Internalization of Methotrexate Conjugates by Folate Receptor-α.

The folate antagonist methotrexate is a cytotoxic drug used in the treatment of several cancer types. The entry of methotrexate into the cell is mediated by two main transport systems: the reduced folate carrier and membrane-associated folate receptors. These transporters differ considerably in their mechanism of (anti)folate uptake, substrate specificity, and tissue specificity. Although the mechanism of action of the reduced folate carrier is fairly well-established, that of the folate receptor has remained unknown. The development of specific folate receptor-targeted antifolates would be accelerated if additional mechanistic data were to become available. In this work, we used two fluorescently labeled conjugates of methotrexate, differently linked at the terminal groups, to clarify the uptake mechanism by folate receptor-α. The results demonstrate the importance of methotrexate amino groups in the interaction with folate receptor-α.

Myeloperoxidase-responsive materials for infection detection based on immobilized aminomethoxyphenol.

There is a strong need for simple and fast diagnostic tools for the detection of wound infection. Immune system-derived enzymes like myeloperoxidase are efficient biomarkers for wound infection that emerge in the early stage infection process. In this study, 5-amino-2-methoxyphenol was functionalized with alkoxysilane to allow visual detection of MPO on carrier materials, for example, in test strips. Indeed, MPO activity was visually detectable in short time in wound background. Oxidation of the substrate was followed spectrophotometrically and proved via HPLC. LC-ESI TOF and NMR analyses unveiled the reaction mechanism and a dimeric reaction product responsible for the visualization of MPO activity. The substrate specificity and sensitivity toward MPO detection was proved and tests with infected wound fluids were successfully performed. The study demonstrates the suitability of the novel MPO substrate for the detection of wound infection and the covalent immobilization on diagnostic carrier materials. Biotechnol. Bioeng. 2016;113: 2553-2560. © 2016 Wiley Periodicals, Inc.

Cellobiohydrolases Produce Different Oligosaccharides from Chitosan.

Chito-oligosaccharides (COSs) are bioactive molecules with interesting characteristics; however, their exploitation is still restricted due to limited amounts accessible with current production strategies. Here we present a strategy for the production of COSs based on hydrolysis of chitosan by using readily available glycosidases. Cellobiohydrolases (EC 3.2.1.91) were compared with chitosanases (EC 3.2.1.132) regarding their ability for COS production, and the resulting fractions were analyzed by MS and NMR. The oligosaccharides had a degree of polymerization between three and six units, and the degree of acetylation (DA) varied depending on the applied enzyme. Different cellobiohydrolases produced COSs with varying DA, and based on comprehensive NMR analysis the preferred cleavage sites of the respective enzymes that show chitosanase and chitinase activity were elucidated. The study reveals the high potential of readily available cellulolytic enzymes besides chitosanases for the production of COSs with distinct structure facilitating access to this bioactive compound class.

Peptide Anchor for Folate-Targeted Liposomal Delivery.

Specific folate receptors are abundantly overexpressed in chronically activated macrophages and in most cancer cells. Directed folate receptor targeting using liposomes is usually achieved using folate linked to a phospholipid or cholesterol anchor. This link is formed using a large spacer like polyethylene glycol. Here, we report an innovative strategy for targeted liposome delivery that uses a hydrophobic fragment of surfactant protein D linked to folate. Our proposed spacer is a small 4 amino acid residue linker. The peptide conjugate inserts deeply into the lipid bilayer without affecting liposomal integrity, with high stability and specificity. To compare the drug delivery potential of both liposomal targeting systems, we encapsulated the nuclear dye Hoechst 34580. The eventual increase in blue fluorescence would only be detectable upon liposome disruption, leading to specific binding of this dye to DNA. Our delivery system was proven to be more efficient (2-fold) in Caco-2 cells than classic systems where the folate moiety is linked to liposomes by polyethylene glycol.

Para-derivatized pybox ligands as sensitizers in highly luminescent Ln(III) complexes.

New complexes of pyridine-bis(oxazoline) derivatized with -H, -OMe, and -Br at the para position of the pyridine ring with Eu(III) and Tb(III) have been isolated. These are highly luminescent in the solid state, regardless of the ligand-to-metal ratio. Several of the metal complexes were isolated and characterized by single crystal X-ray diffraction, showing the rich diversity of structures that can be obtained with this family of ligands. [Eu(PyboxOMe)(3)](NO(3))(3)·3CH(2)Cl(2), 1, crystallizes in the monoclinic space group P2(1)/n and has the cell parameters a = 14.3699(10) Å, b = 13.4059(9) Å, c = 25.8766(18) Å, ß = 95.367(1)°, and V = 4963.1(6) Å(3). The isostructural [Tb(PyboxOMe)(3)](NO(3))(3)·3CH(2)Cl(2), 2, crystallizes with the parameters a = 14.4845(16) Å, b = 13.2998(15) Å, c = 25.890(3) Å, ß = 94.918(2)°, and V = 4969.1(10) Å(3). 3, a 1:1 complex with the formula [Eu(PyboxBr)(NO(3))(3)(H(2)O)], crystallizes in the monoclinic P2(1)/c space group with a = 11.649(2) Å, b = 8.3914(17) Å, c = 20.320(4) Å, ß = 100.25(3)°, and V = 1954.5(7) Å(3). 4, a product of the reaction of PyboxBr with Tb(NO(3))(3), is [Tb(PyboxBr)(2)(η(2)-NO(3))(η(1)-NO(3)](2)[Tb(NO(3))(5)]·5H(2)O. It crystallizes in the monoclinic space group P2(1) with a = 15.612(3) Å, b = 14.330(3) Å, c = 16.271(3) Å, ß = 92.58(3)°, and V = 3636.5(13) Å(3). [Tb(Pybox)(3)](CF(3)SO(3))(3)·3CH(2)CN, 5, crystallizes in the triclinic space group P1̅ with a = 12.3478(2) Å, b = 15.0017(2) Å, c = 16.1476(4) Å, α = 100.252(1)°, ß = 100.943(1)°, γ = 113.049(1)°, and V = 2594.80(8) Å(3). Finally, compound 6, [Tb(Pybox)(2)(NO(3))(H(2)O)](NO(3))(2)·CH(3)OH, crystallizes in the triclinic P1̅ space group with a = 9.7791(2) Å, b = 10.1722(2) Å, c = 15.3368(3) Å, α = 83.753(1)°, ß = 78.307(1)°, γ = 85.630(1)°, and V = 1482.33(5) Å(3). In solution, the existence of 3:1, 2:1, and 1:1 species can be observed through absorption and luminescence speciation measurements as well as NMR spectroscopy. The stability constants in acetonitrile, as an average obtained from absorption and emission titrations, are log ß(11) = 5.4, log ß(12) = 8.8, and log ß(13) = 12.8 with Eu(III) and log ß(11) = 4.5, log ß(12) = 8.4, and log ß(13) = 11.7 for the Tb(III) species with PyboxOMe. Pybox displayed stability constants log ß(11) = 3.6, log ß(12) = 9.1, and log ß(13) = 12.0 with Eu(III) and log ß(11) = 3.7, log ß(12) = 9.3, and log ß(13) = 12.2 for the Tb(III) species. Finally, PyboxBr yielded log ß(11) = 7.1, log ß(12) = 12.2, and log ß(13) = 15.5 for the Eu(III) species and log ß(11) = 6.2, log ß(12) = 11.0, and log ß(13) = 15.4 with Tb(III). Photophysical characterization was performed in all cases on solutions with 3:1 ligand-to-metal ion stoichiometry and allowed determination of quantum yields and lifetimes of emission for PyboxOMe of 23.5 ± 1.6% and 1.54 ± 0.04 ms for Eu(III) and 21.4 ± 3.6% and 1.88 ± 0.04 ms for Tb(III). For Pybox these values were 25.6 ± 1.1% and 1.49 ± 0.04 ms for Eu(III) and 23.2 ± 2.1% and 0.44 ± 0.01 ms for Tb(III) and for PyboxBr they were 35.8 ± 1.6% and 1.46 ± 0.03 ms for Eu(III) and 23.3 ± 1.3% and a double lifetime of 0.79 ± 0.05/0.07 ± 0.01 ms for Tb(III). A linear relationship between the triplet level energies and the Hammett σ constants was found. Lifetime measurements in methanol as well as the NMR data in both methanol and acetonitrile indicate that all complexes are stable in the 3:1 stoichiometry in solution and that there is no solvent coordination to the metal ion.

Smart textiles in wound care: functionalization of cotton/PET blends with antimicrobial nanocapsules.

Management of infected wounds is one of the most costly procedures in the health care sector. Burn wounds are of significant importance due to the high infection risk that can possibly lead to severe consequences such as sepsis. Because antibiotic wound treatments have caused increasing antibiotic resistance in bacteria, there is currently a strong need for alternative strategies. Therefore, we developed new antimicrobial wound dressings consisting of pH-responsive human serum albumin/silk fibroin nanocapsules immobilized onto cotton/polyethylene terephthalate (PET) blends loaded with eugenol, which is an antimicrobial phenylpropanoid. Ultrasound-assisted production of eugenol-loaded nanocapsules resulted in particle sizes (hydrodynamic radii) between 319.73 ± 17.50 and 574.00 ± 92.76 nm and zeta potentials ranging from -10.39 ± 1.99 mV to -12.11 ± 0.59 mV. Because recent discoveries have indicated that the sweat glands contribute to wound reepithelialisation, release studies of eugenol were conducted in different artificial sweat formulas that varied in pH. Formulations containing 10% silk fibroin with lower degradation degree exhibited the highest release of 41% at pH 6.0. After immobilization, the functionalized cotton/PET blends were able to inhibit 81% of Staphylococcus aureus and 33% of Escherichia coli growth. Particle uniformity, silk fibroin concentration, and high surface-area-to-volume ratio of the produced nanocapsules were identified as the contributing factors leading to high antimicrobial activities against both strains. Therefore, the production of antimicrobial textiles using nanocapsules loaded with an active natural compound that will not contribute to antibiotic resistance is seen as a potential future alternative to commercially available antiseptic wound dressings.

Structural insights into pH-responsive drug release of self-assembling human serum albumin-silk fibroin nanocapsules.

Inflammation processes are associated with significant decreases in tissue or lysosomal pH from 7.4 to 4, a fact that argues for the application of pH-responsive drug delivery systems. However, for their design and optimization a full understanding of the release mechanism is crucial. In this study we investigated the pH-depending drug release mechanism and the influence of silk fibroin (SF) concentration and SF degradation degree of human serum albumin (HSA)-SF nanocapsules. Sonochemically produced nanocapsules were investigated regarding particle size, colloidal stability, protein encapsulation, thermal stability and drug loading properties. Particles of the monodisperse phase showed average hydrodynamic radii between 438 and 888 nm as measured by DLS and AFM and a zeta potential of -11.12 ±â€¯3.27 mV. Together with DSC results this indicated the successful production of stable nanocapsules. ATR-FTIR analysis demonstrated that SF had a positive effect on particle formation and stability due to induced beta-sheet formation and enhanced crosslinking. The pH-responsive release was found to depend on the SF concentration. In in-vitro release studies, HSA-SF nanocapsules composed of 50% SF showed an increased pH-responsive release for all tested model substances (Rhodamine B, Crystal Violet and Evans Blue) and methotrexate at the lowered pH of 4.5 to pH 5.4, while HSA capsules without SF did not show any pH-responsive drug release. Mechanistic studies using confocal laser scanning microscopy (CLSM) and small angle X-ray scattering (SAXS) analyses showed that increases in particle porosity and decreases in particle densities are directly linked to pH-responsive release properties. Therefore, the pH-responsive release mechanism was identified as diffusion controlled in a novel and unique approach by linking scattering results with in-vitro studies. Finally, cytotoxicity studies using the human monocytic THP-1 cell line indicated non-toxic behavior of the drug loaded nanocapsules when applied in a concentration of 62.5 µg mL-1. Based on the obtained release properties of HSA-SF nanocapsules formulations and the results of in-vitro MTT assays, formulations containing 50% SF showed the highest requirements arguing for future in vivo experiments and application in the treatment of inflammatory diseases.

Chitosan based substrates for wound infection detection based on increased lysozyme activity.

There is a strong need of point-of-care diagnostics for early detection of wound infection. In this study, substrates based on functionalized chitosan were developed for visual detection of elevated lysozyme activity, an infection biomarker in wound fluids. For efficient hydrolysis by lysozyme, N-acetyl chitosan with a final degree of acetylation of around 50% was synthesized. N-acetylated chitosan and a chitosan-starch composite were labeled with structurally different dyes resulting in lysozyme-responsive biomaterials. Incubation with lysozyme in buffer and artificial wound fluid lead to a release of colored hydrolysis products already after 2h incubation. Tests in human wound fluid from infected wounds indicated a clear visual color change after 2.5h compared to control samples. A higher degree of swelling of the chitosan/starch containing substrate led to faster hydrolysis by lysozyme. This study demonstrates the potential of the lysozyme-responsive materials for diagnosis of wound infection and provides different diagnostic substrates for potential incorporation in point-of-care devices.

Antimicrobial Cellobiose Dehydrogenase-Chitosan Particles.

Increasing prevalence of chronic wounds and microbial infection constitute a severe health challenge. The situation is further complicated by emerging multidrug resistance making the treatment of infections increasingly difficult. Here, a novel antimicrobial system based on in situ release of hydrogen peroxide (H2O2) by cellobiose dehydrogenase (CDH) immobilized on chitosan (CTS) particles is described. Covalent immobilization using carbodiimide coupling lead to a higher amount of protein immobilized on CTS (104 µg CDH/mg CTS) when compared to noncovalent immobilization, which, however, showed highest recovery of CDH activity (0.01 U/mg CTS). The CDH-CTS in situ generated H2O2 completely inhibited growth of Escherichia coli and Staphylococcus aureus over a period of 24 h. This resilient antimicrobial system represents a novel strategy for preventing infection with potential application in counteracting microbial colonization of chronic wounds.

Silk-elastin-like protein biomaterials for the controlled delivery of therapeutics.

INTRODUCTION: Genetically engineered biomaterials are useful for controlled delivery owing to their rational design, tunable structure-function, biocompatibility, degradability and target specificity. Silk-elastin-like proteins (SELPs), a family of genetically engineered recombinant protein polymers, possess these properties. Additionally, given the benefits of combining semi-crystalline silk-blocks and elastomeric elastin-blocks, SELPs possess multi-stimuli-responsive properties and tunability, thereby becoming promising candidates for targeted cancer therapeutics delivery and controlled gene release. AREAS COVERED: An overview of SELP biomaterials for drug delivery and gene release is provided. Biosynthetic strategies used for SELP production, fundamental physicochemical properties and self-assembly mechanisms are discussed. The review focuses on sequence-structure-function relationships, stimuli-responsive features and current and potential drug delivery applications. EXPERT OPINION: The tunable material properties allow SELPs to be pursued as promising biomaterials for nanocarriers and injectable drug release systems. Current applications of SELPs have focused on thermally-triggered biomaterial formats for the delivery of therapeutics, based on local hyperthermia in tumors or infections. Other prominent controlled release applications of SELPs as injectable hydrogels for gene release have also been pursued. Further biomedical applications that utilize other stimuli to trigger the reversible material responses of SELPs for targeted delivery, including pH, ionic strength, redox, enzymatic stimuli and electric field, are in progress. Exploiting these additional stimuli-responsive features will provide a broader range of functional biomaterials for controlled therapeutics release and tissue regeneration.

Size controlled protein nanoemulsions for active targeting of folate receptor positive cells.

Bovine serum albumin (BSA) nanoemulsions were produced by high pressure homogenization with a tri-block copolymer (Poloxamer 407), which presents a central hydrophobic chain of polyoxypropylene (PPO) and two identical lateral hydrophilic chains of polyethylene glycol (PEG). We observed a linear correlation between tri-block copolymer concentration and size - the use of 5mg/mL of Poloxamer 407 yields nanoemulsions smaller than 100nm. Molecular dynamics and fluorescent tagging of the tri-block copolymer highlight their mechanistic role on the size of emulsions. This novel method enables the fabrication of highly stable albumin emulsions in the nano-size range, highly desirable for controlled drug delivery. Folic Acid (FA)-tagged protein nanoemulsions were shown to promote specific folate receptor (FR)-mediated targeting in FR positive cells. The novel strategy presented here enables the construction of size controlled, functionalized protein-based nanoemulsions with excellent characteristics for active targeting in cancer therapy.

HSA nanocapsules functionalized with monoclonal antibodies for targeted drug delivery.

The chronic autoimmune disorder rheumatoid arthritis (RA) affects millions of adults and children every year. Chronically activated macrophages secreting enzymes and inflammatory cytokines play a key role in RA. Distinctive marker molecules on the macrophage surface could be used to design a targeted drug delivery device for the treatment of RA without affecting healthy cells and tissues. Here, different methods for covalent attachment of antibodies (mAb) recognizing MHC class II molecules found on macrophages onto human serum albumin (HSA) nanocapsules were compared. HSA nanocapsules were prepared with a hydrodynamic diameter of 500.7 ± 9.4 nm and a narrow size distribution as indicated by a polydispersity index (PDI) of 0.255 ± 0.024. This was achieved by using a sonochemical process avoiding toxic cross linking agents and emulsifiers. Covalent binding of mAb on the surface of HSA nanocapsules was realized using polyethyleneglycol (PEG)3000 as spacer molecule. The presence of mAb was confirmed by confocal laser scanning microscopy (CLSM) and enzyme-linked immunosorbent assay (ELISA). Specific binding of mAb-HSA nanocapsules to MHC class II molecules on antigen-presenting cells was demonstrated by flow cytometry analysis.

Folic acid-functionalized human serum albumin nanocapsules for targeted drug delivery to chronically activated macrophages.

Activated synovial macrophages play a key role in Rheumatoid Arthritis (RA). Recent studies have shown that folate receptor beta (FRß) is specifically expressed by activated macrophages. Therefore a folate-based nanodevice would provide the possibility of delivering therapeutic agents to activated macrophages without affecting normal cells and tissues. This study shows for the first time the sonochemical preparation of HSA nanocapsules avoiding toxic cross linking chemicals and emulsifiers used in other methods. Production of HSA nanocapsules was optimized leading to a diameter of 443.5 ± 9.0 nm and a narrow size distribution indicated by a polydispersity index (PDI) of 0.066 ± 0.080. Nanocapsules were surface modified with folic acid (FA) and the FA content was determined to be 0.38 and 6.42 molecules FA per molecule HSA, depending on the surplus of FA employed. Dynamic light scattering was used to determine size, PDI and zetapotential of the produced nanocapsules before and after surface modification. FA distribution on the surface of HSA nanocapsules was localized three-dimensionally after fluorescence labeling using confocal laser scanning microscopy (CLSM). Furthermore, specific binding and internalization of HSA nanocapsules by FRß-positive and FRß-negative macrophages, obtained from human peripheral blood mononuclear cells, was demonstrated by flow cytometry. FRß-expressing macrophages showed an increased binding for FA-modified capsules compared with those without FA.

Bioresponsive systems based on polygalacturonate containing hydrogels.

Polysaccharide acid (PSA) based devices (consisting of alginic acid and polygalacturonic acid) were investigated for the detection of contaminating microorganisms. PSA-CaCl(2) hydrogel systems were compared to systems involving covalent cross-linking of PSA with glycidylmethacrylate (PSA-GMA) which was confirmed with Fourier Transformed Infrared (FTIR) analysis. Incubation of PSA-CaCl(2) and PSA-GMA beads loaded with Alizarin as a model ingredient with trigger enzymes (polygalacturonases or pectate lyases) or bacteria lead to a smoothening of the surface and exposure of Alizarin according to Environmental Scanning Electron Microscopy (ESEM) analysis. Enzyme triggered release of Alizarin was demonstrated for a commercial enzyme preparation from Aspergillus niger and with purified polygalacturonase and pectate lyase from S. rolfsii and B. pumilus, respectively. In contrast to the PSA-CaCl(2) beads, cross-linking (PSA-GMA beads) restricted the release of Alizarin in absence of enzymes. There was a linear relation between release of Alizarin (5-348 µM) and enzyme activity in a range of 0-300 U ml(-1) dosed. In addition to enzymes, both PSA-CaCl(2) and PSA-GMA beads were incubated with Bacillus subtilis and Yersinia entercolitica as model contaminating microorganism. After 72 h, a release between 10 µM and 57 µM Alizarin was detected. For protection of the hydrogels, an enzymatically modified PET membrane was covalently attached onto the surface. This lead to a slower release and improve long term storage stability based on less than 1% release of dye after 21 days. Additionally, this allowed simple detection by visual inspection of the device due to a colour change of the white membrane to orange upon enzyme triggered release of the dye.

Covalent immobilisation of protease and laccase substrates onto siloxanes.

Immobilisation of enzyme substrates is a powerful tool in the detection of enzymes in the chemosphere and the environment. A siloxane based strategy for the covalent immobilisation of oxidoreductase and protease substrates was developed involving activation of silica gel and polyethylene terephthalate (PET) as model carriers with (3-aminopropyl)-triethoxysilane or (3-mercaptopropyl)-trimethoxysilane (APTS, MPTS). Ferulic acid and L-Leucine-p-nitroanilide, Gly-Phe p-nitroanilide (GPpNA) and N-Succinyl-Ala-Ala-Pro-Leu p-nitroanilide (SAAPLpNA) as laccase and protein substrates, respectively, were covalently attached using glutaraldehyde or carbodiimide based cross-linking strategies. In contrast to conversion in solution, immobilised SAAPLpNA was hydrolysed much faster by protease than immobilised GPpNA indicating steric hindrance with decreasing chain length between point of attachment and site of enzyme attack. Immobilised ferulic acid was oxidised by laccase both in case of MPTS and APTS-modified silica gel giving clearly visible colour changes with Delta E values of 7.2 and 2.3, respectively after 24h of incubation, where Delta E describes the distance between two colours. Similarly, clearly visible colour changes with a Delta E value of 8.6 were seen after laccase treatment of ferulic acid immobilised on APTS activated PET as carrier. Limited surface hydrolysis of PET with a cutinase enhanced coupling of APTS and ferulic acid due to a larger number of hydroxyl groups available on the surface and consequently led to a higher colour difference of Delta E=12.2 after laccase oxidation. The covalent coupling product between ferulic acid and 1,3-bis(3-aminopropyl)-1,1,3,3-tetramethyldisiloxane was identified by LC-MS (M+1m/z601) and successfully oxidised with laccase.