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This preclinical proof-of-concept study aimed to evaluate the effectiveness of secretome therapy in diabetic mice with pressure ulcers. We utilized a custom-made hyaluronic acid (HA)-based porous sponge, which was rehydrated either with normal culture medium or secretome derived from human mesenchymal stromal cells (MSCs) to achieve a hydrogel consistency. Following application onto skin ulcers, both the hydrogel-only and the hydrogel + secretome combination accelerated wound closure compared to the vehicle group. Notably, the presence of secretome significantly enhanced the healing effect of the hydrogel, as evidenced by a thicker epidermis and increased revascularization of the healed area compared to the vehicle group. Notably, molecular analysis of healed skin revealed significant downregulation of genes involved in delayed wound healing and abnormal inflammatory response in ulcers treated with the hydrogel + secretome combination, compared to those treated with the hydrogel only. Additionally, we found no significant differences in therapeutic outcomes when comparing the use of secretome from fetal dermal MSCs to that from umbilical cord MSCs. This observation is supported by the proteomic profile of the two secretomes, which suggests a shared molecular signature responsible of the observed therapeutic effects.
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Multiple sclerosis (MS) is a complex autoimmune disease with multiple players. In particular, peripheral (myelin-reactive CD4+ T lymphocytes) and central immune cells (microglia) are involved in the neuroinflammatory process and are found in MS brain lesions. New nanotechnological approaches that can cross the blood-brain barrier and specifically target the key players in the disease using biocompatible nanomaterials with low immunoreactivity represent an important challenge. To this end, nanoparticles and nanovesicles have been studied to induce immune tolerance to a wide range of myelin-derived antigens as potential approaches against MS. To this aim, we extracted myelin from bovine brain and produced myelin-based nanovesicles (MyVes) by nanoprecipitation. MyVes have a diameter of about 100 nm, negative zeta potential and contain the typical proteins of the myelin sheath. The results showed that MyVes are not cytotoxic, are hemocompatibile and do not induce an inflammatory response. In vitro experiments showed that MyVes are specifically taken up by microglial cells and are able to induce the expression of the anti-inflammatory cytokine IL-4. In addition, we have used biodistribution experiments to show that MyVes are able to reach the brain after intranasal administration. Finally, MyVes induced the production of the anti-inflammatory cytokines IL-10 and IL-4 in peripheral blood mononuclear cells isolated from MS patients. Taken together, these data provide proof of concept that MyVes may represent a safe nanosystem capable of promoting anti-inflammatory effects by modulating both central and peripheral immune cells to treat neuroinflammation in MS. STATEMENT OF SIGNIFICANCE: Recently, nanoparticles and nanovesicles have been investigated as potential approaches for the treatment of neurodegenerative diseases. We propose the use of myelin nanovesicles (MyVes) as a potential application to counteract neuroinflammation in multiple sclerosis (MS). Approximately 2.8 million people worldwide are estimated to live with MS. It is an autoimmune disease directed toward various myelin-derived antigens. Both peripheral immune cells (lymphocytes) and central immune cells (microglia) actively contribute to MS brain lesions. MyVes, due to their myelin nature, specific characteristics (size, zeta potential, and presence of myelin proteins), biocompatibility, and ability to cross the blood-brain barrier, could represent the first nanosystem capable of promoting anti-inflammatory actions by modulating both central and peripheral immune cells to treat neuroinflammation in MS.
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An amine derivative of hyaluronic acid (HA) was crosslinked to obtain a 3D dried sponge. The sponge was subsequently rehydrated using secretome from human mesenchymal stromal cells (MSCs), resulting in the formation of a hydrogel. The release kinetics analysis demonstrated that the hydrogel effectively sustained secretome release, with 70% of the initially loaded wound-healing-associated cytokines being released over a 12-day period. Tuning the hydrogel properties through heparin crosslinking resulted in a biomaterial with a distinct mechanism of action. Specifically, the presence of heparin enhanced water uptake capacity of the hydrogel and increased its sensitivity to enzymatic degradation. Notably, the heparin crosslinking also led to a significant retention of cytokines within the hydrogel matrix. Overall, the secretome-rehydrated HA hydrogel holds promise as a versatile device for regenerative medicine applications: the non-heparinized hydrogel may function as a biomaterial with low reabsorption rates, sustaining the release of bioactive molecules contained in MSC secretome. In contrast, the heparinized hydrogel may serve as a depot of bioactive molecules with faster reabsorption rates. Given its patch-like characteristic, the HA-based hydrogel appears suitable as topical treatment for external organs, such as the skin.
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Hidrogéis , Células-Tronco Mesenquimais , Humanos , Hidrogéis/farmacologia , Ácido Hialurônico/farmacologia , Secretoma , Células-Tronco Mesenquimais/metabolismo , Heparina , Materiais Biocompatíveis/metabolismo , Citocinas/metabolismoRESUMO
Infected skin wounds represent a serious health threat due to the long healing process and the risk of colonization by multi-drug-resistant bacteria. Silver nanoparticles (AgNPs) have shown broad-spectrum antimicrobial activity. This study introduces a novel approach to address the challenge of infected skin wounds by employing gellan gum-dopamine (GG-DA) as a dual-functional agent, serving both as a reducing and capping agent, for the in situ green synthesis of silver nanoparticles. Unlike previous methods, this work utilizes a spray-drying technique to convert the dispersion of GG-DA and AgNPs into microparticles, resulting in nano-into-micro systems (AgNPs@MPs). The microparticles, with an average size of approximately 3 µm, embed AgNPs with a 13 nm average diameter. Furthermore, the study explores the antibacterial efficacy of these AgNPs@MPs directly and in combination with other materials against gram-positive and gram-negative bacteria. The versatility of the antimicrobial material is showcased by incorporating the microparticles into injectable hydrogels. These hydrogels, based on oxidized Xanthan Gum (XGox) and a hyperbranched synthetic polymer (HB10K-G5-alanine), are designed with injectability and self-healing properties through Shiff base formation. The resulting nano-into-micro-into-macro hybrid hydrogel emerges as a promising biomedical solution, highlighting the multifaceted potential of this innovative approach in wound care and infection management.
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Anti-Infecciosos , Nanopartículas Metálicas , Polissacarídeos Bacterianos , Antibacterianos , Hidrogéis , Prata , Dopamina , Bactérias Gram-Negativas , Bactérias Gram-PositivasRESUMO
Treating wound infections is a difficult task ever since pathogenic bacteria started to develop resistance to common antibiotics. The present study develops hybrid hydrogels based on the formation of a polyelectrolyte complex between the anionic charges of dopamine-functionalized Gellan Gum (GG-DA) and the cationic moieties of the TMP-G2-alanine dendrimer. The hydrogels thus obtained can be doubly crosslinked with CaCl2 , obtaining solid hydrogels. Or, by oxidizing dopamine to GG-DA, possibly causing further interactions such as Schiff Base and Michael addition to take place, hydrogels called injectables can be obtained. The latter have shear-thinning and self-healing properties (efficiency up to 100%). Human dermal fibroblasts (HDF), human epidermal keratinocytes (HaCaT), and mouse monocyte cells (RAW 264.7), after incubation with hydrogels, in most cases show cell viability up to 100%. Hydrogels exhibit adhesive behavior on various substrates, including porcine skin. At the same time, the dendrimer serves to crosslink the hydrogels and endows them with excellent broad-spectrum microbial eradication activity within four hours, evaluated using Staphylococcus aureus 2569 and Escherichia coli 178. Using the same GG-DA/TMP-G2-alanine ratios hybrid hydrogels with tunable properties and potential for wound dressing applications can be produced.
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Dendrímeros , Hidrogéis , Camundongos , Animais , Suínos , Humanos , Hidrogéis/farmacologia , Hidrogéis/química , Dopamina , Dendrímeros/farmacologia , Cicatrização , Antibacterianos/farmacologia , Antibacterianos/química , AlaninaRESUMO
The development of biomedical systems with antimicrobial and antibiofilm properties is a difficult medical task for preventing bacterial adhesion and growth on implanted devices. In this work, a fibrillar scaffold was produced by electrospinning a polymeric organic dispersion of polylactic acid (PLA) and poly(α,ß-(N-(3,4-dihydroxyphenethyl)-L-aspartamide-co-α,ß-N-(2-hydroxyethyl)-L-aspartamide) (PDAEA). The pendant catechol groups of PDAEA were used to reduce silver ions in situ and produce silver nanoparticles onto the surface of the electrospun fibers through a simple and reproducible procedure. The morphological and physicochemical characterization of the obtained scaffolds were studied and compared with virgin PLA electrospun sample. Antibiofilm properties against Pseudomonas aeruginosa, used as a biofilm-forming pathogen model, were also studied on planar and tubular scaffolds. These last were fabricated as a proof of concept to demonstrate the possibility to obtain antimicrobial devices with different shape and dimension potentially useful for different biomedical applications. The results suggest a promising approach for the development of antimicrobial and antibiofilm scaffolds.
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Anti-Infecciosos , Nanopartículas Metálicas , Prata/farmacologia , Prata/química , Nanopartículas Metálicas/química , Pseudomonas aeruginosa , Biofilmes , Antibacterianos/farmacologia , Antibacterianos/químicaRESUMO
A microfibrous tubular scaffold has been designed and fabricated by electrospinning using poly (1,4-butylene succinate) as biocompatible and biodegradable material. The scaffold morphology was optimized as a small diameter and micro-porous conduit, able to foster cell integration, adhesion, and growth while avoiding cell infiltration through the graft's wall. Scaffold morphology and mechanical properties were explored and compared to those of native conduits. Scaffolds were then seeded with adult normal human dermal fibroblasts to evaluate cytocompatibility in vitro. Haemolytic effect was evaluated upon incubation with diluted whole blood. The scaffold showed no delamination, and mechanical properties were in the physiological range for tubular conduits: elastic modulus (17.5 ± 1.6 MPa), ultimate tensile stress (3.95 ± 0.17 MPa), strain to failure (57 ± 4.5%) and suture retention force (2.65 ± 0.32 N). The shown degradation profile allows the graft to provide initial mechanical support and functionality while being colonized and then replaced by the host cells. This combination of features might represent a step toward future research on PBS as a biomaterial to produce scaffolds that provide structure and function over time and support host cell remodelling.
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The development of wound dressings with combined antioxidant, antibacterial and tissue adhesion functions has been a difficult medical task for the treatment of wound infections. We synthetized a dopamine and PEG functionalized Gellan Gum (GG) to produce an injectable hydrogel with radical scavenging activity having both specific and aspecific antibiotic/antimicrobial properties. Using starting GG with different molecular weights, we obtained two derivatives that have been used to prepare the gel precursor dispersion, that undergoes gelation in the presence of colistin and dried microparticles (MPs) functionalized on the surface with polydopamine (pDA). Both were used to dope the hydrogel, increase the radical scavenger activity and impart near-infrared light (NIR) responsiveness. Indeed, with an irradiation of 810 nm, the incorporated microparticles exhibit photothermal transformation properties and improve the release of antibiotics on demand. The combination of photothermal and antibiotic therapy with synergistic antibacterial action acts on Pseudomonas aeruginosa and leads to a bactericidal effect in a few hours, while on Staphylococcus aureus there is an effect of inhibition of growth over time due only to the hyperthermic effect. We believe this study provides a promising method for fabricating a multifunctional injectable hydrogel for the potential treatment of infected skin wounds.
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Hipertermia Induzida , Infecção dos Ferimentos , Humanos , Hidrogéis/farmacologia , Antioxidantes/farmacologia , Dopamina , Colistina/farmacologia , Antibacterianos/farmacologia , Cicatrização , Infecção dos Ferimentos/tratamento farmacológicoRESUMO
In this work a synthetic protocol for the functionalization of hyaluronic acid with diethylenetriamine (DETA) was standardized. HA-DETA derivatives were characterized by NMR and proton carbon correlation analysis HSQC and HMBC to confirm chemical structure. A selected derivative was used to set up a green fabrication procedure for HA-DETA capped silver nanoparticles with the aim to achieve a polymeric based coating with potential application in the treatment of medical devices associated infections. Data from UV-visible spectroscopy, electron scanning and transmission microscope (STEM), photoelectric spectroscopy (XPS) and rheological characterization were combined to characterize the HA-DETA/Ag nanocomposites. HA-DETA stabilized Ag nanoparticles (10-30 nm) were obtained through an UV accelerated production. The viability of MC3T3-E1 was analyzed with the aim of designing a cytocompatible antimicrobial coating. Antibacterial and antibiofilm activity of HA-DETA/Ag nanocomposites have been tested in vitro against Staphylococcus aureus and Pseudomonas aeruginosa both in culture plates than on titanium specimens.
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Nanopartículas Metálicas , Prata , Antibacterianos/química , Antibacterianos/farmacologia , DEET , Ácido Hialurônico , Nanopartículas Metálicas/química , Poliaminas , Prata/química , Prata/farmacologiaRESUMO
Here we reported the fabrication of an electrospun membrane based on a hyaluronic acid derivative (HA-EDA) to be used as a bandage for the potential treatment of chronic wounds. The membrane, loaded with graphene oxide (GO) and ciprofloxacin, showed photothermal properties and light-triggered drug release when irradiated with a near-infrared (NIR) laser beam. Free amino groups of HA-EDA derivative allowed autocrosslinking of the electrospun membrane; thus, a substantial enhancement in the hydrolytic resistance of the patch was obtained. In vitro antibacterial activity studies performed on Staphylococcus aureus and Pseudomonas aeruginosa revealed that such electrospun membranes, due to the synergistic effect of the antibiotic and NIR-mediated hyperthermia, reduced the viability of both pathogens. Specific in vitro experiment demonstrated also that is possible to disrupt, through laser irradiation, the biofilms formed onto the membrane.
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Grafite , Infecções Estafilocócicas , Infecção dos Ferimentos , Antibacterianos/farmacologia , Grafite/farmacologia , Humanos , Ácido Hialurônico/farmacologia , Pseudomonas aeruginosa , Staphylococcus aureus , Infecção dos Ferimentos/tratamento farmacológicoRESUMO
Here, for the production of a bioink-based gellan gum, an amino derivative of this polysaccharide was mixed with a mono-functionalized aldehyde polyethyleneglycol in order to improve viscoelastic macroscopic properties and the potential processability by means of bioprinting techniques as confirmed by the printing tests. The dynamic Schiff base linkage between amino and aldehyde groups temporally modulates the rheological properties and allows a reduction of the applied pressure during extrusion followed by the recovery of gellan gum strength. Rheological properties, often related to printing resolution, were extensively investigated confirming pseudoplastic behavior and thermotropic and ionotropic responses. The success of bioprinting is related to different parameters. Among them, cell density must be carefully selected, and in order to quantify their role on printability, murine preostoblastic cells (MC3T3-E1) and human colon tumor cells (HCT-116) were chosen as cell line models. Here, we investigated the effect of their density on the bioink's rheological properties, showing a more significant difference between cell densities for MC3T3-E1 compared to HCT-116. The results suggest the necessity of not neglecting this aspect and carrying out preliminary studies to choose the best cell densities to have the maximum viability and consequently to set the printing parameters.
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An alkyl functionalized gellan gum derivative is here used to produce hydrogels containing hydroxyapatite and tricalcium phosphate nanoparticles as injectable nanostructured scaffolds for bone regeneration. The amphiphilic nature of the polysaccharide derivative along with its thermotropic behavior and ionotropic crosslinking features make possible to produce injectable bone mimetic scaffolds that can be used to release viable cells and osteoinductive biomolecules. The influence of different nanoparticles concentration on the rheological and physicochemical properties of the injectable systems is studied. It is found that the presence of inorganic nanoparticles reinforces the 3D hydrated polymeric networks without influencing their injectability but improving the physicochemical properties of ionotropic crosslinked hydrogels produced with two different curing media. Preliminary cytocompatibility tests performed with murine preosteoblast cells revealed that gellan gum based hydrogels can safely encapsulate viable cells. Loading and release experiments for dexamethasone and stromal cell-derived factor-1 demonstrate the drug delivery features of the obtained injectable systems.
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Hidrogéis , Nanopartículas , Animais , Regeneração Óssea , Fosfatos de Cálcio , Durapatita/farmacologia , Hidrogéis/química , Hidrogéis/farmacologia , Camundongos , Polissacarídeos Bacterianos , Engenharia TecidualRESUMO
Neurodegenerative diseases affect millions of people worldwide and the presence of various physiological barriers limits the accessibility to the brain and reduces the efficacy of various therapies. Moreover, new carriers having targeting properties to specific brain regions and cells are needed in order to improve therapies for the brain disorder treatment. In this study, for the first time, Myelin nanoVesicles (hereafter defined MyVes) from brain-extracted myelin were produced. The MyVes have an average diameter of 100-150 ânm, negative zeta potential, spheroidal morphology, and contain lipids and the key proteins of the myelin sheath. Furthermore, they exhibit good cytocompatibility. The MyVes were able to target the white matter and interact mainly with the microglia cells. The preliminary results here presented allow us to suppose the employment of MyVes as potential carrier to target the white matter and microglia in order to counteract white matter microglia-related diseases.
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In this work, with the aim to obtain a wound dressing hydrogel, an amine derivative of gellan gum was crosslinked in the presence of 4arm-polyethylenglycole-vinylsulfone. Through this easy and reproducible chemical procedure, a hydrogel with advanced elastic properties and hydrolytic resistance under physiological conditions was obtained. The incorporation of different quantities of polydopamine in the gelling solutions allows to obtain different hydrogels with marked photothermal properties when irradiated with a laser in the near infrared at 810 nm. The organic nanoparticles, reacting with the amino groups of the polysaccharide derivative, contribute to increase the storage moduli of the hydrogels. Ciprofloxacin was loaded into the hydrogel with higher amount of polydopamine and drug delivery experiments were performed to investigate the effect of irradiation on the antibiotic release profile. Antimicrobial studies, evaluated against S. aureus and P. aeruginosa, revealed that generated hyperthermia exerts a direct inhibition on the pathogens growth and, in the case of S. aureus, adjuvates the ciprofloxacin antimicrobial effect.
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Ciprofloxacina , Hidrogéis , Indóis , Polímeros , Polissacarídeos Bacterianos , Staphylococcus aureusRESUMO
A series of four different low molecular weight gellan gum products was obtained by alkaline hydrolysis with the aim to investigate the impact of the molecular weight on the rheological properties of the polysaccharide aqueous dispersions and on the physicochemical characteristics of derived ionotropic crosslinked hydrogels. In particular, thermo-rheological analysis was conducted on aqueous dispersions to study the influence of molecular weight on the thermogelation properties typical of the native polysaccharide while strain sweep experiments were conducted to establish if aqueous dispersion shows a viscoelastic behavior. The effect of different Ca2+ on the rheological properties of hydrogels were studied. Furthermore, ionotropic crosslinked hydrogels were analyzed in terms of morphology on the dried state and swelling behavior, while their viscoelastic properties were studied by means of rheological analysis conducted in frequency sweep regime after different time points of incubation in phosphate buffer at pH 7.4. Release experiments conducted using fluorescein isothiocyanate labelled dextran as a model diffusion agent and was performed to investigate the possibility of using the low molecular weight GG-derived hydrogels as an active molecule-releasing device. Finally, the cytocompatibility of hydrolysis products was investigated, as well as the capacity of hydrogels to encapsulate viable MC3T3-E1 preosteoblastic cells.
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Localized delivery of anticancer drugs is often the most useful therapeutic approach for the treatment of solid tumors. The use of injectable polymeric systems that maximize drug concentration in the proximal area of the tumor represents an extremely advantageous therapeutic strategy. Here, the development of an injectable in situ forming hydrogel was accomplished by exploiting the azo-type Michael reaction between an amine derivative of hyaluronic and vinylsulfone functionalized ß-cyclodextrins complexing doxorubicin. This injectable system can be easily prepared and administered with timelines compatible with normal operating room procedures, as demonstrated by rheological tests. In vitro experiments revealed that the peculiar physicochemical properties of the hydrogel guarantee a sustained release of the anticancer drug that blocks the growth of colorectal carcinoma micromasses cultured in 3D conditions. In vivo studies have confirmed that the medicated hydrogel can drastically reduce the tumor mass in the animal model without causing cytotoxic side effects in other areas of the body such as the heart. Overall, the proposed system has shown promising characteristics that make it an interesting useful device for localized chemotherapy of solid tumors.
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Ciclodextrinas , Neoplasias , Animais , Doxorrubicina , Sistemas de Liberação de Medicamentos , Ácido Hialurônico , Hidrogéis , Neoplasias/tratamento farmacológicoRESUMO
This research focused on the aim of tackling the urgent demand of printable biomaterials, hence we synthetized and characterized three gellan gum-graft-poly(d,l-lactide-co-glycolide) copolymers (GGm-PLGA a, b and c) which differed in the graft substitution degree. We investigated the effect of the polyester chain grafted onto hydrophilic backbone of gellan gum in terms of physicochemical properties and the ability of the system to print 3D cell laden constructs. In particular, we evaluated thermo-rheological, ionotropic crosslinking, shear thinning, swelling and stability properties of these copolymers and their derived biomaterials and findings related to the degree of functionalization. Moreover, the optimization of the 3D process parameters and the effect of different water/DPBS mixtures was investigated, demonstrating the feasibility of the system to print 3D constructs. Finally, biological tests revealed that fibroblasts and chondrocytes remained viable after printing and over a culture period of seven days into scaffolds.
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Materiais Biocompatíveis/química , Tinta , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Polissacarídeos Bacterianos/química , Células 3T3 , Animais , Teste de Materiais , Fenômenos Mecânicos , Camundongos , Reologia , Engenharia TecidualRESUMO
The production of an amine derivative of gellan gum, named GG-EDA, was here obtained by functionalizing the polysaccharide backbone with pendant ethylenediamine moieties. The obtained derivative was characterized by spectroscopic, colorimetric, chromatographic and rheological analyses to study the effect of the free amino groups on the physicochemical properties of the macromolecule. A titration experiment was conducted to study the acid-base dissociation constants in aqueous media for the carboxylic and amino groups in the GG-EDA and to shed light on the possibility that the derivative shows a polyampholyte structure under physiological conditions. The rheological analysis conducted on both physical and chemical hydrogels based on GG-EDA revealed that the presence of amino groups plays a fundamental role in influencing the viscoelastic properties and stability of the produced samples.
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The purpose of this review is to make a summary of high quality research trends using gellan gum (GG) as a polymeric constituent for the design of innovative drug delivery systems and devices for biomedical applications, such as cell therapy and regenerative medicine. The use of gellan gum is described both in its native form and as chemically functionalized derivatives or physically mixed with natural or synthetic materials. Starting from a systematic study of recent research works, the main properties of the native polysaccharide have been highlighted and therefore some improvements have been focused thanks to the design of chemically functionalized derivatives and the use of composite materials.
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Terapia Baseada em Transplante de Células e Tecidos , Sistemas de Liberação de Medicamentos , Polissacarídeos Bacterianos/administração & dosagem , Animais , HumanosRESUMO
Microfluidics is emerging as an innovative technique for the "on chip" fabrication of nanoparticles for drug delivery applications. Here, by using an amphiphilic derivative of hyaluronic acid as a starting macromolecule, nanohydrogels loaded with Imatinib were produced by the microfluidic procedure in order to develop an innovative therapeutic tool for the treatment of retinal neovascularization. Both cyRGDC functionalized and non-functionalized nanohydrogels were designed and fabricated by using the same technique. The targeting efficiency of the obtained nanosystems was studied in vitro on human retinal pigment epithelial cells (HRPEpiC) and human umbilical vein endothelial cells (HUVEC), the latter chosen as generic cellular model to assay inhibiting effect on cellular sprouting of Imatinib loaded nanohydrogels. The suitability of microfluidic approach for nanohydrogel production and drug loading was demonstrated. The cyRGDC functionalized nanosystems loaded with Imatinib, showed in vitro an enhanced ability to inhibit HUVEC organization into a capillary like structure.