RESUMEN
A 3-benzylmorpholine-2,5-dione monomer is synthesized from the natural amino acid l-phenylalanine and characterized by means of nuclear magnetic resonance and infrared spectroscopy, electrospray ionization mass spectrometry, and elemental analysis. Subsequent to preliminary polymerization studies, a well-defined poly(ester amide) homopolymer is synthesized via ring-opening polymerization using a binary catalyst system comprising 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and a 1-(3,5-bis(trifluoromethyl)phenyl)-3-cyclohexylthiourea (TU) cocatalyst with a feed ratio of M/I/DBU/TU = 100/1/1/10. Kinetic studies reveal high controllability of the dispersities and molar masses up to conversions of almost 80%. Analysis by mass spectrometry hints toward excellent end-group fidelity at these conditions. In consequence, utilization of hydroxyl-functionalized poly(ethylene glycol) and poly(2-ethyl-2-oxazoline) as macroinitiators results in amphiphilic block copolymers. Bulk miscibility of the building blocks is indicated by differential scanning calorimetry investigations. As more and more promising new drugs are based on hydrophobic molecules featuring aromatic moieties, the novel polyesteramides seem highly promising materials to be used as potential drug delivery vehicles.
Asunto(s)
Polietilenglicoles , Polímeros , Polimerizacion , Cinética , Polímeros/química , Polietilenglicoles/química , AminoácidosRESUMEN
Interleukin-4 (IL-4) is a potentially interesting anti-inflammatory therapeutic, which is rapidly excreted. Therefore, serum half-life extension by polymer conjugation is desirable, which may be done by PEGylation. Here, we use PEtOx as an alternative to PEG for bioconjugate engineering. We genetically extended murine IL-4 (mIL-4) with the d-domain of insulin-like growth factor I (IGF-I), a previously identified substrate of transglutaminase (TG) Factor XIIIa (FXIIIa). Thereby, engineered mIL-4 (mIL-4-TG) became an educt for TG catalyzed C-terminal, site-directed conjugation. This was deployed to enzymatically couple an azide group containing peptide sequence to mIL-4, allowing C-terminal bioconjugation of polyethylene glycol or poly(2-ethyl-2-oxazoline). Both bioconjugates had wild-type potency and alternatively polarized macrophages.
Asunto(s)
Interleucina-4RESUMEN
Influenza A viruses (IAV), including the pandemic 2009 (pdm09) H1N1 or avian influenza H5N1 virus, may advance into more pathogenic, potentially antiviral drug-resistant strains (including loss of susceptibility against oseltamivir). Such IAV strains fuel the risk of future global outbreaks, to which this study responds by re-engineering Interferon-α2a (IFN-α2a) bioconjugates into influenza therapeutics. Type-I interferons such as IFN-α2a play an essential role in influenza infection and may prevent serious disease courses. We site-specifically conjugated a genetically engineered IFN-α2a mutant to poly(2-ethyl-2-oxazoline)s (PEtOx) of different molecular weights by strain-promoted azide-alkyne cyclo-addition. The promising pharmacokinetic profile of the 25 kDa PEtOx bioconjugate in mice echoed an efficacy in IAV-infected ferrets. One intraperitoneal administration of this bioconjugate, but not the marketed IFN-α2a bioconjugate, changed the disease course similar to oseltamivir, given orally twice every study day. PEtOxylated IFN-α2a bioconjugates may expand our therapeutic arsenal against future influenza pandemics, particularly in light of rising first-line antiviral drug resistance to IAV.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Subtipo H5N1 del Virus de la Influenza A , Virus de la Influenza A , Gripe Humana , Animales , Antivirales/farmacología , Hurones , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/genética , Gripe Humana/tratamiento farmacológico , Ratones , Oseltamivir/farmacología , Oseltamivir/uso terapéuticoRESUMEN
BACKGROUND: Insufficient solubility and stability of bioactive small molecules as well as poor biocompatibility may cause low bioavailability and are common obstacles in drug development. One example of such problematic molecules is 6-bromoindirubin-3'-glycerol-oxime ether (6BIGOE), a hydrophobic indirubin derivative. 6BIGOE potently modulates the release of inflammatory cytokines and lipid mediators from isolated human monocytes through inhibition of glycogen synthase kinase-3 in a favorable fashion. However, 6BIGOE suffers from poor solubility and short half-lives in biological aqueous environment and exerts cytotoxic effects in various mammalian cells. In order to overcome the poor water solubility, instability and cytotoxicity of 6BIGOE, we applied encapsulation into poly(D,L-lactide-co-glycolide) (PLGA)-based nanoparticles by employing formulation methods using the sustainable solvents Cyrene™ or 400 g/mol poly(ethylene glycol) as suitable technology for efficient drug delivery of 6BIGOE. RESULTS: For all preparation techniques the physicochemical characterization of 6BIGOE-loaded nanoparticles revealed comparable crystallinity, sizes of about 230 nm with low polydispersity, negative zeta potentials around - 15 to - 25 mV, and biphasic release profiles over up to 24 h. Nanoparticles with improved cellular uptake and the ability to mask cytotoxic effects of 6BIGOE were obtained as shown in human monocytes over 48 h as well as in a shell-less hen's egg model. Intriguingly, encapsulation into these nanoparticles fully retains the anti-inflammatory properties of 6BIGOE, that is, favorable modulation of the release of inflammation-relevant cytokines and lipid mediators from human monocytes. CONCLUSIONS: Our formulation method of PLGA-based nanoparticles by applying sustainable, non-toxic solvents is a feasible nanotechnology that circumvents the poor bioavailability and biocompatibility of the cargo 6BIGOE. This technology yields favorable drug delivery systems for efficient interference with inflammatory processes, with improved pharmacotherapeutic potential.
Asunto(s)
Indoles , Sistema de Administración de Fármacos con Nanopartículas , Nanopartículas/química , Oximas , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Adolescente , Adulto , Anciano , Animales , Supervivencia Celular/efectos de los fármacos , Fluoresceína/química , Fluoresceína/farmacocinética , Humanos , Indoles/química , Indoles/farmacocinética , Indoles/toxicidad , Leucocitos/efectos de los fármacos , Persona de Mediana Edad , Sistema de Administración de Fármacos con Nanopartículas/química , Sistema de Administración de Fármacos con Nanopartículas/farmacocinética , Sistema de Administración de Fármacos con Nanopartículas/farmacología , Nanopartículas/toxicidad , Nanotecnología , Oximas/química , Oximas/farmacocinética , Oximas/toxicidad , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/toxicidad , Solventes/química , Adulto JovenRESUMEN
Conjugation of biologics with polymers modulates their pharmacokinetics, with polyethylene glycol (PEG) as the gold standard. We compared alternative polymers and two types of cyclooctyne linkers (BCN/DBCO) for bioconjugation of interferon-α2a (IFN-α2a) using 10 kDa polymers including linear mPEG, poly(2-ethyl-2-oxazoline) (PEtOx), and linear polyglycerol (LPG). IFN-α2a was azide functionalized via amber codon expansion and bioorthogonally conjugated to all cyclooctyne linked polymers. Polymer conjugation did not impact IFN-α2a's secondary structure and only marginally reduced IFN-α2a's bioactivity. In comparison to PEtOx, the LPG polymer attached via the less rigid cyclooctyne linker BCN was found to stabilize IFN-α2a against thermal stress. These findings were further detailed by molecular modeling studies which showed a modulation of protein flexibility upon PEtOx conjugation and a reduced amount of protein native contacts as compared to PEG and LPG originated bioconjugates. Polymer interactions with IFN-α2a were further assessed via a limited proteolysis (LIP) assay, which resulted in comparable proteolytic cleavage patterns suggesting weak interactions with the protein's surface. In conclusion, both PEtOx and LPG bioconjugates resulted in a similar biological outcome and may become promising PEG alternatives for bioconjugation.
Asunto(s)
Polietilenglicoles , Polímeros , Glicerol , Interferón alfa-2 , Proteínas Recombinantes/genéticaRESUMEN
The incorporation of an amino group into a bifunctional initiator for the cationic ring-opening polymerization (CROP) is achieved in a two-step reaction. Detailed kinetic studies using 2-ethyl-2-oxazoline demonstrate the initiators' eligibility for the CROP yielding well-defined polymers featuring molar masses of about 2000 g mol-1 . Deprotection of the phthalimide moiety subsequent to polymerization enables the introduction of a cyclooctyne group in central position of the polymer which is further exploited in a strain-promoted alkyne-azide click reaction (SpAAC) with a Fmoc-protected azido lysine representing a commonly used binding motif for site specific polymer-protein/peptide conjugation. In-depth characterization via electrospray ionization mass spectrometry (ESI) confirms the success of all post polymerization modification steps.
Asunto(s)
Oxazoles , Cinética , Poliaminas , PolimerizacionRESUMEN
During the last decades, poly(2-oxazoline)s (POx) have gained increased interest due to their versatility. In particular, cationic ring-opening polymerization (CROP) enables the synthesis of well-defined polymers bearing quantitative α- and ω-functionalities. In contrast to small initiating groups, the introduction of more sophisticated, respectively demanding groups remains challenging. To fulfill this challenge, the initiator should comply with one major requirement in order to yield well-defined polymers: a fast and complete initiation. The straight forward two-step synthesis of a novel initiator containing a 4-(trifluoromethyl)benzenesulfonate (fluorylate, TosCF3 ) counter-ion is herein presented to accomplish the introduction of a sophisticated functional 3-(2-(2-ethoxy)ethoxy)ethoxy)prop-1-ene (TEG) initiating group. Kinetic studies are conducted in acetonitrile and chlorobenzene using the hydrophilic 2-ethyl-2-oxazoline (EtOx) as well as the hydrophobic 2-octyl-2-oxazoline (OctOx) as monomers to examine the influences of the solvent as well as the different monomers. In particular, the initiator efficiency is determined by 1 H and 19 F nuclear magnetic resonance spectroscopy and compared to the corresponding tosylate (TEGTos) and triflate (TEGTf). It is shown that the fluorylate combines the stability of the tosylate and an enhanced propagation rate comparable to the triflate.
Asunto(s)
Oxazoles/síntesis química , Sulfonamidas/química , Iones/química , Espectroscopía de Resonancia Magnética , Estructura Molecular , Oxazoles/químicaRESUMEN
In a two-step synthesis, five different alkyl-substituted morpholine-2,5-dione monomers were synthesized from the natural amino acids glycine, alanine, valine, leucine, and isoleucine. The heterocyclic compounds crystallize in a boat-like conformation and are polymerized via 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD)-catalyzed ring-opening polymerization (ROP) in tetrahydrofuran. Well-defined polymers could be obtained from the monomers based on valine, leucine, and isoleucine at a feed ratio of M/I/TBD = 100/1/0.5. Kinetic studies of the ROP reveal that the molar masses and dispersities (D < 1.2) could be well controlled, as confirmed by size exclusion chromatography and 1 H NMR spectroscopy. At conversions above 50%, the polymerization rate decreases and the dispersity slightly increases, presumably due to transesterification. Matrix-assisted laser desorption time-of-flight mass spectrometry indicates the presence of polymer chains with α-end groups derived from the initiator.
Asunto(s)
Compuestos de Azabiciclo/química , Morfolinas/síntesis química , Catálisis , Estructura Molecular , Morfolinas/química , Tamaño de la Partícula , Polimerizacion , Propiedades de SuperficieRESUMEN
Detailed kinetic studies during the cationic ring-opening polymerization (CROP) of 2-ethyl-2-oxazoline (EtOx) are conducted using four bifunctional bromo-type initiators in N,N-dimethylformamide (DMF) at 140 °C. Serving as models to quantify chain transfer to monomer occurring during the CROP initiated by monofunctional initiators, size exclusion chromatography (SEC) resolves a second molar mass distribution with lower molar mass at initial [monomer] to [initiation site] ratios ([M]0 /[I]0 ) of 25, while the resolution is insufficient at [M]0 /[I]0 of 10. Slightly slow initiation is revealed at [M]0 /[I]0 = 25, which prohibits the derivation of chain transfer rates by fitting of the size exclusion chromatography (SEC) data. Although conventional kinetic plots give no indication of significant amounts of chain transfer, the molar mass distributions resolved by SEC can unambiguously be identified as such by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) in both the high as well as the low m/z regions of the mass spectra.
Asunto(s)
Dimetilformamida/química , Sustancias Macromoleculares/química , Oxazoles/química , Polímeros/química , Cationes/química , Cromatografía en Gel , Cinética , Peso Molecular , PolimerizacionRESUMEN
The in-depth analytical characterization of polymers, in particular regarding intended biomedical applications, is becoming increasingly important to elucidate their structure-property relationships. Specifically, end group analysis of e.g. polymers featuring a 'stealth effect' towards the immune system is of particular importance because of their use in coupling reactions to bioactive compounds. Herein, we established a liquid chromatography (LC) protocol to analyse bicyclo[6.1.0]nonyne-functionalized poly(2-alkyl-2-oxazoline)s (POx)s as promising functional polymers that can be applied in strain-promoted click reactions. This work involved the synthesis of poly(2-methyl-2-oxazoline) (PMeOx) and poly(2-ethyl-2-oxazoline) (PEtOx) by living cationic ring-opening polymerization (CROP) with different molar masses ranging from 2 up to 17.5 kDa and, to our knowledge, the first liquid chromatographic analysis of PMeOx. The developed analytical protocol enables the quantitative determination of post-polymerization reaction sequences with respect to the conversion of the ω-end groups. All synthesized polymers were straightforwardly analysed on a C18-derivatized silica monolithic column under reversed-phase chromatographic conditions with a binary mobile phase gradient comprising a mixture of acetonitrile and water. Subsequent mass spectrometry of collected elution fractions enabled the confirmation of the desired ω-end group functionalities and the identification of synthetic by-products.
RESUMEN
Hydrophilic poly(2-oxazoline)s represent a promising alternative to replace poly(ethylene glycol) in the biomedical field. For that purpose, reliable analytical protocols to confirm identity and quantity of impurities are required. In particular, side products deriving from chain transfer reactions occurring during the cationic ring-opening polymerization and incomplete end-capping processes may be present. The analytical approach must hence be capable of separating polymers according to minor changes regarding their end group. We demonstrate that liquid chromatography, relying on a monolithic C18-modified silica column and isocratic as well as gradient elution using water / acetonitrile mixtures and varying detectors, can accomplish such demanding high resolution separations. Poly(2-ethyl-2-oxazoline)s (PEtOx) with acetyl, hydroxyl, and phthalimide ω-end groups were investigated. Identification of side products was achieved through coupling with electrospray ionization mass spectrometry. UV / Vis detection was applied to quantify chain transfer products in PEtOx comprising biphenyl moieties. In addition, gradient elution enabled the separation of PEtOx into macromolecules according to their specific degrees of polymerization in molar mass ranges around 2,000 g mol-1.
Asunto(s)
Cromatografía Liquida , Polímeros , Peso Molecular , Polímeros/síntesis química , ProtonesRESUMEN
A new monomer, 2-ferrocene-ethyl-2-oxazoline, was copolymerized with 2-alkyl-2-oxazolines. The cationic ring opening polymerization (CROP) of 2-oxazolines allows the synthesis of well-defined copolymers with adjustable molar masses as well as end-group control, which was also evident from kinetic studies. The utilization of this new comonomer led to redox-active polymers as proven by UV-VIS-measurements and cyclic-voltammetry.