Activated Polyethylene Glycol Research Articles

Highly active polyethylene glycol grafted cellulose supported Pd nanoparticles for glucose electrooxidation

In this study, poly(ethylene glycol) (PEG) grafted cellulose (CE) composite catalyst support material (PEG-CE) was obtained by chemically cross-linking PEG at varying molecular weight and CE with isophorone diisocyanate (IPDI) and 2,4-toluylene diisocyanate (TDI). PEG-CE supported Pd (Pd/PEG-CE) catalyst was prepared by the chemical reduction method. The crystal size of the Pd/PEG-CE catalyst is 4.79 nm and its crystal structure has been determined to be face-centered cubic Pd. Elemental mapping results indicate that Pd was reduced onto PEG-CE successfully and uniformly. Pd exists in the elemental and Pd-O form in the catalyst system. The Pd loading rate of Pd/PEG-C_IPDI catalyst was determined as 18.8% by mass. Among the PEG-CEs prepared with TDI and IPDI, PEG4000-CE_TDI and PEG6000-CE_IPDI displayed the highest specific activities of 1.03 and 1.50 mA cm−2 for glucose electrooxidation. With Pd reduction on PEG4000-CE_TDI and PEG6000-CE_IPDI, the specific activities increased to 1.62 mA cm−2 and 6.97 mA cm‐2. Pd/PEG6000-CE_IPDI has the highest electrocatalytic activity and stability in this study for glucose electrooxidation and is an encouraging anode catalyst for direct glucose fuel cells.

A study on activation of polyethylene glycol and its characterization by infrared spectroscopy and thin layer chromatography

Polyethylene Glycol (PEG) is the most popular polymeric material used for alteration and control of biodistribution. PEG may increase the lifetime of “drug carrier” assembly which helps in administering lower concentration of the “drug carrier” composite. It has been used widely for the modification of carriers used in therapeutics because PEG offers a shielding characters that avoids rapid renal clearance from the body. This study was carried out at Dolphin Institute of Biomedical and Natural Sciences, Dehradun and Indian Institute of Technology (IIT), Roorkee, India during May-July 2005. Activation of PEG of different molecular weight (400 Da, 4000 Da, 8000 Da and 20,000 Da) was done using dry benzene, Triethylamine, Ethylene dichloride and 4-nitrophenyl chloroformate. Reaction mixture was monitored on TLC using EDC: Methanol (7:3). Then reaction mixture was portioned between EDC & water. The lower fraction in separating funnel of EDC was collected & concentrated on rota evaporator to get activated PEG. From the above reaction and structure it was clear that 4-Nitrophenyl Chloroformate contains two highly electronegative groups i.e. NO2 and Cl. These two groups interact with each other resulting in neutralization of polarity. It then acts as non-polar molecule and shows high affinity for mobile phase (methanol & EDC). Therefore, 4-Nitrophenylchloroformate has the highest mobility and PEG has lowest mobility. From IR Spectroscopy it was found that the peak of Hydroxyl group- (OH) of PEG was at 3400- 3450cm-1. The peak of C-Cl bond was found at 746 cm-1. But after the reaction between PEG and 4-Nitrophenyl Chloroformate the- OH peak was found not so deep as in PEG. The peak was somewhat short and broad.

PH/enzyme dual sensitive and nucleus-targeting dendrimer nanoparticles to enhance the antitumour activity of doxorubicin

Direct delivery of drugs into the nucleus is a promising nanotechnology therapy, since the nucleus is one of the most important organelles controlling cell proliferation and apoptosis. Here, we report a nucleus-targeting nanocarrier for nuclear drug delivery using a pH/enzyme dual sensitive strategy. The specific ligand PGM (PKKKRKV-GFLG-Mp), composed of nuclear localization sequence (PKKKRKV), enzyme-sensitive tetrapeptide (Gly-Phe-Leu-Gly, GFLG), and pH-sensitive molecules morpholine (Mp), was modified on poly (amidoamine) (PAMAM) by maleimide active polyethylene glycol ester (NHS-PEG-MAL) to form PAMAM-PEG-PGM. Doxorubicin (DOX) was loaded into the cavity of PAMAM to prepare DOX/PAMAM-PEG-PGM. In vitro release study suggested DOX release from DOX/PAMAM-PEG-PGM nanoparticles followed pH and enzyme-triggered manner. In vitro studies showed DOX/PAMAM-PEG-PGM nanoparticles could not only promote cell internalization through the charge switching of morpholine, but also achieve nuclear internalization by the mediation of composite formed by NLS and importin α/β receptor. Further, employing H22 tumour-bearing BALB/c mice as a model, the systemic distribution and anticancer effects of nanoparticles were studied in vivo. The results indicated the nanoparticles could preferentially accumulate in the tumour site in vivo, and the tumour inhibition rate was 88.47%. In short, the nanoparticles developed could be promising in application to nucleus-targeting therapy to enhance antitumour activity.

Synthesis and Biological Evaluation of Polyethylene Glycol-Hyaluronic Acid-Melphalan as a Potential Therapy for Tumor

Melphalan has been shown to effectively improve cancers by inhibiting deoxyribonucleic acid synthesis. However, it has some obvious drawbacks such as short plasma half-life, non-target selectivity and serious adverse reactions. Because of these shortcomings, its clinical application is limited. In the present research work, we have tried to obtain polyethylene glycol-hyaluronic acid by modifying hyaluronic acid with activated polyethylene glycol; polyethylene glycol-hyaluronic acid was then connected to melphalan to prepare the polyethylene glycol-hyaluronic acid-melphalan prodrug. Polyethylene glycol-hyaluronic acid was obtained by modifying hyaluronic acid with activated polyethylene glycol and it was then connected with melphalan to prepare the polyethylene glycol-hyaluronic acid-melphalan prodrug. The prodrug generates micelles by self-assembly in aqueous solution and was characterized by nuclear magnetic resonance and infrared spectroscopy. Changes of tumor cell morphology and cytotoxicity were observed by an inverted microscope. Dynamic light scattering and transmission electron microscope showed that the prodrug formed micelle and was evenly distributed in 100-130 nm. Tumor cells treated with polyethylene glycol-hyaluronic acid-melphalan (10−7 and 10−6 mol/l respectively) showed significant changes in cell proliferation compared with the control group (p<0.05, p<0.01) with half-maximal inhibitory concentration values of 0.53 μM/48 h. Tumor cells treated with polyethylene glycol-hyaluronic acid-melphalan (10−6 mol/l) were condensed and rounded, aggregated into clumps, and the number of cells was significantly reduced at both 24 h and 48 h after treatment. The polyethylene glycol-hyaluronic acid-melphalan prodrug was synthesized successfully and characterized by fourier-transform infrared spectroscopy, proton nuclear magnetic resonance, dynamic light scattering and transmission electron microscope. It can form micelle with the average size of about 100-130 nm. It was demonstrated that these natural, biocompatible and biodegradable particles can be used as drug delivery system and found that this compound play potent inhibitory role on tumor cells, and indicated that the polyethylene glycol-hyaluronic acid-melphalan prodrug could be very useful on release of control targeted drug.

Innovational combination of hetero-bifunctional N-PEG quinoline scaffolds derivatives with improved anticancer activity against breast and colon cancer cell lines and P-glycoprotein, cytochrome p450 enzyme activity prediction.

Polyethylene glycol (PEG) is a polymer that is widely used as a carrier for drug delivery systems (DDS). A library of N-PEGylated quinoline derivatives of PEG molecular weight 200 was prepared rapidly after the activation of PEGs using maleic anhydride. Quinoline with a polymer backbone is essential as new material. PEG is a water-soluble nonionic polymer approved by food and drug organizations for medicine applications. Because of its nontoxic grapheme, it is widely utilized in numerous biochemical, cosmetic, pharmaceutical, and industrialized applications. The modern SwissADME is a web tool that stretches free admittance to a pool of hasty, yet solid, clarifying models for physicochemical properties, pharmacokinetics, and therapeutic science. The present facile synthetic strategy can be a practical approach for incorporating polymeric carriers conjugated with drug moieties, either in the backbone of the polymer or as a terminal and pendant group on the polymer chains.

N-PEGylated Thiazolium Salt: A Green and Reusable Homogenous Organocatalyst for the Synthesis of Benzoins and Acyloins

N-PEGylated-thiazolium salt is used as efficient catalyst for the benzoin condensation. The catalyst was synthesized by reaction of activated polyethylene glycol 10,000 (PEG-10000) with 4-methyl-5-thiazoleethanol (sulfurol). Reaction mixture undergoes temperature-assisted phase transition and catalyst separated by simple filtration. After reaction course, catalyst can be recycled and reused without any apparent loss of activity which makes this process cost effective and hence ecofriendly. Synthesized benzoins and acyloins by this method have been characterized on the basis of melting point and 1H-NMR spectral studies.

Investigating folate-conjugated combinatorial drug loaded ZnO nanoparticles for improved efficacy on nasopharyngeal carcinoma cell lines

Zinc oxide (ZnO) nanoparticles, due to their well-defined physico-chemical and antimicrobial attributes have been presented lately as favourable theranostic nanoplatforms for drug delivery. We hypothesized that surface modification of ZnO nanoparticle with activated polyethylene glycol and folic acid might be of great importance in targeted drug delivery in nasopharyngeal carcinoma since carcinoma cells possess folate receptors absent in normal cells. Therefore in this study, we have prepared and characterized activated PEG and folate conjugated dual drug loaded ZnO nanoparticles for increased cellular uptake efficiency in nasopharyngeal cancer cell lines. We synthesized the ZnO nanoparticles using sol-gel technique, conjugated them with activated PEG and folate and loaded them with two conventional anti-cancer drugs, namely cisplatin and docetaxel. The nanoparticle formulations were characterized by electron microscopy, stability studies, in-vitro release studies(acidic and normal pH), encapsulation & loading efficiency studies, FTIR and cytotoxicity studies. Additionally, cellline studies were done to assess cellular uptake efficiency using HNE-1 and CNE-2 cells. The dual drug loaded, folate & PEG activated ZnO nanoparticle were found to be extremely small size with high zeta potential & polydispersity index and high entrapment efficiency (82%) too. The nanoparticle formulations had stability and did not aggregate when kept in dispersed form for a long time. Moreover, they were found to be cytocompatible with negligible side-effects when incubated with normal cells. They also exhibited higher cellular uptake efficiencies when compared with free form. Therefore, it may safely concluded that combinatorial drug loaded, folate & PEG activated ZnO nanoparticle may result in better uptake of chemotherapeutic drugs with lower toxicity to normal cells.

The effects of the treatment conditions on the dissolution profile of ethylcellulose coated pellets

Due to the additional particle coalescence in the coating, changes in the dissolution profile occur over time in the formulations coated by aqueous ethylcellulose latex. Dry thermal treatment (DT) of the coating can be used as a prevention of this process. Alternatively, it is advisable to take advantage of the synergistic effect of high humidity during wet treatment (WT), which substantially accelerates the film formation. This can be a problem for time-controlled systems, which are based on the coating rupture due to the penetration of water into the core causing the increase in the system volume. This process can begin already during the WT, which may affect the coating adversely. The submitted work was focused on the stability testing of two pellet core compositions: pellets containing swelling superdisintegrant sodium carboxymethyl starch (CMS) and pellets containing osmotically active polyethylene glycol (PEG). Another objective was to identify the treatment/storage condition effects on the pellet dissolution profiles. These pellets are intended to prevent hypoglycemia for patients with diabetes mellitus and therefore, besides the excipients, pellet cores contain 75% or 80% of glucose. The pellet coating is formed by ethylcellulose-based latex, which provides the required lag time (120–360 min). The sample stability was evaluated depending on the pellet core composition (PEG, CMS) for two types of final pellet coating treatment (DT or WT). Scanning electron microscopy and Raman microspectroscopy revealed the penetration of glucose and polyethylene glycol from the core to the PEG pellet surface after WT. For the CMS sample, significant pellet swelling after WT (under the conditions of elevated humidity) was statistically confirmed by the means of stereomicroscopic data evaluation. Therefore, the acceleration of dissolution rate during the stress tests is caused by the soluble substance penetration through the coating in the case of PEG pellets or by dosage form volume increase in the case of CMS pellets. The observed mechanisms can be generally anticipated during the stability testing of the ethylcellulose coated dosage forms. The aforementioned processes do not occur after DT and the pellets are stable in the environment without increased humidity.

Effectiveness of the Biophysical Barriers to the Peridural Fibrosis in Rat Laminectomy Model

ABSTRACTPurpose: Peridural fibrosis which could occur after the spinal surgery could adhere neural tissue closely and may cause to neural entrapment symptoms and require surgical reintervention. Aim of the study: Present study was designed to reduce occurrence of peridural fibrosis in rat laminectomy model by using biophysical barriers called hyaluronic acid (HAS) dural barrier, activated polyethylene glycol and polyethylene imine (PEG) dural barrier, and platelet-rich plasma (PRP). Materials and methods: In this study, 2 of 26 male Wistar albino rats (325–350 g body weight), which were not included into study groups were sacrificed by removing their total blood and their blood was used for preparation of PRP, and remaining rats were randomly delivered into four groups called SHAM, HAS, PEG, and PRP groups. Then L3-4-5 laminectomy was performed to all animals and experimental agents were administered to the selected groups mentioned above. Spinal colons of all animals were removed gross total after 6-week period and investigated histopathologically. Additionally, real-time-polymerase chain reaction was used to obtain collagen type I and type III, transforming growth factor-1β, and tumor necrosis factor-α gene expressions. Results: All results demonstrated that polyethylene glycol and polyethylene imine dural barrier and PRP could decrease peridural fibrosis formation efficiently in rat. Conclusion: Present study results suggested that to reduce or block formation of peridural fibrosis, either polyethylene glycol and polyethylene imine dural barrier or PRP could be used effectively in human subjects after they will be closely investigated in future studies.

PEGylated recombinant L-asparaginase from Erwinia carotovora: Production, properties, and potential applications

N-hydroxysuccinimide ester of monomethoxy polyethylene glycol hemisuccinate was synthesized. It acylated amino groups in a molecule of recombinant L-asparaginase from Erwinia carotovora. A method of L-asparaginase modification by the obtained activated polyethylene glycol derivative was developed. The best results were produced by modification of the enzyme with a 25-fold excess of reagent relative to the enzyme tetramer. The modified L-asparaginase was isolated from the reaction mixture by gel filtration on Sepharose CL-6B. The purified bioconjugate did not contain PEG unbound to the protein, demonstrated high catalytic activity, and exhibited antiproliferative action on cell cultures.

Quantification of PEGylated proteases with varying degree of conjugation in mixtures: An analytical protocol combining protein precipitation and capillary gel electrophoresis

PEGylation, i.e. the covalent attachment of chemically activated polyethylene glycol (PEG) to proteins, is a technique commonly used in biopharmaceutical industry to improve protein stability, pharmacokinetics and resistance to proteolytic degradation. Therefore, PEGylation represents a valuable strategy to reduce autocatalysis of biopharmaceutical relevant proteases during production, purification and storage. In case of non-specific random conjugation the existence of more than one accessible binding site results in conjugates which vary in position and number of attached PEG molecules. These conjugates may differ considerably in their physicochemical properties. Optimizing the reaction conditions with respect to the degree of PEGylation (number of linked PEG molecules) using high-throughput screening (HTS) technologies requires a fast and reliable analytical method which allows stopping the reaction at defined times.In this study an analytical protocol for PEGylated proteases is proposed combining preservation of sample composition by trichloroacetic acid (TCA) precipitation with high-throughput capillary gel electrophoresis (HT-CGE). The well-studied protein hen egg-white lysozyme served as a model system for validating the newly developed analytical protocol for 10kDa mPEG-aldehyde conjugates. PEGamer species were purified by chromatographic separation for calibrating the HT-CGE system. In a case study, the serine protease Savinase® which is highly sensitive to autocatalysis was randomly modified with 5kDa and 10kDa mPEG-aldehyde and analyzed. Using the presented TCA protocol baseline separation between PEGamer species was achieved allowing for the analysis of heterogeneous PEGamer mixtures while preventing protease autocatalysis.

Covalent stretokinase-polyethylene glycol conjugates with increased stability and decreased side effects

By variation of incubation time of streptokinase (SK) with activated polyethylene glycol (M 2 and 5 kDa, PEG2 and PEG5) it was obtained covalent SK-PEG2 and SK-PEG5 conjugates with different modification degrees of amino groups of protein and their properties were studied in vitro as compared with free SK. It was shown, that maximal stable and retaining 80% fibrinolytic activity SK-PEG2 and SK-PEG5 conjugates are formed when the modification degrees of amino groups of protein are 54 and 52%, respectively. At interaction of the given conjugates with equimolar plasminogen concentration it were formed the plasmin (Pm)·SK-PEG2 and Pm·SK-PEG5 activator complexes, the maximal amidase activity of which is equal to activity of unmodified Pm·SK complex. It was found, that the catalytic efficiency of plasminogen activation (kPg/KPg) by Pm·SK-PEG2 complex is some higher (2.84 min(-1) μM(-1)) and by Pm·SK-PEG5 complex is lower (1.17 min(-1) μM(-1)), than that by unmodified Pm·SK complex (2.1 min(-1) μM(-1)). Investigation of lysis kinetics of human plasma clots and depletion of plasminogen and fibrinogen in plasma under the action of free SK and SK-PEG2 and SK-PEG5 conjugates showed, that the latter's have high thrombolytic activity (89 and 72%, respectively) and cause 3.5-4 fold lower side effects, than free SK. Obtained by us SK-PEG2 and SK-PEG5 conjugates with increased stability and decreased side effects may be used in the therapy of thrombotic disorders.

Enzymatic Formation of PEGylated Oligonucleotides

Gene therapy, siRNA, and therapeutic aptamers attract great interest owing to their versatility to treat a wide range of diseases and their potential high selectivity. Unfortunately, oligonucleotide-based therapeutics suffer rapid degradation by nucleases, scarce cell internalization, and fast kidney clearance. To address these limitations, the covalent attachment by mild chemical reactions of an activated polyethylene glycol (PEG) is widely used to obtain PEGylated nucleic acids showing a more favorable pharmacokinetic profile. We describe here a method for the enzymatic formation of PEGylated nucleic acids employing T4 DNA ligase: the ligation protocol was set up and optimized allowing the complete achievement of PEGylated oligonucleotides amenable to further enzymatic reactions. The feasibility of this approach for bioconjugation was demonstrated employing a set of PEG-donors and oligonucleotide acceptors, differing in the chemical link between PEG and the oligonucleotide donor, and in the length, sequence, and structure of the oligonucleotides employed. The ligase reaction allowed us to obtain double-stranded as well as single-stranded oligonucleotides, thus demonstrating the applicability of the method to a variety of substrates suitable for diagnostic and therapeutic applications.

Evaluation of the neurotoxicity of the polyethylene glycol hydrogel dural sealant

Although polyethylene glycol (PEG) is a neutral, biocompatible hydrophilic polymer recognized for its lack of interaction with biological barrier, its neurotoxicity has not been clearly identified in neurosurgery. This study is constructed to evaluate the possible neurotoxicity of a PEG hydrogel dural sealant. After a burrhole was opened in the left parietal bone of the twenty five Wistar albino rats, the dura mater and cerebral cortex were incised and the experimental material (activated polyethylene glycol and polyethylene imine) was sprayed into the burrhole. Then brain tissues were harvested for histopathological and biochemical studies at 72 hours to investigate the acute stage changes and on 15th day to evaluate the chronic stage changes. There were statistically significant differences among the groups regarding the comparison of the values of the PMNL cell infiltration grades, gliosis and congestion in both acute and chronic stages. However, the values of the MNL cell infiltration grades, edema and fibrin formation, lipid peroxidation levels of harvested brain tissues were similar in all groups. Although this study did not present the detailed histopathological and biochemical evaluation results, it indicated that the application of the PEG-based hydrogel sealant was not associated with neurotoxicity, delayed healing, or degenerative changes.

Hemoglobin Conjugated with a Band 3 N-terminus Derived Peptide as an Oxygen Carrier

A peptide composed of 9 amino acids, 7 residues from N-terminus of human erythrocytic Band 3 protein (AcMEELQDD) followed by cysteine and glutamic acids, was conjugated to hemoglobin (Hb) serving as an allosteric effector for oxygen release. The activated polyethylene glycol (PEG), maleimide-PEG-N-hydroxysuccinimidyl, was used to crosslink Hb with the peptide. The putative conjugation site on Hb for effective enhancement of oxygen release was characterized as Lys-beta95 by liquid chromatography-tandem mass spectrometry. In addition, the conjugated peptide causes a rightward shift of the oxygen dissociation curve as compared to that of its parent Hb when the degree of oxygen saturation is higher than 50%. Furthermore, this conjugated peptide remains effective on lowering Hb's oxygen affinity after Hb polymerization by another PEG crosslinker. The allosteric properties of the peptide-conjugated Hb may provide a new aspect of Hb-based oxygen carriers.

The pH-Dependent Formation of PEGylated Bovine Lactoferrin by Branched Polyethylene Glycol (PEG)-N-Hydroxysuccinimide (NHS) Active Esters

PEGylation is an established method for improving the pharmacokinetic properties and pharmacodynamic effects of therapeutic proteins. Amino conjugation by N-hydroxysuccinimide activated polyethylene glycol (PEG-NHS) represents the most common modification of the target proteins. In this study, we compared the reaction velocities of pH-dependent preparations of bovine lactoferrin modified with branched PEG-NHS conducted at pH 7.4 to pH 9.0. PEGylation reaction rates were dependent on pH value, but not on PEG molecular mass. At pH 7.4, reactions advanced gradually and reached steady state by 2 h, whereas at pH 9.0, reactions advanced very quickly and reached steady state within 10 min. Hydrolysis half-life of PEG-NHS exceeded 120 min at pH 7.4, but was below 9 min at pH 9.0. Thus, the pH value is one of the most important factors to obtain the optimal condition for PEGylation by NHS esters.

PEG-modified biopharmaceuticals

PEGylation is a process in which one or more units of chemically activated polyethylene glycol reacts with a biomolecule, usually a protein, peptide, small molecule or oligonucleotide, creating a putative new molecular entity possessing physicochemical and physiological characteristics that are distinct from its predecessor molecules. In recent years, PEGylation has been used not only as a drug delivery technology but used also as a drug modification technology to transform existing biopharmaceuticals clinically more efficacious than before their PEGylation. PEGylation bestows several useful properties upon the native molecule, resulting in improved pharmacokinetic and pharmacodynamic properties, which in turn enable the native molecule to achieve maximum clinical potency. In addition, PEGylation results in sustained clinical response with minimal dose and less frequency of dosing, leading to improved quality of life via increased patient compliance and reduced cost. During the course of development of various pegylated protein therapeutics, several new insights have been gained. This review article focuses on the approaches, strategies and the utilization of modern PEGylation concepts in the design and development of well-characterized pegylated protein therapeutics.

Cell Surface Modification by Activated Polyethylene Glycol Prevents Allosensitization after Islet Transplantation

The necessity to transplant islet tissue without the need for immunosuppressant therapy has led to the development of materials for immune modulation. Pegylation makes islets antigenically silent, protecting them from the adsorption of foreign protein and thus avoiding immune injury. The aim of this study is to determine whether pegylation of islets prolongs islet survival and function both during tissue culture and posttransplantation. We used cyanuric chloride-activated methoxy-polyethylene glycol for cell surface modification. To detect the pegylation effect on splenocytes, we measured antibody binding inhibition and abrogation of lymphocyte proliferation. To detect the pegylation effect on islet grafts, we performed rodent islet transplantation. Islet viability and function were maintained after pegylation. Pegylated islets showed a 90% decrease in antibody binding and decreased lymphocyte proliferation in a mixed lymphocyte culture. However, when pegylated islets were transplanted, no prolongation of graft survival was observed. When a subtherapeutic dose of immunosuppressant was given at the time of transplantation of pegylated islets, islet graft survival was significantly prolonged. In addition, when rats were sensitized with donor splenocytes, graft survival was prolonged by pegylation. We observed that pegylation of islets, combined with a subtherapeutic dose of immunosuppressant, protects the graft from rejection. Prolonged graft survival in sensitized recipients showed that pegylation of islets shifted the pattern of rejection from an acute humoral response to a less aggressive cellular alloresponse.

Precise and comparative pegylation analysis by microfluidics and mass spectrometry

Standard SDS–PAGE analysis of a pegylated protein was able to confirm an increase in its molecular size after reaction with an activated polyethylene glycol (PEG) but could do little to identify the extent of pegylation or to support characterization of the consistency of the modified protein. In this article, we demonstrate the utility of the capillary electrophoresis technology (using a microfluidic system) in analyzing the pegylation pattern of a recombinant protein over a range of 1–12 PEGs per polypeptide. Confirmatory data from mass spectrometry analysis of pegylated adducts are also presented. These allowed independent confirmation of the extent of pegylation. This electrophoretic analysis gives a robust, reproducible, and direct characterization of PEG adducts. We found that traditional estimation of PEG adducts by an indirect colorimetric (trinitrobenzene sulfonic acid) reaction, which detects loss of free amino groups, was quite erroneous for the recombinant protein in our study as well as several commercially available pegylated proteins. These results support the use of this capillary electrophoresis device for precise characterization of pegylated proteins.

Tissue-on-Tissue Testing of Dry Eye Formulations for Reduction of Bioadhesion

Glutaraldehyde-preserved, human umbilical cord vein graft (UCVG) was selected as a stable surrogate tissue source for testing of bioadhesion-reducing lubricants. Bioadhesion, as manifested in tissue-on-tissue friction coefficients of 0.2–0.4 for saline-lubricated UCVG, was quantitatively and persistently reduced after the instillation of a single aliquot of an ophthalmic “artificial tears” formulation containing active demulcents polyethylene glycol (PEG400) and propylene glycol (PG), as well as a gellable hydroxypropyl guar (HP Guar) in a borate-buffered solution between the “blinking” tissues. Reduced adhesion was maintained (was “substantive”), even after rinsing excess lubricant from the surfaces. Comparative tests with tissue-on-solid, and solid-on-solid, similarly lubricated couples point to a potentially unique mechanism that involves macromolecules modifying the tissue phases to provide rinse-resistant lubricity and surface protection in articulated tissue-to-tissue interfaces. Results for tissue-on-tissue couples were obtained in laboratory trials utilizing a reciprocating pin-on-disc type friction/wear test device articulating preserved human umbilical cord vein segments under increasing loads, and again after saline rinsing to determine persistence of the friction-reducing effects. A single confirmatory test using donated human cornea against vein graft tissue showed the lowest coefficient of friction, below 0.05, for the “artificial tears” formulation. Mechanistic studies employing the same test device and protocol for metal oxide (germanium)-on-metal oxide couples, as well as for metal oxide-on-tissue couples, indicated that simple increases in viscosity were not the likely sources of friction reduction, and revealed frictional values higher than measured for the similarly lubricated tissue-on-tissue couples. Thus, formulation development to minimize bioadhesion requires that appropriate simulations be used to obtain clinically predictive data for circumstances of liquid uptake into the tissues, resultant tissue swelling, and binding to impermeable adjacent materials.