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1.  A molecular dynamics study of the structure and inter-particle interactions of polyethylene glycol-conjugated PAMAM dendrimers 
The journal of physical chemistry. B  2009;113(40):13202-13207.
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.
doi:10.1021/jp906497e
PMCID: PMC2772150  PMID: 19754139
2.  Single-Step Surface Functionalization of Polymeric Nanoparticles for Targeted Drug Delivery 
Biomaterials  2008;30(5):859-866.
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.
doi:10.1016/j.biomaterials.2008.09.056
PMCID: PMC2637351  PMID: 19019427
Targeted delivery; surface functionalization; chemotherapy; polymeric systems; multivalent
3.  Theory of Polymer-Nanopore Interactions Refined Using Molecular Dynamics Simulations 
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.
doi:10.1021/ja4026193
PMCID: PMC3704344  PMID: 23590258
Molecular dynamics; nanopore; alpha-hemolysin; PEG; DNA sequencing
4.  Enhanced EGFP Fluorescence Emission in Presence of PEG Aqueous Solutions and PIB1000-PEG6000-PIB1000 Copolymer Vesicles 
BioMed Research International  2013;2013:329087.
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.
doi:10.1155/2013/329087
PMCID: PMC3723060  PMID: 23936792
5.  Synthesis and characterization of thermoresponsive polyamidoamine-polyethylene glycol-poly (D, L-lactide) (PAMAM-PEG-PDLLA) core-shell nanoparticles 
Acta biomaterialia  2009;6(3):1131.
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.
doi:10.1016/j.actbio.2009.08.036
PMCID: PMC2815164  PMID: 19716444
dendrimers; drug delivery; nanomedicine; PEG-PLA copolymers; sol-gel phase transition; thermoresponsiveness
6.  A coarse-grained model for PEGylated lipids: the effect of PEGylation on size and shape of self-assembled structures 
The journal of physical chemistry. B  2011;115(24):7830-7837.
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.
doi:10.1021/jp2020148
PMCID: PMC3462017  PMID: 21618987
7.  Optimization of Serine Protease Purification from Mango (Mangifera indica cv. Chokanan) Peel in Polyethylene Glycol/Dextran Aqueous Two Phase System 
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.
doi:10.3390/ijms13033636
PMCID: PMC3317732  PMID: 22489172
purification; polyethylene glycol (PEG); serine protease; mango peel; yield
8.  Optimization of single-walled carbon nanotube solubility by noncovalent PEGylation using experimental design methods 
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.
doi:10.2147/IJN.S17626
PMCID: PMC3084320  PMID: 21556348
phospholipid-PEG; D-optimal design; loading efficiency; Raman spectroscopy; scanning electron microscopy; theromogravimetric analysis; carbon nanotubes
9.  Effective extraction of elastase from Bacillus sp. fermentation broth using aqueous two-phase system*  
This paper presents the evaluation of an aqueous two-phase system (ATPS) for extracting elastase produced by Bacillus sp. EL31410. The elastase and cell partition behavio r in polyethylene glycol (PEG)/salt systems was investigated. The suitable system for elastase extraction was PEG/KH2PO4-K2HPO4, in which elastase is mainly partitioned into the PEG-rich phase, while the cells remained in the other phase. The influence of defined system parameters (e.g. PEG molecular mass, pH, NaCl addition) on the partitioning behavior of elastase is described. The concentration of phase forming components, PEG and KH2PO4-K2HPO4, was optimized for elastase recovery by means of response surface methodology, and it was found that they greatly influenced extraction recovery. The optimal ATPS was 23.1% (w/w) PEG 2 000 and 11.7% (w/w) KH2PO4-K2HPO4. The predicted recovery was about 89.5%, so this process is suggested to be a rapid and convenient method for elastase extraction.
doi:10.1631/jzus.2005.B1087
PMCID: PMC1390656  PMID: 16252343
Elastase; Aqueous two-phase system (ATPS); Bioseparation; Purification; Optimization; Response surface methodology (RSM)
10.  Characterizing the modification of surface proteins with poly(ethylene glycol) to interrupt platelet adhesion 
Biomaterials  2006;27(16):3125-3135.
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.
doi:10.1016/j.biomaterials.2006.01.012
PMCID: PMC2857701  PMID: 16457880
Platelet adhesion; Thrombosis; Protein adsorption; Fibrinogen; Poly(ethylene glycol); Protein modification
11.  Hybridization-based affinity partitioning of nucleic acids using PEG-coupled oligonucleotides. 
Nucleic Acids Research  1994;22(10):1880-1884.
Polyethylene glycol (PEG)-coupled oligonucleotides are partitioned in an aqueous two-phase system PEG/dextran. The affinity of the oligonucleotide for the PEG-rich phase increases proportionally to the length of the coupled PEG polymer. After hybridization, the PEG-coupled oligonucleotide is able to force a complementary nucleic acid strand into the PEG-rich phase. This property can be used for the sequence-specific isolation of nucleic acids through hybridization-based affinity partitioning. The dependence of the partition coefficient in this system on various parameters is described. The application of this principle to multistage chromatographic separations is demonstrated.
PMCID: PMC308088  PMID: 8208613
12.  Systematic Investigation of Polyamidoamine Dendrimers Surface-Modified with Poly(ethylene glycol) for Drug Delivery Applications: Synthesis, Characterization, and Evaluation of Cytotoxicity 
Bioconjugate chemistry  2008;19(8):1660-1672.
Surface-modification of amine-terminated polyamidoamine (PAMAM) dendrimers by poly(ethylene glycol) (PEG) groups generally enhances water-solubility and biocompatibility for drug delivery applications. In order to provide guidelines for designing appropriate dendritic scaffolds, a series of G3 PAMAM-PEG dendrimer conjugates was synthesized by varying the number of PEG attachments and chain length (shorter PEG550 and PEG750 and longer PEG2000). Each conjugate was purified by size exclusion chromatography (SEC) and the molecular weight (MW) was determined by 1H NMR integration and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). NOESY experiments performed in D2O on selected structures suggested no penetration of PEG chains to the central PAMAM domain, regardless of chain length and degree of substitution. CHO cell cultures exposed to PAMAM-PEG derivatives (≤ 1 µM) showed a relatively high cell viability. Generally, increasing the degree of PEG substitution reduced cytotoxicity. Moreover, compared to G3 PAMAM dendrimers that were N-acetylated to varying degrees, a lower degree of surface substitution with PEG was needed for a similar cell viability. Interestingly, when longer PEG2000 was fully incorporated on the surface, cell viability was reduced at higher concentrations (32 µM), suggesting increased toxicity potentially by forming intermolecular aggregates. A similar observation was made for anionic carboxylate G5.5 PAMAM dendrimer at the same dendrimer concentration. Our findings suggest that a lower degree of peripheral substitution with shorter PEG chains may suffice for these PAMAM-PEG conjugates to serve as efficient universal scaffolds for drug delivery, particularly valuable in relation to targeting or other ligand-receptor interactions.
doi:10.1021/bc700483s
PMCID: PMC2583177  PMID: 18610944
13.  Hydrophobic pharmaceuticals mediated self-assembly of β-cyclodextrin containing hydrophilic copolymers: Novel chemical responsive nano-vehicles for drug delivery 
Double hydrophilic copolymers with one polyethylene glycol (PEG) block and one β-cyclodextrin β-CD) flanking block (PEG-b-PCDs) were synthesized through the post-modification of macromolecules. The self-assembly of PEG-b-PCDs in aqueous solutions was initially studied by a fluorescence technique. This measurement together with AFM and TEM characterization demonstrated the formation of nanoparticles in the presence of lipophilic small molecules. The host-guest interaction between the β-CD unit of a host copolymer and the hydrophobic group of a guest molecule was found to be the driving force for the observed self-assembly. This spontaneous assembly upon loading of guest molecules was also observed for hydrophobic drugs with various chemical structures. Relatively high drug loading was achieved by this approach. Desirable encapsulation was also achieved for the hydrophobic drugs that cannot efficiently interact with free β-CD. In vitro release studies suggested that the payload in nano-assemblies could be released in a sustained manner. In addition, both the fluorescence measurement and the in vitro drug release studies suggested that these nano-assemblies mediated by the inclusion complexation exhibited a chemical sensitivity. The release of payload can be accelerated upon the triggering by hydrophobic guest molecules or free β-CD molecules. These results support the potential applications of the synthesized copolymers for the delivery of hydrophobic drugs.
doi:10.1016/j.jconrel.2010.04.019
PMCID: PMC2902716  PMID: 20417674
Host-guest interactions; β-Cyclodextrin; Nano-assemblies; Chemical sensitivity; Drug delivery
14.  Competitive Adsorption of Thiolated Polyethylene Glycol and Mercaptopropionic acid on Gold Nanoparticles Measured by Physical Characterization Methods 
Competitive adsorption kinetics between thiolated polyethylene glycol (SH-PEG) and mercaptopropionic acid (MPA) on gold nanoparticles (Au-NPs) were studied using a prototype physical characterization approach that combines dynamic light scattering (DLS) and electrospray differential mobility analysis (ES-DMA). The change in hydrodynamic particle size (intensity-average) due to the formation of SH-PEG coatings on Au-NPs were measured by DLS in both two component (Au-NP + MPA or Au-NP + SH-PEG) and three component (Au-NP +MPA + SH-PEG) systems. ES-DMA was employed to quantify the surface coverage of SH-PEG and establish a correlation between surface coverage and the change in particle size measured by DLS. A change in equilibrium binding constant for SH-PEG on Au-NPs at various concentrations of SH-PEG and MPA showed that the presence of MPA reduced the binding affinity of SH-PEG to the Au-NP surface. Kinetic studies showed SH-PEG was desorbed from the Au-NP surface following a second-order desorption model after subsequently introducing MPA. The desorption rate constant of SH-PEG from the Au-NP surface by MPA displacement was strongly affected by the concentration of MPA and the excess SH-PEG in solution.
doi:10.1021/la100484a
PMCID: PMC2935271  PMID: 20465235
dynamic light scattering; electrospray; differential mobility analysis; gold; nanoparticle; polyethylene glycol; mercaptopropionic acid; binding constant; kinetics; adsorption; desorption
15.  Synthesis and Characterization of Star Poly(ε-caprolactone)-b-Poly(ethylene glycol) and Poly(l-lactide)-b-Poly(ethylene glycol) Copolymers: Evaluation as Drug Delivery Carriers 
Bioconjugate chemistry  2008;19(7):1423-1429.
Two types of 32 arm star polymers incorporating amphiphilic block copolymer arms have been synthesized and characterized. The first type, stPCL-PEG32, is composed of a polyamidoamine (PAMAM) dendrimer as the core with radiating arms having poly(ε-caprolactone) (PCL) as an inner lipophilic block in the arm and poly(ethylene glycol) (PEG) as an outer hydrophilic block. The second type, stPLA-PEG32, is similar but with poly(l-lactide) (PLA) as the inner lipophilic block. Characterization with SEC, 1H NMR, FTIR, and DSC confirmed the structure of the polymers. Micelle formation by both star copolymers was studied by fluorescence spectroscopy. The stPCL-PEG32 polymer exhibited unimolecular micelle behavior. It was capable of solubilizing hydrophobic molecules, such as pyrene, in aqueous solution, while not displaying a critical micelle concentration. In contrast, the association behavior of stPLA-PEG32 in aqueous solution was characterized by an apparent critical micelle concentration of ca. 0.01 mg/mL. The hydrophobic anticancer drug etoposide can be encapsulated in the micelles formed from both polymers. Overall, the stPCL-PEG32 polymer exhibited a higher etoposide loading capacity (up to 7.8 w/w % versus 4.3 w/w % for stPLA-PEG32) as well as facile release kinetics and is more suitable as a potential drug delivery carrier.
doi:10.1021/bc7004285
PMCID: PMC2711207  PMID: 18564869
16.  Dendrimer-Based MRI Contrast Agents: The Effects of PEGylation on Relaxivity and Pharmacokinetics 
Polyethylene glycol (PEG)-surface modification can make nano-materials highly hydrophilic, reducing sequestration in the reticuloendothelial system. In this study, polyamidoamine (PAMAM) dendrimers bearing gadolinium chelates were PEGylated with different PEG-chain lengths and the effects on paramagnetic and pharmacokinetic properties were evaluated. Specifically, gadolinium chelate-bearing PAMAM dendrimers (G4 and G5) were conjugated with two different PEG chains (2k and 5k). Long PEG chains (5k) on the smaller (G4) dendrimer resulted in reduced relaxivity compared to unPEGylated dendrimer whereas short PEG (2k) and larger (G5) dendrimer produced comparable relaxivities to unPEGylated G4 dendrimer. The relaxivity of all PEGylated or lysine conjugated dendrimers increased at higher temperature, while that of intact G4 Gd-PAMAM-dendrimer decreased. All PEGylated dendrimers had minimal liver and kidney uptake and remained in circulation for at least 1 hour. Thus, surface-PEGylated Gd-PAMAM-Dendrimers showed decreased plasma clearance and prolonged retention in the blood pool. Shorter PEG, higher generation conjugates led higher relaxivity.
doi:10.1016/j.nano.2011.03.007
PMCID: PMC3159711  PMID: 21515406
MRI; dendrimer; polyethylene glycol (PEG); relaxivity; pharmacokinetics
17.  Cryoprotection mechanisms of polyethylene glycols on lactate dehydrogenase during freeze-thawing 
The AAPS Journal  2004;6(3):45-54.
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.
doi:10.1208/aapsj060322
PMCID: PMC2751247  PMID: 15760107
lactate dehydrogenase (LDH); freeze-thaw; circular dichroism (CD); SDS-PAGE; 14C-PEG 4000 binding
18.  Molecular crowding stabilizes folded RNA structure by the excluded volume effect 
Crowder molecules in solution alter the equilibrium between folded and unfolded states of biological macromolecules. It is therefore critical to account for the influence of these other molecules when describing the folding of RNA inside the cell. Small angle x-ray scattering experiments are reported on a 64 kDa bacterial group I ribozyme in the presence of polyethylene-glycol 1000 (PEG-1000), a molecular crowder with average molecular weight 1000 Da. In agreement with expected excluded volume effects, PEG favors more compact RNA structures. Firstly, the transition from the unfolded to the folded (more compact) state occurs at lower MgCl2 concentrations in PEG. Secondly, the radius of gyration of the unfolded RNA decreases from 76 Å to 64 Å as the PEG concentration increases from 0 to 20 % wt./vol. Changes to water and ion activities were measured experimentally, and theoretical models were used to evaluate the excluded volume. We conclude that the dominant influence of the PEG crowder on the folding process is the excluded volume effect.
doi:10.1021/ja101500g
PMCID: PMC2906142  PMID: 20521820
19.  Partitioning and purification of extracellular β-1,3-1,4-glucanase in aqueous two-phase systems*  
The partition behaviors of β-1,3-1,4-glucanase, α-amylase and neutral proteases from clarified and whole fermentation broths of Bacillus subtilis ZJF-1A5 were investigated. An aqueous two-phase system (polyethylene glycol (PEG)/MgSO4) was examined with regard to the effects of PEG molecular weight (MW) and concentration, MgSO4 concentration, pH and NaCl concentration on enzyme partition and extraction. The MW and concentration of PEG were found to have significant effects on enzyme partition and extraction with low MW PEG showing the greatest benefit in the partition and extraction of β-glucanase with the PEG/MgSO4 system. MgSO4 concentration influenced the partition and extraction of β-glucanase significantly. pH had little effect on β-glucanase or proteases partition but affected α-amylase partition when pH was over 7.0. The addition of NaCl had little effect on the partition behavior of β-glucanase but had very significant effects on the partitioning of α-amylase and on the neutral proteases. The partition behaviors of β-glucanase, α-amylase and proteases in whole broth were also investigated and results were similar to those obtained with clarified fermentation broth. A two-step process for purifying β-glucanase was developed, which achieved β-glucanase recovery of 65.3% and specific activity of 14027 U/mg, 6.6 times improvement over the whole broth.
doi:10.1631/jzus.2005.B0825
PMCID: PMC1389866  PMID: 16052718
Aqueous two-phase system; Partition; β-1,3-1,4-glucanase
20.  Novel PEG-graft-PLA nanoparticles with the potential for encapsulation and controlled release of hydrophobic and hydrophilic medications in aqueous medium 
This study concerns the encapsulation and controlled release of both hydrophobic and hydrophilic medications with one polymer, which are delivered together as a combined therapy to treat diseased tissue. To test our hypothesis that the novel PEG-graft-PLA (PEG, polyethylene glycol; PLA, polylactic acid) can deliver both the hydrophobic and hydrophilic medications on account of its amphiphility, charge, and graft structure, PEG-graft-PLA (molecular weight of PEG = 1900) with very low critical micelle concentration was synthesized. One hydrophilic (insulin) and one hydrophobic (naproxen) model medication were loaded in separately during its self-assembly in aqueous solution. The resulting nanoparticles (NPs) were narrowly distributed and spherical, with average particle size around 200 nm, zeta potential >−10 mV, and encapsulation efficiency >50%. The NPs realized controlled release of insulin and naproxen for over 24 and 160 hours, respectively. Specifically, the bioactivity of the insulin released from the NPs was maintained. Owing to encapsulation, both for hydrophobic and hydrophilic medicines, and NPs obtained with similar size and zeta potential, as well as maintenance of bioactivity of loaded protein, we expect the applications of PEG-graft-PLA NPs in combination therapy.
doi:10.2147/IJN.S19765
PMCID: PMC3141871  PMID: 21796246
NPs; insulin; naproxen; controlled release; combination therapy
21.  Synthesis and Characterization of Photocurable Polyamidoamine Dendrimer Hydrogels as a Versatile Platform for Tissue Engineering and Drug Delivery 
Biomacromolecules  2010;11(3):666-673.
In this work, we describe a novel polyamidoamine (PAMAM) dendrimer hydrogel (DH) platform with potential for tissue engineering and drug delivery. With PAMAM dendrimer G3.0 being the underlying carrier, polyethylene glycol (PEG) chains of various lengths (MW=1500, 6000, or 12000 gmol−1) were coupled to the dendrimer to different extents, and the resulting PEGylated PAMAM dendrimers were further coupled with acrylate groups to yield photoreactive dendrimer macromonomers for gel formation. It was found that gelation based on photoreactive PAMAM G3.0 macromonomers was restricted by the degree of PEGylation, PEG chain length, and the distribution of acrylate groups on the dendrimer surface. Further, the architecture of the photoreactive macromonomers affects the structural stability and swelling of the resultant networks. A completely crosslinked network (DH-G3.0–12000H) with a high water swelling ratio was created by UV-curing of PAMAM dendrimer G3.0 coupled with 28 PEG 12000 chains in the presence of the eosin Y-based photoinitiating system. The disintegration of DH-G3.0–12000H was pH-insensitive. DH-G3.0–12000H was found to have similar cytocompatibility to uncrosslinked G3.0–12000H but have a significantly lower cellular uptake by macrophages. With PAMAM dendrimer G3.5 being the underlying carrier, the dendrimer modified with 43 PEG 1500 chains was able to form a completely crosslinked network (DH-G3.50–1500H) by UV-curing in the presence of the eosin Y-based photoinitiating system. DH-G3.50–1500H exhibited pH-dependent disintegration. Its disintegration ratio increased with pH. PAMAM dendrimer hydrogels uniquely express the structural characteristics of both PEG hydrogel and PAMAM dendrimer and have potential for various applications in tissue engineering and drug delivery.
doi:10.1021/bm901240g
PMCID: PMC2849659  PMID: 20108892
amphiphilic hydrogel network; dendrimer; dendritic PEG acrylate; PEGylation; tissue engineering
22.  Tailoring the degradation kinetics of mesoporous silicon structures through PEGylation 
SYNOPSIS
Injectable and implantable porosified silicon (pSi) carriers and devices for prolonged and controlled delivery of biotherapeutics offer great promise for treatment of various chronic ailments and acute conditions. Polyethylene glycols (PEGs) are important surface modifiers currently used in clinic mostly to avoid uptake of particlulates by reticulo-endothelial system (RES). In this work we show for the first time that covalent attachment of PEGs to the pSi surface can be used as a means to finely tune degradation kinetics of silicon structures. Seven PEGs with varying molecular weights (245, 333, 509, 686, 1214, 3400 and 5000Da) were employed and the degradation of PEGylated pSi hemispherical microparticles in simulated physiological conditions was monitored by means of ICP-AES, SEM and fluorimetry. Biocompatibility of the systems with human macrophages in vitro was also evaluated. The results clearly indicate that controlled PEGylation of silicon microparticles can offer a sensitive tool to finely tune their degradation kinetics and that the systems do not induce release of proinflammatory cytokines IL-6 and IL-8 in THP1 human macrophages.
doi:10.1002/jbm.a.32807
PMCID: PMC2920054  PMID: 20694990
mesoporous silicon; polyethylene glycol; biodegradation; biocompatibility
23.  Chemically defined polyethylene glycol siRNA conjugates with enhanced gene silencing effect 
Bioorganic & Medicinal Chemistry  2014;22(7):2320-2326.
Graphical abstract
The therapeutic application of siRNA suffers from poor bioavailability caused by rapid degradation and elimination. The covalent attachment of PEG is a universal concept to increase molecular size and enhance the pharmacokinetic properties of biomacromolecules. We devised a facile approach for attachment of PEG molecules with a defined molecular weight, and successful purification of the resulting conjugates. We directly conjugated structurally defined PEG chains with twelve ethylene glycol units to the 3′-terminal hydroxyl group of both sense and antisense strands via an aminoalkyl linker. The conjugates were easily purified by HPLC and successful PEGylation and molecule integrity were confirmed by ESI-MS. The evaluation of in vitro gene knockdown of two different targets in MCF-7 breast cancer cells showed stable pharmacologic activity when combined with a standard transfection reagent. Sense strand PEGylation even increased the silencing potency of a CRCX4-siRNA which had modest activity in its wild-type form. The results indicate that PEG chains at the 3′-terminus of both strands of siRNA are well tolerated by the RNAi effector. The attachment of short, chemically defined PEG chains is a feasible approach to improve the pharmacokinetic properties of siRNA, and can be combined with other targeted and untargeted delivery vehicles.
doi:10.1016/j.bmc.2014.02.004
PMCID: PMC3994277  PMID: 24613624
Synthetic oligonucleotides; PEGylation; RNA interference; Oligonucleotide conjugates; Gene silencing
24.  Rapid and simplified purification of recombinant adeno-associated virus 
Journal of Virological Methods  2012;183(2):139-146.
Preclinical gene therapy studies both in-vitro and in-vivo require high purity preparations of adeno-associated virus (AAV). Current methods for purification of AAV entail the use of centrifugation over either a CsCl or iodixanol gradient, or the use of chromatography. These methods can be cumbersome and expensive, necessitating ultrahigh speed gradient centrifugation or, for chromatography the use of other expensive equipment. In addition, these methods are time consuming, and the viral yield is not high. Currently no commercial purification kits are available for other than AAV serotype 2. A simplified method was used for the purification of AAV, with a viral yield that is able to be used effectively in adult and embryo mice. The method does not require ultrahigh speed gradient centrifugation nor chromatography. Instead, polyethylene glycol (PEG) / aqueous two-phase partitioning is used to remove soluble proteins from the PEG8000 precipitated virus-protein mixture. The procedure obtained rapidly up to 95% recovery of high quality purified AAV. The entire purification process, including HEK-293 cell transfection, can be completed readily within one week, with purity seemingly higher than that obtained after one round of CsCl gradient purification.
doi:10.1016/j.jviromet.2012.04.004
PMCID: PMC3374034  PMID: 22561982
AAV8; Aqueous two-phase partition system (ATPs)
25.  Quantifying the Coverage Density of Poly(ethylene glycol) Chains on the Surface of Gold Nanostructures 
ACS Nano  2011;6(1):512-522.
The coverage density of poly(ethylene glycol) (PEG) is a key parameter in determining the efficiency of PEGylation, a process pivotal to in vivo delivery and targeting of nanomaterials. Here we report four complementary methods for quantifying the coverage density of PEG chains on various types of Au nanostructures by using a model system based on HS-PEG-NH2 with different molecular weights. Specifically, the methods involve reactions with fluorescamine and ninhydrin, as well as labeling with fluorescein isothiocyanate (FITC) and Cu2+ ions. The first two methods use conventional amine assays to measure the number of unreacted HS-PEG-NH2 molecules left behind in the solution after incubation with the Au nanostructures. The other two methods involve coupling between the terminal –NH2 groups of adsorbed -S-PEG-NH2 chains and FITC or a ligand for Cu2+ ion, and thus pertain to the “active” –NH2 groups on the surface of a Au nanostructure. We found that the coverage density decreased as the length of PEG chains increased. A stronger binding affinity of the initial capping ligand to the Au surface tended to reduce the PEGylation efficiency by slowing down the ligand exchange process. For the Au nanostructures and capping ligands we have tested, the PEGylation efficiency decreased in the order of citrate-capped nanoparticles > PVP-capped nanocages ≈ CTAC-capped nanoparticles ≫ CTAB-capped nanorods, where PVP, CTAC, and CTAB stand for poly(vinyl pyrrolidone), cetyltrimethylammonium chloride, and cetyltrimethylammonium bromide, respectively.
doi:10.1021/nn2038516
PMCID: PMC3265621  PMID: 22148912
Au nanostructure; PEGylation; ligand exchnage; capping ligand

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