Linear Poly Ethylene Glycol Research Articles

Fine-tuning vitamin E-containing telodendrimers for efficient delivery of gambogic acid in colon cancer treatment.

Certain natural products such as gambogic acid (GA) exhibit potent antitumor effects. Unfortunately, administration of these natural products is limited by their poor solubility in conventional pharmaceutical solvents. In this study, a series of telodendrimers, composed of linear polyethylene glycol (PEG)-blocking-dendritic oligomer of cholic acid (CA) and vitamin E (VE), have been designed with architectures optimized for efficient delivery of GA and other natural anticancer compounds. Two of the telodendrimers with segregated CA and VE domains self-assembled into stable cylindrical and/or spherical nanoparticles (NPs) after being loaded with GA as observed under transmission electron microscopy (TEM), which correlated with the dynamic light scattering (DLS) analysis of sub-30 nm particle sizes. A very high GA loading capacity (3:10 drug/polymer w/w) and sustained drug release were achieved with the optimized telodendrimers. These novel nanoformulations of GA were found to exhibit similar in vitro cytotoxic activity against colon cancer cells as the free drug. Near-infrared fluorescence small animal imaging revealed preferential accumulation of GA-loaded NPs into tumor tissue. The optimized nanoformulation of GA achieved superior antitumor efficacy compared to GA-Cremophor EL formulation at equivalent doses in HT-29 human colon cancer xenograft mouse models. Given the mild adverse effects associated with this natural compound and the enhanced anticancer effects via tumor targeted telodendrimer delivery, the optimized GA nanoformulation is a promising alternative to the traditional chemotherapy in colon cancer treatment.

Encapsulation and covalent binding of molecular payload in enzymatically activated micellar nanocarriers.

The high selectivity and often-observed overexpression of specific disease-associated enzymes make them extremely attractive for triggering the release of hydrophobic drug or probe molecules from stimuli-responsive micellar nanocarriers. Here we utilized highly modular amphiphilic polymeric hybrids, composed of a linear hydrophilic polyethylene glycol (PEG) and an esterase-responsive hydrophobic dendron, to prepare and study two diverse strategies for loading of enzyme-responsive micelles. In the first type of micelles, hydrophobic coumarin-derived dyes were encapsulated noncovalently inside the hydrophobic core of the micelle, which was composed of lipophilic enzyme-responsive dendrons. In the second type of micellar nanocarrier the hydrophobic molecular cargo was covalently linked to the end-groups of the dendron through enzyme-cleavable bonds. These amphiphilic hybrids self-assembled into micellar nanocarriers with their cargo covalently encapsulated within the hydrophobic core. Both types of micelles were highly responsive toward the activating enzyme and released their molecular cargo upon enzymatic stimulus. Importantly, while faster release was observed with noncovalent encapsulation, higher loading capacity and slower release rate were achieved with covalent encapsulation. Our results clearly indicate the great potential of enzyme-responsive micellar delivery platforms due to the ability to tune their payload capacities and release rates by adjusting the loading strategy.

ChemInform Abstract: Site‐Specific PEGylation of Proteins: Recent Developments

AbstractReview: 69 refs.

PEGylated d-serine dehydratase as a d-serine reducing agent

d-Serine is an endogenous coagonist for N-methyl-d-aspartate (NMDA) receptors and is involved in excitatory neurotransmission. Excessive receptor activation causes excitotoxicity, leading to various acute and chronic neurological disorders. Decrease in d-serine content may provide a therapeutic strategy for the treatment of the neurological disorders in which overstimulation of NMDA receptors plays a pathological role. Saccharomyces cerevisiaed-serine dehydratase (Dsd1p), which acts dominantly on d-serine, may be a useful d-serine reducing agent. We conjugated a linear 5-kDa polyethylene glycol (PEG) to Dsd1p (PEG-Dsd1p) and examined the effects of PEG-conjugation on its biochemical and pharmacokinetic properties. PEG-Dsd1p retained activity, specificity, and stability of the enzyme. The PEG modification extended the serum half-life of Dsd1p in mice 6-fold, from 3.8h to 22.4h. PEG-Dsd1p was much less immunogenic compared to the unmodified enzyme. Intraperitoneal administration of PEG-Dsd1p was effective in decreasing the d-serine content in the mouse hippocampus. These findings suggest that PEG-Dsd1p may be a novel tool for lowering d-serine levels in vivo.

Enzymatically crosslinked dendritic polyglycerol nanogels for encapsulation of catalytically active proteins.

The enormous potential of nanogel scaffolds for protein encapsulation has been widely recognized. However, constructing stable polymeric nanoscale networks in a facile, mild, and controllable fashion still remains a technical challenge. Here, we present a novel nanogel formation strategy using horseradish peroxidase (HRP) catalyzed crosslinking on phenolic derivatized dendritic polyglycerol (dPG) in the presence of H2O2 in an inverse miniemulsion. This "enzymatic nanogelation" approach was efficient to produce stable 200 nm dPG nanogel particles, and was performed under physiological conditions, thus making it particularly beneficial for encapsulating biological proteins. Purification of the nanogels was easy to handle and practical because there was no need for a post-quenching step. Interestingly, the use of dPG resulted in higher HRP laden nanogels than for linear polyethylene glycol (PEG) analogs, which illustrates the benefits of dendritic backbones in nanogels for protein encapsulation. In addition, the mild immobilization contributed to the enhanced thermal stability and reusability of HRP. The nanogel preparation could be easily optimized to achieve the best HRP activity. Furthermore, a second enzyme, Candida antarctica lipase B (CalB), was successfully encapsulated and optimized for activity in dPG nanogels by the same enzymatic methodology, which shows the perspective applications of such techniques for encapsulation of diverse proteins.

SPRi determination of inter-peptide interaction by using 3D supramolecular co-assembly polyrotaxane film

Accurate measurement of inter-peptide interactions is beneficial for in-depth understanding disease-related protein folding and peptide aggregation, and further for designing and selecting potential peptide drugs to the target antigen. Herein, we demonstrate a 3D polyrotaxane (PRX) surface for detecting peptides interactions by surface plasmon resonance imaging (SPRi). This surface is supramolecular self-assembly monolayer (SAM) structure fabricated by threading α-cyclodextrans (α-CD) through a linear polyethylene glycol (PEG) chain fixed on gold chip surface to form pseudopolyrotaxane, and further capping the pseudopolyrotaxane with bulky terminated group to form PRX film. The hydroxyl groups of α-CD can provide more active sites to increase molecules immobilization density, and PEG chain has unique protein non-fouling feature. We chose Alzheimer's disease marker β-amyloid 40 (Aβ40) as model peptide, and detected the interaction between it and its inhibitors KLVFFK6 by SPRi. As a striking result, the specific adsorption of KLVFFK6 solution at the concentration of 352μM on Aβ40-PRX was 700RU, whereas PEG SAM surface gave no significant binding. Interaction between other lower molecular weight peptides was detected via PRX surface, and the relatively weak interactions (KD=1.73×10−4M) between LPFFD (Mw=0.6kDa) and amylin20-29 (Mw=1.0kDa) are successfully detected.

A polymeric prodrug of 5-fluorouracil-1-acetic acid using a multi-hydroxyl polyethylene glycol derivative as the drug carrier.

PurposeMacromolecular prodrugs obtained by covalently conjugating small molecular drugs with polymeric carriers were proven to accomplish controlled and sustained release of the therapeutic agents in vitro and in vivo. Polyethylene glycol (PEG) has been extensively used due to its low toxicity, low immunogenicity and high biocompatibility. However, for linear PEG macromolecules, the number of available hydroxyl groups for drug coupling does not change with the length of polymeric chain, which limits the application of PEG for drug conjugation purposes. To increase the drug loading and prolong the retention time of 5-fluorouracil (5-Fu), a macromolecular prodrug of 5-Fu, 5-fluorouracil-1 acid-PAE derivative (5-FA-PAE) was synthesized and tested for the antitumor activity in vivo.MethodsPEG with a molecular weight of 38 kDa was selected to synthesize the multi-hydroxyl polyethylene glycol derivative (PAE) through an addition reaction. 5-fluorouracil-1 acetic acid (5-FA), a 5-Fu derivative was coupled with PEG derivatives via ester bond to form a macromolecular prodrug, 5-FA-PAE. The in vitro drug release, pharmacokinetics, in vivo distribution and antitumor effect of the prodrug were investigated, respectively.ResultsThe PEG-based prodrug obtained in this study possessed an exceedingly high 5-FA loading efficiency of 10.58%, much higher than the maximum drug loading efficiency of unmodified PEG with the same molecular weight, which was 0.98% theoretically. Furthermore, 5-FA-PAE exhibited suitable sustained release in tumors.ConclusionThis study provides a new approach for the development of the delivery to tumors of anticancer agents with PEG derivatives.

Site-specific PEGylation of Proteins: Recent Developments

The attachment of linear polyethylene glycol (PEG) to peptides and proteins for their stabilization for in vivo applications is a milestone in pharmaceutical research and protein-drug development. However, conventional methods often lead to heterogeneous PEGylation mixtures with reduced protein activity. Current synthetic efforts aim to provide site-specific approaches by chemoselective targeting of canonical and noncanonical amino acids and to improve the PEG architecture. This synopsis highlights recent work in this area, which also resulted in improved pharmacokinetics of peptide and protein therapeutics.

New epoxy thermosets modified with amphiphilic multiarm star polymers as toughness enhancer

The synthesis and characterization of two novel amphiphilic multiarm star polymers with linear polyethylene glycol (PEG) and poly(ε-caprolactone) (PCL) arms and their use as toughening modifiers of epoxy anhydride thermosets are reported. The new star polymers were obtained by partial pegylation of a hyperbranched polyester and subsequent growth of PCL arms. The curing process was studied by calorimetry and thermomechanical analysis, demonstrating the accelerating effect and the influence on gelation of the hydroxyl terminal groups. The curing kinetics was analyzed by model-free and model-fitting methods. The final properties of the resulting materials were determined by thermal and mechanical tests. The addition of the star-like modifiers led only to notable improvement on impact strength in the material containing a 10% of the star with PCL and PEG arms, without compromising glass transition temperature and thermal stability. The morphology of the resulting materials depended on the structure of the toughness modifier used, as demonstrated by electron microscopy, but all modified thermosets obtained showed phase-separated morphologies with nanosized particles.

PEGylated recombinant human interferon-ω as a long-acting antiviral agent: Structure, antiviral activity and pharmacokinetics

Recombinant human interferon-ω (rhIFN-ω) exhibits a potent antiviral activity. Because of poor pharmacokinetics (PK) of rhIFN-ω, frequent dosing of rhIFN-ω is necessitated to achieve the sustained antiviral efficacy. PEGylation can efficiently improve the PK of rhIFN-ω while substantially decrease its bioactivity. The structure, antiviral activity and PK of the PEGylated rhIFN-ω were measured to establish their relationship with PEGylation sites, polyethylene glycol (PEG) mass and PEG structure. Accordingly, N-terminus and the lysine residues were selected as the PEGylation sites. PEGs with Mw of 20kDa and 40kDa were used to investigate the effect of PEG mass. Linear and branched PEGs were used to investigate the effect of PEG structure. PEGylation decreased the antiviral activity of rhIFN-ω and improved its PK. The PEGylation sites determine the bioactivity of the PEGylated rhIFN-ω and the conjugated PEG mass determines the PK. N-terminally PEGylated rhIFN-ω with 40kDa linear PEG maintains 21.7% of the rhIFN-ω antiviral activity with a half-life of 139.6h. Thus, N-terminally PEGylated rhIFN-ω with linear 40kDa PEG is a potential antiviral agent for long-acting treatment of the viral diseases.

Improved production of recombinant human Fas ligand extracellular domain in Pichia pastoris: yield enhancement using disposable culture-bag and its application to site-specific chemical modifications.

BackgroundA useful heterologous production system is required to obtain sufficient amounts of recombinant therapeutic proteins, which are often necessary for chemical characterization and engineering studies on the development of molecules with improved properties. Human Fas ligand extracellular domain (hFasLECD) is an agonistic death ligand protein that has potential applications for medical purposes. Site-specific chemical modifications can provide a powerful means for the development of engineered proteins with beneficial functions. This study aimed to enhance the yield of hFasLECD using a Pichia pastoris secretory expression system suitable for efficient production on a small laboratory scale, and further to provide procedures for its site-specific chemical modification without impairing the biological functions based on the developed production system.ResultsA convenient cultivation system using a disposable plastic bag provided a three-fold increase in purification yield of tag-free hFasLECD as compared with the conventional system using a baffled glass flask. The system was further applied to the production of a mutant, which contains an additional reactive cysteine residue in the N-terminal tag-sequence region. Site-specific conjugations and cross-linking without impairing biological functions were achieved by reaction of the mutant hFasLECD with single maleimide group containing compounds and a linear polyethylene glycol derivative containing two maleimide groups at either end, respectively. All purified tag-free and chemically modified hFasLECDs showed an evident receptor binding activity in co-immunoprecipitation experiments mediated by wild-type and N-glycosylation site deficient mutant human Fas receptor extracellular domain derivatives. An N-Ethylmaleimide conjugated hFasLECD derivative demonstrated a significant cytotoxic activity against human HT-29 colorectal cancer cells.ConclusionsA new, efficient cultivation system for enhanced secretory production of hFasLECD using P. pastoris and an effective strategy for site-specific chemical modifications of hFasLECD were devised. The results obtained constitute the basis for biomedical applications including developments of novel therapeutic proteins and diagnostic tools targeted to related diseases and their biomarkers.

Recent advances and potential applications of the photo‐induced macroscopic response of self‐assembled azopolymer in aqueous media

This mini‐review highlights issues related to the photo‐triggered macroscopic response of azopolymer in supramolecular hydrogel and polymer vesicles. Cyclodextrin (CD) can form an inclusion complex with trans‐azobenzene while cis‐azobenzene is expelled out of the CD's cavity. This photo‐reversible supramolecular host − guest interaction is used to trigger sol − gel transitions and to induce macroscopic assembly. When substituted azobenzene is located at chain ends of linear polyethylene glycol, monodisperse vesicles are formed exhibiting a photo‐induced pulsating behavior. The macroscopic phase transition and assembly or pulsating behavior of azopolymers can be expected to have potential applications as smart biomedical materials. © 2014 Society of Chemical Industry

Determination of polyethylene glycol end group functionalities by combination of selective reactions and characterization by matrix assisted laser desorption/ionization time-of-flight mass spectrometry

End groups play a critical role in macromolecular coupling reactions for building complex polymer architectures, yet their identity and purity can be difficult to ascertain using traditional analytical technique. Recent advances in mass spectrometry techniques have made matrix-assisted laser desorption/ionization time-of-fight (MALDI-TOF) mass spectrometry a rapid and powerful tool for providing detailed information about the identity and purity of homopolymer end groups. In this work, MALDI-TOF mass spectrometry was used to study end groups of linear polyethylene glycols. In particular, the identifications of alcohol, amine and thiol end groups are investigated because these nucleophilic moieties are among the most common within biological and synthetic macromolecules. Through comparative characterization of alcohol, amine, and thiol end groups, the exact identification of these end groups could be confirmed by selective and quantitative modification. The precision of this technique enables the unambiguous differentiation of primary amino groups relative to hydroxyl groups, which differ by only 1 mass unit. In addition, the quantitative conversion of various polyethylene glycol end groups using highly efficient coupling reactions such as the thiol-ene and azide-alkyne click reactions can be confirmed using MALDI-TOF mass spectrometry.

A non-micellar synthesis of mesoporous carbon via spinodal decomposition

Mesoporous carbons were prepared via spinodal decomposition of non-amphiphilic linear polyethylene glycol with phloroglucinol–formaldehyde resin under refluxing acidic ethanol conditions.

Supramolecular Hydrogel Capsules Based on PEG: A Step Toward Degradable Biomaterials with Rational Design

Supramolecular microgel capsules based on polyethylene glycol (PEG) are a promising class of soft particulate scaffolds with tailored properties. An approach to fabricate such particles with exquisite control by droplet-based microfluidics is presented. Linear PEG precursor polymers that carry bipyridine moieties on both chain termini are gelled by complexation to iron(II) ions. To investigate the biocompatibility of the microgels, living mammalian cells are encapsulated within them. The microgel elasticity is controlled by using PEG precursors of different molecular weights at different concentrations and the influence of these parameters on the cell viabilities, which can be optimized to exceed 90% is studied. Reversion of the supramolecular polymer cross-linking allows the microcapsules to be degraded at mild conditions with no effect on the viability of the encapsulated and released cells.

Side-by-side comparison of dendritic-linear hybrids and their hyperbranched analogs as micellar carriers of chemotherapeutics

Amphiphilic block copolymers are successfully synthesized possessing a hydrophobic dendritic component based on the bis-MPA monomer and a hydrophilic linear polyethylene glycol (PEG) component. The ...

Controlling the diameter, monodispersity, and solubility of ApoA1 nanolipoprotein particles using telodendrimer chemistry

Nanolipoprotein particles (NLPs) are nanometer-scale discoidal particles that feature a phospholipid bilayer confined within an apolipoprotein "scaffold," which are useful for solubilizing hydrophobic molecules such as drugs and membrane proteins. NLPs are synthesized either by mixing the purified apolipoprotein with phospholipids and other cofactors or by cell-free protein synthesis followed by self-assembly of the nanoparticles in the reaction mixture. Either method can be problematic regarding the production of homogeneous and monodispersed populations of NLPs, which also currently requires multiple synthesis and purification steps. Telodendrimers (TD) are branched polymers made up of a dendritic oligo-lysine core that is conjugated to linear polyethylene glycol (PEG) on one end, and the lysine "branches" are terminated with cholic acid moieties that enable the formation of nanomicelles in aqueous solution. We report herein that the addition of TD during cell-free synthesis of NLPs produces unique hybrid nanoparticles that have drastically reduced polydispersity as compared to NLPs made in the absence of TD. This finding was supported by dynamic light scattering, fluorescence correlation spectroscopy, and cryo transmission electron microscopy (Cryo-EM). These techniques demonstrate the ability of TDs to modulate both the NLP size (6-30 nm) and polydispersity. The telodendrimer NLPs (TD-NLPs) also showed 80% less aggregation as compared to NLPs alone. Furthermore, the versatility of these novel nanoparticles was shown through direct conjugation of small molecules such as fluorescent dyes directly to the TD as well as the insertion of a functional membrane protein.

Interaction Mechanism between Bovine Serum Albumin and Polyethylene Glycol Revealed with Elastic Light Scattering Spectroscopy

In order to gain deeper insight into the interaction mechanism between bovine serum albumin (BSA) and polyethylene glycol (PEG), the present work applied elastic light scattering (ELS) spectroscopy to investigate the interaction between BSA and PEG, and explore the effects of concentration and molecular weight of PEG on the interaction at physiological pH. The results showed that the interaction force existed between linear PEG and spherical BSA molecules was mainly hydrogen bonding. In addition, the apparent binding constant of system was evaluated by model calculation.

Quantification of a pegylated interferon-alpha2a product by a customised and validated reverse phase-high performance liquid chromatography method

There is increasing development of pegylated proteins as clinical products for therapeutic interventions in human disease. Quantification of such products relies on appropriately calibrated traditional methods, including reverse phase-high performance liquid chromatography (RP-HPLC). However, currently available pharmacopoeia calibrants, e.g., chemical reference substances (CRS), are highly purified non-pegylated proteins of known concentration. These are uncertain to be suitable for calibration purposes where the precise quantification of the mass of pegylated proteins, often heterogeneous with respect to polyethylene glycol (PEG) chain size, structure, attachment sites and isomer numbers and proportions, is required. In this study, a customised RP-HPLC method was developed and validated for the analysis of a pegylated IFN-α2a product having a linear 20kDa PEG chain (PEG20-IFN-α2a; Reiferon Retard®). Since the PEG20 moiety generated no signal at the detection wavelength of 210nm, the concentration of the base IFN-α2a molecules in PEG-IFN-α2a could be determined. By calculating the UV absorbance at 210nm of peak areas in their respective chromatographic profiles, a high correlation (r2≥0.995) of PEG20-IFN-α2a concentrations with equal concentrations of the CRS of IFN-α2a, or of a well-characterised PEG20-IFN-α2a internal reference substance (IRS) was found. This finding confirmed the suitability of this CRS as a primary calibrant for mass determinations of PEG20-IFN-α2a by the customised RP-HPLC method. Application of this method to the quantitative analysis of 10 batches of Reiferon Retard® yielded accurate and consistent results, indicating its utility for mass determinations of current and future Reiferon Retard® batches.

Cysteine-Specific PEGylation of rhG-CSF via Selenylsulfide Bond

A new PEGylation reagent enabling selective modification of free thiol groups is described in this article. The reagent was synthesized by attaching linear polyethylene glycol (PEG) N-hydroxysuccinimide to selenocystamine. The reaction was very fast, resulting in over 95% conversion yield. The active group of this new PEG-Se reagent is a diselenide, reacting with thiols via thiol/diselenide exchange reaction. Recombinant human granulocyte colony-stimulating factor (rhG-CSF) with an unpaired cysteine at the position 18 (Cys18) was used as a model protein. It was comparatively PEGylated with the new PEG-Se reagent, as well as with commercially available maleimide (PEG-Mal) and ortho-pyridyl disulfide (PEG-OPSS) PEG reagents. The highest PEGylation yield was obtained with PEG-Mal, followed by PEG-OPSS and PEG-Se. The reaction rates of PEG-Mal and PEG-Se were comparable, while the reaction rate of PEG-OPSS was lower. Purified monoPEGylated rhG-CSF conjugates were characterized and compared. Differences in activity, stability, and in vivo performance were observed, although all conjugates contained a 20 kDa PEG attached to the Cys18. Minor conformational changes were observed in the conjugate prepared with PEG-Mal. These changes were also reflected in low in vitro biological activity and aggregate formation of the maleimide conjugate. The conjugate prepared with PEG-Se had the highest in vitro biological activity, while the conjugate prepared with PEG-OPSS had the best in vivo performance.