Simultaneous refolding and purification of recombinant proteins by macro-(affinity ligand) facilitated three-phase partitioning

A strategy called macro-(affinity ligand) facilitated three-phase partitioning (MLFTPP) is described for refolding of a diverse set of recombinant proteins starting from the solubilized inclusion bodies. It essentially consists of: (i) binding of the protein with a suitable smart polymer and (ii) precipitating the polymer-protein complex as an interfacial layer by mixing in a suitable amount of ammonium sulfate and t-butanol. Smart polymers are stimuli-responsive polymers that become insoluble on the application of a suitable stimulus (e.g., a change in the temperature, pH, or concentration of a chemical species such as Ca 2+ or K +). The MLFTPP process required approximately 10min, and the refolded proteins were found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The folded proteins were characterized by fluorescence emission spectroscopy, circular dichroism spectroscopy, biological activity, melting temperature, and surface hydrophobicity measurements by 8-anilino-1-naphthalenesulfonate fluorescence. Two refolded antibody fragments were also characterized by measuring K D by Biacore by using immobilized HIV-1 gp120. The data demonstrate that MLFTPP is a rapid and convenient procedure for refolding a variety of proteins from inclusion bodies at high concentration. Although establishing the generic nature of the approach would require wider trials by different groups, its success with the diverse kinds of proteins tried so far appears to be promising.

A fluorescent molecularly-imprinted polymer gate with temperature and pH as inputs for detection of alpha-fetoprotein

In this work, we have reported a new approach on the use of stimuli-responsive molecularly imprinted polymer (MIP) for trace level sensing of alpha-fetoprotein (AFP), which is a well know cancer biomarker. The stimuli-responsive MIP is composed of three components, a thermo-responsive monomer, a pH responsive component (tyrosine derivative) and a highly fluorescent vinyl silane modified carbon dot. The synthesized AFP-imprinted polymer possesses excellent selectivity towards their template molecule and dual-stimuli responsive behavior. Along with this, the imprinted polymer was also explored as `OR' logic gate with two stimuli (pH and temperature) as inputs. However, the non-imprinted polymers did not have such `OR' gate property, which confirms the role of template binding. The imprinted polymer was also used for estimation of AFP in the concentration range of 3.96-80.0 ng mL(-1), with limit of detection (LOD) 0.42 ng mL(-1). The role of proposed sensor was successfully exploited for analysis of AFP in real human blood plasma, serum and urine sample. (C) 2015 Elsevier B.V. All rights reserved.

Recent developments in intelligent biomedical polymers

Intelligent polymers or stimuli-responsive polymers may exhibit distinct transitions in physical-chemical properties, including conformation, polarity, phase structure and chemical composition in response to changes in environmental stimuli. Due to their unique 'intelligent' characteristics, stimuli-sensitive polymers have found a wide variety of applications in biomedical and nanotechnological fields. This review focuses on the recent developments in biomedical application of intelligent polymer systems, such as intelligent hydrogel systems, intelligent drug delivery systems and intelligent molecular recognition systems. Also, the possible future directions for the application of these intelligent polymer systems in the biomedical field are presented.

Synthesis and Characterization of Hyperbranched Poly(ether amide)s with Thermoresponsive Property and Unexpected Strong Blue Photoluminescence

A facile and efficient strategy for the syntheses of novel hyperbranched poly(ether amide)s (HPEA) from multihydroxyl primary amines and (meth)acryloyl chloride has been developed. The chemical structures of the HPEAs were confirmed by IR and NMR spectra. Analyses of SEC (size exclusion chromatography) and viscosity characterizations revealed the highly branched structures of the polymers obtained. The resultant hyperbranched polymers contain abundant hydroxyl groups. The thermoresponsive property was obtained from in situ surface modification of abundant OH end groups with N-isopropylacrylamide (NIPAAm). The study oil temperature-dependent characteristics has revealed that NIPAAm-g-HPEA exhibits an adjustable lower critical solution temperature (LCST) of about 34-42 degrees C depending on the grafting degree. More interestingly, the work provided an interesting phenomenon where the HPEA backbones exhibited strong blue photoluminescence.

Direct formation of giant vesicles from synthetic polypeptides

This report describes direct formation of giant vesicles from a series of poly(L-lysine)-block-poly(L-phenylalanine) (PLL-b-PPA) block copolymers from their water solution. These polymers are prepared by successive ring-opening polymerization (ROP) of the two alpha-amino acid N-carboxyanhydrides and then removing the side chain protecting groups by acidolysis. The structures of the copolymers are confirmed by nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), and size exclusion chromatography ( SEC). The vesicles are studied by atomic force microscopy (AFM), field emission scanning electron microscopy (ESEM), and confocal laser scanning microscopy (CLSM). Rhodamine B is used as a fluorescent probe to confirm the existence of the vesicle with an aqueous interior. The vesicle size is in the range 0.55-6 mu m, depending on the absolute and relative lengths of the two blocks, on initial polymer concentration, and on solution pH. The vesicles are still stable in water for 2 months after preparation. Addition of the copolymer to DNA solution results in complex formation with it. The complex assumes the morphology of irregular particles of less than 2 mu m. It is expected to be used in drug and gene delivery.

Adipose stromal vascular fraction cell sheets as vascular strategies for tissue engineering and regenerative medicine applications

Tese de mestrado em Engenharia Biomédica e Biofísica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2015

Perylene as an electron-rich moiety in healable, complementary π–π stacked, supramolecular polymer systems

A two-component, supramolecular polymer blend has been designed using a novel π-electron rich bisperylene- terminated polyether. This polymer is able to self-assemble through electronically complementary π–π stacking interactions with a π-electron-deficient chain-folding polydiimide to afford thermally healable polymer blends. Model compounds were developed to assess the suitability of the deep green complexes formed between perylene residues and chain-folding bis-diimides for use in polymer blends. The polymer blends thus synthesised were elastomeric in nature and demonstrated healable properties as demonstrated by scanning electron microscopy. Healing was observed to occur rapidly at ca. 75 degC, and excellent healing efficiencies were found by tensometric and rheometric analyses. These tuneable, stimuli-responsive, supramolecular polymer blends are compared to related healable blends featuring pyrene-terminated oligomers.

Lipid merging, protrusion and vesicle release triggered by shrinking/swelling of poly(N-isopropylacrylamide) microgel particles

Cell membrane changes its morphology during many physiological processes with the assistance of a solid support, such as the cytoskeleton, under an environmental stimulus. Here, a novel type of stimuli-responsive lipogel was fabricated, mimicking the changes of cell membrane. The lipogel was prepared from poly(N-isopropylacrylamide) (pNIPAM) microgel particle and phospholipid by a solvent-exchange method. The temperature dependent volume phase transition of pNIPAM triggers reversible transformation of the lipogel between a lipid vesicle-coated sun-like structure and a contracted hybrid sphere, through lipid merging and protrusion processes, respectively. By contrast, the salt induced pNIPAM phase transition leads to an irreversible vesicle release behaviour. The lipogel creates a unique platform for studying cell membrane behaviour and provides promising candidates in drug delivery and controlled release applications. © 2014 Elsevier B.V. All rights reserved.

Polymeric Systems as Nanodevices for siRNA Delivery

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estudo das propriedades estímulo responsivas dos polímeros escovas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Defects in thermosensitive colloidal crystals

Poly-N-Isopropylacrylamide (PNIPAM) colloidal particles form crystal phases that show a thermosensitive behaviour and can be used as atomic model systems. This polymer has both hydrophilic and hydrophobic character and has interesting stimuli-responsive properties in aqueous solution, of which the most important is the temperature response. Above a certain temperature, called Lower Critical Solution Temperature (LCST), the system undergoes a volume phase transition (VPT). Above the LCST, the water is expelled from the polymer network and the swollen state at low temperature transforms into a shrunken state at high temperature. The thermoresponsive behaviour of PNIPAM can be influenced by pH and ionic strength, as well as by the presence of copolymers, such as acrylic acid. In a system formed both by particles of PNIPAM and PNIPAM doped with acrylic acid, one can control the size ratio of the two components by changing the temperature of the mixture, while keeping particle interactions relatively the same. It is therefore possible to obtain thermoresponsive colloidal crystal in which temperature changes induce defects whose formation processes and dynamics can be analysed in an optical microscope at a convenient spatial and temporal scale. The goal of this thesis project was to find the conditions in which such a system could be formed, by using characterization techniques such as Static Light Scattering, Dynamic Light Scattering and Confocal Laser Scanning Microscopy. Two PNIPAM-AAc systems were available, and after characterization it was possible to select a suitable one, on the basis of its low polydispersity and the lack of a VPT, regardless of the external conditions (system JPN_7). The synthesis of a PNIPAM system was attempted, with particles of dimensions matching the JPN_7 system and, unlike JPN_7, displaying a VPT, and one suitable candidate for the mixed system was finally found (system CB_5). The best conditions to obtain thermoresponsive crystal were selected, and the formation and healing of defects were investigated with CLSM temperature scans. The obtained results show that the approach is the correct one and that the present report could represent a useful start for future developments in defect analysis and defect dynamics studies.

Advanced schemes for surface plasmon resonance and plasmon-enhanced fluorescence biosensors

Advanced optical biosensor platforms exploiting long range surface plasmons (LRSPs) and responsive N-isopropylacrylamide (NIPAAm) hydrogel binding matrix for the detection of protein and bacterial pathogen analytes were carried out. LRSPs are optical waves that originate from coupling of surface plasmons on the opposite sites of a thin metallic film embedded between two dielectrics with similar refractive indices. LRSPs exhibit orders of magnitude lower damping and more extended profile of field compared to regular surface plasmons (SPs). Their excitation is accompanied with narrow resonance and provides stronger enhancement of electromagnetic field intensity that can advance the sensitivity of surface plasmon resonance (SPR) and surface plasmon-enhanced fluorescence spectroscopy (SPFS) biosensors. Firstly, we investigated thin gold layers deposited on fluoropolymer surface for the excitation of LRSPs. The study indicates that the morphological, optical and electrical properties of gold film can be changed by the surface energy of fluoropolymer and affect the performance of a SPFS biosensor. A photo-crosslinkable NIPAAm hydrogel was grafted to the sensor surface in order to serve as a binding matrix. It was modified with bio-recognition elements (BREs) via amine coupling chemistry and offered the advantage of large binding capacity, stimuli responsive properties and good biocompatibility. Through experimental observations supported by numerical simulations describing diffusion mass transfer and affinity binding of target molecules in the hydrogel, the hydrogel binding matrix thickness, concentration of BREs and the profile of the probing evanescent field was optimized. Hydrogel with a up to micrometer thickness was shown to support additional hydrogel optical waveguide (HOW) mode which was employed for probing affinity binding events in the gel by means of refractometric and fluorescence measurements. These schemes allow to reach limits of detection (LODs) at picomolar and femtomolar levels, respectively. Besides hydrogel based experiments for detection of molecular analytes, long range surface plasmon-enhanced fluorescence spectroscopy (LRSP-FS) was employed for detection of bacterial pathogens. The influence of capture efficiency of bacteria on surfaces and the profile of the probing field on sensor response were investigated. The potential of LRSP-FS with extended evanescent field is demonstrated for detection of pathogenic E. coli O157:H7 on sandwich immunoassays . LOD as low as 6 cfu mL-1 with a detection time of 40 minutes was achieved.rn

Specific enzymatic cleavage and payload release from peptide-based hybrid nanocapsules

Within this thesis, new approaches for the concepts of peptide-polymer conjugates and peptide-based hybrid nanomaterials are investigated. In the first part, the synthesis of a triblock polymer-peptide-polymer is carried out following a typical peptide coupling reaction, both in solution and on solid-phase. The peptide sequence is chosen, so that it is cleaved by an enzyme preparation of trypsin. End-functionalized polystyrene is used as a model hydrophobic polymer and coupled to the peptide sequence. The results show successful coupling reactions in both methods, while the solid phase method produced a more defined product. Suspensions, consisting of peptide-polymer conjugates particles, are prepared in water by ultrasonication. In contact with the enzyme, the peptide constituting the conjugated particles is cleaved. This demonstrates the enzymatic cleavage in heterophase of enzymatic sequence bond to hydrophobic polymers, and is of great interest for the encapsulation and delivery of hydrophobic molecules.rnA second approach is the preparation of peptide-based hybrid nanocapsules. This is achieved by interfacial polyaddition in inverse miniemulsion with the peptide sequence functionalized with additional amino acids. A method suitable to the use of a peptide sequence for interfacial polyaddition was developed. It is shown that, the polarity of the dispersed phase influences the structures prepared, from particle-like to polymeric shell with a liquid core.rnThe peptide sequence is equipped with a FRET pair (more exactly, an internally-quenched fluorescent system) which allows the real-time monitoring of the enzymatic cleavage of the recognition site. This system shows the successful cleavage of the peptide-based nanocapsules when trypsin preparation is added to the suspensions. A water-soluble fluorescent polymer is efficiently entrapped and its possible use as marker for the capsules is highlighted. Furthermore, a small water-soluble fluorescent dye (SR-101) is successfully encapsulated and the encapsulation efficiency as a function of the functionality of the peptide and the amount of comonomer equivalent (toluene diisocyanate) is studied. The dye is encapsulated at such a high concentration, that self-quenching occurs. Thus, the release of the encapsulated dye triggered by the enzymatic cleavage of the peptide results in a fluorescence recovery of the dye. The fluorescence recovery of the FRET pair in the peptide and of the encapsulated dye correlate well.rnFinally, nanocapsules based on a hepsin-cleavable peptide sequence are prepared. Hepsin is an enzyme, which is highly upregulated in prostate cancer cells. The cleavage of the nanocapsules is investigated with healthy and “cancerous” (hepsin-expressing) cell cultures. The degradation, followed via fluorescence recovery of the FRET system, is faster for the suspensions introduced in the hepsin expressing cell cultures.rnIn summary, this work tackles the domain of responsive nanomaterials for drug delivery from a new perspective. It presents the adaptation of the miniemulsion process for hybrid peptide-based materials, and their successful use in preparing specific enzyme-responsive nanoparticles, with hydrophilic payload release properties.rn

Functional silicones and silicone-containing block copolymers

Polysiloxanes can be synthesized and subsequently modified (i) by the attachment of small molecules that change the properties of the silicone in such a way that it becomes more hydrophilic, but under the premise that this does not go together with a loss of the silicone-specific features. This can be done by adding hydrophilic sidechains to a polysiloxane. Polyethers like poly(ethylene glycol) or hyperbranched polyether-polyols are suitable in this regard. In order to assure that the silicone properties retain, these side groups can be attached to only one part of the polysiloxane backbone, which results in a block copolymer that consists of a common polysiloxane and a second block of the modified structure. (ii) Polysiloxanes can be equipped with functional groups that are capable of initializing polymerization of a different monomer (macroinitiator approach). For example, hydroxyl groups are used to initiate the ring opening polymerization of cyclic esters, or ATRP macroinitiators can be synthesized to add a second block via controlled radical polymerization. Stimuli responsive polymers like poly(oligoethylene glycol methacrylate) (POEGMA) can be added via this route to create “smart” siloxane-containing block copolymers that respond to certain stimuli. rnAn important premise for all synthetic routes is to achieve the targeted structure in a process as simple as possible, because facile availability of the material is crucial with regard to industrial applicability of the invented products. rnConcerning characterization of the synthesized macromolecules, emphasize is put on their (temperature dependent) aggregation behavior, which can be investigated by several microscopic and scattering methods, their behavior at the interface between silicone oils and water and their thermal properties.rnrn

Synthese und Charakterisierung von Polymeren mit peptidischen Seitenketten

Die Forschung im Bereich der Drug Delivery-Systeme konzentriert sich auf biokompatible und wenig immunogene Trägermoleküle. Eine Klasse vielversprechender Trägersysteme stellen Peptid basierte Polymere dar, die neben einer hohen Biokompatibilität auch eine Sensitivität gegenüber externen Einflüssen aufweisen. Der zwitterionische Charakter von Aminosäuren und Peptiden verhindert die Adsorption von Serumproteinen und ein „antifouling“ Verhalten kann festgestellt werden, sodass diese Moleküle für den Einsatz als Wirkstoffträgersystem sehr geeignet scheinen. In Kombination mit einer bürstenartigen Struktur entstehen Systeme mit einer einzigartigen Peptidarchitektur, die sich durch eine hohe Dichte funktioneller Gruppen für Konjugationsreaktionen auszeichnen und deren formabhängige Zellaufnahme sie besonders attraktiv für die Anwendung als „Nanocarrier“ macht.rnrnDas zwitterionische Poly-(ε-N-Methacryloyl-L-Lysin) (Mw = 721,000 g∙mol 1) wurde durch freie radikalische Polymerisation dargestellt und seine Konformation in Abhängigkeit von Ionenstärke und pH-Wert untersucht. Die Biokompatibilität des Systems konnte durch Toxizitätstests und dynamische Lichtstreuung in humanem Blutserum nachgewiesen werden. Zusammen mit der vernachlässigbaren unspezifischen Aufnahme in dendritische Zellen aus Knochenmark erfüllt das System damit alle Bedingungen, die an ein polymeres Wirkstoffträgersystem gestellt werden. Darüber hinaus können Komplexe des Polymers mit DNA in Gegenwart von divalenten Metallionen für die Gentransfektion verwendet werden.rnrnDurch Kopplung von ε-N-Methacryloyl-L-Lysin mit der Elastin-ähnlichen Polypeptid Pentasequenz Valin-Prolin-Glycin-Glycin-Glycin konnte ein Hexapeptid-Makromonomer dargestellt werden, welches anschließend mittels „grafting through“ Polymerisation zur Polymerbürste umgesetzt wurde. Die wurmartige Struktur der Polymerbürsten wurde in AFM-Aufnahmen gezeigt und eine hohe Kettensteifigkeit der Polymerbürsten über dynamische und statische Lichtstreuung nachgewiesen. Zirkulardichroismus-Messungen lieferten Informationen über struktur-, salz- und temperaturabhängige Veränderungen der Konformation. Toxizitätstests und dynamische Lichtstreuung in humanem Blutserum bestätigten die erwartete Biokompatibilität.rnrnBasierend auf zwei Elastin-ähnlichen Polypeptiden mit ähnlicher Peptidsequenz wurden insgesamt vier unterschiedliche Makromonomere mit jeweils 20 Pentapeptid-Wiederholungseinheiten dargestellt. Über anschließende „grafting through“ Polymerisation entstanden molekulare Bürstenmoleküle mit variierenden externen funktionellen Gruppen, die für zukünftige Konjugationsreaktionen verwendet werden können. Der Einfluss von Ionenstärke und Temperatur auf die Konformation der Makromonomere und Polymere wurde mittels Zirkulardichroismus- und Trübungskurven-Messungen untersucht und ein starker Einfluss der hohen Seitenkettendichte auf das Verhalten der Polymerbürsten wurde festgestellt. Über dynamische Lichtstreuung konnte ein von den externen funktionellen Gruppen abhängiges Aggregationsverhalten in humanem Blutserum nachgewiesen werden.rnrnDie in dieser Arbeit synthetisierten Polymerbürsten mit peptidischen Seitenketten stellen damit biokompatible und vielversprechende Trägersysteme für die Konjugation mit Biomolekülen dar, die zukünftig als Drug Delivery-Systeme ihren Einsatz finden können.rn