Biopolymer-stabilized zein nanoparticles for pressure ulcer management: Formulation, optimization, and preliminary in vivo assessment.

Abstract[2‐(Diethylamino)ethyl]dextran hydrochloride (EA) was reacted with a mixture of sodium dextran sulfate (DS) and poly(styrene sulfonate) (NaSS) to form a novel water‐water precipitate, a so‐so polyelectrolyte complex (PEC), comprising three biomedical materials. The mole ratios of (DS + NaSS)/EA in PEC's prepared at higher [H+] were greater than at lower [H+] except for those PEC's prepared in 5% NaOH solution. IR, elemental analyses, solubility measurements, and blood clotting tests for PEC's revealed that the molecular structure of the various PEC's differed according to the [H+], order of mixing in solution, though all PEC's were prepared from the same starting materials. It appears that the degree of dissociation and the conformation of DS, NaSS, and EA change with [H+] and that a different neutralization reaction occured in terms of different order of mixing. Blood clotting tests were performed on PEC tablets. It was found that PEC's suppressed the coagulation of blood compared with glass.

Antibiotic prophylaxis after swan bite

Each year, thousands of patients suffer from sacral decubitus ulcers, also known as pressure ulcers or sores. The current standard of care for sacral decubitus ulcer treatment is expensive and suboptimal, ranging in cost from a 15-dollar tube of Neosporin Ointment to 240,000 dollars for a skin flap surgery. Grade II pressure sores inevitably progress to stage III and IV if not addressed aggressively and early. Late-stage pressure sores present a unique challenge to physicians, particularly when they are deep, tunneling, and have tendon or bone involvement, as is the case for the two patients in this case study. The first patient in this study (referred to as patient 1) was afflicted with a mid-sacral pressure sore with exposed tendon, bone, and tunneling of ten years duration. The second patient in this study (referred to as patient 2) suffered from an ischial pressure sore with exposed tendon, bone, and tunneling for 30 months duration. Both patients exhausted conservative measures, including wound vac placement, oral and IV antibiotic treatment, multiple episodes of sharp debridement, wet-to-dry dressings, silver sulfadiazine dressings, and dehydrated amniotic membrane allograft placements. After failing conservative management, both patients received several applications of Wharton’s Jelly, a Mesenchymal Connective Tissue allograft (MCT), to accelerate wound closure. Conservative management, including sharp debridement, oral antibiotics, and electrical stimulation, was used in conjunction with the WJ allograft applications. At the time of consultation with Dr. Michael Lavor, both patients had Sacral Decubitus Ulcers (SDU) classified as Stage IV with tissue loss and involvement of bone or tendon, according to the National Pressure Ulcer Advisory Panel (NPUAP). After eight months of standardized wound care treatment combined with six Wharton’s Jelly allograft applications, both patients had wounds showing over 90% contraction in depth, tunneling, and diameter. This case study demonstrates a precedent for applying Wharton’s Jelly allografts in late-stage sacral decubitus ulcers with associated tunneling in combination with standard of care. Future research efforts with Wharton’s Jelly allografts applied to recalcitrant wounds may be directed at the frequency and combination of procedural techniques that best promote granulation tissue formation and volumetric contracture of deep wounds by secondary intention.

In this study, zein nanoparticles coated with carboxymethyl chitosan (CMCS) were prepared to encapsulate vitamin D3 (VD3). VD3 was first encapsulated into zein nanoparticles using a low-energy phase separation method and coated with CMCS simultaneously. Then, calcium was added to cross-link CMCS to achieve thicker and denser coatings. The nanoparticles with CMCS coatings had a spherical structure with particle size from 86 to 200 nm. The encapsulation efficiency was greatly improved to 87.9% after CMCS coating, compared with 52.2% for that using zein as a single encapsulant. The physicochemical properties were characterized by differential scanning calorimetry and Fourier transform infrared spectroscopy. Nanoparticles with coatings provided better controlled release of VD3 in both PBS medium and simulated gastrointestinal tract. Photostability against UV light was significantly improved after encapsulation. Encapsulation of hydrophobic nutrients in zein nanoparticles with CMCS coatings is a promising approach to enhance chemical stability and controlled release property.

This study aims to evaluate the effect of combined use of the racemic flavanone Naringenin (NRG) and the protein sericin as TNF-α blockers. Sericin (SMs) and (R/S) NRG-loaded Sericin (SNRGMs) microparticles were prepared by spray-drying, characterized in terms of morphology and particle size distribution, and encapsulation efficiency was determined. Concerning morphology and particle size distribution of microparticles, results indicated that they were not affected by the presence of NRG. The encapsulation efficiency was almost quantitative (93%), thus proving that sericin can be advantageously loaded with (R/S) NRG. Biological evaluation of (R/S) NRG, SMs and SNRGMs was then performed in lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (hPBMC). SNRGMs resulted cytotoxic at the higher dose used (200 μg/mL) and the effect was greater than (R/S) NRG alone. Moreover, even if sericin alone was not effective in suppressing LPS-induced serum TNF-α levels, SNRGMs loaded with 9.3% of (R/S) NRG were significantly more potent than (R/S) NRG alone. In summary, this study provides the proof of concept that sericin-based microspheres loaded with TNF-α-blockers could contribute to the down regulation of the cytokine and represents the starting point for the development of new topical formulations for the treatment of middle-stage psoriasis.

Investigation of wound healing potential of photo-active curcumin-ZnO-nanoconjugates in excisional wound model

Madhu ghrita is a herbal formulation containing honey and ghee (clarified butterfat) as its constituents. The aim of present study is to verify the wound healing and antiinflammatory claims of Madhu ghrita. Incision and excision wound models were used for evaluation of wound healing activity followed by histopathological study in which healing markers like keratinization, epithelization, fibrosis, neovascularisation and collagenation were evaluated in male Wistar rats. The results of Madhu ghrita were compared with the results of untreated control group and results of framycetine sulphate cream, considered as a positive control. The formulation was also tested for antiinflammatory activity, using carrageenan-induced paw oedema in male Wistar rats. The test formulation Madhu ghrita enhanced the tensile strength, which significantly improved over untreated wounds. The tensile strength of untreated control wound was 281±5.82, while with the Madhu ghrita and framycetine sulphate cream 1% w/w, it was 328±8.9 and 398±6.32, respectively. Treatment with Madhu ghrita alone promoted wound contraction and reduced the wound closure time, so increase in tensile strength and wound contraction shows the wound healing potential of Madhu ghrita . Histopathological study shows that proliferation of epithelial tissue promotes angiogenesis, multiplication of fibrous connective tissue due to treatment with Madhu ghrita . The test formulation Madhu ghrita also shows significant antiinflammatory activity when the results are compared with the activity of ibuprofen gel as reference standard. The present study demonstrates the wound healing and antiinflammatory potential of Madhu ghrita .

Naringenin (NR), a bioactive flavonoid with antioxidant and therapeutic potential, has limited pharmaceutical and nutraceutical applications owing to its poor water solubility and stability. To overcome these challenges, this study presents a novel core-shell nanocarrier system comprising zein (Zn) nanoparticles coated with calcium-crosslinked gellan gum (GG) for enhanced NR stability and sustained release. Zn-NR-GG nanoparticles were formulated via anti-solvent precipitation and calcium-mediated gelation, resulting in a uniform gel network with an average size of 234 nm. The GG shell provides structural integrity, preventing nanoparticle aggregation and enzymatic degradation under simulated gastrointestinal conditions. The nanoparticles exhibited high encapsulation efficiency (>91%), enhanced antioxidant activity, and a controlled NR release profile of 56.66 ± 0.11% over 4 h. The stability assessments demonstrated resistance to thermal, ionic, and pH variations, underscoring their robustness for bioactive delivery. The integration of protein-polysaccharide interactions with calcium-induced gelation offers an effective strategy for improving NR solubility, bioavailability, and therapeutic efficacy. This study highlights Zn-NR-GG nanoparticles as promising polymeric biomaterials for controlled nutraceutical and pharmaceutical applications.

A non-thermal modification method to enhance the encapsulation efficiency, stability, and slow-release performance of zein-based delivery systems – Cold plasma

This paper evaluated the potential of zein–resins nanoparticles to act as a carrier for water-insoluble oil carvacrol. The rosin and shellac were used as natural resins in different mass ratios to plant protein zein. Rosin is one of the natural gums. Shellac is a resinous secretion of the insect. The influence of nanoparticle composition on colloidal properties, encapsulation efficiency, and release of oil was studied. Zeta potential and size of thus prepared nanoparticles were studied. Carvacrol encapsulation efficiency and release were determined by the HPLC method developed for these compounds. Results showed that the resin type and share affect encapsulation efficiency and release of carvacrol. It was also shown that the addition of resins enhanced release from nanoparticles, compared to plain zein nanoparticles. Findings in the present work will help further understanding the interaction between alcohol–soluble biopolymers and provide new insight into the development of natural carriers for bioactive compounds. Novelty impact statement Natural polymers derived from food are considered desirable materials for constructing delivery systems to encapsulate, protect, and release bioactive components in nutraceuticals, pharmaceuticals, and food. Therefore, this paper is approached to fabricate the carvacrol-loaded zein/rosin (Z/R) and zein/shellac (Z/S) composite nanoparticles (NPs). Findings in the present work will help further understanding of the interaction between alcohol–soluble biopolymers (e.g., zein, rosin, and shellac) and provide new insight into the development of natural carriers for bioactive compounds.

Fabrication and characterization of curcumin-loaded albumin/gum arabic coacervate

Fragrances, a mixture of various aromatic compounds, usually have low aqueous solubility and instability to the environment. This research aimed to fabricate the encapsulation of two fragrances in zein nanoparticles and apply it in fabric softener to sustain the fabric with long‐lasting fragrance. Pink fruity fragrances (PF) and white floral fragrances (WF) encapsulated zein nanoparticles (PF‐ZNs and WF‐ZNs) were prepared by the liquid–liquid dispersion method. Zein and fragrances were mixed and immediately poured under high‐speed homogenizer into citrate buffer containing Tween 20 as a surfactant, and ethanol was removed using a rotary vacuum evaporator. PF‐ZNs and WF‐ZNs obtained exhibited spherical shape with average sizes at 219.1 ± 7.7 and 301.1 ± 9.8 nm, respectively. The encapsulation efficiency of PF‐ZNs, at 69.15 ± 1.31 %, was significantly higher than that of the WF‐ZNs, at 63.91 ± 1.40% (P < .05). Thermal gravimetric analysis (TGA) indicated that both fragrances' thermal stability was improved after the encapsulation. The PF‐ZNs and WF‐ZNs were added to fabric softeners and applied to three types of fabrics: cotton, polyester (TK), and blends of cotton/polyester (TC), and the fragrance remaining in the fabrics for 30 days of storage were determined by gas chromatography. The retention of both PF and WF was improved in all fabrics, and significant effects were performed in the cotton fabric with PF‐ZNs. The results showed the potential to use the fragrances encapsulated in zein nanoparticles as fabric softener to improve fabrics' long‐lasting properties, especially for cotton.

<p dir="ltr">Tissue repair following inflammatory damage is a critical process, essential for restoring tissue homeostasis. Nowhere is this more crucial than in the intestine, where the gut epithelium endures constant assaults from physical, chemical, and microbial threats. These challenges are ever-present, whether under normal conditions or during severe episodes of chronic intestinal diseases like inflammatory bowel disease (IBD). When an IBD flare-up occurs, the body is thrust into a dual struggle: it must urgently calm the overactive immune response while simultaneously mending the compromised epithelial barrier, a critical step toward achieving true mucosal healing.</p><p dir="ltr">Current treatments aim to suppress inflammation by blocking inflammatory pathways, which, while necessary to quell the immediate immune response, may also promote the regenerative processes required for healing. Yet, the reality is stark: up to 50% of IBD patients either don't respond to these therapies or suffer relapses after an initial improvement. Despite the clear link between mucosal healing and long-term remission, no therapies currently exist that specifically promote epithelial regeneration to facilitate this healing process.</p><p dir="ltr">We believe that the signals triggered by inflammation and tissue damage are intricately intertwined with the body's repair mechanisms. Effective communication between various cellular components-including but not limited to epithelial, immune, and stromal cells-is crucial for maintaining a healthy intestinal barrier under normal conditions and restoring it after injury and inflammation. In my thesis, I set out to unravel these complex interactions to deepen our understanding of the cellular and molecular mechanisms that drive intestinal inflammation damage and tissue repair.</p><p dir="ltr">In Study I, we used spatial transcriptomics to reveal a transcriptomics profile in mouse colon at steady state and during mucosal healing with a previously unachieved spatial resolution. By integrating tissue bulk RNA sequencing and single cell RNA sequencing datasets from mouse and human, we identified distinct molecular regionalization and compartmentalized transcriptional programs with human relevance. This study provides a useful methodology and resource for the followings studies to understand mechanism involved in tissue regeneration.</p><p dir="ltr">In Study II, B cell expansion was found to be a major event within the immune compartment during the tissue regeneration phase following withdrawal of Dextran Sulfate Sodium (DSS). Depletion of B cells from mice during recovery phase resulted in an improved outcome in experimental colitis. Mechanistic study using tissue and single cell RNA sequencing and immunostaining validation revealed that expanded B cells interfered with the interaction between stromal cells and epithelium by physically reducing their proximity. The findings emphasize the impact of cellular interactions and the local microenvironment on tissue regeneration. They suggest that targeting B cells during the regenerative phase may be a promising approach to improve the management of colitis and related intestinal disorders.</p><p dir="ltr">In Study III, Liver X receptor (LXR) was identified as a common pathway induced by tissue damage in the intestine. Activation of LXR by feeding a synthetic LXR ligand GW3965 in diet resulted in enhanced crypt cell proliferation in vivo with an improved histological outcome in DSS and irradiation induced damage. Mechanistically, the pro-regenerative effect of LXR is achieved by inducing amphiregulin expression in epithelial cells and regulated by the upstream endogenous ligands producing enzyme Cyp27a1. Surprisingly, the elevated regenerative capacity did not result in increased tumor burden, in contrast, activating LXR modulates the anti-tumor immunity and suppressed tumorigenesis in both AOM-DSS induced and ApcMin/+ tumor models. This study unveiled the bi- directional modulation of LXR in enhancing regeneration while controlling tumorigenesis.</p><p dir="ltr">In Study IV, we observed that LXR activation dampened tuft cell and Type 2 innate lymphoid cells (ILC2) abundance in the small intestine of wild type C57BL/6 mice during homeostasis. The decreased tuft cell and ILC2 number failed to be rescued by an inducer of tuft cell expansion, i.e. succinate but was rescued by rIL-25 in vitro and in vivo and. This negative regulation resulted in impaired anti-helminth response in the mouse infected by Nippostrongylus brasiliensis and Heligmosomoides polygyrus, as well as impaired mast cell activation, indicating a role of LXR in suppressing type 2 immunity. Similarly, LXR activation in BALB/c mice was found to inhibit mast cells and showed a protective role by preventing weight loss in an OVA-induced food allergy model. This study underscores the function of LXR in controlling innate type 2 immunity during mucosal homeostasis and in limiting type 2 inflammation upon challenges.</p><p dir="ltr">Collectively, this thesis contributes to spatially resolved transcriptomic profile of murine intestine in naïve and challenged conditions, underscoring the novel finding of LXR modulation in intestinal inflammation and mucosal healing. The findings in thesis further our understanding of restoring balance, enhancing tissue resilience, and paving the way for novel treatments that could transform the management of inflammatory diseases in the intestine, such as IBD.</p><h3>List of scientific papers</h3><p dir="ltr">I. Sara M. Parigi#, Ludvig Larsson#, Srustidhar Das#, Ricardo O. Ramirez Flores, Annika Frede, Kumar P. Tripathi, Oscar E. Diaz, Katja Selin, Rodrigo A. Morales, <b>Xinxin Luo</b>, Gustavo Monasterio, Camilla Engblom, Nicola Gagliani, Julio Saez-Rodriguez, Joakim Lundeberg & Eduardo J. Villablanca*</p><p dir="ltr">The spatial transcriptomic landscape of the healing mouse intestine following damage Nature Communication. 13, 828 (2022). <a href="https://doi.org/10.1038/s41467-022-28497-0" rel="noreferrer" target="_blank">https://doi.org/10.1038/s41467-022-28497-0</a></p><p dir="ltr">II. Annika Frede#, Paulo Czarnewski#, Gustavo Monasterio#, Kumar P Tripathi, David A Bejarano, Ricardo O Ramirez Flores, Chiara Sorini, Ludvig Larsson, <b>Xinxin Luo</b>, Laura Geerlings, Claudio Novella-Rausell, Chiara Zagami, Raoul Kuiper, Rodrigo A Morales, Francisca Castillo, Matthew Hunt, Livia Lacerda Mariano, Yue O O Hu, Camilla Engblom, Ana-Maria Lennon- Duménil, Romy Mittenzwei, Astrid M Westendorf, Nadine Hövelmeyer, Joakim Lundeberg, Julio Saez-Rodriguez, Andreas Schlitzer, Srustidhar Das, Eduardo J Villablanca*</p><p dir="ltr">B cell expansion hinders the stroma-epithelium regenerative cross talk during mucosal healing Immunity. 2022 Dec 13;55(12):2336-2351.e12. <a href="https://doi.org/10.1016/j.immuni.2022.11.002" rel="noreferrer" target="_blank">https://doi.org/10.1016/j.immuni.2022.11.002</a></p><p dir="ltr">III. Srustidhar Das*#, S. Martina. Parigi#, <b>Xinxin Luo</b>#, Jennifer Fransson, Bianca Carola Kern, Ali Okhovat, Oscar E. Diaz, Chiara Sorini, Paulo Czarnewski, Anna T. Webb, Rodrigo A. Morales, Sacha Lebon, Gustavo Monasterio, Francisca Castillo, Kumar P. Tripathi, Ning He, Penelope Pelczar, Nicola Schaltenberg, Marjorie De la Fuente, Francisco López-Köstner, Susanne Nylén, Hjalte List Larsen, Raoul Kuiper, Per Antonson, Marcela A. Hermoso, Samuel Huber, Moshe Biton, Sandra Scharaw, Jan-Åke Gustafsson, Pekka Katajisto, Eduardo J. Villablanca*</p><p dir="ltr">Liver X receptor unlinks intestinal regeneration and tumorigenesis. Nature. [Accepted]</p><p dir="ltr">IV. <b>Xinxin Luo</b>, Qilin Zhu, Jennifer Fransson, Tilde Andersson, Ning He, Marta Campillo Poveda, Claire Ciancia, Ricardo O. Ramírez Flores, Ludvig Larsson, Sara Martina Parigi, Rodrigo A. Morales Castro, Bianca C. Kern, Francisca Castillo, Joakim Lundeberg, Julio Saez-Rodriguez, Srustidhar Das, Rick M. Maizels, Christoph Schneider, and Eduardo J. Villablanca*</p><p dir="ltr">Liver X Receptor modulates type 2 immune responses in parasitic infection and food allergy. [Manuscript]</p><p dir="ltr"># Equal contribution</p><p dir="ltr">* Corresponding author</p>

The white water lily (Nymphaea alba) is a traditional medicinal plant recognized for its diverse array of bioactive properties. However, its potential in wound healing remains largely unexplored. This study aimed to evaluate the phytochemical profile, cytotoxicity, and wound healing efficacy of Nymphaea alba flower extract (NAFE) using both in vitro and in vivo models, as well as computational network analysis. Qualitative phytochemical screening of NAFE was conducted using standard techniques. Cytotoxicity was assessed on HaCaT keratinocyte cells at concentrations ranging from 0 to 1000 μg/ml. In vivo wound healing was evaluated using excision wound models in Wistar albino rats treated with 2.5% and 5% NAFE ointments, measuring wound contraction, epithelialization time, and breaking strength. In vitro scratch assays were used to assess cell migration at selected concentrations of NAFE. A wound-healing-associated network analysis was performed using IMPPAT, STRING, GeneCards, and OMIM databases to explore the molecular targets and interactions of bioactive compounds. Phytochemical analysis confirmed the presence of alkaloids, flavonoids, phenolics, tannins, and glycosides. NAFE was found to be non-cytotoxic with an IC50 of 245 μg/ml. In vivo, 5% NAFE ointment showed 98.92% wound closure by day 14 and complete closure by day 21, comparable to betadine. Epithelialization time (15.83±0.16 days) was nearly equivalent to the standard drug. In vitro assays demonstrated enhanced HaCaT cell migration at concentrations of 122.5 and 245 μg/ml. Network analysis identified kaempferol and quercetin as key compounds interacting with wound-healing proteins, notably AKT1, ESR1, and EGFR. The findings suggest that NAFE promotes wound healing by enhancing wound contraction, epithelialization, and cell migration, likely through the modulation of molecular pathways involved in tissue repair. The presence of bioactive compounds such as kaempferol and quercetin underpins the extract's pharmacological potential. Nymphaea alba flower extract exhibits promising wound-healing activity through multiple mechanisms, including enhancement of cell migration and regulation of key proteins involved in tissue regeneration. These results support its potential as a natural therapeutic agent in wound management.

Chronic wounds may lead to the development of various pathological conditions such as diabetic foot ulcers and pressure sores. The current study evaluated wound healing and anti-inflammatory potentials of methanolic extract of Ephedra ciliata using series of in vivo models. Methanolic extract of Ephedra ciliata was prepared by maceration (Ec.Me). Qualitative and quantitative (HPLC) phytochemical and metal analyses were conducted to explore the chemical and metal profiles of Ec.Me. Safety profile (behavioural)and, antimicrobial, antioxidant, wound healing, anti-inflammatory and anti-angiogenic potentials of Ec.Me were evaluated using well-established in vitro and in vivo models. ELISA assay was performed to estimate the effects of Ec.Me treatment on serum levels of TNF-α. HPLC analysis identified quercetin as one of the major compounds in Ec.Me. Safety study data showed that Ec.Me was safe up to the dose of 2000mg/kg. Antimicrobial assay data showed that Ec.Me was active against bacterial (Staphylococcus aureus) as well as fungal (Candida albicans and Aspergillus niger) strains. Ec.Me showed modertate antioxidant potential in in vitro and in vivo models. Data of excision and burn woundhealing models showed that Ec.Me, promoted wound closure in a dose and time-dependent manner. Treatment with 20% Ec.Me cream and heparin showed almost the same effects with no statistical differences (p > 0.05). Ec.Me also showed time-dependent anti-inflammatory activities in both acute and chronic models. In carrageenan model, treatment with 200mg/kg of Ec.Me showed comparable anti-inflammatory effects (p > 0.05) with quercetin and indomethacin throughout the study. In cotton pellet granuloma model treatment with 200mg/kg of Ec.Me and indomethacin inhibited granuloma formation significantly better (p < 0.05) as compared with the rest of the treatment groups. Histopathological examination of skin samples showed marked improvement in architecture with minimal infiltration of inflammatory cells. Data of in vivo angiogenesis assay showed marked improvement in vessels length, density, branching points, total segments and total nets after treatment with Ec.Me, indicating no toxic effects towards vasculature development. Significant (p < 0.05) downregulation of TNF-α was observed in serum samples of animals treated with Ec.Me. Based on data of the current study, it is concluded that quercetin-rich extract of Ephedra ciliata has wound healing and anti-inflammatory potentials via downregulation of TNF-α. Moreover, it is suggested that the antimicrobial activity of Ec.Me prevented microbial invasion, thus promoted natural woundhealing mechanismsas well.