Functional biopolymer-based matrices for modulation of chronic wound enzyme activities

Collagen, collagen/hyaluronic acid (HA) and collagen/HA/chitosan (CS) sponges loaded with epigallocatechin gallate (EGCG), catechin (CAT) and gallic acid (GA) were developed and evaluated as active chronic wound dressings. Their physico-mechanical properties, biostability, biocompatibility and ability to inhibit in vitro myeloperoxidase (MPO) and collagenase—major enzymes related with the persistent inflammation in chronic wounds—were investigated as a function of the biopolymer composition and the polyphenolic compound used. The results demonstrated that the molecular weight of HA influences significantly the bulk properties of the obtained materials: higher elastic modulus, swelling ability and biostability against collagenase were measured when HA with higher molecular weights (830 and 2000 kDa) were added to the collagen matrices. The addition of CS and the polyphenols increased further the biostability of the sponges. Preliminary in vitro tests with fibroblasts revealed that the cells were able to adhere to all sponges. Cell viability was not affected significantly by the addition of the polyphenols; however, the presence of CS or high molecular weight HA in the sponge composition was associated with lower cellular viability. Finally, all specimens containing polyphenols efficiently inhibited the MPO activity. The highest inhibition capacity was observed for EGCG (IC50 = 15 ± 1 lM) and it was coupled to the highest extent of binding to the biopolymers (>80%) and optimal release profile from the sponges that allowed for prolonged (up to 3–5 days) effects.Spanish Ministerio de Ciencia e Innovació

GAGs-thiolated chitosan assemblies for chronic wounds treatment: control of enzyme activity and cell attachment

Multilayered polyelectrolyte coatings comprising thiolated chitosan (TC) and glycosaminoglycans (GAGs), chondroitin sulphate and hyaluronic acid, were built using a layer-by-layer approach. The surface activity of these coatings for binding and inhibition of enzymes related to chronic inflammation, such as collagenase and myeloperoxidase, was assessed. The build-up of five bi-layers of TC/GAGs onto gold surfaces was monitored in situ by QCM-D. All experimental groups showed exponential growth of the coatings controlled by the degree of chitosan thiolation and the molecular weight of the GAGs. The degree of chitosan modification was also the key parameter influencing the enzyme activity: increasing the thiols content led to more efficient myeloperoxidase inhibition and was inversely proportional to the adsorption of collagenase. Enhanced fibroblast attachment and proliferation were observed when the multilayered polyelectrolyte constructs terminated with GAGs. The possibility to control either the activity of major wound enzymes by the thiolation degree of the coating or the cell adhesion and proliferation by proper selection of the ultimate layer makes these materials potentially useful in chronic wounds treatment and dermal tissue regeneration.EU projects Lidwine (contract no. FP6-026741)Ministerio de Ciencia e Innovaci on de España - (MICINN) - bolsa BES-2008-0037

Sulfation degree of glycosaminoglycans triggers distinct cytoskeleton organisation in mesenchymal stem cells

Glycosaminoglycans (GAGs) comprise the closest cellular environment: they are building elements of the ECM and can be also found on cells surface. Their biological activity depends on several parameters among which the negative charge is of prime importance[1]. This charge is generally associated with the presence of sulfate groups (-OSO3H). Sulfation is a dynamic modification: it can occur at various positions within the glycan and different sulfation patterns have been identified for the same organs and cells during their development. However, the mechanisms of coding and transferring information by these functionalities are not yet complete understood, mainly because of (i)the complex physiological microenvironment in which GAGs interactions occur and (ii)the inability to access homogeneous GAGs[2]. In this work, we propose model surfaces bearing GAGs with different sulfation degree as platform to investigate the pathways by which mesenchymal stem cells (MSCs) sense and respond to this peculiar functionality: the -OSO3H. We have selected two natural GAGs for this study: hyaluronic acid (HA) because it is the only non-sulfated glycan and heparin (HEP) as it is the GAG with the highest degree of sulfation. To obtain a larger range of sulfation degrees, we have also prepared a synthetic analogue of HA with a sulfation degree of 1.4 (sHA). All these GAGs were covalently bonded to aminothiols deposited on gold surfaces. MSCs, both from bone marrow and adipose tissue, adhered well to all surfaces. Formation of focal adhesions was observed after only 1h of culture for bone marrow derived MSCs regardless the used substrate. The presence of –OSO3H groups induced different morphology and cytoskeleton organisation: formation of longer filopodia and well pronounced actin fibers were visible for the MSCs from both sources. Moreover, cells were more spread after 24h in contact with – OSO3H containing surfaces. Cells behaved similarly on both sulfated surfaces (sHA and HEP) and differences in cell morphology were less obvious: higher sulfation degree induced less lamellipodia formation while filopodia number and length increased. In summary, the present study provides evidence that sulfation degree of GAGs triggers distinct cytoskeleton organisation in mesenchymal stem cells that may be related with the differentiation of those cells. However, further studies at the molecular level about the exact mechanism of these processes need to be carried out

Hyaluronan functionalized pH-responsive calcium carbonate nanoparticles for local treatment of breast cancer

[Excerpt] Introduction: Current radio- and chemotherapies are not efficient and many tumors remain resistant to conventional cancer treatments. Specific properties and signaling molecules from tumor microenvironment (TME) have been explored to increase treatment efficacy. An example is the acidic pH of the TME that has been explored to develop stimuli responsive release systems. Herein, we obtained biocompatible calcium carbonate (CaCO3) nanoparticles that are stable at neutral pH but dissolve at acidic conditions and evaluated their potential as a drug carrier for local cancer treatment. METHODS: CaCO3 nanoparticles were produced by co-precipitation of calcium chloride (CaCl2) and sodium carbonate (Na2CO3) in the presence of ethylene glycol [1]. Rhodamine was encapsulated as a model drug. The morphology and diameter of the nanoparticles were determined by scanning electron microscopy and dynamic laser scattering. The particles were coated by layer-by-layer (LbL) assembly of poly-L-lysine and hyaluronic acid (HA). The release was studied in phosphate buffered saline at pH 6.3 and 7.4. Cultures of two breast cancer cell lines (MDA-MB-231 and SK-BR-3) and a healthy epithelial cell line MCF10A (control) were observed under confocal laser scanning microscopy to assess particles internalization and their effect on cell viability (live/dead staining) and metabolic activity (Alamar Blue). [...]Portuguese Fundação para a Ciência e Tecnologia (FCT, grants CEECIND/02842/2017, SFRH/BPD/85790/2012; project PTDC/CTM-REF/0022/2020)

Sulfonic groups induce formation of filopodia in mesenchymal stem cells

Glycosaminoglycans (GAGs) are an integral part of the extracellular matrix and glycocalix, i.e. the closest cellular environment. They are abundant in –OH groups and their bioactivity is also associated with the presence of negatively charged –SO3H functionalities. Therefore, we have investigated and discussed the influence of these functional units on mesenchymal stem cells (MSCs) behaviour using single component and mixed self-assembled monolayers of alkanethiols with –SO3H and –OH end groups. In the absence of serum, MSCs attachment, spreading, cytoskeleton organisation and motility were significantly influenced by the surface chemistry. We found that the sulfonic groups induce starlike cell shape with very intense actin staining and a high density net of filopodia that enlarge from the base of lamellipodia structures. Moreover, this response is concentration dependent and is apparent only for very short culture time in the presence of serum.Fundação para a Ciência e a Tecnologia (FCT

Layer-by-layer coated calcium carbonate nanoparticles for targeting breast cancer cells

Breast cancer is resistant to conventional treatments due to the specific tumour microenvironment, the associated acidic pH and the overexpression of receptors that enhance cells tumorigenicity. Herein, we optimized the synthesis of acidic resorbable calcium carbonate (CaCO3) nanoparticles and the encapsulation of a low molecular weight model molecule (Rhodamine). The addition of ethylene glycol during the synthetic process resulted in a particle size decrease: we obtained homogeneous CaCO3 particles with an average size of 564 nm. Their negative charge enabled the assembly of layer-by-layer (LbL) coatings with surface-exposed hyaluronic acid (HA), a ligand of tumour-associated receptor CD44. The coating decreased Rhodamine release by two-fold compared to uncoated nanoparticles. We demonstrated the effect of nanoparticles on two breast cancer cell lines with different aggressiveness â SK-BR-3 and the more aggressive MDA-MB-231 â and compared them with the normal breast cell line MCF10A. CaCO3 nanoparticles (coated and uncoated) significantly decreased the metabolic activity of the breast cancer cells. The interactions between LbL-coated nanoparticles and cells depended on HA expression on the cell surface: more particles were observed on the surface of MDA-MB-231 cells, which had the thickest endogenous HA coating. We concluded that CaCO3 nanoparticles are potential candidates to carry low molecular weight chemotherapeutics and deliver them to aggressive breast cancer sites with an HA-abundant pericellular matrix. This work was supported by the Fundação para a Ciência e Tecnologia (project OncoNeoTreat, grant number PTDC/CTM-REF/0022/2020, co-financed by FCT – OE component); and the European program FEDER/FEEI. R.R.C. acknowledges FCT for support through grants 2022.00764.CEECIND and CEECIND/02842/2017. D.S.C. thanks FCT for the grant SFRH/BPD/85790/2012. Parts of Fig. A.1 were drawn by using pictures from Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/)

Fucoidan-based hydrogels particles as versatile carriers for diabetes treatment strategies

There is a current lack of fully efficient therapies for diabetes mel-litus, a chronic disease where the metabolism of blood glucose isseverely hindered by a deficit in insulin or cell resistance to thishormone. Therefore, it is crucial to develop new therapeutic strat-egies to treat this disease, including devices for the controlleddelivery of insulin or encapsulation of insulin-producing cells. Inthis work, fucoidan (Fu)â a marine sulfated polysaccharide exhib-iting relevant properties on reducing blood glucose and antioxi-dant and anti-inflammatory effectsâ was used for thedevelopment of versatile carriers envisaging diabetes advancedtherapies. Fu was functionalized by methacrylation (MFu) using8% and 12% (v/v) of methacrylic anhydride and further photo-crosslinked using visible light in the presence of triethanolamineand eosin-y to produce hydrogel particles. Degree of methacryla-tion varied between 2.78 and 6.50, as determined by1HNMR, andthe produced particles have an average diameter ranging from0.63 to 1.3mm (dry state). Insulin (5%) was added to MFu solutionto produce drug-loaded particles and the release profile wasassessed in phosphate buffer solution (PBS) and simulated intes-tinal fluid (SIF) for 24h. Insulin was released in a sustained man-ner during the initial 8 h, reaching then a plateau, higher in PBSthan in SIF, indicating that lower pH favors drug liberation.Moreover, the ability of MFu particles to serve as templates forthe culture of human pancreatic cells was assessed using 1.1B4cell line during up to 7 days. During the culture period studied,pancreatic beta cells were proliferating, with a global viabilityover 80% and tend to form pseudo-islets, thus suggesting thatthe proposed biomaterial could be a good candidate as versatilecarrier for diabetes treatment as they sustain the release of insulinand support pancreatic beta cells viability.We acknowledge ERDF for the financial support through POCTEP Project 0687_NOVOMAR_1_P, under the scope of INTERREG 2007-2013, and project 0302_CVMAR_I_1_P, under the scope of INTERREG Espana-Portugal 2014-2020, and Structured Projects NORTE-01-0145-FEDER-000021, NORTE-01-0145-FEDER-000023 and ATLANTIDA (ref. NORTE-01–0145-FEDER-000040), under the scope of Programa Operacional Regional do Norte (Norte 2020). Funding from the Portuguese Foundation for Science and Technology for doctoral grant (SFRH/BD/112139/2015) and post-doctoral grant (SFRH/BPD/85790/2012) is also acknowledge

Surfaces mimicking glycosaminoglycans trigger different response of stem cells via distinct fibronectin adsorption and reorganization

We report on the utility of a platform created by self-assembled monolayers to investigate the influence of the degree of sulfation of glycosaminoglycans (GAGs) on their interactions with fibronectin (Fn) and the impact of these interactions on the adhesion and morphology of human adipose derived stem cells (ASCs). We used the label-free QCM-D, AFM and SPR to follow the changes in the protein adlayer in close proximity to the substrates surface and QCM-D in combination with live imaging to characterize the adherent cells. Our results suggest that Fn interactions with GAGs are governed by both H-bonding and electrostatic forces. Strong electrostatic interactions cause irreversible change in the protein conformation, while the weaker H-bonding only partially restricts the protein flexibility, allowing Fn reorganization and exposure of its binding sites for ASC adhesion. These findings imply that a delicate balance between these two types of forces must be considered in the design of biomaterials that mimic GAGs.We acknowledge EU for financial support (FP7/2007-2013, under Grants REGPOT-CT2012-316331-POLARIS and ERC- 2012-ADG-20120216-321266 for the project ComplexiTE). I.P. is thankful to the Portuguese Foundation for Science and Technology (IF/00032/2013). The authors are very grateful to Dr. Raquel Teixeira for her help with the treatment and discussion of the MP-SPR data

Thiolated chitosan/glycosaminoglycans multilayered films : QCM-D study on the films formation and their biological properties

Layer-by-layer technique is widely used to produce polyelectrolyte multilayered films for material surface functionalization. The technique simplicity coupled with the biological potential of biopolymers, e.g. polysaccharides, make such assemblies a suitable choice for many biomedical applications. In this study the formation of the films comprising of thiolated chitosan and glycosaminoglycans (GAGs) by alternate deposition was assessed in situ using QCM-D. Thiolated chitosan was used under the hypothesis of disulfide formation between its molecules to increase stability and/or stiffness of the films. The effects of the chitosan modification degree and GAGs molecular weight on the film thickness were investigated. All experimental groups showed exponential film growth, while the thickness increased with the chitosan thiolation degree and molecular weight of GAGs. Cellular behavior on the assemblies was found to be tunable by the appropriate selection of the terminate layer. Antimicrobial activity and protein adsorption on the new constructs are also commented