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Lipopolymers from New 2-Substituted-2-Oxazolines for Artificial Cell Membrane Constructs
Abstract
We present the synthesis of novel 2-oxazoline monomers with different 2-substituents and their consecutive conversion into lipopolymers by living cationic polymerization. The side functions of these monomers were varied to realize different steric needs and hydrogen bonding interactions of the polymer side chains. 2-(2'-N-pyrrolidonyl-ethyl)-2-oxazoline, 2-(3'-methoxymonoethyleneglycol)propyl-2-oxazoline, and 2-(3'-methoxytriethyleneglycol)propyl-2-oxazoline were synthesized. All of the monomers could be converted into the corresponding lipopolymers by living cationic polymerization using 2,3-di-O-octadecyl-1-trifluormethansulfonyl-sn-glycerol as the initiator. The characterization of the 2,3-di-O-octadecyl-glycerol-poly(2-oxazoline) lipopolymers by NMR spectroscopy, IR spectroscopy, and gel permeation chromatography revealed that the targeted molar masses and compositions can be controlled by the initial initiator/monomer ([M](0)/[I](0)) ratio for all the synthesized lipopolymers. The polydispersities were found to be narrow (polydispersity indices from 1.06-1.3). The amphiphilic lipopolymers were spread at the air-water interface (Langmuir-Blodgett film balance) and the effect of the polymer side groups and chain lengths upon the Pi-area (A) isotherms of the corresponding lipopolymer monolayers were compared and analyzed. The impact of the polymer side functionalities on a 2D gel formation was examined using an interfacial rheometer operated in an oscillating stress-strain mode. Interestingly enough, none of the newly synthesized lipopolymers showed a rheological transition. This somewhat surprising result not only verified that these 2D gels are not established by hydrogen bonding among hydrophilic polymer moieties, as earlier proposed, but also supported the concept of jammed surface micelles as the more likely origin for the gelation phenomenon. [Diagram: see text]
... The CROP of 2-oxazoline monomers that contain heteroatoms in the side chain is more scarce. Monomers with oxygen heteroatoms have been reported with, for example, oligo ethylene glycol (199), ketal (66,76), and ester (181) functionalities. The latter two being protected functional groups for aldehydes or carboxylic acids and alcohols, respectively (Fig. 18) (181). ...
... Functional 2-oxazolines containing nitrogen entities in the side chain that are utilized for CROP include Boc-protected amines (66), pyrrolidone (199) and even azide-based 2-oxazoline monomers (188,200) (Fig. 19). Less commonly reported is the CROP of 2-oxazolines with side-chain functionalities containing silica (179,201), sulfur (180,189,202), and phosphoric ester (66) moieties (Fig. 19). ...
... A last class of functional 2-oxazoline monomers is the macromonomers with a polymeric side chain reported with polystyrene (203)(204)(205), PEG (199) and poly(ϵ-caprolactone) (206) side chains, which can be used to prepare graft copolymers with a PAOx backbone by CROP. ...
Research in the field of poly(2-oxazoline)s (PAOx) is rapidly expanding as this polymer class combines high synthetic versatility and biocompatibility, opening up the way to highly functional (biocompatible) materials. PAOx are prepared by living cationic ring-opening polymerization (CROP) of 2-oxazolines. The variety of 2-oxazoline monomers that are readily available or can easily be synthesized allows for tuning of polymer properties and introduction of diverse functionalities. Moreover, thanks to the living nature of CROP, well-defined polymers with narrow molar mass distribution and high end group fidelity can be obtained. This article covers all aspects of PAOx ranging from the synthesis of 2-oxazoline monomers, via in-depth discussion on the CROP mechanism to the synthesis and properties of functional PAOx (co)polymers. The presented research demonstrates that owing to their structural adaptability and the so-called “stealth” behavior, PAOx are well suited for a range of biomedical applications, including polymer therapeutics, scaffolds for three-dimensional cell culture, surface modification, matrix excipient for solid dispersions, and antimicrobial agents.Keywords:poly(2-oxazoline);2-oxazoline;biocompatible;cationic ring-opening polymerization;living polymerization
... In the specific case of castor oil the synthesis of tosylate initiator from the native alcohol group produces a similar structure of ramified LipoPOx as represented in Scheme 5d (Giardi et al., 2009). In other cases, ramified LipoPOx results from diacylglycerol, DAG, converted into tosylate initiator (Einzmann and Binder, 2001;Lüdtke et al., 2005) (Scheme 5e). The last class of ramified LipoPOx concerns the brush polymers obtained by the copolymerization of hydrophilic oxazoline and 2-R L -2-oxazolines (Baekmark et al., 1998) (Scheme 5f). ...
... Used at lipopolymer tethers, the lipid head group of the LipoPOx incorporates into the lipid bilayer by hydrophobic interactions to support phospholipid bilayers (Förtig et al., 2004). The morphology, stability and physical properties (lipid diffusivity, mobile lipid fraction) of the supported lipid membrane can be modulated with the LipoPOx HLB (Lüdtke et al., 2005). Indeed, it is possible to fine-tune the LipoPOx HLB depending on the choice of the monomer, the degree of polymerisation and the lipid head group (Förtig et al., 2004). ...
Facing the growing demand in nano drug delivery systems (nDDS), hybrid excipients based on natural molecules and well-defined synthetic polymers are intensively investigated. Lipopolyoxazolines (LipoPOx) composed of a polyoxazoline block (POx) and a lipid or lipid-like derivative are detailed in this review. The nature of lipids used, the route to synthesize LipoPOx and their advantages for the formulation of drugs are reported. The place of POx family in nanomedicine is discussed compared to PEG, considered as the gold standard of hydrophilic polymers. LipoPOx nanoformulations including liposomes, mixed micelles, lipid nanocapsules are provided alongside discussion of the nDDS for intravenous or topical administration.
... POx ailesi oldukça işlevsel bir yapı sergilemektedir, örneğin; Her iki zincir ucu kimyasal (örn; -OH, -NH vb.) ya da yapısal (örn; lipit) işlevsellik ile donatılmıştır 17 . Bununla birlikte, 2-oksazolin monomerlerinin bir kaçı ticari olarak (örn, 2-metil-, 2-etil-, 2-isopropil, 2-fenil-2-oksazolin) ve diğer alifatik yan zincir ve fonksiyonel yan zincirler taşıyan 2-oksazolin monomerlerinin büyük bir kısmı da sentez sırasında monomerin 2 pozisyonundaki fonksiyonel grubun değiştirilmesiyle kolaylıkla elde edilebilir. ...
... Bu materyallerin çözelti ortamında ya da yüzeyler üzerinde kendiliğinden düzenlendiği belirtilmiştir. Polimer yan grupları ve zincir sonlarına fonksiyonel ve reaktif grupların kolaylıkla eklenmesi (uç-grup fonksiyonelleştirme) bu polimer sınıfını terapötik özellikte yüksek miktarda genetik materyal ve ilaç yükleme, proteinlere konjuge edilme, lipozomal çift tabaka üzerine bağlanma, miseller yapı oluşturma ve yüzeyler üzerine uygulanma gibi avantajlı özellikleri nedeniyle taşıyıcı sistem geliştirilmesi için ideal bir platform haline getirir [14][15][16][17][18][19][20][21][22][23][24][25] . POx tabanlı polimerlerin büyük bir kısmında in vitro sitotoksisite çalışılmış ve genellikle insan nöral ata hücreleri, Madin-Darby Canine böbrek hücreleri, MCF-7, HEPG2 ve CATH-a-hücrelerinde sitotoksisite oldukça düşük bulunmuştur 31 . ...
Sentetik polimerler taşıyıcı sistemler için umut verici materyaller olma yolunda hızla ilerlemektedir. Son zamanlarda biyomateryal olarak kullanı-lan çevresel şartlara duyarlı polimerlerin sentezi için oldukça yoğun bir ilgi bulunmaktadır. İstenilen kimyasal, fiziksel ve biyolojik özelliklere sahip çe-şitli polimer formülasyonları geliştirilmiştir. Bu özellikler pH, sıcaklık, ışık ve iyonik güç gibi bir uyarıcının etkisine bağlı olarak etkin maddenin kontrollü salımı için oldukça önemlidir. Bu bahsedilen özellikler polimerik materyalle-rin, etkin madde ve gen taşıma, biyomembran teknolojisi ve biyokataliz gibi biyomedikal uygulamalarda kullanımı için gereklidir. Polimerik materyalin biyouyumluluk ve immünotoksisitesi, taşıyıcı sistemler için uygun materyal-lerin keşfedilmesi açısından önemli bir konudur 1. Polimer tabanlı kendiliğin-den düzenlenen sistemler, etkin madde taşınması, ayırma teknolojileri, da-ğılım stabilizasyonu gibi geniş uygulama alanlarında gerekli çok moleküllü yapıların oluşumunda önemli bir ilerlemeyi temsil eder. Bu bağlamda, farklı taşıyıcı sistemler için uygun sentetik polimerlerin bir çeşidi olarak, 2-oksa-zolinler çok yönlü ve umut verici materyallerdir. 2-oksazolin tabanlı polimerler, fonksiyonel gruplar ile işlevselleştirilerek kendi alanlarında biyolojik ve medikal kullanım için önemli bir rol üstlen-mekle birlikte özellikle biyolojik ve tıbbi olarak etkin ve genetik madde taşın-ması gibi temel uygulama alanlarında etkinlik göstermektedirler.
... Jordan et al. prepared lipopolymers by using 2,3-di-O-octadecyl-1-trifluormethanesulfonyl-snglycerol as initiator for the CROP of (Py)EtOx, (MeO-EO 1 ) n PrOx, and (MeO-EO 3 ) n PrOx [56] (Scheme 3). The influence of the side-chain functionalities in terms of 2D-gel formation was investigated. ...
... For both systems, the poly(ethylene glycol)-as well as the pEtOx-based one, similar results were obtained in terms of preserving the enzymatic activity, increasing the drug hydrodynamic volume and being protein-repellant. Scheme 3. Structural formula of telechelic (MeO-EO 1 ) n PrOx mimicking a lipopolymer structure (for details, see reference [56]). ...
The polymer class of poly(2-oxazoline)s currently is under intensive investigation due to the versatile properties that can be tailor-made by the variation and manipulation of the functional groups they bear. In particular their utilization in the biomedic(in)al field is the subject of numerous studies. Given the mechanism of the cationic ring-opening polymerization, a plethora of synthetic strategies exists for the preparation of poly(2-oxazoline) s with dedicated functionality patterns, comprising among others the functionalization by telechelic end-groups, the incorporation of substituted monomers into (co)poly(2-oxazoline)s, and polymeranalogous reactions. This review summarizes the current state-of-the-art of poly(2-oxazoline) preparation and showcases prominent examples of poly(2-oxazoline)-based materials, which are retraced to the desktop-planned synthetic strategy and the variability of their properties for dedicated applications.
... Water-soluble polyoxazolines have been studied for various biomedical applications, because of their amphiphilic and self-assembling nature [114][115][116][117]. These polymers, with various chemical functionalities and architectures, can be synthesized by cationic ring-opening polymerization in a controlled manner [116,[118][119][120]. Poly(2-methyl-2-oxazoline) (PMOZ)-and poly(2-ethyl-2-oxazoline) (PEOZ)-linked DSPE (8) have been investigated for surface modification of liposomes as an alternative to PEG-DSPE [121]. ...
... Water-soluble polyoxazolines have been studied for various biomedical applications, because of their amphiphilic and self-assembling nature [114][115][116][117]. These polymers, with various chemical functionalities and architectures, can be synthesized by cationic ring-opening polymerization in a controlled manner [116,[118][119][120]. Poly(2-methyl-2-oxazoline) (PMOZ)-and poly(2-ethyl-2-oxazoline) (PEOZ)-linked DSPE (8) have been investigated for surface modification of liposomes as an alternative to PEG-DSPE [121]. ...
Liposomes are used as a delivery vehicle for drug molecules and imaging agents. The major impetus in their biomedical applications comes from the ability to prolong their circulation half-life after administration. Conventional liposomes are easily recognized by the mononuclear phagocyte system and are rapidly cleared from the blood stream. Modification of the liposomal surface with hydrophilic polymers delays the elimination process by endowing them with stealth properties. In recent times, the development of various materials for surface engineering of liposomes and other nanomaterials has made remarkable progress. Poly(ethylene glycol)-linked phospholipids (PEG-PLs) are the best representatives of such materials. Although PEG-PLs have served the formulation scientists amazingly well, closer scrutiny has uncovered a few shortcomings, especially pertaining to immunogenicity and pharmaceutical characteristics (drug loading, targeting, etc.) of PEG. On the other hand, researchers have also begun questioning the biological behavior of the phospholipid portion in PEG-PLs. Consequently, stealth lipopolymers consisting of non-phospholipids and PEG-alternatives are being developed. These novel lipopolymers offer the potential advantages of structural versatility, reduced complement activation, greater stability, flexible handling and storage procedures and low cost. In this article, we review the materials available as alternatives to PEG and PEG-lipopolymers for effective surface modification of liposomes.
... Interestingly enough, the C 16 chain DPPE–PEG2000 displayed a 10 mN m −1 higher π high relative to the C 18 chain DSPE–PEG2000, and the C 14 chain DMPE–PEG2000 never displayed π high at all, thus indicating a sensitive relationship between acyl chain length of the lipid moiety and π high . In order to explore further the importance of the lipid tail to the high-pressure transition, [7, 8]) pressure isotherms were undertaken with the partially unsaturated 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine–PEG2000 (DOPE–PEG2000), and compared to DSPE–PEG2000 [7]. No high pressure transition was found in the pressure–area isotherm of the unsaturated-lipid lipopolymer Langmuir monolayer. ...
... Overall , these data indicate that the location of the high-pressure transition depends on the subtle interplay of several factors, including the polymer structure and molecular weight and the nature of the hydrophobic anchor. Using a synthetic approach to understanding the nature of the acyl chain condensation , a number of novel oxazoline lipopolymers were synthesized with the same lipid backbone, two 18 carbon chains attached to glycerol, but with polymers characterized by having different sidechains including a methoxymonoethylene glycol and an isopropylmethoxymonoethylene glycol on the ethyl end of the ethyloxazoline polymer (DiC 18 PMOGOx 21 and DiC 18 PTEGOx 18 ) [8]. These polymers are collected inFig. ...
Polymer-tethered membranes combine fascinating structural, dynamic, and viscoelastic properties. Many important insights into
these peculiar supramolecular systems can be obtained from studies on polymer-tethered monolayers. This chapter discusses
recent experimental findings on polymer-tethered monolayers at the air–water interface. In particular, Langmuir monolayers
which are comprised of pure lipopolymers and of binary phospholipid–lipopolymer mixtures are considered. Thermodynamic data
as well as structural data based on a host of experimental techniques including X-ray and neutron reflectrometry, infrared
reflection absorption spectroscopy, and sum frequency generation spectroscopy provide information on how lipopolymers organize
at the air–water interface. This information is followed by a review of the viscoelastic properties of these systems, including
the remarkable gelation transition that can be observed in lipopolymers and mixed phospholipid–lipopolymer monolayers. The
diffusion properties are also discussed at length, and show that lipid diffusivity is critically dependent on the strength
of inter-polymer interactions of lipopolymers.
... [24,25] Recently, we investigated self-association and gelation of the polymer by a combination of end-functionalization of the polymer with a lipid moiety (lipopolymer) and by a variation of the steric demands and hydrogen bonding sites in the polymer side group. [26] By a double end-functionalization with a lipid and a coupling group, surface-bond lipopolymer scaffolds for the construction of biomimetic membranes can be produced. [27][28][29][30][31][32] Since amphiphilicity, as well as the chemical functionality can be combined by copolymerization of the various 2-oxazoline monomers, the solubility and the chemistry of the polymer can be fine-tuned to a desired application. ...
... Other commercially available chemicals (Aldrich) were used without further purification. [26,35] The same purification procedures were used in the NMR and ATR FT-IR spectroscopy and gel permeation chromatography (GPC) experiments. NMR spectra were recorded in CDCl 3 , unless otherwise stated, at 250 or 300 MHz. ...
A new 2-oxazoline monomer with a Boc protected amino function, 2-[N-Boc-5-aminopentyl]-2-oxazoline; (Boc-AmOx), was synthesized from commercially available Compounds. With an initiator salt system (N-methyl-2-methyl-2-oxazolinium triflate: MeOxOTf), the monomer could be converted via living cationic ring-opening polymerization to well-defined homopolymers with narrow molar mass distributions and targeted polymer chain length. After a quantitative deprotection, poly(2-oxazoline)s with pendant amino functions were obtained. In order to vary the polymer functional group density and solubility of the polymer, copolymerization with different monomer ratios of BocAmOx and 2-ethyl-2-oxazoline (EtOx) was performed. Exsitu NMR spectroscopy studies verified the randomness of the cationic copolymerization. The accessibility of the pendant amino side functions was confirmed in polymer analog thiourea formation with different isothiocyanates, such as benzyl isothiocyanate (BzNCS), or a fluorescence dye, tetramethyl rhodamine isothiocyanate (TRITC). A cross-linking reaction with a bifunctional isothiocyanate (Ph(NCS)(2)) resulted in poly(2-oxazoline) hydrogels.
... [19][20][21][22] Furthermore, the living cationic ring-opening polymerization (LCROP) of 2-substituted 2-oxazoline provides synthetic possibilities to tailor macromolecules with a broad variety of architectures, composition, and numerous side and end functions. [23][24][25][26][27][28][29][30][31][32] Additionally, the LCST of hydrophilic POx can be tailored over a broad temperature range. [33][34][35][36][37][38][39] For the preparation of POx cylindrical molecular brushes all three routes (grafting-from, -through, or -to) are possible; however, e.g., the grafting through of POx macromonomers was employed with comonomers to realize sufficient degrees of polymerization. ...
... Although a targeted degree of polymerization of m ) 25 for the side chains, in all cases slightly shorter chains of about m ) 20 were determined (Table 1). An incomplete grafting from or termina- 24 1.20 220 P(IPOx n -g-iPrOx m ) A 49 d 125 1.33 220 18 25 a Calculated against initial initiator feed. b As calculated from GPC traces. ...
We report on the synthesis and characterization of cylindrical molecular brushes based on poly(2-oxazoline)s (Pox). The dual-functional monomer, 2-isopropenyl-2-oxazoline (IPOx), was first converted to a poly (2-isopropenyl-2-oxazoline), backbone by free radical (PIPOX(R)) or living anionic polymerization (PIPOX(A)). Quantitative reaction with methyl triflate yields a macroinitiator salt (PIPOxOTf(R/A)) for the preparation of molecular brushes via the grafting from approach by living cationic polymerization of 2-oxazolines (2-methyl-, 2-ethyl-, and 2-isopropyl-2-oxazoline). Characterization of the resulting molecular brushes by NMR and FTIR spectroscopy indicates a very high side chain grafting density and quantitative reactions. Visualization of adsorbed molecular brushes by AFM corroborates this assumption. Furthermore, the lower critical solution temperatures of the POx molecular brushes were determined. The transition temperatures were found to be very defined, reversible. and with no noticeable hysteresis.
... The choice of monomers is only limited by the incompatibility of nucleophilic reactive groups with the CROP process, therefore other functionalities like amines, alcohols, thiols or carboxylic acids rely on the use of protection techniques. [463] Alcohols, aldehydes [211] and carboxylic acids [512,[582][583] are usually protected by esters [512] or acetal groups [502,584] , whereas amines and thiols are protected with ...
In this work, a toolbox was provided to create three-component polymer conjugates with a defined architecture, designed to bear different biocomponents that can interact with larger biological systems in biomacromolecular recognition experiments. The target architecture is the attachment of two biomolecule ‘arms’ to the alpha telechelic end point of a polymer and fixating the conjugate to the gold surface of SAW and SPR sensor chips with the polymer’s other omega chain end. This specific design of a conjugate will be implemented by using a strategy to yield novel double alpha as well as omega telechelic functionalized POx and the success of all cascade reaction steps leading to the final conjugation product will be proven through affinity measurements between covalently bound mannose and ConA. All reactions were performed on a low molecular model level first and then transferred to telechelic and also side chain functionalized polymer systems.
... The phospholipids DOPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and CHOL were purchased from Avanti Polar Lipids (Alabaster, AL). The lipopolymer 1,2-dioctadecyl-sn-glycero-3-N-poly(2-methyl-2oxazoline) 50 was synthesized as reported previously (31). The dye-labeled phospholipids N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadec-anoylsn-glycero-3-phosphoethanolamine, triethylammonium salt (NBD-DHPE) and N-(6-tetramethylrhodamine-thiocarbamoyl)-1, 2-dihexadecanayl-snglycero-3-phosphoethanolamine, triethylammonium salt (TRITC-DHPE) were obtained from Invitrogen (Carlsbad, CA). ...
The influence of cholesterol (CHOL) level on integrin sequestration in raft-mimicking lipid mixtures forming coexisting liquid-ordered (lo) and liquid-disordered (ld) lipid domains is investigated using complementary, single-molecule-sensitive, confocal detection methods. Systematic analysis of membrane protein distribution in such a model membrane environment demonstrates that variation of CHOL level has a profound influence on lo-ld sequestration of integrins, thereby exhibiting overall ld preference in the absence of ligands and lo affinity upon vitronectin addition. Accompanying photon-counting histogram analysis of integrins in the different model membrane mixtures shows that the observed changes of integrin sequestration in response to variations of membrane CHOL level are not associated with altering integrin oligomerization states. Instead, our experiments suggest that the strong CHOL dependence of integrin sequestration can be attributed to CHOL-mediated changes of lipid packing and bilayer thickness in coexisting lo and ld domains, highlighting the significance of a biophysical mechanism of CHOL-mediated regulation of integrin sequestration. We envision that this model membrane study may help clarify the influence of CHOL in integrin functionality in plasma membranes, thus providing further insight into the role of lipid heterogeneities in membrane protein distribution and function in a cellular membrane environment.
... 8,[18][19][20] Furthermore, multiple functionalization and structural versatility are possible using respective initiators, monomers and terminating agents. 7,[21][22][23][24][25][26][27][28][29][30] After early approaches 31,32 yielding a variety of POx-based comb polymers, [33][34][35][36][37][38][39][40][41][42][43][44][45][46] POx molecular brushes have been synthesized recently by several routes. The majority of the applied methods focuses on the polymerization of macromonomers by group transfer polymerization(GTP), free radical polymerization (FRP) or reversible addition/fragmentation chain transfer polymerization (RAFT). ...
Molecular brushes of poly(2-oxazoline)s (POx) are an intriguing class of polymers as they combine a unique architecture with the properties of POx as a biomaterial. Here, the synthesis of several POx macromonomers with methacrylate end groups and consecutive grafting through polymerization by aqueous atom transfer radical polymerization (ATRP) at room temperature is reported. 1H-NMR spectroscopy and size exclusion chromatography (SEC) confirmed the synthesis of POx molecular brushes with maximum side chain grafting densities, narrow molar mass distributions (Ð ≤ 1.16) and final molar masses corresponding to the initial macromonomer : initiator ratio. Chain extension experiments show high end group fidelity, formation of block copolymer molecular brushes and kinetic studies revealed a polymerization behavior of oligo(2-methyl-2-oxazoline methacrylate) very similar to the frequently used oligo(ethylene glycol) methacrylate (OEGMA475). Aqueous solutions of POx molecular brushes with poly(2-ethyl- and 2-isopropyl-2-oxazoline) side chains exhibit the typically defined thermoresponsive behavior with a tunable, very narrow and reversible phase transition.
... The phospholipids 1,2-dioleoyl-sn-glycro-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), as well as CHOL were obtained from Avanti Polar Lipids (Alabaster, AL). The lipopolymer 1,2-dioctadecyl-sn-glycero-3-N-poly(2-methyl-2-oxazoline) 50 (diC 18 M 50 ) was synthesized as reported previously (33). The dye-labeled phospholipids N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadec-anoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (NBD-DHPE) and N-(6-tetramethylrhodamine-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TRITC-DHPE) were purchased from Invitrogen (Carlsbad, CA). ...
Lipid heterogeneities, such as lipid rafts, are widely considered to be important for the sequestering of membrane proteins in plasma membranes, thereby influencing membrane protein functionality. However, the underlying mechanisms of such sequestration processes remain elusive, in part, due to the small size and often transient nature of these functional membrane heterogeneities in cellular membranes. To overcome these challenges, here we report the sequestration behavior of urokinase receptor (uPAR), a glycosylphosphatidylinositol-anchored protein, in a planar model membrane platform with raft-mimicking lipid mixtures of well-defined compositions using a powerful optical imaging platform consisting of confocal spectroscopy XY-scans, photon counting histogram, and fluorescence correlation spectroscopy analyses. This methodology provides parallel information about receptor sequestration, oligomerization state, and lateral mobility with single molecule sensitivity. Most notably, our experiments demonstrate that moderate changes in uPAR sequestration are not only associated with modifications in uPAR dimerization levels, but may also be linked to ligand-mediated allosteric changes of these membrane receptors. Our data show that these modifications in uPAR sequestration can be induced by exposure to specific ligands (urokinase plasminogen activator, vitronectin), but not via adjustment of the cholesterol level in the planar model membrane system. Good agreement of our key findings with published results on cell membranes confirms the validity of our model membrane approach. We hypothesize that the observed mechanism of receptor translocation in the presence of raft-mimicking lipid mixtures is also applicable to other glycosylphosphatidylinositol-anchored proteins.
... The phospholipids DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), and CHOL were purchased from Avanti Polar Lipids (Alabaster, AL). The procedure for synthesizing the lipopolymer diC 18 M 50 (1,2-dioctadecyl-sn-glycero-3-n-poly(2-methyl-2-oxazoline) 50 ) has been described previously in Lüdtke et al. (25). The dye-labeled lipids NBD-DHPE (n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadec-anoyl-sn-glycero-3-phosphoethanolamine), TRITC-DHPE (n-(6-tetramethylrhodamine-thiocarbamoyl)-1, 2-dihexadecanayl-sn-glycero-3-phosphoethanolamine, triethylammonium salt), DID (1,1 0 -dioctadecyl-3,3,3 0 ,3 0 -tetramethylindodicarbocyanine,4chlorobenzenesulfonate salt), and DiI (1,1 1 -dioctacadecayl-3,3,3 1 ,3 1 -tetramethylindocarbocyanine perchlorate), as well as the kits for fluorescently labeling antibodies with Alexa-555 were obtained from Invitrogen (Carlsbad, CA). ...
There is growing recognition that lipid heterogeneities in cellular membranes play an important role in the distribution and functionality of membrane proteins. However, the detection and characterization of such heterogeneities at the cellular level remains challenging. Here we report on the poorly understood relationship between lipid bilayer asymmetry and membrane protein sequestering in raft-mimicking model membrane mixtures using a powerful experimental platform comprised of confocal spectroscopy XY-scan and photon-counting histogram analyses. This experimental approach is utilized to probe the domain-specific sequestering and oligomerization state of αvβ3 and α5β1 integrins in bilayers, which contain coexisting liquid-disordered/liquid-ordered (ld/lo) phase regions exclusively in the top leaflet of the bilayer (bottom leaflet contains ld phase). Comparison with previously reported integrin sequestering data in bilayer-spanning lo-ld phase separations demonstrates that bilayer asymmetry has a profound influence on αvβ3 and α5β1 sequestering behavior. For example, both integrins sequester preferentially to the lo phase in asymmetric bilayers, but to the ld phase in their symmetric counterparts. Furthermore, our data show that bilayer asymmetry significantly influences the role of native ligands in integrin sequestering.
... ring-opening polymerization (LCROP) of 2-substituted-2oxazolines allows for the synthesis of defi ned macromolecules with a broad variety of architectures, [38][39][40][41][42][43] composition, [ 44 , 45 ] side and end functionalities, [46][47][48][49][50][51][52][53][54][55][56] able to cope with the various requirements for biomaterials development. [ 5 , 57 , 58 ] As the LCROP allows for defi ned random, gradient as well as block copolymerization and thus, that is, the amphiphilic contrast including the thermoresponsiveness of POx can be tuned in a wide range. ...
Molecular brushes (MBs) of poly(2-oxazoline)s were prepared by living anionic polymerization of 2-isopropenyl-2-oxazoline to form the backbone and living cationic ring-opening polymerization of 2-n-propyl-2-oxazoline and 2-methyl-2-oxazoline to form random and block copolymers. Their aqueous solutions displayed a distinct thermoresponsive behavior as a function of the side-chain composition and sequence. The cloud point (CP) of MBs with random copolymer side chains is a linear function of the hydrophilic monomer content and can be modulated in a wide range. For MBs with block copolymer side chains, it was found that the block sequence had a strong and surprising effect on the CP. While MBs with a distal hydrophobic block had a CP at 70 $^circ$C, MBs with hydrophilic outer blocks already precipitated at 32 $^circ$C.
... [107] The absence of physical gelation with other hydrophilic lipopoly(2-oxazoline)s with more bulky side chains indicated that hydrogen bonding between the polymer chains does not play an important role. [108] More recently, the two-dimensional diffusion of the center of mass of a single lipoPMeOx molecule in monolayers at the airwater interface was investigated by single-molecule fluorescence microscopy, which revealed different diffusion mechanisms at different surface concentrations of the polymer. [109] Besides the self-assembly of pure lipopoly(2-oxazoline)s, the coassembly of phospholipids and silane-functionalized lipopoly(2-oxazoline)s at the air-water interface followed by transfer of the (condensed) monolayer onto a glass slide was reported by Jordan and co-workers. ...
Die lebende kationische Ringöffnungspolymerisation von 2-Oxazolinen ist seit ihrer Entdeckung 1966 eingehend untersucht worden. Die Vielseitigkeit der lebenden Polymerisation ermöglicht die Copolymerisation verschiedener 2-Oxazolin-Monomere zu Polymeren mit einstellbaren Eigenschaften; so lassen sich z. B. hydrophile, hydrophobe, fluorophile sowie harte und weiche Materialien erhalten. Allerdings geriet diese Polymerklasse wegen der langen Reaktionszeiten und ihrer beschränkten Anwendungsmöglichkeiten in den 1980er und 1990er Jahren fast in Vergessenheit. Im neuen Jahrtausend erlebten die Poly(2-oxazoline) jedoch einen neuen Aufschwung wegen ihrer möglichen Verwendung als Biomaterialien oder thermoresponsive Stoffe sowie dank der Tatsache, dass sie einen leichten Zugang zu definierten amphiphilen Strukturen für die (hierarchische) Selbstorganisation eröffnen. In diesem Aufsatz werden neue Entwicklungen beschrieben, die das Potenzial der Poly(2-oxazoline) demonstrieren. Außerdem wird die vielversprechende Kombination von Poly(2-oxazolinen) mit der Klickchemie diskutiert.
... 94 Lipopolymers were prepared as a potential candidate for constructing tailored model cell membranes, in which a lipid trifrate was used as initiator for CROP of hydrophilic monomers, MeOZO and EtOZO, to produce an amphiphilic polymers [Scheme 16(b)]. 95 Glycoproteins play important functions in mechanism like bioadhesion, cell-cell interactions, and recognition phenomena. A glycoprotein analog was newly prepared by combining a linear polysaccharide block and a PROZO block (psuedopeptide), for which hyaluronic acid (hyaluronan, HA) and PEtOZO were used, respectively. ...
Chemistry of 2-oxazolines is involved in the polymer synthesis fields of cationic ring-opening polymerization (CROP) and enzymatic ring-opening polyaddition (EROPA), although both polymerizations look like a quite different class of reaction. The key for the polymerization to proceed is combination of the catalyst (initiator) and the design of monomers. This article describes recent developments in polymer synthesis via these two kinds of polymerizations to afford various functional polymers having completely different structures, poly(N-acylethylenimine)s via CROP and 2-amino-2-deoxy sugar unit-containing oligo and polysaccharides via EROPA, respectively. From the viewpoint of reaction mode, an acid-catalyzed ring-opening polyaddition (ROPA) is considered to be a crossing where CROP and EROPA meet. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 1251–1270, 2010
... For biomedical applications the POx system is especially interesting as hydrophilic POx are nontoxic [31][32][33][34][35] and display an advantageous biodistribution and excretion behavior [36]. Thus, POx are currently under intense study for biological and biomedical applications [37][38][39] such as drug-delivery, biohybrid [40,41], and biomimetic [42][43][44][45][46][47][48][49][50][51][52] systems such as artificial cell membranes. ...
We have studied different thermo-responsive poly(2-oxazoline)s with iso-propyl (iPrOx) and n-propyl (nPrOx) pendant groups in aqueous solutions, where they exhibit LCST behavior. This paper focuses on the effect of the degree of polymerization, n, the concentration, c, in the dilute regime, and the presence of hydrophobic moieties. The cloud points were investigated as a function of the degree of polymerization, n, and of the polymer concentration, c. The aggregation behavior near the cloud point was studied by temperature-resolved small-angle neutron scattering and fluorescence correlation spectroscopy, i.e. a combination of ensemble and single molecule methods. We found that at the cloud points large aggregates are formed and that the cloud points depend strongly on both, n and c. Diblock copolymers from iPrOx and nPrOx form large aggregates already at the cloud point of PnPrOx, and, unexpectedly, no micelles are observed between the cloud points of the two blocks. Gradient copolymers from iPrOx and n-nonyl-2-oxazoline (NOx) display a complex aggregation behavior resulting from the interplay between intra- and intermolecular association mediated by the hydrophobic NOx blocks. Above the cloud point, an intermediate temperature regime with a width of a few Kelvin was found with small but stable polymer aggregates. Only at higher temperatures, larger aggregates are found in significant number.
... lipid) functionality. [3] A number of 2-oxazoline monomers are commercially available (e.g. 2-methyl-, 2-ethyl-, 2-isopropyl, 2phenyl-2-oxazoline) and a wide range of 2-oxazoline monomers carrying other aliphatic side chains and functional side chains are readily accessible. ...
The family of poly(2-oxazoline)s (POx) is being increasingly investigated in the context of biomedical applications. We tested the relative cytotoxicity of POx and were able to confirm that these polymers are typically not cytotoxic even at high concentrations. Furthermore, we report structure-uptake relationships of a series of amphiphilic POx block copolymers that have different architectures, molar mass and chain termini. The rate of endocytosis can be fine-tuned over a broad range by changing the polymer structure. The cellular uptake increases with the hydrophobic character of the polymers and is observed even at nanomolar concentrations. Considering the structural versatility of this class of polymers, the relative ease of preparation and their stability underlines the potential of POx as a promising platform candidate for the preparation of next-generation polymer therapeutics.
... Studies of the interfacial properties of amphiphilic diblock copolymers with a (2-alkyl-2-oxazoline) block-indicate the propensity of poly(2-alkyl-2-oxazoline) to adsorb at the A/W interface [21]. A poly(2-methyl-2-oxazoline) or a poly(2-ethyl-2-oxazoline) carrying a dialkyl phospholipid on one chain end (lipopolymer) also adsorb at the A/W interface, forming a quasi two-dimensional physical network as a result of strong hydrophobic interactions between the phospholipid-groups aligned in the air-phase, coupled with water mediated organization of the tethered poly(2-alkyl-2-oxazoline) chains in the subphase [22][23][24][25][26]. ...
We describe herein the properties at the air/water (A/W) interface of hydrophobically end-modified (HM) poly(2-isopropyl-2-oxazoline)s (PiPrOx) bearing an n-octadecyl chain on both termini (telechelic HM-PiPrOx) or on one chain end (semitelechelic HM-PiPrOx) for different subphase temperatures and spreading solvents using the Langmuir film balance technique. The polymer interfacial properties revealed by the pi-A isotherms depend markedly on the architecture and molecular weight of the polymer. On cold water subphases (14 degrees C), diffusion of PiPrOx chains onto water takes place for all polymers in the intermediate compressibility region (5mNm(-1)). At higher subphase temperatures (36 and 48 degrees C), the HM-PiPrOx film exhibited remarkable stability with time. Brewster angle microscopy (BAM) imaging of the A/W interface showed that the polymer assembly was not uniform and that large domains formed, either isolated grains or pearl necklaces, depending on the polymer structure, the concentration of the spreading solution and the subphase temperature. The Langmuir films were transferred onto hydrophilic substrates (silica) by the Langmuir-Blodgett (LB) technique and onto hydrophobic substrates (gold) by Langmuir-Schaefer (LS) film deposition, resulting in the formation of adsorbed particles ranging in size from 200 to 500nm, depending on the polymer architecture and the substrate temperature. The particles presented "Janus"-like hydrophilic/hydrophobic characteristics.
Poly(2‐alkyl/aryl‐2‐oxazoline)s (PAOxs) have gained immense attraction in the polymer research community because of their relative ease of microwave‐assisted synthesis, tailor‐made properties and, most importantly, recent recognition as an upcoming polymer platform for many medical and biological applications (next generation polymer therapeutics/biomaterials). The materials and solution properties of PAOx can be easily tuned by changing the side chain functionalities. This review describes very recent advances in structures and side‐chain‐dependent properties of PAOx that are reported in the literature from 2017 and onwards. The review starts with providing an encyclopaedic overview of newly reported PAOx homopolymers, including their design strategies and limitations. Subsequently, their side‐chain‐dependent thermal and solution properties are discussed. © 2022 Society of Industrial Chemistry.
Liposome surface modifications serve great potential applications of liposomes, for instance, increasing stability, bioactive liposome conjugates, and targeted drug, gene, and image agent delivery. In this study, novel targeted lipopolymers, peptide 18/peptide 563-poly(2-ethyl-2-oxazoline)-dioleoylphosphatidyl-ethanolamine (P18/P563-PEtOx-DOPE), have been demonstrated to be successfully synthesized. The structures of P18/P563-PEtOx-DOPE were confirmed by FT-IR spectroscopy, GPC, and ¹H-NMR. In this strategy, poly(2-ethyl 2-oxazoline)-modified liposomes were firstly constructed with molecular weights of 3,500 and 5,800 Da. Then, we chose PEtOx5800-DOPE because it has been obtained better particle size (88.74 ± 0.6816) according to the DLS results. Then, peptides- and dye-PEtOx lipid-based nanovesicle (LN) were prepared by peptide-18, peptide-563, and 7-mercapto-4-methyl coumarin. Genetic material (pDNA) was encapsulated into the liposomes and evaluated the encapsulation of plasmid DNA with migration by using agarose gel electrophoresis. In vitro cytotoxicity experiment results on prostate cancer and breast cancer cell lines, parallelly with the healthy prostate (PNT1A) and breast (MCF10A) epithelial cell lines, cells showed insignificant toxic effects. Thus, we can suggest a novel PEtOx phospholipid thanks to this article and its integration with ligands, which great potential for gene transfer system.
Poly(2-oxazoline)s have been investigated for decades as biomaterials. Pioneering early work suggested that hydrophilic poly(2-oxazoline)s are comparable to poly(ethylene glycol) regarding their potential as biomaterials, but the ready commercial availability of the latter has led to its meteoric rise to become the gold standard of hydrophilic synthetic biomaterials. In contrast, poly(2-oxazoline)s almost fell into oblivion. However, in the last decade, this family of polymers has gained much more interest in general and as biomaterials in particular. The rich chemistry and comparably straightforward synthesis of poly(2-oxazoline)s gives many opportunities for tailoring the properties of the resulting biomaterials, allowing the chemist to explore new conjugation chemistry, and to fine-tune the molar mass, hydrophilic-lipophilic balance as well as architecture. Thus, the wide range of demands for various applications of biomaterials can be suitably addressed. This review aims to give a comprehensive and critical update of the development of poly(2-oxazoline) based biomaterials, focusing on the last 5 years, which have seen an explosive increase of interest. We believe that the research regarding this diverse family of polymers will remain strong and will keep growing, in particular after the promising first-in-human studies of a poly(2-oxazoline) drug conjugate. This review aims at researchers and students new to this polymer family and seasoned poly(2-oxazoline) experts alike and attempts to showcase how the chemical diversity of poly(2-oxazoline)s allows a relatively facile and broad access to biomaterials of all kinds.
Poly(2-oxazoline)s (PAOx) are of increasing importance for a wide range of applications, mostly in the biomedical field. This review describes the synthesis of 2-oxazoline monomers, their cationic ring-opening polymerization (CROP) and gives a comprehensive overview of all reported PAOx homopolymers. In the second part of the review, the polymer properties of these PAOx homopolymers with varying side-chain structures are discussed. Altogether, this review intends to serve as encyclopedia for poly(2-oxazoline)s enabling the straightforward selection of a polymer structure with the desired properties for a certain application.
Polymer-tethered membranes combine fascinating structural, dynamic, and viscoelastic properties. Many important insights into these peculiar supramolecular systems can be obtained from studies on polymer-tethered monolayers. This chapter discusses recent experimental findings on polymer-tethered monolayers at the air-water interface. In particular, Langmuir monolayers which are comprised of pure lipopolymers and of binary phospholipid-lipopolymer mixtures are considered. Thermodynamic data as well as structural data based on a host of experimental techniques including X-ray and neutron reflectrometry, infrared reflection absorption spectroscopy, and sum frequency generation spectroscopy provide information on how lipopolymers organize at the air-water interface. This information is followed by a review of the viscoelastic properties of these systems, including the remarkable gelation transition that can be observed in lipopolymers and mixed phospholipid- lipopolymer monolayers. The diffusion properties are also discussed at length, and show that lipid diffusivity is critically dependent on the strength of inter-polymer interactions of lipopolymers.
This chapter deals with ring-opening polymerization of oxazolines and their derivatives, in particular, the developments of 2-oxazoline (OZO) polymerization chemistry. Polymerization of oxazolines undergoes in a variety of reaction modes. Ring-opening polymerization of OZOs is the major and divided mainly into two modes: cationic ring-opening polymerization (CROP) and ring-opening polyaddition (ROPA). They are further divided into submodes of reaction like enzymatic ROPA (EROPA) reactions of sugar oxazolines for synthesizing various polysaccharides. All these reaction modes are mentioned. Living polymerization of CROP is readily achieved, giving often a versatile method to lead to various functional polymers and polymer architectures. Product polymers are of amide-type structure in almost all cases. Since the polymers derived from CROP are of no or low toxicity, their application studies in biomedical or pharmacological areas are beneficial and actively conducted recently.
The self-assembly in solution of original structures of amphiphilic partially natural copolymers based on polyoxazoline [more precisely poly (2-methyl-2-oxazoline) (POx)] and grape seed vegetable oil derivatives (linear, T-, and trident-structure) is investigated. The results show that such systems are found, using dynamic light scattering (DLS), to spontaneously self-organize into monomodal, narrow-size, and stable nanoparticles in aqueous medium. The obtained hydrodynamic diameters (D-h) range from 8.6 to 32.5 nm. Specifically, such size increases strongly with increasing natural block (i.e., lipophilic species) length due to higher hydrophobic interactions (from 10.1 nm for C-19 to 19.2 nm for C-57). Furthermore, increasing the polyoxazoline (i.e., hydrophilic block) length leads to a moderate linear increase of the D-h-values. Therefore, the first-order size effect comes from the natural lipophilic block, whereas the characteristic size can be tuned more finely (i.e., in a second-order) by choosing appropriately the polyoxazoline length. The DLS results in terms of characteristic size are corroborated using nanoparticle tracking analysis (NTA), and also by atomic force microscopy (AFM) and transmission electron microscopy (TEM) imaging where well-defined spherical and individual nanoparticles exhibit a very good mechanical resistance upon drying. Moreover, changing the lipophilic block architecture from linear to T-shape, while keeping the same molar mass, generates a branching and thus a shrinking by a factor of 2 of the nanoparticle volume, as observed by DLS. In this paper, it is clearly shown that the self-assemblies of amphiphilic block copolymer obtained from grape seed vegetable oil derivatives (sustainable renewable resources) as well as their tunability are of great interest for biomass valorization at the nanoscale level [continuation of the article by Stemmelen et al. (Polym Chem 4:1445-1458, 2013)].
A new 2‐oxazoline monomer with a protected thiol group, 2‐[2‐(4‐methoxybenzylsulfanyl)ethyl]‐2‐oxazoline, MOB‐SOx , was synthesized from commercially available compounds. MOB‐SOx and 2‐ethyl‐2‐oxazoline (EtOx) were simultaneously polymerized yielding well defined copolymers with narrow molar mass distributions and target polymer chain length. The copolymerization was initiated by N ‐methyl‐2‐methyl‐2‐oxazolinium triflate ( MeOxOTf ). After quantitative deprotection, poly(2‐oxazoline) with pendant thiol groups was obtained. The thiol groups were quantitatively added to the double bond of N ‐phenyl‐acrylamide ( PhA ) and benzylmaleimide ( BzM ). Graft copolymers were obtained by reaction of those SH containing polymers with poly(2‐methyl‐2‐oxazoline)s bearing acrylamide ( PMeOx 10 A ) and maleimide ( PMeOx 10 M ) as terminal reactive groups.
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CROP has been used to synthesize well-defined POXZ with a monofunctional (iodomethane) or a bifunctional (1,3-diiodopropane) initiator. POXZ has been functionalized with an azido group at one (alpha-azido-POXZ, (M) over bar (n) = 3.58 x 10(3) g.mol(-1)) or both ends (alpha,omega-azido-POXZ, (M) over bar (n) = 6.21 x 10(3) g.mol(-1)) of the macromolecular chain. The Huisgen 1,3-dipolar cycloaddition has been investigated between azido-POXZ and a terminal alkyne on a small or larger molecule (PEG). In each case, the click reaction has been successful and quantitative. In this way, different telechelic polymers (polymers bearing different functions such as acrylate, epoxide, or carboxylic acid) and block copolymers of POXZ and PEG have been prepared. The polymers have been characterized by means of FTIR, (1)H NMR, and SEC.
The prevention of surface fouling is becoming increasingly important for the development of anti-infective medical implants, biosensors with improved signal-to-noise ratios, and low-fouling membranes to name a few examples. We review a direct comparison of poly(ethylene glycol), the gold standard polymer to impart surfaces with nonfouling properties, to an alternative polymer, poly(2-methyl-2-oxazoline) (PMOXA), and show that both polymers are equally excellent in rendering surfaces nonfouling while PMOXA coatings are more stable in oxidative environments. We discuss prerequisites for the fabrication of nonfouling surface coatings and implications for the polymer choice according to application requirements.
Poly(2-oxazoline)s (POx) are currently discussed as an upcoming polymer platform for biomaterials design and especially for polymer therapeutics. POx meet specific requirements needed for the development of next-generation polymer therapeutics such as biocompatibility, high modulation of solubility, variation of size, architecture as well as chemical functionality. Although in the early 1990s first and promising POx-based systems were presented, the field lay dormant for almost two decades. Only very recently, POx-based polymer therapeutics came back into the focus of very intensive research. In this review, we give an overview on the chemistry and physicochemical properties of POx and summarize the research of POx-protein conjugates, POx-drug conjugates, POx-based polyplexes and POx micelles for drug delivery.
IntroductionPolyethersPolyoxazolinesSummary and ProspectsAcknowledgmentsReferences
Polyoxazolines (POx) are increasingly being studied as polymeric building blocks due to the possibility of affording tunable properties. Additionally, as the biocompatibility and stealth behavior of POx are similar to that of poly(ethylene glycol) (PEG), it has become challenging to develop polyoxazoline-based (co)polymers. Even if POx have a lot of advantages, they also show an important drawback, as it has been impossible, to date, to prepare high-molecular-weight polyoxazolines with a low polydispersity index. Thus, it appears important that they be judiciously functionalized. This review covers the multiple ways that polyoxazolines can be functionalized. The use of functional initiators, functional terminating agents, and 2-R-2-oxazolines with an R functional side group is detailed. In conclusion, some perspectives on POx functionalizations are also reported, with functions permitting selective "click" reactions.
Numerous non-ionic thermally responsive homopolymers phase separate from their aqueous solutions upon heating. Far fewer neutral
homopolymers are known to phase separate upon cooling. A systematic compilation of the polymers reported to exhibit thermoresponsive
behaviour is presented in this review, including N-substituted poly[(meth)acrylamide]s, poly(N-vinylamide)s, poly(oxazoline)s, protein-related polymers, poly(ether)s, polymers based on amphiphilic balance, and elastin-like
synthetic polymers. Basic properties of aqueous solutions of these polymers are briefly described.
KeywordsAmphiphilic-LCST-Polymer-Solution-Thermoresponsive-Water
Molecular brushes of poly(2-oxazoline)s were prepared by living anionic polymerization of 2-iso-propenyl-2-oxazoline to form the backbone and subsequent living cationic ring-opening polymerization of 2-n- or 2-iso-propyl-2-oxazoline for pendant chain grafting. In situ kinetic studies indicate that the initiation efficiency and polymerization rates are independent from the number of initiator functions per initiator molecule. This was attributed to the high efficiency of oxazolinium salt and the stretched conformation of the backbone, which is caused by the electrostatic repulsion of the oxazolinium moieties along the macroinitiator. The resulting molecular brushes showed thermoresponsive properties, that is, having a defined cloud point (CP). The dependence of the CP as a function of backbone and side chain length as well as concentration was studied.
Novel $alpha$,$ømega$-functionalized amphiphilic lipopolymers were prepared which are composed of a proximal transmembrane lipid moiety and a hydrophilic poly(2-oxazoline)-based (POx) polymer chain. The synthesis is started from bifunctional lipoinitiators which are asymmetrically protected as tert.butyldiphenylsilyl (TBDPS) ethers followed by cationic living ring-opening polymerization of 2-oxazolines in a one-pot multistep reaction. This results in polymers with defined terminal end groups and narrow molar mass distributions. All protective groups involved can be readily cleaved in a single step reaction keeping the structure of the polymer intact. This gives access to (lipo)polymers with a variety of defined identical or chemical orthogonal $alpha$,$ømega$-functionalities. The described synthetic strategy is a versatile tool for the preparation of defined polymer-drug or polymer-protein conjugates or asymmetric functionalized modeled lipid membranes for the quantitative study of membrane-associated phenomena such as transmembrane transport and cell adhesion / recognition.
The current study reports the compartmentalization of a physisorbed polymer-tethered phospholipid bilayer built using subsequent LangmuirBlodgett (LB) and LangmuirSchaefer (LS) transfers, where compartmentalization is due to buckling in the bottom (LB) monolayer and polymer-specific stress relaxation processes. Buckling arises from lateral stress within the membrane induced by a high (1530 mol%) concentration of poly(2-ethyl-2-oxazoline) lipopolymers in the LB monolayer. Epifluorescence microscopy (EPI) and fluorescence recovery after photobleaching (FRAP) experiments confirm the formation of a homogeneous bilayer at low lipopolymer molar concentrations (low lateral stress), but demonstrate the compartmentalization of the bilayer into m size compartments at elevated lipopolymer concentrations (high lateral stress). Quantitative EPI of the LB monolayer as well as additional atomic force microscopy (AFM) experiments show that bilayer compartmentalization, buckling and partial delamination of the LB monolayer occur without causing notable phospholipidlipopolymer phase separations, but do preclude bilayer formation above buckled/delaminated regions after LS transfer. As long-time tracking experiments of photostable quantum dot-conjugated lipids in the compartmentalized bilayer system confirm, our membrane system enables the facile adjustment of the permeability of diffusion boundaries between bilayer compartments, thus providing an excellent experimental tool to mimic length-scale dependent diffusion processes observed in cellular membranes. We expect that the fundamental concept of lateral stress regulation and buckling-associated membrane compartmentalization can also be applied to other polymerlipid composite materials than the one studied in this work.
Thermo-sensitive poly(2-isopropyl-2-oxazoline)s (PiPrOx) were functionalized with end groups of different polarity by living cationic ring-opening polymerization using the initiator and/or termination method as well as sequential block copolymerization with 2-methyl-2-oxazoline. As end groups, methyl, n-nonyl, piperidine, piperazine as well as oligo(ethylenglygol) and oligo(2-methyl-2-oxazoline) were introduced quantitatively. The lower critical solution temperature (LCST) of the aqueous solutions was investigated. The introduction of hydrophobic end groups decreases the LCST, while hydrophilic polymer tails raise the cloud point. In comparison to poly(N-isopropyl acrylamide), the impact of the end group polarity upon the modulation of the LCST was found to be significantly stronger. Surprisingly, terminal oligoethylenegycol units also decrease the LCST of PiPrOx, thus acting as moieties of higher hydrophobicity as compared to the poly(2-oxazoline) main chain. Together with the possible variation of the side group polarity, this allows a broad modulation of the LCST of poly(2-oxazoline)s.
Abstract Living cationic copolymerization of 2-isopropyl-2-oxazoline with 2-n-propyl-, 2-n-butyl-, and 2-n-nonyl-2-oxazoline results in gradient copolymers of defined composition, narrow molar mass distributions (PDI$permil$=1.09-1.3), and defined overall degree of polymerization, set to n=25 for all polymers. The introduction of monomer units of stronger amphiphilic character results in a systematic decrease of the lower critical solution temperature (LCST). The LCST modulation can be controlled by the choice of the comonomer as well as the comonomer ratio and was tuned in the temperature range from 46 to 9 Â$^circ$C.
In our ongoing efforts to develop poly(2‐oxazoline)s (POx) for biomedical applications, we report on the preparation of defined, star‐like hydrophilic POx. Using pluritriflate initiators, we show, through online kinetic measurements by gas chromatography, that multiple initiating groups are of equal reactivity for the initiation of the polymerization of 2‐oxazolines. The overall polymerization rate increases linearly with the number of initiator functions per molecule. Thus, all initiating moieties are of the same reactivity and all arms grow at the same rate. This is crucial for the establishment of a meaningful structure‐property relationship for polymers of star architectures.
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The micelle formation of five poly(2‐oxazoline) diblock, triblock and gradient copolymers in water was investigated using fluorescence correlation spectroscopy. The polymers were synthesized by consecutive or simultaneous living cationic polymerization of 2‐methyl‐2‐oxazoline for the hydrophilic and 2‐nonyl‐2‐oxazoline for the hydrophobic polymer segments. Fractions of the polymers were fluorescence‐labeled at the polymer termini with TRITC for the FCS measurements. The hydrodynamic radii of solubilized polymer unimers and of the aggregates (micelles) were determined in a concentration range of 10 ⁻⁸ –10 ⁻³ M and were found to depend in a characteristic way on the polymer architecture.
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A new 2-oxazoline with a pendant alkyne moiety, 2-(pent-4-ynyl)-2-oxazoline, 1 (PynOx), was synthesized from commercial available compounds. Polymerization of PynOx with methyl triflate as initiator and copolymerization with 2-methyl- or 2-ethyl-2-oxazoline (MeOx or EtOx) as comonomers results in well-defined water-soluble polymers of narrow molar mass distributions and predefined degrees of polymerization. Since the alkyne moiety is compatible with the living cationic polymerization, no protection group was needed. The consecutive copper-catalyzed Huisgen 1,3-dipolar cycloadditions of two different azides with the polymer bound alkynes to the 1,2,3-triazoles were quantitative.
Distinct lipid environments, including lipid rafts, are increasingly recognized as a crucial factor affecting membrane protein function in plasma membranes. Unfortunately, an understanding of their role in membrane protein activation and oligomerization has remained elusive due to the challenge of characterizing these often small and transient plasma membrane heterogeneities in live cells. To address this difficulty, we present an experimental model membrane platform based on polymer-supported lipid bilayers containing stable raft-mimicking domains (type I) and homogeneous cholesterol-lipid mixtures (type II) into which transmembrane proteins are incorporated (α vβ 3 and α 5β 1 integrins). These flexible lipid platforms enable the use of confocal fluorescence spectroscopy, including the photon counting histogram method, in tandem with epifluorescence microscopy to quantitatively probe the effect of the binding of native ligands from the extracellular matrix ligands (vitronectin and fibronectin for α vβ 3 and α 5β 1, respectively) on domain-specific protein sequestration and on protein oligomerization state. We found that both α vβ 3 and α 5β 1 sequester preferentially to nonraft domains in the absence of extracellular matrix ligands, but upon ligand addition, α vβ 3 sequesters strongly into raft-like domains and α 5β 1 loses preference for either raft-like or nonraft-like domains. A corresponding photon counting histogram analysis showed that integrins exist predominantly in a monomeric state. No change was detected in oligomerization state upon ligand binding in either type I or type II bilayers, but a moderate increase in oligomerization state was observed for increasing concentrations of cholesterol. The combined findings suggest a mechanism in which changes in integrin sequestering are caused by ligand-induced changes in integrin conformation and/or dynamics that affect integrin-lipid interactions without altering the integrin oligomerization state.
With the advancement of medical and investigative science, it is somewhat surprising that although it is possible to stabilise medical patients with hypertension and the associated kidney dysfunction, obesity, diabetes and even cancer, there is still no clear method of significantly reducing these chronic disease pathologies, and thus, extending life expectancy. There is one hormone common to these pathologies, the antagonism of which goes some way to clinical improvements, and this is angiotensin, which is released during hypovolaemia. Angiotensin antagonists are used to treat many of these pathologies, and it has been shown in the obesity literature that angiotensin antagonists decrease weight, but also increase the drinking of water. Increased cellular hydration, and hence, improved mitochondrial metabolism could be one of the mechanisms for the reduction in weight seen in these studies, as well as for reducing the other pathologies, all showing metabolic dysfunction. It appears that the application of straightforward physiological regulation might be an appropriate medical approach to the prevention of hypertension, kidney disease, obesity, diabetes and cancer, and thus, to an increased life expectancy.
We report on the preparation of microstructured poly(2-oxazoline) bottle-brush brushes (BBBs) on nanocrystalline diamond (NCD). Structuring of NCD was performed by photolithography and plasma treatment to result in a patterned NCD surface with oxidized and hydrogenated areas. Self-initiated photografting and photopolymerization (SIPGP) of 2-isopropenyl-2-oxazoline (IPOx) resulted in selective grafting of poly(2-isopropenyl-2-oxazoline) (PIPOx) polymer brushes only at the oxidized NCD areas. Structured PIPOx brushes were converted by methyl triflate into the polyelectrolyte brush macroinitiator for the living cationic ring-opening polymerization (LCROP) of 2-oxazolines. The LCROP was performed with 2-ethyl-2-oxazoline (EtOx) as well as 2-(carbazolyl)ethyl-2-oxazoline (CarbOx) as monomers, resulting in structured bottle-brush brushes (BBB) with different pendant side chains and functionalities. FT-IR spectroscopy, fluorescence microscopy, and AFM measurements indicated a high side chain grafting density as well as quantitative and selective reactions. Poly(2-oxazoline) BBBs containing hole conducting carbazole moieties on NCD as electrode material may open the way to advanced amperometric biosensing systems.
The diffusional behavior of amphiphilic poly(2-oxazoline) diblock copolymers in aqueous solution is studied using photon correlation spectroscopy (PCS) and fluorescence correlation spectroscopy (FCS). The polymers were synthesized by living cationic polymerization and were fluorescence-labeled with tetramethyl rhodamine isothiocyanate either at the end of the hydrophilic or the hydrophobic block. Temperature-resolved PCS showed that, at room temperature, large metastable aggregates are present along with unimers and micelles. An annealing above similar to40 degreesC resulted in stable equilibrium micellar solutions. By means of FCS, the hydrodynamic radii of the unimers and the micelles were measured simultaneously in a broad concentration range, and the critical micelle concentration could be determined. Comparison of the results from conventional PCS measurements with this first FCS study showed excellent agreement and the high potential of the FCS technique.
A novel class of amphiphilic silane functionalized linear poly(N-propionylethylenimine)s (PPEIs) are synthesized by living polymerization of 2-ethyloxazoline and grafted onto silica substrates to form stable polymer supported alkyl monolayers (PSM) which may mimic biological membranes.
Complete structures of nearly 40 ether polar lipids from seven species of methanogens have been elucidated during the past 10 years. Three kinds of variations of core lipids, macrocyclic archaeol and two hydroxyarchaeols, were identified, in addition to the usual archaeol and caldarchaeol (for the nomenclature of archaeal [archaebacterial] ether lipids, see the text). Polar head groups of methanogen phospholipids include ethanolamine, serine, inositol, N-acetylglucosamine, dimethyl- and trimethylaminopentanetetrol, and glucosaminylinositol. Glucose is the sole hexose moiety of glycolipids in most methanogens, and galactose and mannose have been found in a few species. Methanogen lipids are characterized by their diversity in phosphate-containing polar head groups and core lipids, which in turn can be used for chemotaxonomy of methanogens. This was shown by preliminary simplified analyses of lipid component residues. Core lipid analysis by high-pressure liquid chromatography provides a method of determining the methanogenic biomass in natural samples. There has been significant progress in the biosynthetic studies of methanogen lipids in recent years. In vivo incorporation experiments have led to delineation of the outline of the synthetic route of the diphytanylglycerol ether core. The mechanisms of biosynthesis of tetraether lipids and various polar lipids, and cell-free systems of either lipid synthesis, however, remain to be elucidated. The significance and the origin of archaeal ether lipids is discussed in terms of the lipid composition of bacteria living in a wide variety of environments, the oxygen requirement for biosynthesis of hydrocarbon chains, and the physicochemical properties and functions of lipids as membrane constituents.
Scientific and practical applications of supported lipid-protein bilayers are described. Membranes can be covalently coupled to or separated from solids by ultrathin layers of water or soft polymer cushions. The latter systems maintain the structural and dynamic properties of free bilayers, forming a class of models of biomembranes that allow the application of a manifold of surface-sensitive techniques. They form versatile models of low-dimensionality complex fluids, which can be used to study interfacial forces and wetting phenomena, and enable the design of phantom cells to explore the interplay of lock-and-key forces (such as receptor-ligand binding) and universal forces for cell adhesion. Practical applications are the design of (highly selective) receptor surfaces of biosensors on electrooptical devices or the biofunctionalization of inorganic solids.
Soft biofunctional and biocompatible interfaces on solids designed by the deposition of ultrathin soft polymer films or supported membranes have numerous scientific and practical applications. These include the immobilization of glycolipids, membrane receptors and proteins to generate models of cell and tissue surfaces. Powerful surface-sensitive techniques can be applied to study protein-protein recognition processes at membranes and the control of cell adhesion by the interplay of specific 'lock-and-key' forces and universal interfacial forces. Potential practical applications include the design of smart biosensors based on electro-optical devices and the fabrication of biofunctional surfaces for the stimulation of cell proliferation and tissue growth, or for the suppression of apoptosis.
Obstructed long-range lateral diffusion of phospholipids (TRITC-DHPE) and membrane proteins (bacteriorhodopsin) in a planar polymer-tethered 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer is studied using wide-field single molecule fluorescence microscopy. The obstacles are well-controlled concentrations of hydrophobic lipid-mimicking dioctadecylamine moieties in the polymer-exposed monolayer of the model membrane. Diffusion of both types of tracer molecules is well described by a percolating system with different percolation thresholds for lipids and proteins. Data analysis using a free area model of obstructed lipid diffusion indicates that phospholipids and tethered lipids interact via hard-core repulsion. A comparison to Monte Carlo lattice calculations reveals that tethered lipids act as immobile obstacles, are randomly distributed, and do not self-assemble into large-scale aggregates for low to moderate tethering concentrations. A procedure is presented to identify anomalous subdiffusion from tracking data at a single time lag. From the analysis of the cumulative distribution function of the square displacements, it was found that TRITC-DHPE and W80i show normal diffusion at lower concentrations of tethered lipids and anomalous diffusion at higher ones. This study may help improve our understanding of how lipids and proteins in biomembranes may be obstructed by very small obstacles comprising only one or very few molecules.
Liposomes are spherical, single- or multi-lamellar, structures in which lipid bilayer(s) entrap part of the solvent, in which they freely float, into their interior. Because of their special physical, chemical, and biological properties such as small size, hydrophilicity and hydrophobicity, possibility of surface modifications, capacity to encapsulate/bind, transport and protect various (macro) molecules, and ability to transfer the entrapped material into living cells, they can be used as drug delivery vehicles. The first generation of liposomes have been used predominantly as a solubilizer of water insoluble drugs and as a system for systemic or localized sustained drug release. Liposomes also alter the pharmacokinetics and biodistribution of the encapsulated drugs. Upon parenteral administration they are cleared by the cells of the RE system and this can improve the therapy of the diseases of the phagocytes and may be very useful for enhancement of vaccine activity. Liposomes do not accumulate in heart, kidneys, or the tissue of the nervous system very strongly and therefore the toxicity of many drugs can be significantly reduced. However, despite very significant improvements in several therapies, the majority of applications is still severely limited due to the rapid clearance of liposomes. The second generation of liposomes are sterically stabilized liposomes which can evade the uptake by the RE system (Stealth liposomes) and circulate longer and may be therefore targeted to other tissues more efficiently. Many tumors and sites of infection/inflammation are characterized by leaky vasculature and small liposomes which are not removed from the blood circulation may extravasate out of the capillaries only at these sites and deliver the encapsulated drug more efficiently to the target. After describing some of the successful applications of the first generation liposomes the most recent data of the antitumor potential of the second generation liposomes will be presented. Briefly, complete remissions of conventional treatment resistant subcutaneous tumors in mice were observed by Doxorubicin and Epirubicin encapsulated into Stealth liposomes.
Certain amphiphilic water-soluble polymers including amphiphilic derivatives of polyvinyl pyrrolidone (PVP) were found to be efficient steric protectors for liposomes in vivo. In this study, we have tried to develop synthetic pathways for preparing amphiphilic PVP and to investigate the influence of the hydrophilic/hydrophobic blocks on some properties of resulting polymers and polymer-coated liposomes. To prepare amphiphilic PVP with the end stearyl (S) or palmityl (P) residues, amino- and carboxy-terminated PVP derivatives were first synthesized by the free-radical polymerization of vinyl pyrrolidone in the presence of amino- or carboxy-mercaptans as chain transfer agents, and then modified by interaction of amino-PVP with stearoyl chloride or palmitoyl chloride, or by dicyclohexyl carbodiimide coupling of stearylamine with carboxy-PVP. ESR-spectra of the hydrophobic spin-probe, nitroxyl radical N-oxyl-2-hexyl-2-(10-methoxycarbonyl)decyl-4,4′-dimethyl oxazoline, in the presence of amphiphilic PVP demonstrated good accessibility of terminal P- and S-groups for the interaction with other hydrophobic ligands. Spontaneous micellization and low CMC values (in a low μmolar range) were found for amphiphilic PVP derivatives using the pyrene method. In general, S-PVP forms more stable micelles than P-PVP (at similar MW, CMC values for S-PVP are lower than for P-PVP). It was found that amphiphilic PVP incorporated into negatively charged liposomes effectively prevents polycation(poly-ethylpyridinium-4-vinylchloride)-induced liposome aggregation, completely abolishing it at ca. 10mol% polymer content in liposomes. Additionally, the liposome-incorporated PVP prevents the fluorescence quenching of the membrane-incorporated hydrophobic fluorescent label [N-(4-fluoresceinthiocarbamoyl)dipalmitoyl-PE] by the free polycation. PVP-modified liposomes were loaded with a self-quenching concentration of carboxyfluorescein, and their destabilization in the presence of mouse serum was investigated following the release of free dye. Amphiphilic PVP with MW between 1500 and 8000 provides good steric protection for liposomes. The degree of this protection depends on both polymer concentration and molecular size of the PVP block.
Durch Umsetzung der Nitrile 1 und 6 mit den Aminoalkoholen 2 und 3 in Gegenwart von katalytisch wirkenden Metallsalzen wurden die 2-Oxazoline 4 und 7 sowie die 4H-5,6-Dihydro- 1,3-oxazine 5 und 8 erhalten. Analog dargestellte 2-Aminoalkyl-2-oxazoline 13 und 15 wurden bei 190–230°C zu den 2-Alkenyl-2-oxazolinen 14 gespalten.Formation of Cyclic Imidic Esters by Reaction of Nitriles with Amino Alcohols**)The 2-oxazolines 4 and 7 and the 4H-5,6-dihydro-1,3-oxazines 5 and 8 were prepared by reaction of the nitriles 1 and 6 with amino alcohols 2 and 3 in the presence of catalytic amounts of certain metal salts. 2-Aminoalkyl-2-oxazolines 13 and 15, which have been prepared analogously, cleave at 190–230°C to give 2-alkenyl-2-oxazolines 14.
We describe the synthesis of lipopolymers suitable for conformation and mobility studies by neutron scattering methods and H-NMR spectroscopy. Starting with 2-methyl-2-oxazoline MeOx monomers, we developed two synthetic routes for the preparation of 2-trideuteromethyl-2-oxazoline D3-MeOx. Both rely on cheap and easily available deuterated sources, but the yield of the products and their degree of deuteration as well as efficiency differ considerably. Using a lipid initiator, we efficiently introduced the lipid end-group into the polymer. Sequential addition of deuterated and non-deuterated monomers led to the segmentally deuterated poly(acetyliminoethylene) lipopolymers [diblock or triblock copolymers consisting of poly(acetyliminoethylene) and poly(trideuteroacetyliminoethylene)]. We also achieved good control of the position and length of the blocks with a narrow molecular mass distribution. Two sets of substances were synthesized differing in molecular mass and degree of deuteration. The physical properties of these substances are described separately.
Here we report the synthesis of end functionalized lipopolymers. For this purpose different lipoinitiators with a bromo group have been prepared, based on monoalkyl and dialkyl N-substituted 2-bromo-propionamides. Theses lipoinitiators could then be used as initiators for atom transfer radical polymerization of acrylamides. Different lipopolymers were synthezised. The polymer back bone was either a homopolymer or a copolymer. Thin polymer films of 20 Å thickness of the copolymers were self-assembled onto surfaces and, finally, vesicle fusion on these polymer cushions was studied.
We have investigated the molecular conformations of a lipopolymer with a polyoxazoline headgroup at air/water interfaces as a function of lateral area per molecule with X-ray and neutron reflectometry. The polymer 1,2-dioctadecanyl-sn-glycero-3-poly(2-methyl-2-oxazoline), PMO−(C18)2, forms stable surface monolayers. Pressure/area isotherms around room temperature show a plateau region, indicative of a phase transition whose origin was examined. For data evaluation, a novel approach was used that acts on explicit quasi-molecular ensemble conformations of the polymer [Politsch et al., preceding paper in this issue]. At lower surface pressure, the polymer density distribution exhibits a maximum near the interface, indicative of attractive interaction between the predominantly hydrophilic polymer chains and the hydrophobic surface. Across the plateau region of the isotherm, a change in the volume density distribution of the alkyl chains was observed which is indicative of a partial immersion of the lipid moieties into the aqueous subphase. In contrast, no major structural change across the phase transition was detected in the polymer volume density profiles which comply with scaling predictions at both sides of the phase transition if deviations due to nonidealities are neglected. We interpret these observations as an alkyl chain ordering induced by the steric interference between the PMO: Immersion of alkyl chains into the subphase relaxes the strain on the hydrophobic anchors which derives from a reduction of the configurational entropy of the PMO chains due to their confinement to the interface.
Lipopolymers are lipids with a polymer chain covalently attached to the lipid. We systematically studied the monolayer behavior of a series of lipopolymers by use of infrared reflection−absorption spectroscopy (IRRAS) and classical Langmuir film balance techniques. The lipopolymers differed in chemical composition both in the lipid chains and in the polymers. However, the lipid chains contained 18 carbon atoms in all cases. We find the lipopolymers to show a very complex phase behavior at the air−water interface, depending on such diverse parameters as polymer hydrophilicity, length of polymer, and saturation of the lipid chains. We were able to measure up to two different plateau regimes in the monolayer isotherms. One is observed for lipopolymers where the polymer part is only slightly water-soluble and is correlated with the desorption of the polymer from the water surface. This desorption transition can be termed a pancake−mushroom transition. The origin of the second plateau regime is more uncertain. Baekmark et al. (Langmuir 1995, 11, 3975) interpreted this plateau as a transition within the polymer (mushroom-to-polymer brush conformation), but in two recent publications, by Gonçalves da Silva et al. (Langmuir 1996, 12, 6547) and Baekmark et al. (Langmuir 1997, 13, 5521) this interpretation has been questioned. In the present work we show that this second transition is native to lipopolymers in the sense that it can only be observed when a lipid and polymer are present in the same molecule. On the same basis of the film balance and the IRRAS data, we are able to show that the lipid alkyl chains must be involved in the molecular processes constituting the “native” transition. To account for the origin of the “native” transition, we suggest that a process of alkyl chain condensation coupled to a strong reduction in the number of gauche isomers within the alkyl chains supply the driving force for the “native” transition.
Lipopolymers are lipids with a polymer chain covalently attached to the lipid. We studied the infrared reflection absorption behavior as a function of molecular area of Langmuir monolayers of an ether lipopolymer, DC18Gly-M35 (35 monomer units). A plateau region was observed in the monolayer isotherm. Within this region the CH2 asymmetric and symmetric stretching mode absorptions shifted toward lower absorption frequencies (6 and 4 cm-1, respectively). This indicates that the plateau is accompanied by strong local ordering in the lipopolymer, which contradicts previous suggestions that the plateau correlates with a mushroom−brush transition in the polymer.
We investigated monolayers of the diblock copolymer poly(2-ethyl-2-oxazoline)−poly(2-nonyl-2-oxazoline) (E60N60) at the air−water interface using classical Langmuir film balance techniques and transmission electron microscopy on transferred Langmuir−Blodgett films. The isotherms exhibit three distinct regions. At low pressures the monolayer is relatively incompressible and the moderately water-soluble ethyloxazoline block is adsorbed to the interface. At intermediate pressures of approximately 10 mN/m the compressibility increases temporarily and decreases again at higher lateral pressures. Desorption of the ethyloxazoline block takes place in the intermediate high-compressibility region. This desorption process cannot be described as a phase transition. In the water-insoluble nonyloxazoline block the polymer backbone remains confined to the interface at all pressures. Electron micrographs of monolayers transferred onto mica at lateral pressures below approximately 10 mN/m showed no structures. In the high-compressibility region structures appeared that persisted at higher pressures. These structures can be described as two-dimensional micelles. The driving force for the micelle formation is the only moderate water solubility of the ethyloxazoline block. For this reason, desorption of the moderately water-soluble ethyloxazoline block and the formation of surface micelles are coupled.
We studied conformational changes in monolayers of lipopolymers consisting of poly(ethylene oxide) head groups coupled to L-alpha-distearoylphosphatidylethanolamine (DSPE-EO(n); n = 45, 110) and of mixtures of these lipopolymers with L-alpha-dimyristoylphosphatidylethanolamine (DMPE) with the aim to deposit composite polymer-lipid films on solid substrates. Film balance experiments performed with pure DSPE-EO(n) over areas ranging from 0.5 to 60 nm(2) per molecule revealed two conformational changes which are interpreted as a transition from a pancake-like to a mushroom-like conformation and a mushroom-to-brush transition, as predicted by the Alexander-de Gennes theory of grafted polymers. The mixtures of DSPE-EO(n) (less than or equal to 10 mol %) with DMPE exhibit in general lateral phase separation as demonstrated by film-balance and microfluorescence studies. The very complex behavior is determined (I) by the well-known tendency of two-dimensional polymer solutions for phase segregation at very low polymer molar fractions, (2) by the interaction of the polymer head groups, and (3) by the chain melting transition of DMPE. A homogeneous phase was found for the case of 10 mol % DSPE-EO(45) in DMPE in the lateral; pressure range between about 3 and 5 mN/m. This is interpreted in terms of a pancake-like state with the lipopolymer head groups starting to overlap. In this region the monolayer is below the DMPE main transition. Finally we studied the interaction of DSPE-EO(45) monolayers with Si/SiO2 wafers by ellipsometry. The thickness of the lipopolymer monolayers was determined as a function of the relative humidity of the atmosphere surrounding the sample in a hydration chamber connected to a film balance. The measurements yield film thickness vs disjoining pressure curves. These distance vs disjoining pressure measurements show interaction regimes which are determined by steric and electrostatic forces, respectively, in analogy to recent force apparatus measurements.
Δ2-Oxazoline und 4H-5, 6-Dihydrooxazine werden, ausgehend von Carbonsäuren und 2- bzw. 3-Aminoalkoholen, durch Gasphasendehydratisierung, 4H-Dihydrooxazine auch durch Kondensation von N-Hydroxymethylcarbonsäureamiden mit Olefinen leicht zugänglich. Diese cyclischen Imidsäureester können unter Ringöffnung reagieren; zu diesen Umsetzungen gehört auch eine neuartige Polymerisation. Das Verhalten 2-ständiger Alkylgruppen sowie die Cycloadditionen in 2, 3-Stellung wurden ebenfalls untersucht. Weiterhin wird über Umsetzungen an der 2-ständigen Seitenkette und über Additionsversuche an den Schwefelanaloga der cyclischen Imidsäureester berichtet. Eine Tabelle unterrichtet über die wichtigsten IR-Banden substituierter Δ2-Oxazoline.
In order to build up tether-supported membranes, we have focused on the synthesis of lipopolymers. Different lipid analog initiators based on 2-bromopropionamide have been synthesized. In addition, fluorescence-labeled lipoinitiators using pyrene were synthesized. These initiators were used for the synthesis of polyacrylamide copolymers by atom-transfer radical polymerization. A final surface-anchor end group was attached to the polymers by polymer analogous reaction. In this way, α,β-functionalized polyacrylamides were obtained. The interaction of these lipopolymers with different lipid bilayer structures was investigated in several experiments. The lipopolymers adsorb onto the surface of vesicles as shown by photon correlation spectroscopy and fluorescence measurements. The fixation of these lipopolymers on flat surfaces was studied using surface plasmon spectroscopy. The film thickness of the adsorbed lipopolymer films is about 12 to 20 Å. The surfaces thus modified can be used for the fixation of lipid vesicles to form polymer-supported bilayers. This lead to an additional thickness increase of 41-62 Å.
Several mono-, bi- and trifunctional oxetanes were synthesized and polymerized in bulk and in solution cationically. Selected photoinitiators have been applied. It was found that sulfonium salts are very efficient due to good solubility, almost no discolouration of the product and storage stability in the monomer in the absence of light. The conversion was determined by quantitative IR-spectroscopy. Conversion between 75% and 85% was found in all cases. The shrinkage during polymerization was much lower than for vinyl monomers. No inhibition by oxygen was recognized. Monomer layers thicker than 5.5 mm could be polymerized. The products are transparent and almost colourless. The glass transition temperature of the crosslinked polymers was above the temperature of a human body.
We present a new data refinement technique for the evaluation of X-ray and neutron reflectometry measurements of interface layers comprising linear molecules. The new method is particularly well-suitedsbut not limited tosmodeling the structure of surface-grafted linear polymers organized in "polymer brushes". In this paper, we discuss technical details of the data inversion technique and demonstrate its capabilities by recovering the structure of a simulated molecular ensemble for which synthetic data sets have been constructed. In the following paper, we utilize this machinery to reveal the nature of a phase transition pertinent to surface-anchored lipopolymers at high molecular densities.
Lipopolymers are known to form a two-dimensional physical network (physical gel) at the air-water interface that is stabilized by two different types of associative interactions: microcondensation of alkyl chains of lipopolymers to form small clusters and physical junction zones linking neighboring polymer chains stabilized by hydrogen bonding of water molecules. In this study we present surface rheology and film balance experiments on amphiphilic PEG lipopolymers of different molecular weights of their polymer moiety (MW: 750, 1000, 2000, 3000, 5000) at the air-water interface. Our experimental data show that the gelation transition shifts within the MW range of 1000-5000 to smaller areas per molecule as the polymer chain length gets shorter until it reaches a specific minimum area where no further change is observed (MW) 1000). Surface rheology data of the storage modulus indicate a qualitative difference between higher MW species (MW: 2000, 3000, 5000) and lower MW species (MW: 750, 1000). While the well-known power law dependence above the gelation point can be observed in the first case, a breakdown of the network after an initial power law-like behavior is found in the latter one. Our data suggest that a sufficient area mismatch between polymer and lipid moieties of these peculiar amphiphiles is essential for a stable two-dimensional physical network to form.
Amphiphilic lipopolymers are known to form 2D thermoreversible gels at the air- water interface. Recently, we have reported surface rheology and film balance experiments on poly(ethylene glycol) (PEG) lipopolymers of different molecular weights, which indicated that a sufficient cross-sectional area mismatch between polymer and lipid moieties is necessary to form stable 2D gels (J. Coffman and C. Naumann. Macromolecules 2002, 35, 1835). In the current studies, we have investigated the influence of the hydrophobic anchor on the gelation properties by surface rheology and film balance technique, Experiments on PEG lipopolymers, carrying saturated and partially unsaturated alkyl chains and on poly [(2-n-nonyl -2-oxazoline)(x)-b-(2-methyl-or 2-ethyl-2-oxazoline)(y)] (NxMy or NxEy) diblock copolymers of different block length show that the gel formation is not merely the result of the area mismatch between hydrophilic and hydropliobic Illoieties of the amphiphile (cone shape), but that a sufficient strength of van der Waals interaction within the hydrophobic moiety is necessary for the 2D gel to form, thus verifying carlier predictions that all alkyl chain condensation is a necessary precursor for the gelation process to occur. We also present neutron reflectometry data on PEG lipopolymers above and below the alkyl chain condensation and gelation transitions, which, in agreement to previous neutron and X-ray scattering experiments, reveal that both transitions occur after surface micelles oflipopolymers are formed at the air-water interface. On the basis of these findings, we assume that the gelation process of lipopolymers at the air-water interface is Mused by a surface micellization of lipopolymers, which can be seen as the 2D analogue to the 3D gel formation observed for polymeric colloids with grafted polymer chains, such as, copolymers and star polymers.
Stable lipid membranes with controlled substrate-membrane spacing can be prepd. using well-defined lipopolymers as a tether. Based on the living cationic ring-opening polymn. of 2-methyl- or 2-ethyl-2-oxazoline, lipopolymers can be synthesized bearing a lipid head group as well as a silanol reactive coupling end group. Using a "grafting onto" procedure these polymers can form dense, brush like monolayers, whose layered structures can be obtained by x-ray reflectivity measurements. By transfer of a pre-organized monolayer that is followed by vesicle fusion, stable polymer supported lipid membranes can be prepd. The substrate-membrane spacing can be controlled via the d.p., while the lateral diffusion of lipids within the membrane depends on the d. of polymer tethers. Preliminary expts. implied that the membrane with long (N = 40) polymer tethers could reside trans-membrane receptors homogeneously, suggesting a large potential of this strategy. [on SciFinder (R)]
Poly(2-methyl-2-oxazoline) (PMOX) and poly(2-ethyl-2-oxazoline) (PEOX) bearing 3-aminopropyltrimethoxysilane end groups for surface coupling were synthesized and grafted onto silicon/silicon dioxide substrates. Static and dynamic hydration of these films by water vapor was quant. studied by ellipsometry. The static swelling response was analyzed by measuring the equil. film thickness as a function of the relative humidity to obtain quant. force-distance curves. The disjoining pressure measures the sum of all operating forces in equil. with the chem. potential of the surrounding atm., where the dominant forces could be detd. The hydration of polymers with different initial thickness was compared by the normalized swelling ratio (r) and the calcd. decay const. (l*). All the measured parameters exhibited a clear dependence upon the chain length. The dynamic swelling response was studied by monitoring the film thickness as a function of time under osmotic shock, switching from dry to humid atm. conditions (relative humidity from .apprx.4% to .apprx.90%). The characteristic time consts. were analyzed for each deriv. semiquant., indicating that the kinetics of water uptake is dependent on the polymer main chain length rather than on the side chains. [on SciFinder (R)]
End-functionalized 3D self-assembled monolayers (SAMs) on gold nanoparticles were used to initiate the living cationic ring-opening polymerization reaction directly on gold nanoparticle surfaces ("grafting-from technique"). In this manner, dense polymer brushes were prepared in a "one-pot multistep" reaction. ER FTIR spectroscopy confirms the successful polymerization reaction as well as the introduction of a terminal functional group. The resulting gold/polymer nanocomposite was very stable. The combination of a "grafting-from reaction" resulting in polymer "brush-type" shells of linear macromolecules and the introduction of a terminal mesogen by means of a quantitative termination reaction resulted in an amphiphilic core-shell material with a well-defined hydrophilic/lipophilic balance (HLB). Ex-situ kinetic studies of the polymerization of 2-phenyl-2-oxazoline using FTIR spectroscopy and MALDI TOF mass spectrometry resulted in a linear relationship between the reaction time and degree of polymerization of the grafted polymer. This as well as the successful end-functionalization by termination with secondary amines corroborates our view of a well-defined. living polymerization mechanism.
A self-assembled monolayer on a planar gold substrate has been used to initiate the living cationic ring-opening polymerization of 2-ethyl-2-oxazoline. The polymer chain end was functionalized with an alkyl moiety by means of the termination reaction in order to form an amphiphilic brush-type layer. The resulting layer (d similar to 10 nm) of linear poly(N-propionylethylenimine) (PPEI) is of uniform thickness and was found to be very stable.
The purpose of this study was to evaluate the circulating properties of liposomes coated with modified polyvinyl alcohol (PVA-R) having different molecular weights (6000, 9000 and 20 000). The size controlled liposomes (egg phosphatidylcholine (or distearoylphosphatidylcholine):cholesterol=7:3 in a molar ratio) were prepared by the hydration method followed by sonication. Polymer coated liposomes were prepared by just mixing the resultant liposomal suspension and a polymer solution. The effects of polymer coating were evaluated by measuring the circulation time of the injected liposomes after i.v. administration in rats and the dispersing property of the liposomes in a biological condition. The circulation of the PVA-R coated liposomes was prolonged with increasing the molecular weight of PVA-R. The aggregation and/or fusion of the liposomes in the presence of serum in vitro was also depressed more by coating the liposomes with PVA-R having higher molecular weight. There was a good correlation between the circulation time and the physical stability of non-coated and the various PVA-R coated liposomes. The prolonged circulation time of PVA-R (molecular weight: 20 000) coated liposomes (ca. 1.3 mol% coating) was comparable to that of a stealth liposome prepared with 8 mol% of DSPE–PEG (molecular weight of PEG: 2000).
Only the coupling of appropriate properties of liposomes and polymers has made possible many of today's liposome applications, leading to the first commercially available liposomal drug in the USA.
Tethered lipid bilayers (tBLMs) were obtained by the fusion of liposomes from diphytanoylphosphatidylcholine (DPhyPC) with self-assembled monolayers (SAMs) of a newly designed archaea analogue thiolipid, 2,3-di-O-phytanyl-sn-glycerol-1-tetraethylene glycol-d,l-α-lipoic acid ester (DPTL) on template stripped gold (TSG) films from silicon wafer as a template. SAMs, as characterized by reflection absorption infrared spectroscopy (RAIRS), show a mixture of different conformations of the tetraethylene segment in air, which appears to rearrange into the fully extended conformation when the SAM is immersed into an aqueous electrolyte solution, as deduced from thickness measurements by surface plasmon resonance spectroscopy (SPR). The fusion of liposomes was followed by SPR, quartz crystal microbalance (QCM), and fluorescence microscopy. Highly resistive tBLMs were obtained, as demonstrated by electrochemical impedance spectroscopy (EIS) results, which are equivalent to those for the BLM. This large resistivity is attributed to the ultraflat surface of TSG, as well as to the distinctive architecture of the newly designed molecule. The roughness of the TSG obtained from mica and silicon wafer as template was determined by AFM and compared to that of a Au(111) surface on mica. The largest roughness features of TSG are shown to be 0.5−1 nm, which is small compared to the vertical dimension of the DPTL molecules. This is regarded to be crucial for the self-assembly process, particularly in the case of amphiphilic molecules.
Poly(ethylene glycol) (PEG) is a molecule that exhibits unique behavior when compared with polymers in its homologous family. Depending on its environment, it may show hydrophilic, hydrophobic, or amphiphilic properties. We have studied several PEG lipopolymers, where a PEG chain with a molecular weight (MW) of 2000 g/mol or 5000 g/mol is covalently attached to 1,2-dipalmitoyl- or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, with a Langmuir film balance and a recently developed interfacial stress rheometer. In particular, we have determined how the rheological properties of PEG molecules anchored at the air-water interface change when the polymer chains are forced into highly stretched brush conformations. Pressure-area isotherms of monolayers of PEG lipopolymers exhibit two phase transitions: a desorption transition of the PEG chains from the air-water interface at 10 mN/m and a high film pressure transition at 20-40 mN/m, but the nature of the latter transition is still poorly understood. We have observed a remarkable change of the viscoelastic properties in the range of the high-pressure transition. The monolayer is fluid below the transition, with the surface loss modulus, G(s)", being larger than the surface storage modulus, Gs', but becomes remarkably elastic above, with G(s)' > G(s)". This indicates that a strong correlation exists between the reversible, first order Like high-pressure transition and the formation of a physical gel. Our surface rheological experiments indicate that formation of a physical network can be understood if water intercalates mediate the interaction between adjacent PEG chains via hydrogen bonding.
The synthesis of polymer monolayers, which are designed to act as “cushions” for lipid membranes attached to solid surfaces, is described. Monolayers of poly(ethyloxazolines) which could act as a polymeric support for biomimetic membranes have been attached to silicon oxide or gold surfaces following different strategies for the surface-attachment of the polymer chains. The polymers are either bound to the surface through a chemisorption process (“grafting-to”) or “grown” on the surface of silicon oxide substrates through a cationic ring-opening polymerization started from the surface of the substrate, creating a surface-attached polymer monolayer in situ (“grafting from”). The characterization of the surface attached films by various surface analytical techniques and the swelling behavior of the surface-attached polymer films in moist air are described. The non-specific adsorption of fibrinogen as a typical example for a strongly adsorbing protein onto the surface attached monolayers is studied.
By a direct combination of the fast initiation technique and a selective quantitative termination reaction, terminally functionalized poly(N-propionylethylenimine)s (PPEIs) were prepared by cationic living ring-opening polymerization of 2-ethyl-2-oxazoline in a one-pot multistep reaction. The chain ends of the linear hydrophilic polymers were functionalized with different lipophilic moieties (methyl-, n-hexadecyl- (C-16-), and 1,2-O-diooctadecyl-sn-glyceryl- ((C-18)(2)-)) and a monofunctional silane coupling group. Varying the length of the double functionalized hydrophilic PPEI chain (n = 10, 20), surface active amphiphilic lipopolymers of controlled hydrophilic lipophilic balance could be obtained in high overall yields. The spectroscopic, chromatographic, and mass spectrometric characterization confirmed that each reaction step was nearly quantitative and narrow molecular weight distributions (M-w/M-n = 1.07-1.24) of all polymers.
Many recent reports have demonstrated that rapid uptake of liposomes in vivo by cells of the mononuclear phagocytic system (MPS), which has restricted their therapeutic utility, can be overcome by incorporation of lipids derivatized with the hydrophilic polymer polyethylene glycol (PEG). The structure-function relationship of PEG-derivatized phosphatidylethanolamine (PEG-PE) has been examined by measurement of blood lifetime and tissue distribution in both mice and rats. The results are reviewed and contrasted with those from liposomes without PEG-PE or other surface modifications. With a PEG molecular weight in the range of 1000 to 5000, prolonged circulation and reduced MPS uptake is achieved. After 24 h, up to 35% of the injected dose remains in the blood and less than 10% is taken up by the two major organs of the MPS, liver and spleen, compared with 1% and up to 50%, respectively, for liposomes without PEG-PE. Other important advantages of PEG-PE have been identified: prolonged circulation is independent of liposome cholesterol content, degree of hydrocarbon chain saturation in either the PC or the PE lipid anchor, lipid dose, or addition of most other negatively charged lipids. This versatility in lipid composition and dose is important for controlling drug release in a liposome-based therapeutic agent. Steric stabilization has been proposed as a theoretical basis for the results and some initial results testing this hypothesis have been reported. A description of a theoretical model is presented here and evaluated with the data available. The results are compared with other particulate drug carriers and the range of potential applications are considered.
Lipid-conjugates of two amphipatic polymers, poly(2-methyl-2-oxazoline) (PMOZ) and poly(2-ethyl-2-oxazoline) (PEOZ) (degree of polymerization approximately 50) were synthesized by linking glutarate esters of the polymers to distearoylphosphatidylethanolamine (DSPE) or alternatively by termination of the polymerization process with DSPE. Surface-modified liposomes (90 +/- 5 nm) prepared from either conjugate (5 mol % of total lipid) were injected into rats and followed by blood level and tissue distribution measurements. Both polymers PEOZ and PMOZ were found to convey long circulation and low hepatosplenic uptake to liposomes to the same extent as polyethylene glycol (PEG), the best known material for this purpose. This is the first demonstration of protection from rapid recognition and clearance conveyed by alternative polymers, which is equal to the effect of PEG.
For the purpose of providing a summary of current clinical trials to determine whether povidone-iodine is beneficial or detrimental to wound healing, an integrated review was completed. Clinical trials were defined as any study that uses some concentration and form of povidone-iodine in a comparison or evaluation with other products or treatments resulting in an impact of povidone-iodine on wounds. The use of povidone-iodine for cleansing, irrigating, and dressing wounds is controversial. Wound healing is complex and requires safe and effective treatment modalities. Numerous in vitro and in vivo studies have been done with conflicting results on bactericidal effects and cytotoxicity of this antimicrobial agent. Human and animal in vivo studies in the last 10 years were used for this review because often the relevance of in vitro data in clinical conditions are questioned. The varied studies provide evidence that in most instances, povidone-iodine did not effectively promote good wound healing; in fact, most studies showed either impaired wound healing, reduced wound strength, or infection.
There is increasing interest in supported membranes as models of biological membranes and as a physiological matrix for studying the structure and function of membrane proteins and receptors. A common problem of protein-lipid bilayers that are directly supported on a hydrophilic substrate is nonphysiological interactions of integral membrane proteins with the solid support to the extent that they will not diffuse in the plane of the membrane. To alleviate some of these problems we have developed a new tethered polymer-supported planar lipid bilayer system, which permitted us to reconstitute integral membrane proteins in a laterally mobile form. We have supported lipid bilayers on a newly designed polyethyleneglycol cushion, which provided a soft support and, for increased stability, covalent linkage of the membranes to the supporting quartz or glass substrates. The formation and morphology of the bilayers were followed by total internal reflection and epifluorescence microscopy, and the lateral diffusion of the lipids and proteins in the bilayer was monitored by fluorescence recovery after photobleaching. Uniform bilayers with high lateral lipid diffusion coefficients (0.8-1.2 x 10(-8) cm(2)/s) were observed when the polymer concentration was kept slightly below the mushroom-to-brush transition. Cytochrome b(5) and annexin V were used as first test proteins in this system. When reconstituted in supported bilayers that were directly supported on quartz, both proteins were largely immobile with mobile fractions < 25%. However, two populations of laterally mobile proteins were observed in the polymer-supported bilayers. Approximately 25% of cytochrome b(5) diffused with a diffusion coefficient of approximately 1 x 10(-8) cm(2)/s, and 50-60% diffused with a diffusion coefficient of approximately 2 x 10(-10) cm(2)/s. Similarly, one-third of annexin V diffused with a diffusion coefficient of approximately 3 x 10(-9) cm(2)/s, and two-thirds diffused with a diffusion coefficient of approximately 4 x 10(-10) cm(2)/s. A model for the interaction of these proteins with the underlying polymer is discussed.
The composition of the Earth's early atmosphere is a subject of continuing debate. In particular, it has been suggested that elevated concentrations of atmospheric carbon dioxide would have been necessary to maintain normal surface temperatures in the face of lower solar luminosity in early Earth history. Fossil weathering profiles, known as palaeosols, have provided semi-quantitative constraints on atmospheric oxygen partial pressure (pO2) before 2.2 Gyr ago. Here we use the same well studied palaeosols to constrain atmospheric pCO2 between 2.75 and 2.2 Gyr ago. The observation that iron lost from the tops of these profiles was reprecipitated lower down as iron silicate minerals, rather than as iron carbonate, indicates that atmospheric pCO2 must have been less than 10(-1.4) atm--about 100 times today's level of 360 p.p.m., and at least five times lower than that required in one-dimensional climate models to compensate for lower solar luminosity at 2.75 Gyr. Our results suggest that either the Earth's early climate was much more sensitive to increases in pCO2 than has been thought, or that one or more greenhouse gases other than CO2 contributed significantly to the atmosphere's radiative balance during the late Archaean and early Proterozoic eons.
Certain amphiphilic water-soluble polymers including amphiphilic derivatives of polyvinyl pyrrolidone (PVP) were found to be efficient steric protectors for liposomes in vivo. In this study, we have tried to develop synthetic pathways for preparing amphiphilic PVP and to investigate the influence of the hydrophilic/hydrophobic blocks on some properties of resulting polymers and polymer-coated liposomes. To prepare amphiphilic PVP with the end stearyl (S) or palmityl (P) residues, amino- and carboxy-terminated PVP derivatives were first synthesized by the free-radical polymerization of vinyl pyrrolidone in the presence of amino- or carboxy-mercaptans as chain transfer agents, and then modified by interaction of amino-PVP with stearoyl chloride or palmitoyl chloride, or by dicyclohexyl carbodiimide coupling of stearylamine with carboxy-PVP. ESR-spectra of the hydrophobic spin-probe, nitroxyl radical N-oxyl-2-hexyl-2-(10-methoxycarbonyl)decyl-4,4'-dimethyl oxazoline, in the presence of amphiphilic PVP demonstrated good accessibility of terminal P- and S-groups for the interaction with other hydrophobic ligands. Spontaneous micellization and low CMC values (in a low micromolar range) were found for amphiphilic PVP derivatives using the pyrene method. In general, S-PVP forms more stable micelles than P-PVP (at similar MW, CMC values for S-PVP are lower than for P-PVP). It was found that amphiphilic PVP incorporated into negatively charged liposomes effectively prevents polycation(poly-ethylpyridinium-4-vinylchloride)-induced liposome aggregation, completely abolishing it at ca. 10 mol% polymer content in liposomes. Additionally, the liposome-incorporated PVP prevents the fluorescence quenching of the membrane-incorporated hydrophobic fluorescent label [N-(4-fluoresceinthiocarbamoyl)dipalmitoyl-PE] by the free polycation. PVP-modified liposomes were loaded with a self-quenching concentration of carboxyfluorescein, and their destabilization in the presence of mouse serum was investigated following the release of free dye. Amphiphilic PVP with MW between 1,500 and 8,000 provides good steric protection for liposomes. The degree of this protection depends on both polymer concentration and molecular size of the PVP block.
We present a new molecular engineering approach in which a polymer-supported phospholipid bilayer is vertically stabilized by controlled covalent tethering at both the polymer-substrate and polymer-bilayer interfaces. This approach is based on lipopolymer molecules, which not only form a polymer cushion between the phospholipid bilayer and a solid glass substrate but also act as covalent connections (tethers) between the bilayer and cushion. Our approach involves Langmuir-Blodgett transfer of a phospholipid-lipopolymer monolayer followed by Schaefer transfer of a pure phospholipid monolayer and is capable of varying the tethering density between the polymer layer and the phospholipid bilayer in a very controlled manner. Further stabilization is achieved if the glass substrate is surface-functionalized with a benzophenone silane. In this case, a photocross-linking reaction between the polymer and benzophenone group allows for the covalent attachment of the polymer cushion to the glass substrate. This approach is similar to that recently reported by Wagner and Tamm in which double tethering is achieved via lipopolymer silanes (Wagner, M. L.; Tamm, L. K. Biophys. J. 2000, 79, 1400). To obtain a deeper understanding of how the covalent tethering affects the lateral mobility of the bilayer, we performed fluorescence recovery after photobleaching (FRAP) experiments on polymer-tethered bilayers at different tethering densities (lipopolymer/phospholipid molar ratios). The FRAP data clearly indicate that the hydrophobic lipopolymer moieties act as rather immobile obstacles within the phospholipid bilayer, thereby leading to hindered diffusion of phospholipids. Whereas the high lateral diffusion coefficient of D = 17.7 mum(2)/s measured at low tethering density (5 mol % lipopolymer) indicates rather unrestricted motion within the bilayer, corresponding values at moderate (10 mol % lipopolymer) and high (30 mol % lipopolymer) tethering densities of D = 9.7 mum(2)/s and D = 1.1 mum(2)/s, respectively, show significant hindered diffusion. These results are contrary to the recent findings on similar membrane systems reported by Wagner and Tamm in which no significant change in phospholipid diffusion was found between 0 and 10 mol % lipopolymer. Our experimental report leads to a deeper understanding of the complex problem of interlayer coupling and offers a path toward a compromise between stability of the whole system and lateral mobility within the bilayer. Furthermore, the FRAP measurements show that polymer-tethered membranes are very interesting model systems for studying problems of restricted diffusion within two-dimensional fluids.
Simple vesicle fusion allowed the formation of a bilayer lipid membrane from the self-assembled monolayer of lipoic acid ester 1 (see formula). Film thicknesses determined by surface plasmon resonance (SPR) spectroscopic measurements agree with the theoretical thicknesses of the stretched conformation. Electrochemical impedance spectroscopy (EIS) of the model system shows that the latter exhibits similar electrical properties to biological membranes.
We report the design of supported lipid membranes attached to the surface by tailored lipopolymer tethers. A series of well-defined lipopolymers were synthesized by means of living cationic polymerization of 2-methyl-2-oxazolines. The polymers were equipped with a silane coupling group on the proximal, and lipid anchors on the distal chain ends. The length of the intermediate hydrophilic polymer tether was varied (n = 14, 18, 33) to change the distance between the membrane and the substrate. Supported membranes have been prepared in two-steps. First, a suitable lipopolymer/lipid mixture was deposited by Langmuir-Blodgett transfer, and annealed to establish the covalent coupling to the surface. On the dry lipopolymer/lipid monolayer, the upper leaflet was deposited by vesicle fusion. Optimization of both preparation steps resulted in the formation of stable and defect-free membranes. Impacts of the spacer length and the lipopolymer fraction upon the lateral diffusivity of the lipids were systematically compared by fluorescence recovery after photobleaching (FRAP). First experiments on the incorporation of a large transmembrane cell receptor (integrin alpha IIb beta 2) into the polymer-tethered membrane suggested that the length of the polymer tether plays a crucial role in distribution of the proteins on the surface.
We have monitored the environment and dynamics of the membrane interface formed by the ester-linked phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and the ether-linked phospholipid 1,2-dihexadecyl-sn-glycero-3-phosphocholine (DHPC) utilizing the wavelength-selective fluorescence approach and using the fluorescent membrane probe 2-(9-anthroyloxy)stearic acid (2-AS). This interfacially localized probe offers a number of advantages over those which lack a fixed location in the membrane. When incorporated in membranes formed by DPPC and DHPC, 2-AS exhibits red edge excitation shift (REES) of 14 and 8 nm, respectively. This implies that the rate of solvent reorientation, as sensed by the interfacial anthroyloxy probe, in ester-linked DPPC membranes is slow compared to the rate of solvent reorientation in ether-linked DHPC membranes. In addition, the fluorescence polarization values of 2-AS are found to be higher in DHPC membranes than in DPPC membranes. This is further supported by wavelength-dependent changes in fluorescence polarization and lifetime. Taken together, these results are useful in understanding the role of interfacial chemistry on membrane physical properties.
We report a simple method to confine transmembrane cell receptors in stripe micropatterns of a lipid/lipopolymer monolayer, which are formed as result of the transfer onto a solid substrate. The stripes are aligned perpendicular to the meniscus, whose periodicity can systematically be tuned by the transfer velocity. This strongly suggests the dominant role of the cooperative interaction between the film and substrate. Selective fluorescence labeling of lipids and lipopolymers confirms that the observed patterns coincide with the demixing of two species. Covalent coupling of polymer headgroups enables us to use the stripe patterns as a support for a lipid bilayer membrane. Spreading of lipid vesicles with platelet integrin alphaIIbbeta3 on a self-assembled membrane micropattern demonstrates that cell adhesion receptors are selectively incorporated into the lipopolymer-rich region. The method established here provides us with a tunable template for the confinement of receptor proteins to geometrically control the cell adhesion.
Synthetic polymers have long been used to modify various properties of proteins such as activity and solubility. Polyethylene glycol (PEG) has been widely used to form adducts with enzymes and antibodies. In this study, the polyoxazoline family of water-soluble polymers was used to synthesize adducts containing a synthetic peptide recognized by a monoclonal antibody (MAb) directed against human protein C (hPC). This is the first application of direct conjugation of unterminated or "living" polymer to a peptide. The avidity of the antibody for the various adducts was characterized with respect to size and hydrophilicity of methyl- and ethyl-substituted polyoxazoline polymers (POX). Avidity of the adducts was not found to be dependent upon the hydrophilicity and was slightly decreased due to polymer modification. The methyl-POX-peptide adducts were found to be highly water soluble, while the ethyl-POX-peptide adducts showed sporadic problems with aqueous solubility. Because the polymer-peptide adducts retained avidity for the antibody, polyoxazoline polymers may have potential application to protein-adduct chemistry.
- T B Bonné
- K Lüdtke
- R Jordan
- P Těpánek
- C M Papadakis
T. B. Bonné, K. Lüdtke, R. Jordan, P. Š těpánek, C. M.
Papadakis, Colloid Polym. Sci. 2004, 282, 833.
- T R Baekmark
- T Wiesenthal
- P Kuhn
- T M Bayerl
- O Nuyken
- R Merkel
T. R. Baekmark, T. Wiesenthal, P. Kuhn, T. M. Bayerl,
O. Nuyken, R. Merkel, Langmuir 1999, 15, 3616.
- H Takeuchi
- H Kojima
- H Yamamoto
- Y Kawashima
H. Takeuchi, H. Kojima, H. Yamamoto, Y. Kawashima, J.
Controlled Release 2001, 75, 83.