We performed molecular dynamics (MD) simulations of one or two copies of polyethylene glycol of molecular weight 550 (PEG550) and 5000 (PEG5000) Daltons, conjugated to generation 3 (G3) to 5 (G5) polyamidoamine (PAMAM) dendrimers with explicit water using a coarse-grained model. We found the radii of gyration of these dendrimer-PEG molecules to be close to those measured in experiments by Hedden and Bauer (Macromolecules 2003, 36, 1829). Densely grafted PEG ligands (>50% of the dendrimer surface) extend like brushes, with layer thickness in agreement with theory for starlike polymers. Two dendrimer-PEG complexes in the box drift away from each other, indicating that no aggregation is induced by either short or long PEG chains, conflicting with a recent view that the cytotoxicity of some PEGylated particles might be due to particle aggregation for long PEG lengths.
Molecular shape, flexibility, and surface hydrophilicity are thought to influence the ability of nanoparticles to cross biological barriers during drug delivery. In this study, coarse-grained (CG) molecular dynamics (MD) simulations were used to study these properties of a polymer-drug construct in potential clinical development: poly(γ-glutamyl-glutamate)-paclitaxel-poly(ethylene glycol) nonpeptide RGD (PGG-PTX-PEG-npRGD), a linear glutamyl-glutamate polymer with paclitaxel and poly(ethylene glycol)-nonpeptide RGD side groups. It was hypothesized that the PEG molecular weight (MW) (500 Da; 1,000 Da; and 2,000 Da) and nonpeptide RGD ligand density (4, 8, 12, and 16 per molecule), respectively, may have advantageous effects on the shape, flexibility, and surface hydrophilicity of PGG-PTX-PEG-npRGD. Circular dichroism spectroscopy was used to suggest initial structures for the all-atom (AA) models of PGG-PTX-PEG-npRGD, which were further converted to CG models using a commercially available mapping algorithm. Due to its semi-flexibility, PGG-PTX-PEG-npRGD is not limited to one specific conformation. Thus, CG MD simulations were run until statistical equilibrium, at which PGG-PTX-PEG-npRGD is represented as an ensemble of statistically similar conformations. The size of a PGG-PTX-PEG-npRGD molecule is not affected by the PEG MW or the nonpeptide RGD density, but higher PEG MW results in increased surface density of a PGG-PTX-PEG-npRGD molecule. Most PGG-PTX-PEG-npRGD shapes are globular, although filamentous shapes were also observed in the PEG500 and PEG1000 molecules. PEG500 and PEG1000 molecules are more flexible than PEG2000 systems. A higher presence of npRGD ligands results in decrease surface hydrophilicity of PGG-PTX-PEG-npRGD. These results indicate that the PGG-PTX-PEG1000-npRGD4 and PGG-PTX-PEG1000-npRGD8 molecules are the most efficacious candidates and are further recommended for experimental preclinical studies.
Electronic supplementary material
The online version of this article (doi:10.1007/s00894-011-0989-4) contains supplementary material, which is available to authorized users.
Polymer; Paclitaxel; Poly(ethylene glycol); Nonpeptide RGD; Active targeting; Coarse-grained modeling
Molecular dynamics (MD) simulations were used to refine a theoretical model that describes the interaction of single polyethylene glycol (PEG) molecules with α-hemolysin (αHL) nanopores. The simulations support the underlying assumptions of the model, that PEG decreases the pore conductance by binding cations (which reduces the number of mobile ions in the pore) and by volume exclusion, and provide bounds for fits to new experimental data. Estimation of cation binding indicates that four monomers coordinate a single K+ in a crown-ether like structure, with, on average, 1.5 cations bound to a PEG 29-mer at a bulk electrolyte concentration of 4 M KCl. Additionally, PEG is more cylindrical and has a larger cross-section area in the pore than in solution, although its volume is similar. Two key experimental quantities of PEG are quantitatively described by the model: the ratio of single channel current in the presence of PEG to that in the polymer’s absence (blockade depth), and the mean residence time of PEG in the pore. The refined theoretical model is simultaneously fit to the experimentally determined current blockade depth and the mean residence times for PEGs with 15 – 45 monomers, at applied transmembrane potentials of -40 to -80 mV, and for three electrolyte concentrations. The model estimates the free energy of the PEG-cation complexes to be -5.3 kBT. Finally the entropic penalty of confining PEG to the pore is found to be inversely proportional to the electrolyte concentration.
Molecular dynamics; nanopore; alpha-hemolysin; PEG; DNA sequencing
Self-assembly of polyethylene glycol (PEG)-grafted lipids at different sizes and concentrations was simulated using the MARTINI coarse-grained (CG) force field. The interactions between CG PEG and CG dipalmitoylglycerophosphocholine (DPPC)-lipids were parametrized by matching densities of 19-mers of PEG and polyethylene oxide (PEO) grafted to the bilayer from all-atom simulations. Mixtures of lipids and PEG(Mw = 550, 1250, 2000)-grafted lipids in water self-assembled to liposomes, bicelles, and micelles at different ratios of lipids and PEGylated lipids. Average aggregate sizes decrease with increasing PEGylated-lipid concentration, in qualitative agreement with experiment. PEGylated lipids concentrate at the rims of bicelles, rather than at the planar surfaces; this also agrees with experiment, though the degree of segregation is less than that assumed in previous modeling of the experimental data. Charged lipids without PEG evenly distribute at the rim and planar surface of the bicelle. The average end-to-end distances of the PEG on the PEGylated lipids are comparable in liposomes, bicelles (edge or planar surface), and micelles, and only slightly larger than for an isolated PEG in solution. The ability of PEGylated lipids to induce the membrane curvature by the bulky head group with larger PEG, and thereby modulate the phase behavior and size of lipid assemblies, arises from their relative concentration.
A surface density gradient of grafted poly (ethylene glycol) (PEG) chains was prepared using two-phase silanization of a flat silica surface. The first step was to create the surface density gradient of isocyanatopropyldimethylsilyl groups and to hydrolyze the isocyanato moiety into an amine. These surface amines were reacted with an excess of aldehyde-terminated PEG. The PEG–silica surface was characterized by dynamic contact angle measurements, X-ray photoelectron spectroscopy and ellipsometry. The length of the PEG gradient region was approximately 7 mm and the thickness in air ranged from zero to 1.1 nm. The maximum surface density of the PEG layer, as calculated from ellipsometric data, amounted to an average 0.4 PEG (molecular weight Mw = 2000 Da) molecule nm−2, while the surface density average of the amine groups was 1.4 molecules nm−2, indicating that only a fraction of the surface amines reacted with aldehyde-terminated PEG. The PEG segment density profile in the gradient PEG region was computed by a self-consistent mean field theory. The PEG (Mw = 2000 Da) segments profile was not parabolic, but showed a thin depletion zone next to the surface.
The influence of the surface density of the grafted PEG chains on protein repellence was tested by the adsorption of fibrinogen from solution and from a ternary protein solution mixture containing fibrinogen, albumin and immunoglobulin G. Fibrinogen adsorption onto the silica end of the gradient was extremely low, both in the presence of the other two proteins and in their absence. As the surface density of the grafted PEG chains increased, so did the fibrinogen adsorption (up to 0.024 μg cm−2). It is not clear whether this low fibrinogen adsorption resulted from the interactions of the protein with the grafted PEG chains or with residual surface amines that were available due to some imperfections in the grafted PEG layer.
Grafted poly (ethylene glycol); Protein adsorption; Surface density gradient
Mango peel is a good source of protease but remains an industrial waste. This study focuses on the optimization of polyethylene glycol (PEG)/dextran-based aqueous two-phase system (ATPS) to purify serine protease from mango peel. The activity of serine protease in different phase systems was studied and then the possible relationship between the purification variables, namely polyethylene glycol molecular weight (PEG, 4000–12,000 g·mol−1), tie line length (−3.42–35.27%), NaCl (−2.5–11.5%) and pH (4.5–10.5) on the enzymatic properties of purified enzyme was investigated. The most significant effect of PEG was on the efficiency of serine protease purification. Also, there was a significant increase in the partition coefficient with the addition of 4.5% of NaCl to the system. This could be due to the high hydrophobicity of serine protease compared to protein contaminates. The optimum conditions to achieve high partition coefficient (84.2) purification factor (14.37) and yield (97.3%) of serine protease were obtained in the presence of 8000 g·mol−1 of PEG, 17.2% of tie line length and 4.5% of NaCl at pH 7.5. The enzymatic properties of purified serine protease using PEG/dextran ATPS showed that the enzyme could be purified at a high purification factor and yield with easy scale-up and fast processing.
purification; polyethylene glycol (PEG); serine protease; mango peel; yield
This work describes the synthesis and characterization of novel thermoresponsive highly-branched polyamidoamine-polyethylene glycol-poly (D, L-lactide) (PAMAM-PEG-PDLLA) core-shell nanoparticles. A series of dendritic PEG-PDLLA nanoparticles were synthesized through conjugation of PEG of various chain lengths (1500, 6000, and 12000 g/mol) to polyamidoamine (PAMAM) dendrimer G3.0 and subsequent ring-opening polymerization of DLLA. Ninhydrin assay, 1H-NMR, FT-IR, dynamic light scattering, AFM were used to characterize the structure and compositions of dendritic PEG-PDLLA nanoparticles. Sol-gel phase transition of aqueous dendritic PEG-PDLLA solutions was measured using UV-Vis spectroscocopy. According to our results, dendritic PEG-PDLLA nanoparticles in aqueous solutions can self-assemble into sub-micron/micron aggregates and the size of aggregates is dependent on temperature and PEG-PDLLA chain length. Further, dendritic PEG-PDLLA solutions exhibit sol-gel phase transition with increasing temperature. The constructed dendritic PEG-PDLLA nanoparticles possess high cytocompatibility, which is significantly improved as compared to PAMAM dendrimers. The potential of dendritic PEG-PDLLA nanoparticles for encapsulation of water insoluble drugs such as camptothecin was demonstrated. Dendritic PEG-PDLLA nanoparticles we developed offer greater structural flexibility and provide a novel nanostructured thermoresponsive carrier for drug delivery.
dendrimers; drug delivery; nanomedicine; PEG-PLA copolymers; sol-gel phase transition; thermoresponsiveness
Mechanisms underlying the release of paclitaxel (PTX) from poly(ethylene glycol)/poly(lactic-co-glycolic acid) (PEG/PLGA) blends were investigated by coherent anti-Stokes Raman scattering (CARS) microscopy. PLGA, PEG, and PTX were selectively imaged by using the resonant CARS signal from the CH3, CH2, and aromatic CH stretch vibrations, respectively. Phase segregation was observed in PLGA films containing 10 to 40 wt.% PEG in the absence of PTX loading. The PEG phase existed in the form of crystalline fibers in the (8:2, weight ratio) and (7:3) PLGA/PEG films. CARS observation indicated that PTX preferentially partitioned into the PEG domains in the (9:1) and (8:2) PLGA/PTX films, but exhibited a uniform mixing with both PLGA and PEG in the (7:3) PLGA/PEG film. The solid dispersion of PTX into PEG domains was attributed to a strong interaction between PTX and PEG, supported by the disappearance of PEG crystallization in the PTX-loaded PLGA/PEG film evidenced through X-ray diffraction analysis. PTX release was induced by exposing the film to an aqueous solution and monitored in real time by CARS and two-photon fluorescence microscopy. Fast dissolution of both PEG and PTX was observed at the film surface. Upon infiltration of water into the film, the PEG domains rearranged into ring structures enriched by both PTX and PEG. The CARS data provided a visual evidence explaining the accelerated burst release followed by more sustained release of PTX from the PLGA/PEG films as measured by HPLC.
PLGA; paclitaxel; PEG; CARS microscopy; molecular imaging
Surface protein modification with poly(ethylene glycol) (PEG) can inhibit acute thrombosis on damaged vascular and biomaterial surfaces by blocking surface protein–platelet interactions. However, the feasibility of employing protein reactive PEGs to limit intravascular and biomaterial thrombosis in vivo is contingent upon rapid and extensive surface protein modification. To characterize the factors controlling this potential therapeutic approach, the model protein bovine serum albumin was adsorbed onto polyurethane surfaces and modified with PEG-carboxymethyl succinimidyl ester (PEG-NHS), PEG-isocyanate (PEG-ISO), or PEG-diisocyanate (PEG-DISO) in aqueous buffer at varying concentrations and contact times. It was found that up to 5 PEGs could be attached per albumin molecule within one min and that adsorbed albumin PEGylation approached maximal levels by 6 min. The lability of reactive PEGs in aqueous buffer reduced total protein modification by 50% when the PEG solution was incubated for 7 min prior to application. For fibrinogen PEGylation (performed in the solution phase), PEG-NHS was more reactive than PEG-ISO or PEG-DISO. The γ peptide of fibrinogen, which contains several key platelet-binding motifs, was highly modified. A marked reduction in platelet adhesion was observed on fibrinogen-adsorbed polyurethane treated with PEG-NHS or PEG-DISO. Relative differences in platelet adhesion on PEG-NHS and PEG-DISO modified surfaces could be attributed to differences in reactivity towards fibrinogen and the size of the polymer backbone. Taken together, these findings provide insight and guidance for applying protein reactive PEGs for the interruption of acute thrombotic deposition.
Platelet adhesion; Thrombosis; Protein adsorption; Fibrinogen; Poly(ethylene glycol); Protein modification
Polyethylene glycols (PEGs) are widely used to perturb the conformations of nucleic acids, including G-quadruplexes. The mechanism by which PEG alters G-quadruplex conformation is poorly understood. We describe here studies designed to determine how PEG and other co-solutes affect the conformation of the human telomeric quadruplex. Osmotic stress studies using acetonitrile and ethylene glycol show that conversion of the ‘hybrid’ conformation to an all-parallel ‘propeller’ conformation is accompanied by the release of about 17 water molecules per quadruplex and is energetically unfavorable in pure aqueous solutions. Sedimentation velocity experiments show that the propeller form is hydrodynamically larger than hybrid forms, ruling out a crowding mechanism for the conversion by PEG. PEGs do not alter water activity sufficiently to perturb quadruplex hydration by osmotic stress. PEG titration experiments are most consistent with a conformational selection mechanism in which PEG binds more strongly to the propeller conformation, and binding is coupled to the conformational transition between forms. Molecular dynamics simulations show that PEG binding to the propeller form is sterically feasible and energetically favorable. We conclude that PEG does not act by crowding and is a poor mimic of the intranuclear environment, keeping open the question of the physiologically relevant quadruplex conformation.
One-component homopolymers of cationic monomers (polycations) and diblock copolymers comprising poly(ethylene glycol) (PEG) and a polycation block have been the most widely used types of polymers for formulation of polymer-based gene delivery systems. In this study, we incorporate a hydrophobic middle block into the conventional PEG-polycation architecture, and investigate the effects of this hydrophobic modification on the physicochemical and cell-level biological properties of the polymer-DNA complexes that are relevant to gene delivery applications. The ABC-type triblock copolymer used in this study consists of (A) PEG, (B) hydrophobic poly(n-butyl acrylate) (PnBA) and (C) cationic poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) component polymers. The properties of the triblock copolymer/DNA complexes are compared with those of two other, more conventional DNA carriers derived, respectively, using a PDMAEMA homopolymer and a PEG-PDMAEMA diblock copolymer having comparable molecular weights for individual blocks. The PEG-PnBA-PDMAEMA polymer forms, in aqueous solution, positively-charged spherical micelles. The electrostatic complexation of these micelles with plasmid DNA molecules results in the formation of stable small-size DNA particles coated with a micelle monolayer, as confirmed by agarose gel electrophoresis, dynamic light scattering (DLS) and cryogenic transmission electron microscopy (cryo-TEM). Proton nuclear magnetic resonance (1H NMR) spectroscopy measurements indicate that the whole micelle-DNA assembly (named for convenience as “micelleplex”) is shielded predominantly by the PEG chains. DLS and optical microscopy imaging measurements indicate that in comparison with PDMAEMA/DNA polyplexes, the micelleplexes have a significantly lower tendency to aggregate under physiological salt concentrations, and show reduced interactions with negatively-charged components in serum such as albumin and erythrocytes. While the micelleplexes are comparable with the PEG-PDMAEMA-based DNA polyplexes in terms of their stability against aggregation under high salt concentrations and in the presence of the albumin protein, they have a slightly higher tendency to interact with erythrocytes than the diblock copolymer polyplexes. Agarose gel electrophoresis measurements indicate that relative to the PEG-PDMAEMA polyplexes, the micelleplexes provide better protection of the encapsulated DNA from enzymatic degradation, and also exhibit greater stability against disintegration induced by polyanionic additives; in these respects, the PDMAEMA homopolymer-based polyplexes show the best performance. In vitro studies in HeLa cells indicate that the PDMAEMA polyplexes show the highest gene transfection efficiency among the three different gene delivery systems. Between the micelleplexes and PEG-PDMAEMA polyplexes, a higher gene transfection efficiency is observed with the latter system. All three formulations show comparable levels of cytotoxicity in HeLa cells.
Asymmetric cell division is common in biology and plays critical roles in differentiation and development. Unicellular organisms are often used as model systems for understanding the origins and consequences of asymmetry during cell division. Although basic as compared to mammalian cells, these are already quite complex. We report complete budding and asymmetric fission of very simple nonliving model cells to produce daughter vesicles that are chemically distinct in both interior and membrane compositions. Our model cells are based on giant lipid vesicles (GVs, 10–30 μm) encapsulating a polyethylene glycol (PEG)/dextran aqueous two-phase system (ATPS) as a crowded and compartmentalized cytoplasm mimic. Ternary lipid compositions were used to provide coexisting micrometer-scale liquid disordered (Ld) and liquid ordered (Lo) domains in the membranes. ATPS-containing vesicles formed buds when sucrose was added externally to provide increased osmotic pressure, such that they became not only morphologically asymmetric but also asymmetric in both their interior and their membrane compositions. Further increases in osmolality drove formation of two chemically distinct daughter vesicles, which were in some cases connected by a lipid nanotube (complete budding), and in others were not (fission). In all cases, separation occurred at the aqueous–aqueous phase boundary, such that one daughter vesicle contained the PEG-rich aqueous phase and the other contained the dextran-rich aqueous phase. PEGylated lipids localized in the Lo domain resulted in this membrane domain preferentially coating the PEG-rich bud prior to division, and subsequently the PEG-rich daughter vesicle. Varying the mole ratio of lipids resulted in excess surface area of Lo or Ld membrane domains such that, upon division, this excess portion was inherited by one of the daughter vesicles. In some cases, a second “generation” of aqueous phase separation and budding could be induced in these daughter vesicles. Asymmetric fission of a simple self-assembled model cell, with production of daughter vesicles that harbored different protein concentrations and lipid compositions, is an example of the seemingly complex behavior possible for simple molecular assemblies. These compartmentalized and asymmetrically dividing ATPS-containing GVs could serve as a test bed for investigating possible roles for spatial and organizational cues in asymmetric cell division and inheritance.
In this study, noncovalent functionalization of single-walled carbon nanotubes (SWCNTs) with phospholipid-polyethylene glycols (Pl-PEGs) was performed to improve the solubility of SWCNTs in aqueous solution. Two kinds of PEG derivatives, ie, Pl-PEG 2000 and Pl-PEG 5000, were used for the PEGylation process. An experimental design technique (D-optimal design and second-order polynomial equations) was applied to investigate the effect of variables on PEGylation and the solubility of SWCNTs. The type of PEG derivative was selected as a qualitative parameter, and the PEG/SWCNT weight ratio and sonication time were applied as quantitative variables for the experimental design. Optimization was performed for two responses, aqueous solubility and loading efficiency. The grafting of PEG to the carbon nanostructure was determined by thermogravimetric analysis, Raman spectroscopy, and scanning electron microscopy. Aqueous solubility and loading efficiency were determined by ultraviolet-visible spectrophotometry and measurement of free amine groups, respectively. Results showed that Pl-PEGs were grafted onto SWCNTs. Aqueous solubility of 0.84 mg/mL and loading efficiency of nearly 98% were achieved for the prepared Pl-PEG 5000-SWCNT conjugates. Evaluation of functionalized SWCNTs showed that our noncovalent functionalization protocol could considerably increase aqueous solubility, which is an essential criterion in the design of a carbon nanotube-based drug delivery system and its biodistribution.
phospholipid-PEG; D-optimal design; loading efficiency; Raman spectroscopy; scanning electron microscopy; theromogravimetric analysis; carbon nanotubes
The purpose of this study was to explore the cryoprotection mechanisms of high molecular weight polyethylene glycols (PEGs) (eg, PEG 4000 and PEG 8000) on lactate dehydrogenase (LDH). Ultraviolet activity assays, circular dichroism (CD) spectroscopy, gel filtration, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE),14C-PEG 4000 labeling and binding, and cryostage microscopic study were conducted. Different molecular weights and concentrations of PEGs in LDH formulations were treated by freeze-thawing. Higher molecular weights and concentrations of PEGs in LDH-PEG formulations obtained better activity and secondary structure recoveries of LDH after freeze-thawing. Insoluble aggregation of LDH was not observed in gel filtration studies. SDS-PAGE results suggested surface characteristic modifications of LDH by the larger molecular weight PEGs. The14C-PEG 4000 labeling and binding study showed extensive nonspecific interactions between the PEG 4000 and LDH molecules in a concentration-dependent manner. The bound LDH-PEG 4000/free PEG 4000 ratio increased when LDH or PEG 4000 concentrations increased. Cryostage microscopic study showed that PEG 8000 delayed the ice crystallization and eutectic transition of LDH formulation. It appeared that multiple mechanisms were at work during PEGs' cryoprotection of LDH. It was unclear whether the delayed eutectic characteristics of PEGs contributed to LDH cryoprotection. The favorable interaction, rather than preferential exclusion, between LDH and PEGs (eg, 4000) cryoprotected LDH.
lactate dehydrogenase (LDH); freeze-thaw; circular dichroism (CD); SDS-PAGE; 14C-PEG 4000 binding
Targeted drug delivery using nanocarriers is achieved by functionalizing the carrier surface with a tissue-recognition ligand. Current surface modification methods require tedious and inefficient synthesis and purification steps, and are not easily amenable to incorporating multiple functionalities on a single surface. In this report, we describe a versatile, single-step surface functionalizing technique for polymeric nanoparticles. The technique utilizes the fact that when a diblock copolymer like polylactide-polyethylene glycol (PLA-PEG) is introduced in the oil/water emulsion used in polymeric nanoparticle formulation, the PLA block partitions into the polymer containing organic phase and PEG block partitions into the aqueous phase. Removal of the organic solvent results in the formation of nanoparticles with PEG on the surface. When a PLA-PEG-ligand conjugate is used instead of PLA-PEG copolymer, this technique permits a ‘one-pot’ fabrication of ligand-functionalized nanoparticles. In the current study, the IAASF approach facilitated the simultaneous incorporation of biotin and folic acid, known tumor-targeting ligands, on drug-loaded nanoparticles in a single step. Incorporation of the ligands on nanoparticles was confirmed by using NMR, surface plasmon resonance, transmission electron microscopy and tumor cell uptake studies. Simultaneous functionalization with both ligands significantly enhanced nanoparticle accumulation in tumors in vivo, and resulted in greatly improved efficacy of paclitaxel-loaded nanoparticles in a mouse xenograft tumor model. This new surface functionalization approach will enable the development of targeting strategies based on the use of multiple ligands on a single surface to target a tissue of interest.
Targeted delivery; surface functionalization; chemotherapy; polymeric systems; multivalent
The aim of this study was to investigate whether a cationic polyelectrolyte; poly(ethylene glycol) PEG-b-poly(l-histidine) diblock copolymer [PEG-polyHis] can stabilize insulin, at the aqueous/methylene chloride interface formed during the microencapsulation process. Insulin aggregation at this interface was monitored spectrophotometrically at 276 nm. The effects of protein concentration, pH of the aqueous medium, and the presence of poly(lactic-co-glycolic acid) [PLGA] in methylene chloride (MC) on insulin aggregation were observed. For the 2.0 mg/ml insulin solutions in phosphate buffer (PB), the effect of addition of Pluronic F-127 as a positive control and addition of PEG-polyHis as a novel excipient in PB was also evaluated at various insulin/polymeric excipient weight ratios. The conformation of insulin protected by PEG-polyHis and recovered after interfacial exposure was evaluated via circular dichroism (CD) spectroscopy.
Greater loss in soluble insulin was observed with increasing insulin concentrations. pH 6.0 was selected for optimal ionic interactions between insulin and PEG-polyHis based on zeta potential and particle size studies. pH 4.5 and 7.4 (no ionic complexation between insulin and PEG-polyHis) were selected as controls to compare the stabilization effect of PEG-polyHis with that at pH 6.0. Incubation of PEG-polyHis with insulin at pH 6.0 drastically reduced protein aggregation, even in the presence of PLGA. PEG-polyHis and F-127 reduced insulin aggregation in non-complexing pH conditions pointing to the role played by PEG in modulation of insulin adsorption at the interface. Far-UV (205-250 nm) circular dichroism (CD) study revealed negligible qualitative effects on soluble insulin’s secondary structure after interfacial exposure. RP-HPLC and size-exclusion HPLC showed no deamidation of insulin or formation of soluble high molecular weight transformation products respectively. MALDI-TOF mass spectrometry confirmed the results from chromatographic procedures. Radioimmunoassay carried out on select samples showed that recovered soluble insulin had retained its immunoreactivity.
An experimental method to simulate interfacial denaturation of proteins was designed for assessment of protein stability at the interface and screening for novel protein stabilizers. Understanding and manipulation of such polyelectrolyte-insulin complexation will likely play a role in insulin controlled delivery via microspheres formulation(s).
Insulin aggregation; Polymeric excipients; Ionic complexation; PEG-b-poly(l-histidine); Aqueous/organic interface
Iron oxide nanoparticles (NPs) with a diameter 21.6 nm were coated with poly(maleic acid-alt-1-octadecene) (PMAcOD) modified with grafted 5,000 Da poly(ethyelene glycol) (PEG) or short ethylene glycol (EG) tails. The coating procedure utilizes hydrophobic interactions of octadecene and oleic acid tails, while the hydrolysis of maleic anhydride moieties as well as the presence of hydrophilic PEG (EG) tails allows the NP hydrophilicity. The success of the NP coating was found to be independent of the degree of grafting which was varied between 20 and 80% of the –MacOD-units, but depended on the length of the grafted tail. The NP coating and hydrophilization did not occur when the modified copolymer contained 750 Da PEG tails independently of the grafting degree. To explain this phenomenon the micellization of the modified PMAcOD copolymers in water was analyzed by small angle x-ray scattering (SAXS). The PMAcOD molecules with the grafted 750 Da PEG tails form compact non-interacting disk-like micelles, whose stability apparently allows for no interactions with the NP hydrophobic shells. The PMAcOD containing the 5,000 Da PEG and EG tails form much larger aggregates capable of an efficient coating of the NPs. The coated NPs were characterized using transmission electron microscopy, dynamic light scattering, ζ-potential measurements, and thermal gravimetry analysis. The latter method demonstrated that the presence of long PEG tails in modified PMAcOD allows the attachment of fewer macromolecules (by a factor of ~20) compared to the case of non-modified or EG modified PMAcOD, emphasizing the importance of PEG tails in NP hydrophilization. The NPs coated with PMAcOD modified with 60% (towards all –MAcOD- units) of the 5,000 PEG tails bear a significant negative charge and display good stability in buffers. Such NPs can be useful as magnetic cores for virus-like particle formation.
monodisperse nanoparticles; amphiphilic; alternating copolymer; hydrophilic; modification; small angle X-ray scattering
Application of organic nanotubes (ONTs) into drug nanocarriers ultimately requires validation in live animals. For improving the dispersibility in biological media and in vivo distribution, the outer surface of an ONT was functionalized with polyethylene glycol (PEG) via the coassembly of an ONT-forming lipid with 5–20 mol% of a PEG-tethered lipid analogue (PEG-lipid). Firstly, the effect of PEGylation on the psysicochemical properties of ONTs, such as morphology and dispersibility, was investigated. PEGylation of ONTs slightly reduced the average length and effectively prevented the aggregation in phosphate-buffered saline (PBS). The PEGylated ONTs even showed high thermal stability in aqueous dispersion at least up to 95°C. Secondly, differential scanning calorimetry and powder X-ray diffraction indicated that ~10 mol% of PEG-lipid was completely incorporated into the ONTs, while 20 mol% of PEG-lipid encountered a partial phase separation during coassembly. In the heating differential scanning calorimetry runs, the resultant PEGylated ONTs with 5 mol% PEG-lipid showed no sign of phase separation up to 180°C under lyophilized condition, while those with 10 mol% and 20 mol% PEG-lipid showed some phase separation of the PEG-lipid above 120°C. Finally, PEGylation significantly affected the tissue distribution and prolonged the persistence time in the blood in mice. Non-PEGylated ONTs was quickly cleared from the circulation after intravenous infusion and preferentially accumulated in the lung, while PEGylated ONTs was mainly trapped in the liver and could circulate in the blood up to 24 hours. This study provided valuable information of physicochemical properties and the in vivo distribution behavior of PEGylated ONTs for their potential application into drug nanocarriers.
nanostructure; dispersibility; distribution
Hydration- and temperature-induced microphase separations were investigated by simultaneous small- and wide-angle X-ray scattering (SAXS and WAXS) and differential scanning calorimetry (DSC) in a family of copolymers in which hydrophilic poly(ethylene glycol) (PEG) blocks are inserted randomly into a hydrophobic polymer made of either desaminotyrosyl-tyrosine ethyl ester (DTE) or iodinated I2DTE segments. Iodination of the tyrosine rings in I2DTE increased the X-ray contrast between the hydrophobic and hydrophilic segments in addition to facilitating the study of the effect of iodination on microphase separation. The formation of phase-separated, hydrated PEG domains is of considerable significance as it profoundly affects the polymer properties. The copolymers of DTE (or I2DTE) and PEG are a useful model system and the findings presented here may be applicable to other PEG-containing random copolymers as well. In copolymers of PEG and DTE and I2DTE, the presence of PEG depressed the glass transition temperature (Tg) of the copolymer relative to the homopolymer, poly(DTE carbonate), and the DTE/ I2DTE segments hindered the crystallization of the PEG segments. In the dry state, at large PEG fractions (> 70 vol%), the PEG domains self-assembled into an ordered structure with 14–18 nm distance between the domains. These domains gave rise to a SAXS peak at all temperatures in the iodinated polymers, but only above the Tg in non-iodinated polymers, due to the unexpected contrast- match between the crystalline PEG domains and the glassy DTE segments. Irrespective of whether PEG was crystalline or not, immersion of these copolymers in water resulted in the formation of hydrated PEG domains that were 10–20 nm apart. Since both water and the polymer chains must be mobile for the phase separation to occur, the PEG domains disappeared when the water froze, and reappeared as the ice began to melt. This transformation was reversible, and showed hysteresis as did the melting of ice and freezing of the water incorporated into the polymer. PEG-water complexes and PEG-water eutectics were observed in WAXS and DSC scans, respectively.
PEG copolymers; hydrophilic blocks; hydrophobic segments, tyroine-derived polymers; hydration; phase separation; X-ray scattering; SAXS; WAXS; DSC
Highly compacted DNA nanoparticles (DNPs) composed of polyethylene glycol linked to a 30-mer of poly-L-lysine via a single cysteine residue (CK30PEG) have previously been shown to provide efficient gene delivery to the brain, eyes and lungs. In this study, we used a combination of flow cytometry, high-resolution live-cell confocal microscopy, and multiple particle tracking (MPT) to investigate the intracellular trafficking of highly compacted CK30PEG DNPs made using two different molecular weights of PEG, CK30PEG10k and CK30PEG5k. We found that PEG MW did not have a major effect on particle morphology nor nanoparticle intracellular transport. CK30PEG10k and CK30PEG5k DNPs both entered human bronchial epithelial (BEAS-2B) cells via a caveolae-mediated pathway, bypassing degradative endolysosomal trafficking. Both nanoparticle formulations were found to rapidly accumulate in the perinuclear region of cells within 2 h, 37 ± 19 % and 47 ± 8 % for CK30PEG10k and CK30PEG5k, respectively. CK30PEG10k and CK30PEG5k DNPs moved within live cells at average velocities of 0.09 ± 0.04 µm/s and 0.11 ± 0.04 µm/s, respectively, in good agreement with reported values for caveolae. These findings show that highly compacted DNPs employ highly regulated trafficking mechanisms similar to biological pathogens to target specific intracellular compartments.
gene therapy; nonviral; intracellular trafficking; particle tracking; Cystic Fibrosis
An EGFP construct interacting with the PIB1000-PEG6000-PIB1000 vesicles surface reported a ~2-fold fluorescence emission enhancement. Because of the constructs nature with the amphiphilic peptide inserted into the PIB core, EGFP is expected to experience a “pure” PEG environment. To unravel this phenomenon PEG/water solutions at different molecular weights and concentrations were used. Already at ~1 : 10 protein/PEG molar ratio the increase in fluorescence emission is observed reaching a plateau correlating with the PEG molecular weight. Parallel experiments in presence of glycerol aqueous solutions did show a slight fluorescence enhancement however starting at much higher concentrations. Molecular dynamics simulations of EGFP in neat water, glycerol, and PEG aqueous solutions were performed showing that PEG molecules tend to “wrap” the protein creating a microenvironment where the local PEG concentration is higher compared to its bulk concentration. Because the fluorescent emission can be perturbed by the refractive index surrounding the protein, the clustering of PEG molecules induces an enhanced fluorescence emission already at extremely low concentrations. These findings can be important when related to the use of EGFP as reported in molecular biology experiments.
PEGylation is a successful approach to improve potency of a therapeutic protein. The improved therapeutic potency is mainly due to the steric shielding effect of PEG. However, the underlying mechanism of this effect on the protein is not well understood, especially on the protein interaction with its high molecular weight substrate or receptor. Here, experimental study and molecular dynamics simulation were used to provide molecular insight into the interaction between the PEGylated protein and its receptor. Staphylokinase (Sak), a therapeutic protein for coronary thrombolysis, was used as a model protein. Four PEGylated Saks were prepared by site-specific conjugation of 5 kDa/20 kDa PEG to N-terminus and C-terminus of Sak, respectively. Experimental study suggests that the native conformation of Sak is essentially not altered by PEGylation. In contrast, the bioactivity, the hydrodynamic volume and the molecular symmetric shape of the PEGylated Sak are altered and dependent on the PEG chain length and the PEGylation site. Molecular modeling of the PEGylated Saks suggests that the PEG chain remains highly flexible and can form a distinctive hydrated layer, thereby resulting in the steric shielding effect of PEG. Docking analyses indicate that the binding affinity of Sak to its receptor is dependent on the PEG chain length and the PEGylation site. Computational simulation results explain experimental data well. Our present study clarifies molecular details of PEG chain on protein surface and may be essential to the rational design, fabrication and clinical application of PEGylated proteins.
Rf-PEG (fluoroalkyl double-ended poly(ethylene glycol)) hydrogel is potentially useful as a drug delivery depot due to its advanced properties of sol–gel two-phase coexistence and low surface erosion. In this study, 1H molecular diffusion nuclear magnetic resonance (NMR) and 19F spin diffusion NMR were used to probe the drug loading and diffusion properties of the Rf-PEG hydrogel for small anticancer drugs, 5-fluorouracil (FU) and its hydrophobic analog, 1,3-dimethyl-5-fluorouracil (DMFU). It was found that FU has a larger apparent diffusion coefficient than that of DMFU, and the diffusion of the latter was more hindered. The result of 19F spin diffusion NMR for the corresponding freeze-dried samples indicates that a larger portion of DMFU resided in the Rf core/IPDU intermediate-layer region (where IPDU refers to isophorone diurethane, as a linker to interconnect the Rf group and the PEG chain) than that of FU while the opposite is true in the PEG–water phase. To understand the experimental data, a diffusion model was proposed to include: (1) hindered diffusion of the drug molecules in the Rf core/IPDU-intermediate-layer region; (2) relatively free diffusion of the drug molecules in the PEG-water phase (or region); and (3) diffusive exchange of the probe molecules between the above two regions. This study also shows that molecular diffusion NMR combined with spin diffusion NMR is useful in studying the drug loading and diffusion properties in hydrogels for the purpose of drug delivery applications.
Fluoroalkyl double-ended poly(ethylene glycol); Hydrogel; 5-Fluorouracil; 1,3-Dimethyl-5-fluorouracil; Dug delivery; Molecular diffusion; Spin diffusion; NMR
Regulation of gene expression using small interfering RNA (siRNA) is a promising strategy for research and treatment of numerous diseases. In this study, we develop and characterize a delivery system for siRNA composed of polyethylenimine (PEI), polyethylene glycol (PEG), and mannose (Man). Cationic PEI complexes and compacts siRNA, PEG forms a hydrophilic layer outside of the polyplex for steric stabilization, and mannose serves as a cell binding ligand for macrophages. The PEI-PEG-mannose delivery system was constructed in two different ways. In the first approach, mannose and PEG chains are directly conjugated to the PEI backbone. In the second approach, mannose is conjugated to one end of the PEG chain and the other end of the PEG chain is conjugated to the PEI backbone. The PEI-PEG-mannose delivery systems were synthesized with 3.45 – 13.3 PEG chains and 4.7 – 3.0 mannose molecules per PEI. The PEI-PEG-Man-siRNA polyplexes displayed a coarse surface in Scanning Electron Microscopy (SEM) images. Polyplex sizes were found to range from 169nm to 357nm. Gel retardation assays showed that the PEI-PEG-mannose polymers are able to efficiently complex with siRNA at low N/P ratios. Confocal microscope images showed that the PEI-PEG-Man-siRNA polyplexes could enter cells and localized in the lysosomes at 2 hours post-incubation. Pegylation of the PEI reduced toxicity without any adverse reduction in knockdown efficiency relative to PEI alone. Mannosylation of the PEI-PEG could be carried out without any significant reduction in knockdown efficiency relative to PEI alone. Conjugating mannose to PEI via the PEG spacer generated superior toxicity and gene knockdown activity relative to conjugating mannose and PEG directly onto the PEI backbone.
HRPT; luciferase; polyethylenimine; mannose; polyethylene glycol; PEI; PEG; nanoplexes; nanoparticles; polyplexes; siRNA; RNA interference; non-viral; gene medicine
Various PEG-Vitamin E conjugates including D-alpha-tocopheryl polyethylene glycol succinate 1000 (TPGS) have been extensively studied as a nonionic surfactant in various drug delivery systems. However, limited information is available about the structure-activity relationship of PEG-Vitamin E conjugates as a micellar formulation for paclitaxel (PTX). In this study, four PEG-Vitamin E conjugates were developed that vary in the molecular weight of PEG (PEG2K vs PEG5K) and the molar ratio of PEG/Vitamin E (1/1 vs 1/2) in the conjugates. These conjugates were systematically characterized with respect to CMC, PTX loading efficiency, stability, and their efficiency in delivery of PTX to tumor cells in vitro and in vivo. Our data show that PEG5K-conjugates have lower CMC values and are more effective in PTX loading with respect to both loading capacity and stability. The conjugates with two Vitamin E molecules also worked better than the conjugates with one molecule of Vitamin E, particularly for PEG2K-system. Furthermore, all of the PEG-Vitamin E conjugates can inhibit P-gp function with their activity being comparable to that of TPGS. More importantly, PTX-loaded PEG5K-VE2 resulted in significantly improved tumor growth inhibitory effect in comparison to PTX formulated in PEG2K-VE or PEG2K-VE2, as well as Cremophor EL (Taxol) in a syngeneic mouse model of breast cancer (4T1.2). Our study suggests that PEG5K-Vitmin E2 may hold promise as an improved micellar formulation for in vivo delivery of anticancer agents such as PTX.
Nanomicelles; Paclitaxel; TPGS; Controlled and sustained drug delivery; Cancer; Nanotechnology