C to 1000 °C at a heating rate of 10 °C/min, under a nitrogen \natmosphere with a nominal gas flow rate of 30 mL/min. In addition, the water content of the particles was quanti-\nfied by Karl-Fischer titration using an 889 KF Coulometer \n(Metrohm Ltd., Herisau, Switzerland) equipped with an \noven (860 KF Thermoprep). A solution of HYDRANAL \n(HYDRANAL™, Coulomat AG) was used as a titrant. The \nairflow rate was set at 100 mL/min and the oven temperature \nat 120 °C. The extraction time was set at 500 s, and the drift \ntime was 10 µg/min. Each powder was tested in triplicate. 3 1347 Drug Delivery and Translational Research (2023) 13:1343–1357 MTT test The loading of CCM in NEs was calculated as the ratio \nbetween the CCM detected and the total weight of NEs, \nwhile the loading of NEs in AP powders was calculated \nthrough the quantization of CCM as the ratio between the \nCCM detected and the total weight of the powder. Encapsu-\nlation efficiency (E.E.) was calculated as the ratio of CCM \ndetected to the amount of CCM initially loaded in the NE. The analyses were performed in triplicate. Cell culture conditions Immortal keratinocyte (HaCaT) cell line derived from nor-\nmal adult human skin was purchased from CLS Cell Lines \nService GmbH (Eppelheim, Germany, accession number \n300493) [27]. The cells were cultured with Dulbecco’s Mod-\nified Eagle’s Medium (DMEM) containing 10% (v/v) fetal \nbovine serum (FBS) and 1% (v/v) penicillin–streptomycin, \nat 37 °C in a humidified atmosphere of 5% ­CO2/ 95% air. The stability of NEs stored at 4 °C was followed over 28 days. Macroscopic aspects (presence of aggregates, cream formation, 1 3 Drug Delivery and Translational Research (2023) 13:1343–1357 1348 Fig. 1   Stability study of NEs stored at 4 °C for 28 days. Data are shown \nas mean ± S.D., n = 3; ***, **, and * denote P < 0.001, P < 0.01, and \nP < 0.05, respectively, formulations vs control total polymers concentration was fixed at 1% (w/v), two dif-\nferent concentrations of NEs were added to obtain a final \nconcentration in nanocomposites of 0.1% and 0.2% w/w. The main characteristics of nanocomposites were reported \nin Table 1. Both nanocomposites AP-11-NE0.1 and AP-\n11-NE0.2 were characterized by relatively high process \nyield without difference to microparticles AP-11. However, \nthe increase of the NEs concentration determined a reduc-\ntion of E.E., ranging from 57% for AP-11-NE0.1 to 50% for \nAP-11-NE0. 2. The stability of the NEs after spray-drying was assessed \nby monitoring their physicochemical features after encapsu-\nlation in AP microparticles. After solubilization of micro-\nparticles in water, the complete dispersion led to the libera-\ntion of nanoencapsulated material with an increase in NEs \ndiameter in NE0.1 and NE0.2 (viz., 0.1% and 0.2% nanoe-\nmulsions loaded in AP particles) from 103 to 205 nm and \n238 nm, respectively. As shown in Table 2, PDI also showed \nan increase from 0.2 to 0.3 or to 0.4, respectively. Moreover, \nthe ζ-potential of the NEs-loaded AP microparticles moved \nfrom a slightly negative value to a highly negative value \n(from − 15 to − 21 or − 29 mV). On the contrary, ζ-potential \nfor AP-11-NE0.1 and AP-11-NE0.2 remains unchanged \ncompared to AP blank particles. These results suggest a \nphysical interaction between polymers and NEs. Fig. 1   Stability study of NEs stored at 4 °C for 28 days. Cell culture conditions Data are shown \nas mean ± S.D., n = 3; ***, **, and * denote P < 0.001, P < 0.01, and \nP < 0.05, respectively, formulations vs control The water content of the spray-dried powders (Table 2), \nevaluated by Karl Fisher titration, showed that the addition \nof NEs to AP-11 polymers led to a reduction in the amount of \nwater entrapped during the spray-drying process, probably \ndue to the added hydrophobic character. or color changes), physicochemical properties (particle size, \npolydispersity, and zeta potential), and drug leakage were evalu-\nated. No sample degradation or changing of color was observed, \nand mean size, PDI, and surface potential remained stable during \nthe studied period (Fig. 1). In addition, the amount of encapsu-\nlated CCM remained unchanged at the storage condition of 4 °C \nas confirmed by UHPLC analysis. Morphology, as well as the dimensional distribution of \nmicroparticles, was affected by NEs content. AP-11 presented \na morphology “deflated balloons” (Fig. 2a, d) which, in the \nnanocomposites, has been moved to spherical shapes with \npores and small cracks due to the presence of NEs on the \nsurface of the microparticles (Fig. 2b, c, e, f). Moreover, dimensional distribution analyses performed \nby static laser scattering revealed that all the microparticles \npresent a single peak particle size distribution with span \nvalues less than 2, with a mean diameter of about 3.5 μm, in \naccordance with SEM analyses. Production and physicochemical characterization \nof nanocomposites Once assessed the stability and encapsulation efficiency of \nCCM in NEs, an original nanocomposite composed of an \nalginate-pectin blend with NEs was produced using spray-\ndrying. Based on previous work [15], alginate-pectin (AP) \npowder in a ratio 1:1 was chosen as a carrier (AP-11). While The thermal properties of the materials were analyzed \nusing both TGA and DSC. Figure 3 shows the TGA profiles \nof alginate and pectin polymers in comparison with those \nof the different microparticles. The degradation profile of Table 1   Main properties of \nalginate-pectin microparticles \nand nanocomposites\nSample\nPolymer concentration \n% (w/w)\nNE % (w/w)\nProcess \nyield (%)\nDrug content \n(%) ± SD\nE.E. (%) ± SD\nAP-11\n1\n-\n67.0\n-\n-\nAP-11-NE0.1\n1\n0.1\n65.0\n0.08 ± 0.00\n57.60 ± 3.89\nAP-11-NE0.2\n1\n0.2\n65.1\n0.14 ± 0.00\n50.47 ± 0.59 Sample\nPolymer concentration \n% (w/w)\nNE % (w/w)\nProcess \nyield (%)\nDrug content \n(%) ± SD\nE.E. (%) ± SD\nAP-11\n1\n-\n67.0\n-\n-\nAP-11-NE0.1\n1\n0.1\n65.0\n0.08 ± 0.00\n57.60 ± 3.89\nAP-11-NE0.2\n1\n0.2\n65.1\n0.14 ± 0.00\n50.47 ± 0.59 1 3 1349 Drug Delivery and Translational Research (2023) 13:1343–1357 investigated using DSC (Fig. 4). NEs (after water evapora-\ntion) showed a melting peak (Fig. 4-1a) at around 48 °C and \na second peak at lower temperatures (below 40 °C) suggest-\ning the presence of a second crystalline phase, ascribable to \na polymorphic form of the stearic acid of polyoxyethylene \n(40) stearate resulting from its mixing with oleoyl poly-\noxyl-6 glycerides and MCT excipient [33]. In addition, a \nbroad crystallization peak from 0 °C to 22 °C upon cooling \nand a second melting peak at − 50 °C (Fig. 4-2a) referred \nto as MCT polymorphism was present [33, 34]. AP-11-NE \npowders were analyzed to estimate the influence of the dry-\ning process on the structure of NEs loaded. After the water \nevaporation, the melting and crystallization peaks of polyox-\nyethylene (40) stearate were visible according to the amount \nof NEs loaded in the powders (Fig. 4b, c). On the contrary, \nalginate and pectin did not show any signal (Fig. 4d, e, f, g). Also, curcumin (Fig. Production and physicochemical characterization \nof nanocomposites Water vapor transmission rate (WVRT) of the in situ \nformed hydrogels was measured to evaluate if the wound \ndressings are able to maintain proper moisture on the \nwound bed. All hydrogel formulations presented WVTR \nvalues between 85 and 89 g/m2h resulting then as an ade-\nquate barrier able to protect the wound while promoting \ngood transpiration. The rate of water evaporation from \nhydrogels was measured to evaluate their ability to retain \nthe water within the dressings over time even when exu-\ndate is not poured out from the wound anymore. Figure 6 \nshows the fluid loss evaluated as the weight decrease of \nFig. 3   Thermogravimetric \ncurves of alginate raw material \n(black), pectin raw material \n(green), AP before spray-drying \n(light blue) AP-11 (red), AP-\n11-NE0.1 (blue), and AP-\n11-NE0.2 (violet). (Color figure \nonline) Fig. 3   Thermogravimetric \ncurves of alginate raw material \n(black), pectin raw material \n(green), AP before spray-drying \n(light blue) AP-11 (red), AP-\n11-NE0.1 (blue), and AP-\n11-NE0.2 (violet). (Color figure \nonline) values between 85 and 89 g/m2h resulting then as an ade-\nquate barrier able to protect the wound while promoting \ngood transpiration. The rate of water evaporation from \nhydrogels was measured to evaluate their ability to retain \nthe water within the dressings over time even when exu-\ndate is not poured out from the wound anymore. Figure 6 \nshows the fluid loss evaluated as the weight decrease of was very quick, but the water uptake was lower due to the \npresence of NEs which enhanced the hydrophobicity of the \nsystems, as confirmed also by Karl Fisher’s analysis. was very quick, but the water uptake was lower due to the \npresence of NEs which enhanced the hydrophobicity of the \nsystems, as confirmed also by Karl Fisher’s analysis. Water vapor transmission rate (WVRT) of the in situ \nformed hydrogels was measured to evaluate if the wound \ndressings are able to maintain proper moisture on the \nwound bed. All hydrogel formulations presented WVTR Water vapor transmission rate (WVRT) of the in situ \nformed hydrogels was measured to evaluate if the wound \ndressings are able to maintain proper moisture on the \nwound bed. All hydrogel formulations presented WVTR Fig. 4   DSC thermograms of NEs (a), AP-11-NE0.1 (b), AP-11-NE0.2 \n(c), AP in combination before spray-drying (d), AP-11 after spray-drying \n(e), pectin raw material (f), alginate raw material (g), and curcumin raw \nmaterial (h). Production and physicochemical characterization \nof nanocomposites 4h) did not exhibit any signal because \nits typical melting is visible at around 180 °C [35], outside \nthe range of interest.l Table 2   Physicochemical properties of NEs before and after encapsu-\nlation in AP particles, AP-11 microparticles, and moisture content of \nAP-11, AP-11-NE0.1, and AP-11-NE.0.2 *NE.01 and NE.02 represent the nanoemulsions released by a spray-\ndried particle, AP-11-NE0.1 and AP-11-NE0.2, respectively\nSize (nm) ± SD\nPDI\nζ–potential \n(mV) ± SD\nH2o % \n(w/w) ± SD\nNEs\n103.90 ± 2\n0.2\n-15 ± 1\n-\nNE0.1*\n205.13 ± 2\n0.3\n-21 ± 2\n-\nNE0.2*\n238.97 ± 5\n0.4\n-30 ± 2\n-\nAP-11\n3517 ± 90\n0.3\n-52 ± 2\n9.27 ± 0.04\nAP-11-NE0.1\n3338 ± 65\n0.2\n-51 ± 4\n6.07 ± 0.05\nAP-11-NE0.2\n3325 ± 79\n0.3\n-50 ± 3\n5.54 ± 0.09 alginate has shown a three-stage process: below 150 °C, \nthe weight loss is associated with humidity release while at \n237 °C and 750 °C, it is correlated to the rupture of chains, \nfragments, and monomers [32]. Differently, pectin presented \ntwo main weight loss steps. The degradation patterns of the \nother samples were very similar to alginate and pectin raw \nmaterial with a weight loss below 150 °C due to the removal \nof moisture, followed by a sharp weight loss at 230 °C due \nto the degradation of the polymer backbone. To evaluate the influence of NEs on the powders ability to \nbecome a gel in situ, AP-11 and AP-11-NEs were placed in \ncontact with simulated wound fluid (SWF), and, at scheduled \ntime points, the increase in weight was calculated. Figure 5 \nshows that AP-11 became gel in less than 5 min increas-\ning almost 7 times its weight after 15 min. As expected, a \ndifferent trend was observed for AP-11-NEs. The swelling The state of the shell (crystalline or amorphous) of \nthe NEs loaded with curcumin and of the powders was Fig. 2   SEM microphotographs at two different magnifications of AP-11 (a, d), AP-11-NE0.1 (b, e), and AP-11-NE0.2 (c, f) EM microphotographs at two different magnifications of AP-11 (a, d), AP-11-NE0.1 (b, e), and AP-11-NE0.2 (c, f) 1 3 3 Drug Delivery and Translational Research (2023) 13:1343–1357 1350 was very quick, but the water uptake was lower due to the \npresence of NEs which enhanced the hydrophobicity of the \nsystems, as confirmed also by Karl Fisher’s analysis. Production and physicochemical characterization \nof nanocomposites AP-11 showed that approximately \n12% of water was retained, while AP-11-NE0.1 and AP-\n11-NE0.2 retained 10% and 11% water, respectively. It \nsuggests that AP hydrogel, also when loaded with NEs, \nloses most of its water content in a short time, but can \nretain almost a proper amount of humidity even when \nexposed to air in dry conditions. the different formulations after the removal of SWF. It can \nbe observed that the loss of water rapidly increased in the \nfirst 12 h, whereas after 72 h, there was almost no water \nloss from the hydrogels even though hydrogels were not \ncompletely dried yet. AP-11 showed that approximately \n12% of water was retained, while AP-11-NE0.1 and AP-\n11-NE0.2 retained 10% and 11% water, respectively. It \nsuggests that AP hydrogel, also when loaded with NEs, \nloses most of its water content in a short time, but can \nretain almost a proper amount of humidity even when \nexposed to air in dry conditions. showed that G′ was much higher than G″. For the AP-\n11-NEs, an increase in the gap between the storage and loss \nmodulus was reported, according to the NEs concentration, \ndemonstrating the prevalent gel-like behavior. This pattern \ncan be better explained through the loss factor that represents \nthe ratio between viscous and elastic components (purely \nelastic, δ = 0; purely viscous, δ = 90) [36]. For the powder \nloaded with NEs, the loss factor decreased (Fig. 7 panel B) \nindicating an increase in structure formation and gelation. Production and physicochemical characterization \nof nanocomposites Panel 1 represents the heating cycle from + 20 °C to 180 °C \nwhile panel 2 cooling performed from + 180 °C to − 80 °C both at 10 °C/\nmin material (h). Panel 1 represents the heating cycle from + 20 °C to 180 °C \nwhile panel 2 cooling performed from + 180 °C to − 80 °C both at 10 °C/\nmin material (h). Panel 1 represents the heating cycle from + 20 °C to 180 °C \nwhile panel 2 cooling performed from + 180 °C to − 80 °C both at 10 °C/\nmin Fig. 4   DSC thermograms of NEs (a), AP-11-NE0.1 (b), AP-11-NE0.2 \n(c), AP in combination before spray-drying (d), AP-11 after spray-drying \n(e), pectin raw material (f), alginate raw material (g), and curcumin raw 1 3 Drug Delivery and Translational Research (2023) 13:1343–1357 1351 Fig. 5   Simulated wound fluid \nuptake of AP-11, AP-11-NE0.1, \nand AP-11-NE0.2 the different formulations after the removal of SWF. It can \nbe observed that the loss of water rapidly increased in the \nfirst 12 h, whereas after 72 h, there was almost no water \nloss from the hydrogels even though hydrogels were not \ncompletely dried yet. AP-11 showed that approximately \n12% of water was retained, while AP-11-NE0.1 and AP-\n11-NE0.2 retained 10% and 11% water, respectively. It \nsuggests that AP hydrogel, also when loaded with NEs, \nloses most of its water content in a short time, but can \nretain almost a proper amount of humidity even when \nd\ni i d\ndi i\nshowed that G′ was much higher than G″. For the AP-\n11-NEs, an increase in the gap between the storage and loss \nmodulus was reported, according to the NEs concentration, \ndemonstrating the prevalent gel-like behavior. This pattern \ncan be better explained through the loss factor that represents \nthe ratio between viscous and elastic components (purely \nelastic, δ = 0; purely viscous, δ = 90) [36]. For the powder \nloaded with NEs, the loss factor decreased (Fig. 7 panel B) \nindicating an increase in structure formation and gelation. I\nit\nl\nt d\nFig. 5   Simulated wound fluid \nuptake of AP-11, AP-11-NE0.1, \nand AP-11-NE0.2 the different formulations after the removal of SWF. It can \nbe observed that the loss of water rapidly increased in the \nfirst 12 h, whereas after 72 h, there was almost no water \nloss from the hydrogels even though hydrogels were not \ncompletely dried yet. In vitro release study The in situ gelled powders viscoelastic properties were \nassessed through rheological measurements performing \nan amplitude sweep test, which depicts the variation of \ndynamic storage modulus (G′) and loss modulus (G″) \ndepending on the shear strain (γ). All formulations (Fig. 7) In vitro release studies of the NEs from nanocomposites \nwere carried out in SWF. The study was carried out in \nnon-sink conditions, and a cumulative release study was \nperformed. All nanocomposites showed a similar profile. 1 3\nFig. 6   Weight loss from hydro-\ngels made on alginate-pectin \nand alginate-pectin loaded with \nnanoemulsions, after 120 h 1 3 3 AP-11-NE0 1 released about 75% of the encapsulated NEs\nIn vitro cytotoxicity\nFig. 7   Rheological characterization of AP-11, AP-11-NE0.1, and AP-11-NE0.2. Storage G' and loss G\" moduli evaluated via amplitude sweep \ntest (Panel A) and strain dependent tangent of loss factor (Panel B)\n1352\nDrug Delivery and Translational Research (2023) 13:1343–1357 Drug Delivery and Translational Research (2023) 13:1343–1357 1352 Fig. 7   Rheological characterization of AP-11, AP-11-NE0.1, and AP-11-NE0.2. Storage G' and loss G\" moduli evaluated via amplitude sweep \ntest (Panel A) and strain dependent tangent of loss factor (Panel B) In vitro cytotoxicity The in vitro cell \nviability assay was conducted by exposing cells to AP-11 and \nnanocomposites for 24, 48, and 72 h at three different systems \nconcentrations (25, 50, 100 µg/mL). As shown in Fig. 9A, the \nantiproliferative activity for the blank formulation AP-11 was \nfound to be time and dose-dependent. The increase in powder \nconcentration led to an increase in antiproliferative activity. properties, morphology, and size distribution were stud-\nied. As reported in the literature, microparticles obtained \nby spray-drying present different morphologies depending Fig. 9   Antiproliferative activity of in  situ gel powders assessed \nby MTT test on HaCaT cells after 24, 48, and 72  h of treatment. Panel A: blank formulation AP-11 at different concentrations (25, \n50, 100  µg/mL). Panel B: nanocomposites AP-11-NE0.1 and AP-\n11-NE0.2 in comparison with AP-11 at 25 µg/mL. Data are expressed \nas mean ± SEM. Mercaptopurine (1 µM) was used as a positive con-\ntrol. ***, **, and * denote P < 0.001, P < 0.01, and P < 0.05, respec-\ntively, formulations vs control In vitro cytotoxicity AP-11-NE0.1 released about 75% of the encapsulated NEs \nafter 8 h, followed by a prolonged release up to 24 h reach-\ning 100% of NEs release, whereas in AP-11-NE0.2 with \nthe doubled content of the NEs release decreased from 75 \nto 65% after 8 h and from 100 to 98% after 24 h (Fig. 8). AP-11-NE0.1 released about 75% of the encapsulated NEs \nafter 8 h, followed by a prolonged release up to 24 h reach-\ning 100% of NEs release, whereas in AP-11-NE0.2 with \nthe doubled content of the NEs release decreased from 75 \nto 65% after 8 h and from 100 to 98% after 24 h (Fig. 8). Since the biocompatibility of any wound dressing is essential \nto assure the safety of the wounds, the cytotoxicity of the \npowders on HaCaT cells expressed as an antiproliferative 1 3 Drug Delivery and Translational Research (2023) 13:1343–1357 1353 activity through the MTT test was evaluated. The in vitro cell \nviability assay was conducted by exposing cells to AP-11 and \nnanocomposites for 24, 48, and 72 h at three different systems \nconcentrations (25, 50, 100 µg/mL). As shown in Fig. 9A, the \nantiproliferative activity for the blank formulation AP-11 was \nfound to be time and dose-dependent. The increase in powder \nconcentration led to an increase in antiproliferative activity. After 72 h, the powders at the maximum concentration of \nsystems tested (100 µg/mL) exhibited a value of antiprolifera-\ntive activity slightly higher than 30% that according to ISO \n10993–5 is considered cytotoxic [37]. Differently, nanocom-\nposites AP-11-NE0.1 and AP-11-NE0.2, after 72 h, only at \n25 µg/mL did not exhibit toxicity (Fig. 9B). Discussion\nIn this work, we developed a tailored hybrid system made \nof CCM-loaded NEs associated with alginate-pectin matrix \nto vehicle lipophilic drugs and maximize adherence to the \nwound. Hydrophobic drugs, once incorporated into NEs, \ncan penetrate efficiently into the skin and through the sub-\ncutaneous barrier [38]. However, NEs suspensions are not \nviscous enough to be directly applied to the wound bed [13]. The composites here produced by spray-drying in the form \nof microparticles are intended to enhance the contact time \nat the dressing site prolonging the release of NEs from the \nhydrophilic matrix. The first step in the design of adherent nanocomposites \nproperties, morphology, and size distribution were stud-\nied. As reported in the literature, microparticles obtained \nby spray-drying present different morphologies depending \nFig. In vitro cytotoxicity 8   Nanoemulsions released \nfrom the nanocomposite AP-\n11-NE0.1 and AP-11-NE0.2 \nperformed in simulated wound \nfluid activity through the MTT test was evaluated. The in vitro cell \nviability assay was conducted by exposing cells to AP-11 and \nnanocomposites for 24, 48, and 72 h at three different systems \nconcentrations (25, 50, 100 µg/mL). As shown in Fig. 9A, the \nantiproliferative activity for the blank formulation AP-11 was \nfound to be time and dose-dependent. The increase in powder \nconcentration led to an increase in antiproliferative activity. After 72 h, the powders at the maximum concentration of \nsystems tested (100 µg/mL) exhibited a value of antiprolifera-\ntive activity slightly higher than 30% that according to ISO \n10993–5 is considered cytotoxic [37]. Differently, nanocom-\nposites AP-11-NE0.1 and AP-11-NE0.2, after 72 h, only at \n25 µg/mL did not exhibit toxicity (Fig. 9B). properties, morphology, and size distribution were stud-\nied. As reported in the literature, microparticles obtained \nby spray-drying present different morphologies depending \nFig. 8   Nanoemulsions released \nfrom the nanocomposite AP-\n11-NE0.1 and AP-11-NE0.2 \nperformed in simulated wound \nfluid activity through the MTT test was evaluated. The in vitro cell \nviability assay was conducted by exposing cells to AP-11 and \nnanocomposites for 24, 48, and 72 h at three different systems \nconcentrations (25 50 100 µg/mL) As shown in Fig 9A the\nproperties, morphology, and size distribution were stud-\nied. As reported in the literature, microparticles obtained \nby spray-drying present different morphologies depending \nFig. 8   Nanoemulsions released \nfrom the nanocomposite AP-\n11-NE0.1 and AP-11-NE0.2 \nperformed in simulated wound \nfluid Fig. 8   Nanoemulsions released \nfrom the nanocomposite AP-\n11-NE0.1 and AP-11-NE0.2 \nperformed in simulated wound \nfluid activity through the MTT test was evaluated. The in vitro cell \nviability assay was conducted by exposing cells to AP-11 and \nnanocomposites for 24, 48, and 72 h at three different systems \nconcentrations (25, 50, 100 µg/mL). As shown in Fig. 9A, the \nantiproliferative activity for the blank formulation AP-11 was \nfound to be time and dose-dependent. The increase in powder \nconcentration led to an increase in antiproliferative activity. After 72 h, the powders at the maximum concentration of \nsystems tested (100 µg/mL) exhibited a value of antiprolifera-\ntive activity slightly higher than 30% that according to ISO \n10993–5 is considered cytotoxic [37]. Differently, nanocom-\nposites AP-11-NE0.1 and AP-11-NE0.2, after 72 h, only at \n25 µg/mL did not exhibit toxicity (Fig. 9B). activity through the MTT test was evaluated. Discussion In this work, we developed a tailored hybrid system made \nof CCM-loaded NEs associated with alginate-pectin matrix \nto vehicle lipophilic drugs and maximize adherence to the \nwound. Hydrophobic drugs, once incorporated into NEs, \ncan penetrate efficiently into the skin and through the sub-\ncutaneous barrier [38]. However, NEs suspensions are not \nviscous enough to be directly applied to the wound bed [13]. The composites here produced by spray-drying in the form \nof microparticles are intended to enhance the contact time \nat the dressing site prolonging the release of NEs from the \nhydrophilic matrix. The first step in the design of adherent nanocomposites \nwas the optimization of NEs loaded with curcumin. NEs \nobtained via the emulsion phase inversion technique coupled \nwith high stirring energy input presented physicochemical \ncharacteristics in accordance with the results reported in a \nprevious study [29]. Then, NEs have been combined with \nthe alginate-pectin matrix to obtain the nanocomposites, and \nthe impact of oil and surfactants on the microparticulate Fig. 9   Antiproliferative activity of in  situ gel powders assessed \nby MTT test on HaCaT cells after 24, 48, and 72  h of treatment. Panel A: blank formulation AP-11 at different concentrations (25, \n50, 100  µg/mL). Panel B: nanocomposites AP-11-NE0.1 and AP-\n11-NE0.2 in comparison with AP-11 at 25 µg/mL. Data are expressed \nas mean ± SEM. Mercaptopurine (1 µM) was used as a positive con-\ntrol. ***, **, and * denote P < 0.001, P < 0.01, and P < 0.05, respec-\ntively, formulations vs control 1 Drug Delivery and Translational Research (2023) 13:1343–1357 1354 carried out at 37 °C to simulate the body temperature. With the \ntemperature rising, the particles tend to move disorderly due \nto a higher Brownian thermal energy, and consequently, while \nthe mobility of macromolecule segments increases, the dura-\ntion of relaxation processes is reduced [44, 45]. As shown in \nFig. 7A the addition of NEs led to an increase of both moduli \nproportionally to the NEs concentration, whereby the growth \nof G' is significantly stronger than that of G\". This effect is \ncorrelated to the influence of the nanoparticles on the mobil-\nity of the polymers chains that might determine an increased \nrigidity of the structure and, as a consequence, stability on the \nwound [45]. Moreover, as reported in Fig. Discussion A rapid hydrogel formation is a crucial charac-\nteristic for the in situ gelling powders intended to be applied \nto the wound with high exudate production. As expected, \nfluid uptake was strictly correlated to NEs concentration. Compared to the blank formulation, nanocomposites showed \nlower water uptake (Fig. 5) probably due to the presence of \nthe NEs that physically impaired the movement of the poly-\nmer chains during the uncoiling connected to the interaction \nwith water. Modifications such as the addition of hydropho-\nbic nanoparticles in a hydrogel system have been reported \nto influence the swelling properties of hydrogel. Nanoparti-\ncles, replacing the space occupied by water molecules, led to \na reduction of water uptake into the polymer structure [41]. The release profiles of the NEs from nanocomposites \n(Fig. 8) are due to the coexistence of different condi-\ntions, including the porosity of the particles, heteroge-\nnous NEs distribution, and the hydrophilic properties of \nthe polymers, which once in contact with SWF diffused \ninto the system, swell and form a gel, causing the system \ndissolution. After the formation of the gel, which takes \nplace in a few minutes, the dissolved NEs molecules dif-\nfuse through the hydrated polymeric network out of the \nsystem resulting in their continuous release. In addition \n(after 8 h), the bioerosion of the polymer might be the \nmain driving force of the sustained release of NEs [47, \n49]. CCM, as any other possible drug, loaded into NEs \nis protected by hydrolysis and various other enzymatic \ndegradations while the environment of the wound would \nlead to the NEs dissociation and the consequent delivery \nof CCM [50]. p\np y\n[\n]\nDressing transpiration is another key parameter of wound \ndressing needed to both prevent an excessive wet condition \nduring wound care and reduce pain removal of the dressing \nafter its use. All hydrogels formed in situ showed WVTR val-\nues between 85 and 89 g/m2h. These values are in the appropri-\nate range to prevent wound dehydration or occlusion phenom-\nena [42]. Moreover, the amount of water retained by gels even \nafter 72 h with the wound in the dry state is enough to avoid \ncomplete gel drying, thus allowing its easy removal at the end \nof the treatment. WWTR and water loss from hydrogels loaded \nwith NEs were similar to those of the unloaded AP hydrogels \n(Fig. 6). Discussion 7B, the addition of \nNEs causes a reduction of the loss factor which, when lower \nthan 1, indicates a clear prevalence of gel behavior [46]. This \nreduction determines an increase in the elasticity of the gels, a \nvery useful property for wound healing devices. on the features of the initial raw material and processing \nconditions [39]. In presence of NEs, microparticles did not \nshow the typical morphology of “deflated balloons” but \nexhibited good sphericity and porous surface due to the \nglobal increase of droplet loading with NEs before drying. The encapsulation of NEs in AP did not affect nanocompos-\nites size distribution and surface charge, contrarily to what \nwas previously reported [40]. In fact, they exhibited a mean \ndiameter of around 3.5 µm characterized by a single peak \nwith a span value of less than 2.i To verify possible particles internal structure modifica-\ntions due to the interaction with the NEs, the thermal behav-\nior of AP-11 microparticles and nanocomposites was investi-\ngated by DSC (Fig. 4). In nanocomposites, the characteristic \nmelting and crystallization peaks of NEs were shifted to \nlower temperatures. This result suggests the presence of NEs \nin the alginate-pectin matrix as supported by previous work \n[40], confirming the hypothesis of an interaction between \nNEs and polymer matrix, also highlighted by the variation \nof the NEs physicochemical properties after the dissolution \nof the microparticles in which they were embedded. In vitro release studies were carried out in SWF in order \nto evaluate the ability of the polymer matrix to effectively \nrelease NEs loaded with CCM in a sustained manner. This \nkind of study is generally carried out in sink conditions, \nwhere the maximum drug concentration in the bulk fluid \ndoes not exceed about 20% of the drug’s solubility [47]. For poorly water-soluble drugs, high dissolution medium \nvolumes are required to maintain a low ratio of particles to \nmedium volume, which however results in a loss of accuracy \nfor the measurement of drug content [48]. In some cases, \nsink conditions do not guarantee that no saturation occurs, \ntherefore, the release of NEs via curcumin from nanocom-\nposites was performed in non-sink conditions, to avoid \nunderestimation of the drug. Following the physicochemical studies and the analyses \nof the physical interactions characterizing the nanocom-\nposite, the gelling properties, and fluid permeability were \ninvestigated. Competing interests  The authors declare no competing interests. Open Access  This article is licensed under a Creative Commons Attri-\nbution 4.0 International License, which permits use, sharing, adapta-\ntion, distribution and reproduction in any medium or format, as long \nas you give appropriate credit to the original author(s) and the source, \nprovide a link to the Creative Commons licence, and indicate if changes \nwere made. The images or other third party material in this article are \nincluded in the article's Creative Commons licence, unless indicated \notherwise in a credit line to the material. If material is not included in \nthe article's Creative Commons licence and your intended use is not \npermitted by statutory regulation or exceeds the permitted use, you will \nneed to obtain permission directly from the copyright holder. To view a \ncopy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. Discussion This similarity suggests that although the presence of \na lipophilic portion, nanocomposites could be able to maintain \na suitable fluid balance in the wound bed, which might enhance \nboth cellular migration and reepithelization [43]. Concerning \nthe rheological study (Fig. 7), the amplitude sweep test was Finally, the biocompatibility of nanocomposites on human \nkeratinocytes (HaCaT) was assessed by monitoring cell meta-\nbolic activity (MTT assay) to ensure the safety of the formula-\ntions when applied to the wound. HaCaT cells are widely used to \ntest the compatibility of wound dressings as skin consists of dif-\nferent types of cells: keratinocytes, melanocytes, and fibroblasts 1 3 3 1355 Drug Delivery and Translational Research (2023) 13:1343–1357 [51]. In a previous work, we demonstrate that AP-11 powders \nwere not toxic in the range of 0.01–10 µg/mL [24]. In this work, \nwe tested the concentrations ranging from 25 to 100 µg/mL to \nverify the possibility to use a larger amount of powders on the \nwound. AP-11 did not show any significant cytotoxicity after \n72 h for any of the tested concentrations, except at 100 µg/mL, \nresulting well tolerated by keratinocytes (Fig. 9A). On the con-\ntrary, AP-11-NE0.1 and AP-11-NE0.2 reduced the metabolic \nactivity of cells after 48 h at the 50 and 100 µg/mL in a dose-\ndependent manner (Fig. S1). At 25 µg/mL and up to 72 h of \nexposure, both nanocomposites were non cytotoxic (Fig. 9B), \nindicating the possibility to apply the hydrogels for a prolonged \ntime on the wound bed, thus reducing the patients’ pain caused \nby frequent removal of the formulations. However, since cel-\nlular respiration might be altered by the shielding effect of the \nhydrogels, further studies will be needed to assess wound heal-\ning properties in vivo. has received funding from the National Research Agency (ANR), HyDNano \nproject (ANR-18-CE18-0025- 01). This work was supported by a grant from \nRegione Campania (I), POR Campania FESR 2014/2020, “Fighting Cancer \nresistance: Multidisciplinary integrated Platform for a technological Inno-\nvative Approach to Oncotherapies (Campania Oncotherapies)” [Project N. B61G18000470007]. has received funding from the National Research Agency (ANR), HyDNano \nproject (ANR-18-CE18-0025- 01). This work was supported by a grant from \nRegione Campania (I), POR Campania FESR 2014/2020, “Fighting Cancer \nresistance: Multidisciplinary integrated Platform for a technological Inno-\nvative Approach to Oncotherapies (Campania Oncotherapies)” [Project N. B61G18000470007]. Data availability  Not applicable. Conclusions Nanocomposites combining NEs with alginate-pectin pow-\nders intended for the controlled release of lipophilic drugs \nto be delivered directly to the wound bed were successfully \ndesigned. This study proposed a novel approach using a \nspray-drying process to load nanosystems into in situ gel-\nling microparticles composed of a natural polymeric blend. Nanocomposites could improve wound management thanks \nto their ability to rapidly form a hydrogel when in contact \nwith simulated wound fluid providing optimal water vapor \ntransmission rate. The combination of polymers and NEs \nenhanced the elastic properties of the in situ formed gel \nand lead to the sustained release of the drug-loaded NEs. In vitro cytocompatibility demonstrated that nanocomposites \nwere well tolerated by keratinocytes, allowing a prolonged \napplication of the powder on the wound bed. Globally, the \nunique combination of NEs loaded into an in situ forming \ngel microparticulate powder can be considered a promising \ncandidate for novel wound dressings. Ethics approval and consent to participate  Not applicable. Consent for publication  All authors have read and agreed to the pub-\nlished version of the manuscript. References 1. Hamdan S, Pastar I, Drakulich S, Dikici E, Tomic-Canic M, Deo \nS, Daunert S. Nanotechnology-driven therapeutic interventions \nin wound healing: potential uses and applications. ACS Cent Sci. 2017;3:163–75. 2. Kung M-L, Lin P-Y, Peng S-W, Wu D-C, Wu W-J, Yeh B-W, \nTai M-H, Hung H-S, Hsieh S. Biomimetic polymer-based Ag \nnanocomposites as a antimicrobial platform. Appl Mater Today. 2016;4:31–9. https://​doi.​org/​10.​1016/j.​apmt.​2016.​05.​003. 3. Teixeira MA, Paiva MC, Amorim MTP, Felgueiras HP. 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ERROR: type should be string, got "https://doi.org/10.1051/shsconf/20197009001 https://doi.org/10.1051/shsconf/20197009001 SHS Web of Conferences 70, 09001 (2019)\nICTDPP-2019 Research of psychological features of the \npersonality of students exhibiting addictive \nbehavior Natalia N. Bessonova¹, Vera S. Butenko2, Natalia A. Gudz3, Anna M. Kukulyar4* Natalia N. Bessonova¹, Vera S. Butenko2, Natalia A. Gudz3, Anna M. Kukulyar4* 1 «Elephant»,344010 Rostov-on-Don, Russian Federation \n2 Rostov State Medical University, 344022 Rostov-on-Don, Russian Federation \n3 Maikop city polyclinic №2, 385000 Maykop, Russian Federation \n4 Southern federal university, Academy of psychology and pedagogy, 344006 Rostov-on-\nDon, Russian Federation 1 «Elephant»,344010 Rostov-on-Don, Russian Federation \n2 Rostov State Medical University, 344022 Rostov-on-Don, Russian Federation \n3 Maikop city polyclinic №2, 385000 Maykop, Russian Federation \n4 Southern federal university, Academy of psychology and pedagogy, 344006 Rostov-on-\nDon, Russian Federation Annotation. This article is devoted to the study of psychological \ncharacteristics of the personality of students experiencing withdrawal \nsyndrome from amphetamine-type psychostimulants. The study was \nconducted on 45 young men, students of 1-3 courses of universities of \nRostov-on-don, rehabilitated In the center of psychological correction \n\"Elephant\". During the study, we used the following methods: 1) the \nscale of \"Amphetamine Cession Symptom Assessment\" (ACSA), 2) the \nscale of Montgomery and Asberg (MADRS); 3) the scale of EuroQol; 4) \nthe scale of self-report (VAS). The results obtained allow us to conclude \nthat among young men with amphetamine dependence accompanied by \nwithdrawal syndrome in the structure of which affective rather than \nsomatovegetative manifestations predominate almost in the complete \nabsence of algic phenomena. Affective disorders diagnosed in the \nhospital center of the respondents, mainly manifested in the form of \ndepression. The maximum expression of these States, as well as \nuncontrolled attraction to surfactants was achieved in the interval from \nthe third to the seventh day in withdrawal syndrome. Further the \ncondition of rehabilitators was normalized from the first week and till \nthe twenty-first day of stay. We noted a similar dynamics in assessing \nthe state of their own well-being rehabilitated. 1 Introduction In modern transitive society there are radical changes in the structure of society, \ninterpersonal relationships, value-semantic structure of the person, directly affecting the \nCommission of risky actions by the individual. The realities of modern society and various \nsocial problems encourage the person to exercise various destructive behaviors in reaction \nto frustrating events, uncertainty, stressors, caregiving from a traumatic reality (T. V. White, T. G. Bohan, A. Y. Egorov, A. O. Kibito, M. M. Orlov, E. N. Skripacheva, O. N. * Corresponding author: vetkina-anna@mail.ru © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons \nAttribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). 2 Discussion Specificity of action of psychostimulants of this type at the level of physiology consists in \nrelease of neurotransmitters which are capable to provide chemical transfer of a nervous \nimpulse in CNS, on a par with it process of slowdown of their reuptake is carried out. When \nusing psychostimulants, a person experiences General stimulation of VPF, accelerates \nthinking processes and cognitive activity, retreats apathy, fatigue and a feeling of \ndrowsiness, increases efficiency, increases the level of emotional background and \nsociability, improves mood, also against the background of toning the entire muscular \nsystem of the body improves coordination, a strong increase in endurance and physical \nstrength [2]. As a consequence, the use of amphetamine-type psychostimulants by young people \nresults in a surge of vivacity and energy, a General lifting of physical strength, the removal \nof fatigue and a partial improvement of such intellectual abilities as memory, attention and \nthinking processes, as well as mood enhancement, i.e., an optimal physiological and \npsychological state is achieved from their point of view [4,5]. To date, in the youth subculture, the use of these psychostimulants is very common. This \nis primarily due to the easy availability of their purchase. With the development of social \nnetworks, in particular The Telegram, there was no need for personal meetings to buy \ndrugs. At the moment within many SOC. there are closed communities for the sale and \ndistribution of drugs, even work related to the implementation of \"bookmarks\" for potential \nbuyers. Thus, the trade in illicit drugs has long moved to a new level. Secondly, \namphetamine-type psychostimulants are available from a financial point of view, in relation \nto other drugs. Third, these psychostimulants are \"light\" drugs, since they can be used \nsporadically: when visiting Nightclubs, parties, etc. Due to the relevance of the topic, we examined students of 1-3 courses in the amount of \n45 people. The main part of the sample consisted of young men-students who took a course \nof rehabilitation measures In the center of psychological correction «Elephant» (hereinafter \nrehabilitated) from 18 years to 21 years with a diagnosis of \"syndrome of dependence on \npsychostimulants (amphetamine/methamphetamine)\". * Corresponding author: vetkina-anna@mail.ru SHS Web of Conferences 70, 09001 (2019)\nICTDPP-2019 https://doi.org/10.1051/shsconf/20197009001 Tkachev, A. V. Trusova) [1,3]. Man has become quite often to show such protective \nbehavior in situations related to life crises, which entails the emergence of various forms of \ndependent behavior from completely different additive agents (alcohol, drugs, information \ndependence, gambling, etc.). In this article, we would like to draw attention to the growing \nmanifestation of such forms of addictive behavior as the use of amphetamine-type \npsychostimulants (\"salts\") among young people, and students in particular. 2 Discussion The main criteria for inclusion in the study were rehabilitated: 1) the fact of the use of \nsurfactants (amphetamine / methamphetamine) for at least 10 days in the last month before \nadmission To the center \"Elephant»; 2) confirmed diagnosis of psychostimulant \ndependence syndrome (amphetamine/methamphetamine) according to ICD-10; 3) no use of \nother surfactants before the start of rehabilitation activities for at least 10 days. Just note the criteria for which rehabilitated excluded from the study: 1) the presence of a \nsyndrome of dependence on other surfactants, except nicotine; 2) any mental illness \nidentified in the course of the study; 3) identification of suicidal tendencies; 4) diagnosis of \nsevere comorbidity, which requires hospitalization in the specialized Department. SHS Web of Conferences 70, 09001 (2019)\nICTDPP-2019 SHS Web of Conferences 70, 09001 (2019)\nICTDPP-2019 https://doi.org/10.1051/shsconf/20197009001 The respondents were selected on the basis of a list of specified criteria and the \ncompliance of the rehabilitated with them. Inclusion in the study group was based on the \ntime sequence of admission to the center of psychological correction \"Elephant\". It should \nbe noted that upon arrival at the Center, each of the participants was subjected to a thorough \nexamination (surrendered: General blood and urine tests, biochemical tests, as well as tests \nto determine the presence of traces of drugs in the urine). A mandatory requirement for \ndetermining rehabilitated in the Center, was the presence of the conclusion of the therapist \nor other specialized specialists (neurologist, surgeon, infectious diseases, etc.). p\np\n(\ng\ng\n)\nDuring the research we used the following methods: 1) scale \"Amphetamine Cessation \nSymptom Assessment\" (ACSA), assessing the duration and severity of withdrawal \nsyndrome of psychostimulants; 2) the dynamics of depressive disorders was estimated by \nus with the help of the scale Montgomery and Asberg (MADRS); 3) assessment of quality \nof life was performed using the EuroQol scale; 4) evaluation of the craving for stimulants \nwas carried out on 100 mm visual-analog scale self-report (VASS). During the study, according to the scale \"Amphetamine Cession Symptom Assessment\" \n(ACSA) and the Montgomery and Asberg scale (MADRS), we conducted measurements on \nthe first, third, seventh, tenth and twenty-first day, in order to identify the dynamics of the \nduration and severity of psychostimulant withdrawal syndrome and the specifics of the \ncourse of depressive disorder. The quality of life, studied on the EuroQol scale, was \ndiagnosed once a week according to the inpatient stay at the Center, and then every 4 weeks \non an outpatient basis. The degree of attraction to psychostimulants on the VASH scale was \ncarried out every 3 days within the framework of the withdrawal syndrome, then 1 time per \nweek in the hospital and 1 time per week with outpatient observation. 3 Research methods and techniques 2 2 4 Research results We would like to note the fact that the analysis of somatic manifestations in withdrawal \nsyndrome showed almost complete absence of any complaints, as only 3 young men (6.7%) \nfelt pronounced pain. p\np\nThe presence of somatovegetative manifestations of withdrawal syndrome and affective \ndisorders in rehabilitated have a negative impact on their assessment of the quality of life. According to the results of the study obtained on the EuroQol scale, within 1 day of stay in \nthe hospital of the center, rehabilitees assess their condition by 67.29+-0.78 points on a \nvisual 100-point scale. By the end of the first week of stay in the hospital Center with the \ncancellation of amphetamine average score reached 53.38+-2.57. The presented dynamics \nof the results is reliable at p<0.05. After passing stationary events during the 3 weeks the \naverage assessment of quality of life in reabilitologist boys reached 80,91+-1,92. During \nthe outpatient observation of the respondents, we noted a significant increase in the average \non this scale to 92.02+-1.97 points. These results can be regarded as an improvement in the \ndynamics of well-being among young men who were able to refrain from using \namphetamine-type psychostimulants for a longer period. The second block of the EuroQol scale helped us to study in detail the question of which \npredictors have the greatest impact on the self-esteem of the rehabilitated. Note that the \nmobility of the majority of subjects did not suffer in the first days of rehabilitation (the \nabsence of such a problem was noted by 90-93%% of persons), and within 14 days all \nrehabilitated mobility was within the norm. From Fig. 1 we see that in the first day of cancellation 11% (5 people) noted a decrease in \nactivity and 11% (5 people) complained of a sharp decrease in activity. However, on the 7th \nday, the total number undergoing rehabilitation activities and complaining of feeling made \nup almost half (48%) of the subjects. By the 14th day, the total number of people who \nnoticed a decrease in activity was 24 people (53%). But by the 21st day, the number of \npeople who noted a decrease in activity was only 4 people (9%). 4 Research results During the collection of anamnestic data, when rehabilitating in the Center within the \nhospital, we obtained the following results. In persons with identified amphetamine \ndependence, in the study of the characteristics of the course of the withdrawal syndrome, \naffective disorders were diagnosed. The presence of these violations was noted in all 45 \nboys (100% of the total sample). Along with this, 89% of respondents (41 boys) identified \nsomatovegetative disorders. The data obtained are reliable at p<0.05. We also studied the specifics of the manifestation of affective disorders. In the course of \nthe study, we found that the prevailing in the structure of affective disorders during \nwithdrawal syndrome in persons with amphetamine dependence was a reduced emotional \nbackground-62.4% of respondents. Irritability, as a manifestation of affective disorder, \naccounts for 28.8% (13 boys). Increased emotional background was diagnosed only in 8.8% \nof respondents (4 boys). According to the data obtained on the MADRS scale, regarding the dynamics of affective \ndisorders, it can be concluded that the average total index for the first two weeks of stay in \nthe hospital of the Center in the entire group of respondents is 16.2, which exceeds the \npermissible norm (15). The obtained indicator indicates the presence of depressive States in \nthe study group of young men. We consider it important to note that the highest values on \nthe MADRS scale (from 18.48 to 18.71) were obtained in the interval from the third to the \nseventh day after the cancellation of surfactants. After 10 days of stay in the hospital of the \nCenter, there is a decline in the manifestation of depressive States, and upon reaching the \ntwenty-first day, these manifestations among the group of young men are completely \nstopped. When processing the average data on the MADRS scale, we obtained significant \ndifferences at p<0.05 for the indicators of the first and third days (16.2 and 18.48), as well \nas between the indicators of the seventh and twenty-first days (18.71 and 14.0) at p<0.05. 3 https://doi.org/10.1051/shsconf/20197009001 SHS Web of Conferences 70, 09001 (2019)\nICTDPP-2019 We would like to note the fact that the analysis of somatic manifestations in withdrawal \nsyndrome showed almost complete absence of any complaints, as only 3 young men (6.7%) \nfelt pronounced pain. 4 Research results I would like to note the fact that among the entire group of young men at the stage of \nadmission to the hospital Center and during the first day of pain in acute abstinence was \nexpressed quite weakly only in 17.7% of respondents (8 boys), of which only 6.6% (3 boys) \nmanifested significantly. After the first week, the pain symptom in the form of General \nmalaise was diagnosed in only 11% (5 boys). After two weeks, only 4.4% (2 boys) showed \nsome discomfort. By the end of the third week, only 1% (1 youth) experienced minor \nsymptoms of discomfort. Assessing the self-esteem of respondents, we were diagnosed with \nanxiety, which were observed in 28 boys (62%) and almost all of them (26 boys - 58 %) \nreached a significant degree of severity. Fig. 1. Dynamics of self-evaluation activity reabilitologist within the cancellation period \namphetamines (in%) \n35\n21\n19\n42\n5\n15\n18\n1\n5\n9\n8\n2\n0\n10\n20\n30\n40\n50\n1 day\n7 day\n14 day\n21 day\nno problem\nproblem\nsignificant problem Fig. 1. Dynamics of self-evaluation activity reabilitologist within the cancellation period \namphetamines (in%) 4 https://doi.org/10.1051/shsconf/20197009001 SHS Web of Conferences 70, 09001 (2019)\nICTDPP-2019 By the 14th day, the number of respondents experiencing anxiety decreased to 26, and by \nthe end of the third week, 91.1% (41yunosh) felt the absence of any problems in this area. Using the scale \"Amphetamine Cession Symptom Assessment\" (ACSA) there was an \nassessment of the severity and duration of withdrawal syndrome from psychostimulants. As \na result of gamia, it was revealed that the most severe symptoms when canceling surfactant \nare observed on the third day. Significant relief of symptoms was observed only at the onset \nof the tenth day of stay in the hospital Center (Fig. 2). Fig. 2. Dynamics of ACSA scale indices in rehabilitated patients with amphetamine dependence \n41,32\n55,31\n56,17\n41,18\n0\n10\n20\n30\n40\n50\n60\n10 day\n7 day\n3 day\n1 day Fig. 2. Dynamics of ACSA scale indices in rehabilitated patients with amphetamine dependence We were also interested in studying the dynamics of amphetamine cravings among the \nyoung men surveyed, which we identified using the ACSA scale. The study found that the \nmost pronounced attraction to amphetamine-type psychostimulants was observed on the \nthird day of hospital stay at the Center. 4 Research results The decrease in craving for amphetamine-type \nsurfactants was observed only by the tenth day of being in the Center (Fig. 3). The \npresented results are reliable at p<0.05. The analysis of the results on the VASS scale gave us the opportunity to describe in more \ndetail the specifics of the dynamics of attraction to amphetamine-type psychostimulants \namong young men. Note that with withdrawal syndrome, the maximum attraction to \npsychostimulants was recorded on the third day. Fig. 3. Dynamics of craving for amphetamines (average score on a scale of ACSA) \n2,19\n4,07\n3,58\n2,46\n0\n2\n4\n6\n1 day\n3 day\n7 day\n10 day Fig. 3. Dynamics of craving for amphetamines (average score on a scale of ACSA) At the end of the first week in the hospital, this attraction in young men began to decline. When diagnosing the dynamics of attraction to amphetamines from the tenth to the twenty-\nfirst day of stay in the hospital of the Center, young men noted a persistent decrease in \ntraction to a minimum (Fig. 4). 5 SHS Web of Conferences 70, 09001 (2019)\nICTDPP-2019 https://doi.org/10.1051/shsconf/20197009001 Fig. 4. The dynamics of attraction to amphetamines in the framework of the syndrome in individuals \nwith amphetamine dependence (the average score for VASS) \n58,03\n91,69\n90,18\n71,97\n67,01\n34,75\n0\n20\n40\n60\n80\n100\n1 day\n3 day\n7 day\n10 day\n14 day\n21 day Fig. 4. The dynamics of attraction to amphetamines in the framework of the syndrome in individuals \nwith amphetamine dependence (the average score for VASS) 5 Conclusion Thus, it can be concluded that among the young men participating in the study was \ndiagnosed amphetamine dependence accompanied by withdrawal syndrome in the structure \nof which affective rather than somatovegetative manifestations predominate in the almost \ncomplete absence of algic phenomena. Affective disorders diagnosed in the hospital center \nof the respondents, mainly manifested in the form of depression. The maximum expression \nof these States, as well as uncontrolled attraction to amphetamine-type psychostimulants \nwas achieved in the interval from the third to the seventh day in withdrawal syndrome. Further after finding within a week and till the twenty-first day there was a gradual \nnormalization of a condition of young men. We noted a similar dynamics in assessing the \nstate of their own well-being rehabilitated. The worst was from the third to the seventh day, \nand then there was a significant improvement by the twenty-first day. After passing the inpatient rehabilitation program, the subjects underwent an outpatient \nprogram, during which studies were also conducted to determine the state of health. We \nnoted that by the 100th day of abstinence from the use of surfactants, the health of the \nsubjects almost normalized. Significant conditions determined by dysphoria in people who \nrefused to take surfactants are decreased activity, anxiety, depression. With all this, it \nshould be emphasized that the subjects noted a slight degree of discomfort. Refusal to use \namphetamines for a period of 3 months or more as a result of hospital stay in the center \noccurred in 72% of rehabilitated (in the first year of remission). 2. A.A. Yusifova, V.S. Knyazev, Advances in modern natural science. 6, 96-97\n(2014) 5. V. White, T. Williams, Australian secondary school students’ use of tobacco, \nalcohol, and over-the-counter and illicit substances in 2014 (The Cancer Council, \nMelbourne, 2016) 3. «Amphetamine gets the job done: Using drugs to work long hours» (ScienceDaily, \n2015) \nwww.sciencedaily.com/releases/2015/02/150220083916.htm \n(accessed \nAugust 19, 2019) 1. A.E. Primerova, Young scientist. 2, 1000-1003 (2016) References 1. A.E. Primerova, Young scientist. 2, 1000-1003 (2016) 3. «Amphetamine gets the job done: Using drugs to work long hours» (ScienceDaily, \n2015) \nwww.sciencedaily.com/releases/2015/02/150220083916.htm \n(accessed \nAugust 19, 2019) 3. «Amphetamine gets the job done: Using drugs to work long hours» (ScienceDaily,\n2015) \nwww.sciencedaily.com/releases/2015/02/150220083916.htm \n(accessed\nAugust 19, 2019) 4. W. Pedersen, S. Sandberg, H. Copes, Deviant Behavior. 36 (2), (2014) 5. V. White, T. Williams, Australian secondary school students’ use of tobacco, \nalcohol, and over-the-counter and illicit substances in 2014 (The Cancer Council, \nMelbourne, 2016) 6"