RESUMO
A synthetic cell mimic in the form of giant glycosylated polymersomes (GGPs) comprised of a novel amphiphilic diblock copolymer is reported. A synthetic approach involving a poly(dimethylsiloxane) (PDMS) macro-chain transfer agent (macroCTA) and postpolymerization modification was used to marry the hydrophobic and highly flexible properties of PDMS with the biological activity of glycopolymers. 2-Bromoethyl acrylate (BEA) was first polymerized using a PDMS macroCTA ( Mn,th ≈ 4900 g·mol-1, D = 1.1) to prepare well-defined PDMS- b-pBEA diblock copolymers ( D = 1.1) that were then substituted with 1-thio-ß-d-glucose or 1-thio-ß-d-galactose under facile conditions to yield PDMS- b-glycopolymers. Compositions possessing ≈25% of the glycopolymer block (by mass) were able to adopt a vesicular morphology in aqueous solution (≈210 nm in diameter), as indicated by TEM and light scattering techniques. The resulting carbohydrate-decorated polymersomes exhibited selective binding with the lectin concanavalin A (Con A), as demonstrated by turbidimetric experiments. Self-assembly of the same diblock copolymer compositions using an electroformation method yielded GGPs (ranging from 2-20 µm in diameter). Interaction of these cell-sized polymersomes with fimH positive E. coli was then studied via confocal microscopy. The glucose-decorated GGPs were found to cluster upon addition of the bacteria, while galactose-decorated GGPs could successfully interact with (and possibly immobilize) the bacteria without the onset of clustering. This demonstrates an opportunity to modulate the response of these synthetic cell mimics (protocells) toward biological entities through exploitation of selective ligand-receptor interactions, which may be readily tuned through a considered choice of carbohydrate functionality.
Assuntos
Dimetilpolisiloxanos/química , Escherichia coli/química , Polímeros/química , Glicosilação , Interações Hidrofóbicas e Hidrofílicas , Microscopia Eletrônica de Transmissão , Nefelometria e Turbidimetria , Espalhamento de RadiaçãoRESUMO
Novel approaches for culturing primary human cells in vitro are increasingly needed to study cell and tissue physiology and to grow replacement tissue for regenerative medicine. Conventional 2D monolayer cultures of endometrial epithelial and stromal cells fail to replicate the complex 3D architecture of tissue. A fully synthetic scaffold that mimics the microenvironment of the human endometrium can ultimately provide a robust platform for investigating tissue physiology and, hence, take significant steps toward tackling female infertility and IVF failure. In this work, emulsion-templated porous polymers (known as polyHIPEs) were investigated as scaffolds for the culture of primary human endometrial epithelial and stromal cells (HEECs and HESCs). Infiltration of HEECs and HESCs into cell-seeded polyHIPE scaffolds was assessed by histological studies, and phenotype was confirmed by immunostaining. Confocal microscopy revealed that the morphology of HEECs and HESCs is representative of that found in vivo. RNA sequencing was used to investigate transcriptome differences between cells grown on polyHIPE scaffolds and in monolayer cultures. The differentiation status of HEECs and HESCs grown in polyHIPE scaffolds and in monolayer cultures was further evaluated by monitoring the expression of endometrial marker genes. Our observations suggest that a 3D cell culture model that could approximate native human endometrial architecture and function can be developed using tailored polyHIPE scaffolds.
Assuntos
Diferenciação Celular , Endométrio/citologia , Polímeros/farmacologia , Estirenos/farmacologia , Alicerces Teciduais/química , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Feminino , Humanos , Polímeros/química , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Estirenos/química , Alicerces Teciduais/efeitos adversosRESUMO
Since their discovery in 1993, interest in various aspects of cyclic peptides (CPs) has expanded rapidly. Of particular note is their potential to form artificial ion channels in lipid membranes, an attractive characteristic in supramolecular chemistry and biological research. The design and synthesis of cyclic peptide-polymer conjugates (CPPCs) that can self-assemble within lipid bilayers into nanotubes, mimicking naturally occurring membrane channels and pores, has been reported. However, methods that allow direct detection of the transport process with high levels of certainty are still lacking. This work focuses on the development of a simple but reliable approach to verify and quantify proton transport across a bilayer membrane. Giant unilamellar vesicles (GUVs) are created via the electroformation method and CPPCs are incorporated in GUV membranes at varying concentrations (0-10%). Confocal fluorescence microscopy is used to demonstrate full inclusion of fluorescein-labeled CPPCs in the GUV membranes. The pH-sensitive dye carboxyfluorescein is encapsulated within the water pool of the GUVs and used as an indicator of proton transport. This assay is versatile and can be exploited on other existing proton transporter systems, providing a consistent tool to compare their performances. It should also aid the development of novel antineoplastics and drug delivery systems.
Assuntos
Canais Iônicos/química , Nanotubos/química , Peptídeos Cíclicos/química , Prótons , Lipossomas Unilamelares/química , Transporte de Íons , Microscopia de FluorescênciaRESUMO
Synthetic polymer nanoparticles that can be tailored through multivalent ligand display on the surface, while at the same time allowing encapsulation of desired bioactive molecules, are especially useful in providing a versatile and robust platform in the design of specific delivery vehicles for various purposes. Glycosylated nanoparticles (glyco-NPs) of a poly(n-butyl acrylate) (pBA) core and poly(N-2-(ß-d-glucosyloxy)ethyl acrylamide) (p(NßGlcEAM)) or poly(N-2-(ß-D-galactosyloxy)ethyl acrylamide) (p(NßGalEAM)) corona were prepared via nanoprecipitation in aqueous solutions of preformed amphiphilic glycopolymers. Well-defined block copolymers of (poly(pentafluorophenyl acrylate) (pPFPA) and pBA were first prepared by RAFT polymerization followed by postpolymerization functionalization with aminoethyl glycosides to yield p(NßGlcEAM-b-BA) and p(NßGalEAM-b-BA), which were then used to form glyco-NPs (glucosylated and galactosylated NPs, Glc-NPs and Gal-NPs, respectively). The glyco-NPs were characterized by dynamic light scattering (DLS) and TEM. Encapsulation and release of ampicillin, leading to nanoparticles that we have termed "glyconanobiotics", were studied. The ampicillin-loaded glyco-NPs were found to induce aggregation of Staphylococcus aureus and Escherichia coli and resulted in antibacterial activity approaching that of ampicillin itself. This glyconanobiotics strategy represents a potential new approach for the delivery of antibiotics close to the surface of bacteria by promoting bacterial aggregation. Defined release in the proximity of the bacterial envelope may thus enhance antibacterial efficiency and potentially reduce the quantities of agent required for potency.
Assuntos
Anti-Infecciosos/administração & dosagem , Sistemas de Liberação de Medicamentos , Escherichia coli/efeitos dos fármacos , Nanopartículas/química , Polímeros/química , Staphylococcus aureus/efeitos dos fármacos , GlicosilaçãoRESUMO
PEGylation, the covalent modification of proteins with polyethylene glycol, is an abundantly used technique to improve the pharmacokinetics of therapeutic proteins. The drawback with this methodology is that the covalently attached PEG can impede the biological activity (e.g., reduced receptor-binding capacity). Protein therapeutics with "disposable" PEG modifiers have potential advantages over the current technology. Here, we show that a protein-polymer "Medusa complex" is formed by the combination of a hexavalent lectin with a glycopolymer. Using NMR spectroscopy, small-angle X-ray scattering (SAXS), size exclusion chromatography, and native gel electrophoresis it was demonstrated that the fucose-binding lectin RSL and a fucose-capped polyethylene glycol (Fuc-PEG) form a multimeric assembly. All of the experimental methods provided evidence of noncovalent PEGylation with a concomitant increase in molecular mass and hydrodynamic radius. The affinity of the protein-polymer complex was determined by ITC and competition experiments to be in the micromolar range, suggesting that such systems have potential biomedical applications.
Assuntos
Lectinas/química , Polietilenoglicóis/química , Cromatografia em Gel , Espectroscopia de Ressonância Magnética , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
In vitro three-dimensional (3D) models are better able to replicate the complexity of real organs and tissues than 2D monolayer models. The human endometrium, the inner lining of the uterus, undergoes complex changes during the menstrual cycle and pregnancy. These changes occur in response to steroid hormone fluctuations and elicit crosstalk between the epithelial and stromal cell compartments, and dysregulations are associated with a variety of pregnancy disorders. Despite the importance of the endometrium in embryo implantation and pregnancy establishment, there is a lack of in vitro models that recapitulate tissue structure and function and as such a growing demand for extracellular matrix hydrogels that can support 3D cell culture. To be physiologically relevant, an in vitro model requires mechanical and biochemical cues that mimic those of the ECM found in the native tissue. We report a semisynthetic gelatin methacryloyl (GelMA) hydrogel that combines the bioactive properties of natural hydrogels with the tunability and reproducibility of synthetic materials. We then describe a simple protocol whereby cells can quickly be encapsulated in GelMA hydrogels. We investigate the suitability of GelMA hydrogel to support the development of an endometrial model by culturing the main endometrial cell types: stromal cells and epithelial cells. We also demonstrate how the mechanical and biochemical properties of GelMA hydrogels can be tailored to support the growth and maintenance of epithelial gland organoids that emerge upon 3D culturing of primary endometrial epithelial progenitor cells in a defined chemical medium. We furthermore demonstrate the ability of GelMA hydrogels to support the viability of stromal cells and their function measured by monitoring decidualization in response to steroid hormones. This study describes the first steps toward the development of a hydrogel matrix-based model that recapitulates the structure and function of the native endometrium and could support applications in understanding reproductive failure.
Assuntos
Endométrio , Células Epiteliais , Gelatina , Hidrogéis , Metacrilatos , Organoides , Células Estromais , Humanos , Feminino , Gelatina/química , Endométrio/citologia , Células Estromais/citologia , Células Estromais/metabolismo , Organoides/citologia , Organoides/efeitos dos fármacos , Hidrogéis/química , Hidrogéis/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Metacrilatos/química , Células CultivadasRESUMO
Three-dimensional (3D) cell culture is regarded as a more physiologically relevant method of growing cells in the laboratory compared to traditional monolayer cultures. Recently, the application of polystyrene-based scaffolds produced using polyHIPE technology (porous polymers derived from high internal phase emulsions) for routine 3D cell culture applications has generated very promising results in terms of improved replication of native cellular function in the laboratory. These materials, which are now available as commercial scaffolds, are superior to many other 3D cell substrates due to their high porosity, controllable morphology, and suitable mechanical strength. However, until now there have been no reports describing the surface-modification of these materials for enhanced cell adhesion and function. This study, therefore, describes the surface functionalization of these materials with galactose, a carbohydrate known to specifically bind to hepatocytes via the asialoglycoprotein receptor (ASGPR), to further improve hepatocyte adhesion and function when growing on the scaffold. We first modify a typical polystyrene-based polyHIPE to produce a cell culture scaffold carrying pendent activated-ester functionality. This was achieved via the incorporation of pentafluorophenyl acrylate (PFPA) into the initial styrene (STY) emulsion, which upon polymerization formed a polyHIPE with a porosity of 92% and an average void diameter of 33 µm. Histological analysis showed that this polyHIPE was a suitable 3D scaffold for hepatocyte cell culture. Galactose-functionalized scaffolds were then prepared by attaching 2'-aminoethyl-ß-D-galactopyranoside to this PFPA functionalized polyHIPE via displacement of the labile pentafluorophenyl group, to yield scaffolds with approximately ca. 7-9% surface carbohydrate. Experiments with primary rat hepatocytes showed that cellular albumin synthesis was greatly enhanced during the initial adhesion/settlement period of cells on the galactose-functionalized material, suggesting that the surface carbohydrates are accessible and selective to cells entering the scaffold. This porous polymer scaffold could, therefore, have important application as a 3D scaffold that offers enhanced hepatocyte adhesion and functionality.
Assuntos
Meios de Cultura/síntese química , Galactose/química , Hepatócitos/fisiologia , Polímeros/química , Estirenos/química , Acrilatos/química , Albuminas/biossíntese , Animais , Adesão Celular , Células Hep G2 , Humanos , Porosidade , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Polymeric dispersants are useful materials used in many different industries and often derived from oil-based chemicals, for example, in automotive fluids so as to prevent particulates from precipitation and causing potential damage. These are very often polyisobutene derivatives, and there is a growing need to replace these using chemicals using renewable resources such as the use of naturally occurring myrcene. Polymyrcene (PMy), with an ordered microstructure, has been successfully synthesized via both anionic and radical polymerization in different solvents and subsequently subjected to functionalization via photoinduced thiol-ene click reactions with a number of thiols, methyl thioglycolate, 3-mercaptopropionic acid, 3-mercapto-1-hexanol, 2-mercaptoethanol, and 1-thioglycerol, using 2,2-dimethoxy-2-phenylacetophenone as a photoinitiator under UV irradiation (λ = 365 nm) at ambient temperature. The polarity of the solvent has an important impact on the microstructure of the produced polymyrcene and, in particular, 1,2-unit (â¼4%), 3,4-unit (â¼41%), and 1,4-unit (â¼51%) PMy were obtained via anionic polymerization in a polar solvent (THF) at ambient temperature, while 3,4-unit (â¼6%) and 1,4-unit (â¼94%, including cis and trans) PMy were obtained with cyclohexane as the solvent. Subsequently, photochemical thiol-ene reactions were carried out on the resulting PMy with different isomers exhibiting different reactivities of the double bonds. This strategy allows for the introduction of functional/polar groups (-COOH, -OH) into hydrophobic PMy in a controlled process. Hydrogenation of PMy and derivatized PMy was carried out to investigate any effects on the stabilities of the products which are desirable for many applications.
RESUMO
AIM: To assess the changes in the anterior chamber parameters using Pentacam following four different lines of treatment of primary angle-closure glaucoma (PACG). METHODS: A retrospective comparative study included 126 patients (126 eye) presented within 24-48h after acute angle-closure glaucoma (AACG). Patients were divided into 2 groups: group A (68 eyes) with controlled intraocular pressure (IOP) ≤21 mm Hg, which included subgroup A1 (34 eyes) with clear lens underwent Nd:YAG laser peripheral iridotomy (LPI) and subgroup A2 (34 eyes) with cataract underwent standard phacoemulsification; and group B (58 eyes) with uncontrolled IOP, which included subgroup B1 (30 eyes) with clear lens underwent trabeculectomy and subgroup B2 (28 eyes) with cataract underwent combined phacoemulsification and trabeculectomy. Patients were followed up for at least 3mo. Primary outcomes were Pentacam anterior segment measurements [anterior chamber angle (ACA) and depth (ACD)]. Secondary outcomes were changes in IOP, visual acuity (VA) and recorded complications. RESULTS: At the 3rd month, there was significant increase in the ACA values in all studied groups compared to preoperative values (P<0.001). The highest percent of increase in ACA was recorded in phacotrabeculectomy group B2 (128.40%). There was significant increase in ACD values at 3rd month compared with baseline values (P<0.001) for groups A1, A2, and B2; without change in B1 trabeculectomy group. The maximum deepening of ACD was noticed in group B2 with 94.27% increase. Significant decrease in postoperative IOP in groups A2, B1 and B2 (P<0.001, P=0.014, and P<0.001 respectively). In group A1 there was significant increase in 3rd month postoperative IOP from baseline values (P<0.001). The maximum decrease in IOP was noticed in group B2 with 59.54% decrease. VA improvement in 3rd month postoperative was recorded in all studied groups, maximum VA improvement was observed in group B2 up to 0.2 logMAR. CONCLUSION: Pentacam can be a helpful tool in studying and comparing the effect of the different lines of management of PACG on the anterior chamber measures. Phacotrabeculectomy was proved to be an effective line for managing PACG with resultant significant increase in the anterior chamber parameters, IOP reduction as well as maximum VA improvement. LPI has only temporary effect on IOP with significant changes in ACA and ACD. Phacoemuslification alone can be an option in treating PACG. Trabeculectomy resulted in temporary increase in the anterior chamber parameter which subsequently returned to baseline values.
RESUMO
A novel strategy for the surface functionalization of emulsion-templated highly porous (polyHIPE) materials as well as its application to in vitro 3D cell culture is presented. A heterobifunctional linker that consists of an amine-reactive N-hydroxysuccinimide ester and a photoactivatable nitrophenyl azide, N-sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-SANPAH), is utilized to functionalize polyHIPE surfaces. The ability to conjugate a range of compounds (6-aminofluorescein, heptafluorobutylamine, poly(ethylene glycol) bis-amine, and fibronectin) to the polyHIPE surface is demonstrated using fluorescence imaging, FTIR spectroscopy, and X-ray photoelectron spectroscopy. Compared to other existing surface functionalization methods for polyHIPE materials, this approach is facile, efficient, versatile, and benign. It can also be used to attach biomolecules to polyHIPE surfaces including cell adhesion-promoting extracellular matrix proteins. Cell culture experiments demonstrated that the fibronectin-conjugated polyHIPE scaffolds improve the adhesion and function of primary human endometrial stromal cells. It is believed that this approach can be employed to produce the next generation of polyHIPE scaffolds with tailored surface functionality, enhancing their application in 3D cell culture and tissue engineering whilst broadening the scope of applications to a wider range of cell types.
Assuntos
Adesão Celular/fisiologia , Endométrio/citologia , Polímeros/metabolismo , Estirenos/metabolismo , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Materiais Biocompatíveis/metabolismo , Técnicas de Cultura de Células , Movimento Celular/fisiologia , Células Cultivadas , Emulsões , Feminino , Fibronectinas/metabolismo , Humanos , Células EstromaisRESUMO
Identifying probiotics and pathogens is of great interest to the health of the human body. It is critical to develop microbiota-targeted therapies to have high specificity including strain specificity. In this study, we have utilized E. coli MG1655 bacteria as living templates to synthesize glycopolymers in situ with high selectivity. By this bacteria-sugar monomer-aptation-polymerization (BS-MAP) method, we have obtained glycopolymers from the surface of bacteria which can recognize template bacteria from two strains of E. coli and the specific bacteria-binding ability of glycopolymers was confirmed by both bacterial aggregation experiment and QCM-D measurements. Furthermore, the synthesized glycopolymers have shown a powerful inhibitory ability which can prevent bacteria from harming cells in both anti-infection and co-culture tests.
RESUMO
Despite the low complexity of their components, several simple physical systems, including microspheres, coacervate droplets and phospholipid membrane structures (liposomes), have been suggested as protocell models. These, however, lack key cellular characteristics, such as the ability to replicate or to dock with extracellular species. Here, we report a simple method for the de novo creation of synthetic cell mimics in the form of giant polymeric vesicles (polymersomes), which are capable of behavior approaching that of living cells. These polymersomes form by self-assembly, under electroformation conditions, of amphiphilic, glycosylated block copolymers in aqueous solution. The glycosylated exterior of the resulting polymeric giant unilamellar vesicles (GUVs) allows their selective interaction with carbohydrate-binding receptor-functionalized particles, in a manner reminiscent of the cell-surface docking of virus particles. We believe that this is the first example of a simple protocell model displaying cell-like behavior through a native receptor-ligand interaction.
Assuntos
Células Artificiais/metabolismo , Lectinas/química , Lipossomos/metabolismo , Fosfolipídeos/metabolismo , Células Artificiais/química , Biopolímeros/química , Biopolímeros/metabolismo , Membrana Celular , Lectinas/metabolismo , Lipossomos/química , Fosfolipídeos/química , Polissacarídeos/química , Polissacarídeos/metabolismo , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo , Vírion/química , Vírion/genética , Vírion/metabolismoRESUMO
This article describes a versatile method for the modification of 2-hydroxyethyl cellulose (HEC) involving azide-alkyne cycloaddition reaction to impart neutral (ester) and ionic (carboxylic acid and 1(ry) amine) functionalities. The synthetic approach involved, first the introduction of the azide functionality to HEC and then followed by its cycloaddition reaction with several alkyne terminated compounds: namely ethyl propiolate, 5-hexynoic acid and propargyl amine. Sequential Click reactions were also demonstrated to be feasible by the successful synthesis of polydimethylsiloxane (PDMS) grafted HEC containing neutral (ester) and ionic (carboxylic acid and 1(ry) amine) functionalities. The Click chemistry was then further utilized similarly to graft poly(lactic acid) (PLA) and poly(ethylene glycol) (PEG) segments to HEC to access its hydrophobic and hydrophilic analogs, respectively. AFM analysis revealed that while HEC itself formed uniform oval features, the PLA grafted HEC exhibited a brushlike architecture. The formation of these brushlike structures suggested that the HEC backbone exhibits an extended conformation with the side chains stretched out. The resulting polymeric materials were characterized by solution and solid state (13)C NMR and FTIR spectroscopy.