Effect Of Polyethylene Glycol Molecular Weight Research Articles

Partitioning behavior of short DNA fragments in polymer/salt aqueous two-phase systems.

The development of liquid biopsy as a minimally invasive technique for tumor profiling has created a need for efficient biomarker extraction systems from body fluids. The analysis of circulating cell-free DNA (cfDNA) is especially promising, but the low amounts and high fragmentation of cfDNA found in plasma pose challenges to its isolation. While the potential of aqueous two-phase systems (ATPS) for the extraction and purification of various biomolecules has already been successfully established, there is limited literature on the applicability of these findings to short cfDNA-like fragments. This study presents the partitioning behavior of a 160 bp DNA fragment in polyethylene glycol (PEG)/salt ATPS at pH 7.4. The effect of PEG molecular weight, tie-line length, neutral salt additives, and phase volume ratio is evaluated to maximize DNA recovery. Selected ATPS containing a synthetic plasma solution spiked with human serum albumin and immunoglobulin G are tested to determine the separation of DNA fragments from the main plasma protein fraction. By adding 1.5% (w/w) NaCl to a 17.7% (w/w) PEG 400/17.3% (w/w) phosphate ATPS, 88% DNA recovery was achieved in the salt-rich bottom phase while over 99% of the protein was removed.

Penicillium citrinum CFAM 521 Isolated From the Amazon Region: A Novel Source of a Fibrinolytic Enzyme.

Fibrinolytic agents are essential in treating thrombosis, playing a critical role in improving survival rates in cardiovascular diseases. Microbial fibrinolytic proteases have emerged as promising alternatives due to their affordability, specificity, lower toxicity, and reduced side effects. Consequently, the search for microorganisms capable of producing these enzymes has gained significant economic importance in the pharmaceutical industry. This study reports and characterizes a novel fibrinolytic enzyme produced by Penicillium citrinum CFAM 521, a strain isolated from the Amazon region. The enzyme was purified using a polyethylene glycol (PEG)-phosphate salt aqueous two-phase system (ATPS). The effects of PEG molecular weight, PEG concentration, and phosphate concentration on the protease partition coefficient (K) were evaluated through a 22 full factorial design. The enzyme exhibited both fibrinolytic and fibrinogenolytic activities. After partitioning in a two-phase system with 10% (w/w) PEG and 15% (w/w) sodium phosphate, the fibrinolytic proteases were predominantly retained in the salt-rich bottom phase (K = 0.33). The enzyme has a molecular weight of 34 kDa, with optimal pH and temperature at 9°C and 37°C, respectively. Inhibitory analysis confirmed that it is a serine protease, and its activity was enhanced by the addition of Mn2+. Notably, the enzyme exhibited no hemolytic activity. Therefore, P. citrinum CFAM 521 represents a novel source of fibrinolytic enzymes, highlighting its potential as an alternative for the development of thrombolytic agents.

Enrichment and separation of recombinant adeno-associated virus particles by aqueous two-phase extraction

The adeno-associated virus (AAV) has been widely employed as a popular gene therapy vector. However, current methods for its separation and purification exhibit numerous limitations. In this study, polyethylene glycol (PEG)/salt aqueous two-phase system (ATPS) was utilized as an efficient and environmentally-benign approach to separate AAV. The separation efficiency of AAV particles from other cellular components was investigated using various ATPS compositions. The effects of PEG molecular weight, types of phase-forming salts, and concentrations of different components were tested and compared. The optimal separation conditions were determined to be 17 % (w/w) PEG 600, 16 % (w/w) sodium citrate, and 15 % (w/w) crude cell lysate. The AAV particles were predominantly enriched in the interphase, while the impurities were observed in the top and bottom phases. High AAV recovery (>99 %) and efficient impurity removal (>95 %) were achieved. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transmission electron microscopy were used to characterize and verify the separated AAV particles. The results indicated that aqueous two-phase extraction possesses a robust capability for the enrichment and separation of AAV particles directly from crude cell lysates, and it shows promising potential for the separation of other viral particles.

Primary recovery of hyaluronic acid produced in Streptococcus equi subsp. zooepidemicus using PEG\u2013citrate aqueous two-phase systems

Given its biocompatibility, rheological, and physiological properties, hyaluronic acid (HA) has become a biomaterial of increasing interest with multiple applications in medicine and cosmetics. In recent decades, microbial fermentations have become an important source for the industrial production of HA. However, due to its final applications, microbial HA must undergo critical and long purification processes to ensure clinical and cosmetic grade purity. Aqueous two-phase systems (ATPS) have proven to be an efficient technique for the primary recovery of high-value biomolecules. Nevertheless, their implementation in HA downstream processing has been practically unexplored. In this work, polyethylene glycol (PEG)–citrate ATPS were used for the first time for the primary recovery of HA produced with an engineered strain of Streptococcus equi subsp. zooepidemicus. The effects of PEG molecular weight (MW), tie-line length (TLL), volume ratio (VR), and sample load on HA recovery and purity were studied with a clarified fermentation broth as feed material. HA was recovered in the salt-rich bottom phase, and its recovery increased when a PEG MW of 8000 g mol−1 was used. Lower VR values (0.38) favoured HA recovery, whereas purity was enhanced by a high VR (3.50). Meanwhile, sample load had a negative impact on both recovery and purity. The ATPS with the best performance was PEG 8000 g mol−1, TLL 43% (w/w), and VR 3.50, showing 79.4% HA recovery and 74.5% purity. This study demonstrated for the first time the potential of PEG–citrate ATPS as an effective primary recovery strategy for the downstream process of microbial HA.

Development of high flux ultrafiltration polyphenylsulfone membranes applying the systems with upper and lower critical solution temperatures: Effect of polyethylene glycol molecular weight and coagulation bath temperature

A novel phase inversion technique for preparation of high flux polyphenylsulfone (PPSU) ultrafiltration membranes employing the systems which feature upper critical solution temperature (UCST), gel point and lower critical solution temperature (LCST) is proposed. These systems include PPSU, polyethylene glycol (PEG) with molecular weight in the range of 6 ÷ 40 × 103 g mol−1 and N-methyl-2-pyrrolidone (NMP) as a solvent. According to the triangular phase diagrams obtained, the polymer solutions consisting of 20 wt% PPSU – 15 wt% PEG (Mw = 6 ÷ 40 × 103 g mol−1) – NMP are revealed to be two-phase systems at T > 100 °C and gels at T < 38–40 °C. The developed technique involves PPSU solution processing at the temperature region between LCST and UCST (gel point). Meanwhile, the coagulation bath temperature has to be between UCST (gel point) and LCST. The effect of PEG molecular weight, casting solution composition and coagulation bath temperature on membrane structure, performance and hydrophilic-hydrophobic balance of the membrane selective layer was studied. Applying the proposed technique flat-sheet membranes with pure water flux of 486 L m−2 h−1 bar−1 and human serum albumin rejection of 90% were prepared which is the best performance for PPSU ultrafiltration membranes reported up-to-date. It was found that introduction of PEG (Mw = 6 ÷ 40 × 103g mol−1) into the casting solution results in the efficient membrane hydrophilization, which is confirmed by FTIR spectroscopy of the membrane selective layer.

Synergistic inhibition of aggressive breast cancer cell migration and invasion by cytoplasmic delivery of anti-RhoC silencing RNA and presentation of EPPT1 peptide on “smart” particles

Overexpression of RhoC protein in breast cancer patients has been linked to increased cancer cell invasion, migration, and metastases. Suppressing RhoC expression in aggressive breast cancer cells using silencing RNA (siRNA) molecules is a viable strategy to inhibit the metastatic spread of breast cancer. In this report, we describe the synthesis of a series of asymmetric pH-sensitive, membrane-destabilizing polymers engineered to complex anti-RhoC siRNA molecules forming “smart” nanoparticles. Using β-CD as the particle core, polyethylene glycol (PEG) chains were conjugated to the primary face via non-cleavable bonds and amphiphilic polymers incorporating hydrophobic and cationic monomers were grafted to the secondary face via acid-labile linkages. We investigated the effect of PEG molecular weight (2 & 5 kDa) on transfection capacity and serum stability of the formed particles. We evaluated the efficacy of EPPT1 peptides presented on the free tips of the PEG brush to function as a targeting ligand against underglycosylated MUC1 (uMUC1) receptors overexpressed on the surface of metastatic breast cancer cells. Results show that “smart” nanoparticles successfully delivered anti-RhoC siRNA into the cytoplasm of aggressive SUM149 and MDA-MB-231 breast cancer cells, which resulted in a dose-dependent inhibition of cell migration and invasion. Further, EPPT1-targeted nanoparticles demonstrate a synergistic inhibition of cell migration and invasion imparted via RhoC knockdown and EPPT1-mediated signaling via the uMUC1 receptor.

Hydrogel Containing PEG-Coated Fluconazole Nanoparticles with Enhanced Solubility and Antifungal Activity

PurposeThe aim of this study was to prepare fluconazole (FLC) nanoparticles coated with polyethylene glycol (PEG) in the form of FLC-PEG-NPs and optimize the size and entrapment efficiency.MethodsNine formulae were prepared by solvent antisolvent precipitation technique according to full 32 factorial designs. The effects of PEG molecular weight (X1) and the drug polymer ratio (X2) on the particle size (Y1) and entrapment efficiency (Y2) were explored. The prepared FLC-PEG-NPs were investigated for particle size, count rate, PDI, zeta potential, and morphology. Carbopol hydrogel was prepared, loaded with optimized FLC-PEG-NPs, and characterized for pH, FLC content, viscosity, homogeneity and spreadability, in vitro release, skin permeation, and antifungal activity.ResultsThe formulated nanoparticles were uniform in size and spherical in shape with slightly rough surface and free from aggregations. The effect of PEG molecular was antagonistic on the particle size and was agonistic on EE %. The release of drug from hydrogel containing pure FLC was always lower than that from hydrogel containing FLC-PEG-NPs. The kinetic analysis of drug release obeys first-order release model and super case II transport mechanism. The cumulative amount of drug permeated applying hydrogel containing optimized FLC-PEG-NPs was significantly higher than the amount permeated using pure fluconazole containing hydrogel. The antifungal activity of hydrogel containing FLC in the form of optimized PEG-coated nanoparticles was better than hydrogel containing pure drug as indicated by relatively high inhibition zone using agar well-diffusion method.ConclusionSmall spherical FLC nanoparticles with enhanced in vitro drug release as well as improved antifungal activity could be achieved by using PEG-coated fluconazole nanoparticles.

In situ PEGylation of Bi nanoparticles prepared via pulsed Nd:YAG laser ablation in low molecular weight PEG: a potential X-ray CT imaging contrast agent

Recent advances and studies have shown that inorganic nanomaterials based on heavy elements represent a new contrast agent which is highly appropriate for X-ray computed tomography (CT) in comparison with commercial Iohexol contrast agents which have several limitations, including short imaging time, rapid renal clearance and toxicity. In this contribution, elemental bismuth (Bi) nanoparticles with polyethylene glycol (PEG) coating are synthesised by pulsed Nd:YAG laser ablation of Bi target in PEGs with 400 and 600 g/mol molecular weight. The effect of PEG molecular weight as surface coating on colloidal stability and size distribution of Bi nanoparticles were investigated. It is found that by in situ surface modification during the ablation in low molecular PEG final particle size as well as average size decreases and colloidal stability increases. Moreover, X-ray attenuation of prepared PEG coated elemental Bi nanoparticles is higher than the commercial Iohexol contrast agent at the same concentration. Finally, the result demonstrates PEGylated Bi nanoparticles can be an appropriate low toxic X-ray contrast agent for the medical application.

Primary recovery of recombinant human serum albumin from transgenic Oryza sativa with a single-step aqueous biphasic system

Recent advances in genetic engineering technology have provided various methods in recombinant proteins production for pharmaceutical purposes. However, the development of the effective purification strategy remains the major challenge for large-scale production of therapeutic proteins because high purity of the proteins is often demanded. In this study, recombinant Oryza sativa L. cv Tainung 67 human serum albumin (OsrHSA) was expressed and purified with a single-step polyethylene glycol (PEG)/phosphate aqueous biphasic system (ABS). The optimum condition of PEG/phosphate ABS for the purification of OsrHSA was determined by investigating the effects of PEG molecular weight; pH; tie-line length (TLL); and volume ratio (VR) of ABS on the OsrHSA purification efficiency. The purification efficiency of OsrHSA was evaluated based on the partition coefficient (KHSA) and recovery yield (RB) of the OsrHSA in the PEG/phosphate ABS. An RB of 69.8% of OsrHSA was recovered in the optimum PEG/phosphate ABS consists of TLL of 26.0% (w/w) of pH 7.5. In addition, large-scale ABS were attempted and results showed that the recovery efficiency of 3000 g ABS was consistent with a 10 g ABS. This novel finding has demonstrated that the feasibility of ABS as a potential tool to purify HSA protein in one-step operation.

Effect of PEG molecular weight and PEGylation degree on the physical stability of PEGylated lysozyme

During production, purification, formulation, and storage proteins for pharmaceutical or biotechnological applications face solution conditions that are unfavorable for their stability. Such harmful conditions include extreme pH changes, high ionic strengths or elevated temperatures. The characterization of the main influencing factors promoting undesired changes of protein conformation and aggregation, as well as the manipulation and selective control of protein stabilities are crucially important to biopharmaceutical research and process development. In this context PEGylation, i.e. the covalent attachment of polyethylene glycol (PEG) to proteins, represents a valuable strategy to improve the physico-chemical properties of proteins.In this work, the influence of PEG molecular weight and PEGylation degree on the physical stability of PEGylated lysozyme is investigated. Specifically, conformational and colloidal properties were studied by means of high-throughput melting point determination and automated generation of protein phase diagrams, respectively. Lysozyme from chicken egg-white as a model protein was randomly conjugated to 2kDa, 5kDa and 10kDa mPEG-aldehyde and resulting PEGamer species were purified by chromatographic separation. Besides protein stability assessment, residual enzyme activities were evaluated employing a Micrococcus lysodeikticus based activity assay. PEG molecules with lower molecular weights and lower PEGylation degrees resulted in higher residual activities. Changes in enzyme activities upon PEGylation have shown to result from a combination of steric hindrance and molecular flexibility. In contrast, higher PEG molecular weights and PEGylation degrees enhanced conformational and colloidal stability. By PEGylating lysozyme an increase of the protein solubility by more than 11-fold was achieved.

Development of PEG/PMMA Based Binders for Ti-Metal Injection Moulding

As the world is moving towards green manufacturing, there is an increasing demand for the use of clean and environmentally friendly binder systems in metal injection moulding (MIM) industry. One example of these developed binders is polyethylene glycol (PEG) - polymethyl methacrylate (PMMA) based system. We have systematically evaluated and optimized this binder system, and reported some interesting new results. In this article, we reported the effect of PEG molecular weight on rheological properties of the feedstock and its water debinding behaviour. We also investigated the effects of different surfactants on MIM feedstock rheological and mechanical properties, and identified a potential surfactant that enhances compatibility between the binder components and metal powders. Furthermore, we reported an interesting problem – ‘voids formation’, which is associated with PEG crystallization. To minimize this void formation a crystallization inhibitor is incorporated in the PEG/PMMA system, thereby eliminating the void formation while maintaining the clean nature of this system. This paper is concluded with some new thoughts with regard to binder design.

Evaluation of Driving Forces for Protein Partition in PEG-Salt Aqueous Two- Phase Systems and Optimization by Design of Experiments

In the present study, the partitioning of four model proteins (bovine serum albumin, ovalbumin, α-chymotrypsin and lysozyme) was investigated in polyethylene glycol (PEG)-salt aqueous two-phase systems (ATPS) addressing the effects of PEG molecular weight (MW) and concentration, phase-forming salt type (potassium phosphate/sodium citrate (KPP/NaCit)) and concentration, sodium chloride (NaCl) concentration and pH, as well as MW, isoelectric point, charge and hydrophobicity of the proteins. It was found that protein partitioning was influenced by different coexisting effects, such as hydrophobic and electrostatic interactions, related to the system parameters and physicochemical/surface properties of proteins. Moreover, a rigorous analysis and optimization of the significant forces for protein partitioning in PEG-salt ATPS was performed by design of experiments (DoE). Initially, KPP/NaCit, NaCl and PEG concentrations were considered as statistically significant factors by a fractional factorial design. Subsequently, the optimal values of the significant factors were determined by a central composite face-centered design coupled with response surface methodology. Furthermore, linear and quadratic models were obtained in the experimental designs, respectively, and evaluated by statistical regression analysis and analysis of variance. Finally, a validation of the quadratic model confirmed a good correlation between predicted and experimental results, thus verifying the validity of the model.

Intensified fractionation of brewery yeast waste for the recovery of invertase using aqueous two-phase systems.

The potential recovery of high-value products from brewery yeast waste confers value to this industrial residue. Aqueous two-phase systems (ATPS) have demonstrated to be an attractive alternative for the primary recovery of biological products and are therefore suitable for the recovery of invertase from this residue. Sixteen different polyethylene glycol (PEG)-potassium phosphate ATPS were tested to evaluate the effects of PEG molecular weight (MW) and tie-line length (TLL) upon the partition behavior of invertase. Concentrations of crude extract from brewery yeast waste were then varied in the systems that presented the best behaviors to intensify the potential recovery of the enzyme. Results show that the use of a PEG MW 400gmol-1 system with a TLL of 45.0% (w/w) resulted in an invertase bottom phase recovery with a purification factor of 29.5 and a recovery yield of up to 66.2% after scaling the system to a total weight of 15.0g. This represents 15.1mg of invertase per mL of processed bottom phase. With these results, a single-stage ATPS process for the recovery of invertase is proposed.

Effect of PEG molecular weight on rheological properties of Ti-MIM feedstocks and water debinding behaviour

Polyethylene glycol (PEG) based binder systems are garnering increasing attention because of the environmentally friendly nature of the water-soluble PEG. Rheological properties of the feedstock are essential to metal injection moulding (MIM). Intriguingly the effect of PEG molecular weight on rheological properties of the feedstock and its water debinding behaviour has not been well investigated. In this work, four molecular weights of PEG, i.e. 1500, 4000, 10,000 and 20,000g/mol were selected and PEG/polymethyl methacrylate (PMMA) based feedstocks were formulated with titanium metal powder. We reported a systematic investigation of the rheological properties of the prepared feedstocks with capillary rheometry and the study of the effects of shear rate and temperature on viscosity. The water debinding behaviour of each feedstock was also investigated in terms of debinding temperature and PEG molecular weight.

Polyethylene glycol-based ultrasound-assisted extraction of magnolol and honokiol from Cortex Magnoliae Officinalis

In this study, a kind of green solvent named polyethylene glycol (PEG) was developed for the ultrasound-assisted extraction (UAE) of magnolol and honokiol from Cortex Magnoliae Officinalis. The effects of PEG molecular weight, PEG concentration, sample size, pH, ultrasonic power and extraction time on the extraction of magnolol and honokiol were investigated to optimise the extraction conditions. Under the optimal extraction conditions, the PEG-based UAE supplied higher extraction efficiencies of magnolol and honokiol than the ethanol-based UAE and traditional ethanol-reflux extraction. Furthermore, the correlation coefficient (R2), repeatability (relative standard deviation, n = 6) and recovery confirmed the validation of the proposed extraction method, which were 0.9993–0.9996, 3.1–4.6% and 92.3–106.8%, respectively.

Effect of PEG molecular weight on stability, T2 contrast, cytotoxicity, and cellular uptake of superparamagnetic iron oxide nanoparticles (SPIONs)

Superparamagnetic iron oxide nanoparticles (SPIONs) are currently unavailable as MRI contrast agents for detecting atherosclerosis in the clinical setting because of either low signal enhancement or safety concerns. Therefore, a new generation of SPIONs with increased circulation time, enhanced image contrast, and less cytotoxicity is essential. In this study, monodisperse SPIONs were synthesized and coated with polyethylene glycol (PEG) of varying molecular weights. The resulting PEGylated SPIONs were characterized, and their interactions with vascular smooth muscle cells (VSMCs) were examined. SPIONs were tested at different concentrations (100 and 500ppm Fe) for stability, T2 contrast, cytotoxicity, and cellular uptake to determine an optimal formulation for in vivo use. We found that at 100ppm Fe, the PEG 2K SPIONs showed adequate stability and magnetic contrast, and exhibited the least cytotoxicity and nonspecific cellular uptake. An increase in cell viability was observed when the SPION-treated cells were washed with PBS after 1h incubation compared to 5 and 24h incubation without washing. Our investigation provides insight into the potential safe application of SPIONs in the clinic.

Synthesis and properties of aqueous polyurethane dispersions: Influence of molecular weight of polyethylene glycol

Aqueous polyurethane dispersions (PUDs) have recently emerged as important alternatives to their solvent-based counterparts for various applications due to increasing health and environmental awareness. A series of aqueous polyurethane dispersions containing carboxylate anion as hydrophilic pendant groups were synthesized through step growth polymerization reaction using hexamethylene diisocyanate (HDI), 1,4-butanediol (1,4-BDO), dimethylol propionic acid (DMPA) and polyethylene glycol (PEG) of different molecular weight. Effect of PEG molecular weight was investigated on molecular structure, contact angle measurement, and physical and adhesive properties of PU emulsions. Fourier transform infrared spectroscopy (FT-IR) was used to check the completion of polymerization reaction. Contact angle measurement indicated that the hydrophilicity of polymer increases by increasing molecular weight of PEG with a corresponding decrease in contact angle. Results of T-peel test showed a decrease in peel strength by increasing molecular weight of PEG. Moreover, solid contents%, drying time and storage stability suggested fast drying properties and greater stability of aqueous PU dispersions.

Separation of Bromelain by Aqueous Two Phase Flotation

Aqueous two phase flotation (ATPF) system of polyethylene glycol (PEG) and potassium phosphate is studied for the separation and partial purification of bromelain from the pineapple fruit (Annanus comosus L. Merryl). The effect of PEG molecular weight (1500–20000), concentration of phase forming components (PEG 12–18% w/w and potassium phosphate 14–20% w/w), system pH, nitrogen flow rate, and flotation time were studied and optimum conditions for ATPF were obtained. At optimum conditions of the system, i.e., 14% w/w PEG 1500, 18% w/w potassium phosphate, 80 mL/min of nitrogen flow rate and pH 7, maximum yield of 91.47% and purification fold of 4.26 were obtained. ATPF was found to be an effective technique for the purification of bromelain from pineapple fruit with higher extraction yield and purification fold as compared to aqueous two phase extraction (ATPE).

Partition of Pepsinogen from the Stomach of Red Perch (Sebastes marinus) by Aqueous Two Phase Systems: Effects of PEG Molecular Weight and Concentration

Fish processing waste can be used to produce commercially valuable by-products, such as pepsinogen, which has application in food, manufacturing industries, collagen extraction, gelatin extraction, and in regulating digestibility.An important acidic protease, pepsin, is synthesized and secreted in the gastric membrane in an inactive state called pepsinogen (PG). In the present study, the purification of pepsinogen from the stomach of red perch, using aqueous two phase systems (ATPS) formed by polyethylene glycol (PEG) and salt at 4°C, was optimized.The effects of PEG molecular weight (PEG 1000, 1500, 3000 and 4000) and concentration (16, 18, 20, 22 and 24%) on the partitioning of PG were studied, and parameters including total volume (TV), volume ratio (VR), total enzyme activity (AE), protein content (Cp), specific enzyme activity (SA), partition coefficient (Kp), purification fold (PF), and recovery yield (RY) were evaluated. PEG molecular weight and PEG concentration also had significant effects on each parameter. TV and VR decreased with increased salt concentration, since salt formed hydrogen bonds with water molecules and formed a more compact and ordered water structure. PG partitioned predominantly in the PEG-rich top phase due to its negative charge. AE, CP, SA, PF and RY increased with increased salt concentration and then decreased, while KP had an opposite pattern. The PEG 3000 (20%), PEG 1000 (24%), PEG 4000 (16%) and PEG 1000 (18%) concentrations gave the highest TV, VR, CP and KP, respectively. PEG 1500 with 18% concentration gave the highest AE, SA, PF and RY (86.2%). As PEG 1500 at 18% concentration gave the highest RY (86.2%). It was selected as the optimum PEG molecular weight and PEG concentration. (NH4)2SO4 at 15%, which gave the highest RY (71.7%), was selected as the optimum salt type and salt concentration. 15% (NH4)2SO418% PEG 1500 was the optimal ATPS combination, and presented a better partition. The values of SA and PF and RY obtained with ATPS method were much higher (2 fold in case of SA and PF, and 1.2 fold in case of RY), than those obtained with the Ammonium Sulphate Fractionation (ASF) method.

DNA-Functionalized Gold Nanoparticles in Macromolecularly Crowded Polymer Solutions

DNA-functionalized gold nanoparticles (AuNPs) are one of the most commonly used reagents in nanobiotechnology. They are important not only for practical applications in analytical chemistry and drug delivery, but also for fundamental understanding of nanoscience. For biological samples such as blood serum or for intracellular applications, the effects of crowded cellular proteins and nucleic acids need to be considered. The thermodynamic effect of crowding is to induce nanoparticle aggregation. But before such aggregation can take place, there might also be a depletion repulsive barrier. Polyethylene glycol (PEG) is one of the most frequently used polymers to mimic the crowded cellular environment. We show herein that while DNA-functionalized AuNPs are very stable in buffer (e.g., no PEG) and citrate-capped AuNPs are very stable in PEG, DNA-functionalized AuNPs are unstable in PEG and are easily aggregated. Although such aggregation in PEG is mediated by DNA, no sharp melting transition typical for DNA-linked AuNPs is observed. We attribute this broad melting to depletion force instead of DNA base pairing. The effects of PEG molecular weight, concentration and temperature have been studied in detail and we also find an interesting PEG phase separation and AuNP partition into the water-rich phase at high temperature.