Polyethylene Glycol Conjugation Research Articles

Near infrared light‐induced disassembly of polymeric micelles based on methylene blue conjugated polyethylene glycol

AbstractLight‐sensitive drug delivery systems are considered ideal for applications in the biomedical fields for their ability to release the payload in an on‐demand spatiotemporal controlled manner through the manipulation of the light source. Among the broad radiation spectrum, near infrared (NIR) light is considered advantageous compared to UV and visible light, due to its inherently lower photodamage to normal tissues and deeper penetration to lesion areas. In this study, we report a successful synthesis of a polymer capable of undergoing partial degradation upon irradiation with NIR light by conjugating 10‐N‐carbamoyl linkage methylene blue (MB) moiety, a NIR photocleavable ligand, with polyethylene glycol (PEG). Through effective coupling of MB, a hydrophobic moiety, to the hydrophilic PEG molecule, an amphiphilic polymer was synthesized, as demonstrated by a lowered surface tension (55 mN/m at 0.1% wt/vol). Subsequently, photo‐induced reversal of surface activity associated with self‐assembled structure disruption, was displayed by surface tension measurements, size distribution analysis, and burst release profile of paclitaxel (PTX) from polymeric micelles upon the exposure to NIR irradiation.

Multifunctional TPP-PEG-biotin self-assembled nanoparticle drug delivery-based combination therapeutic approach for co-targeting of GRP78 and lysosome

BackgroundIn this study, a multifunctional tetraphenylporphyrin (TPP) conjugated polyethylene glycol with biotin (TPP-PEG-biotin) as a photo-dynamic therapy (PDT) material encapsulating a ruthenium complex 1 (Ru-1) was fabricated as self-assembled nanoparticle (Ru-1@TPP-PEG-biotin SAN) to co-target glucose-regulated protein 78 (GRP78) and the lysosome as a new anti-cancer therapeutic strategy.ResultsThe MTT assay results reveals the enhanced anticancer activity of the Ru-1@TPP-PEG-biotin SANs due to the co-targeting of the GRP78 and lysosome. The Ru-1@TPP-PEG-biotin reduced level of GRP78 and lysosomal ceramide that contributed to the stability of the lysosomal membrane. The endoplasmic reticulum (ER) stress concomitant with the inhibition of GRP78 was clearly monitored by the phosphorylation of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), and inositol-requiring enzyme 1 α (IRE1α) kinases to indicate the activation of the unfolded protein response (UPR) signaling using immunofluorescence assay. On the other hand, the degradation of the lysosome was observed through PDT action by the Ru-1@TPP-PEG-biotin SAN treatment. This was confirmed by the co-localization assay showing the disappearance of cathepsin D and lysosomal-associated membrane protein 1 (LAMP1) in the lysosome.ConclusionsConsidering lysosome-mediated autophagy is an effective cancer cell survival mechanism, the degradation of the lysosome along with GRP78 inhibition by the Ru-1@TPP-PEG-biotin SAN combination therapy is suggested as a new co-targeting cancer treatment.

Impeded Molecular Reorganization by Polyethylene Glycol Conjugation Revealed by X-ray Reflectivity and Diffraction Measurements.

Polyethylene glycol (PEG) coatings have been widely applied in pharmaceutical and biomedical systems to prevent nonspecific protein absorption, increase vesicle blood circulation time, and sustain drug release. This study systematically investigated the planar interfacial organization of phospholipid monolayers containing various amounts of PEG conjugations before and after enzyme-catalyzed degradation of the lipids using X-ray reflectivity and grazing incidence X-ray diffraction techniques. Results showed that attaching PEG to the headgroup of the lipids up to 15 mol % had limited effects on molecular packing of the lipid monolayers in the condensed phase at the gas-liquid interface and negligible effects on the enzyme adsorption to the interface. After enzyme-catalyzed degradation, equimolar fatty acids and lyso PC were generated. The fatty acids together with the subphase Ca2+ self-assembled into highly organized multilayer domains at the interface. The X-ray measurements unambiguously revealed that the densely packed PEG markedly hindered microphase separation and formation of the palmitic acid-Ca2+ complexes.

THU0054 NKTR-358, A NOVEL IL-2 CONJUGATE, STIMULATES HIGH LEVELS OF REGULATORY T CELLS IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS

Background:Impaired IL-2 production and dysfunction of regulatory T cells (Tregs) have been identified as key immunological defects leading to the breakdown of immune self-tolerance in SLE. Low-dose IL-2 can expand Tregs, but the effect is limited by a narrow therapeutic window for Treg selectivity. Furthermore, the short half-life of IL-2 necessitates frequent administration. NKTR-358 is a polyethylene glycol (PEG) conjugate of recombinant human IL-2 (aldesleukin sequence) and is differentiated from native IL-2 by its altered binding to the IL-2 receptor and prolonged biological activity. NKTR-358 resulted in marked and selective stimulation of Tregs when administered as a single SC injection to healthy volunteers.Objectives:This multiple ascending dose study assessed the safety, tolerability, pharmacokinetics (PK), and immune effects of NKTR-358 in patients with SLE after repeated administration of SC doses. The time course and extent of changes in numbers and percentages of Tregs, conventional CD4+ and CD8+ T cells, NK cells, and cytokine levels in peripheral blood were investigated.Methods:In this double-blind, multiple ascending dose study, patients with mild to moderate SLE received 3 SC doses q2w in 4 cohorts ranging from 3.0 to 24.0 µg/kg (9 active:3 placebo per cohort); patients were followed for a total of 79 days.Results:There were no dose-limiting toxicities, deaths, or clinically significant abnormalities in either vital signs or electrocardiograms. Adverse events attributed to NKTR-358 were primarily limited to mild (grade 1) injection site reactions. At the highest dose, one subject had transient and mild (grade 1) symptoms of a flu-like syndrome after administration, without associated elevated cytokine levels, and another subject had dosing stopped due to elevated eosinophil levels. No other individual at any dose level had systemic signs or symptoms known to be associated with IL-2 therapy. No anti-drug antibodies were detected. NKTR-358 demonstrated dose-proportional PK with repeated dosing; plasma levels peaked 3-6 days post-dose and declined with a terminal half-life of ~10-13 days.The primary and consistent effect of NKTR-358 was seen on Tregs. In the four dose cohorts, dose-dependent and sustained increases in absolute numbers and percentages of circulating CD4+FoxP3+CD25brightTregs were observed. Treg levels remained elevated throughout the dosing period, peaking at Day 10 after the first administration of NKTR-358 and returning to baseline ~ 20-30 days following last administration. At 24.0 µg/kg, the mean peak increase in numbers of CD25brightTregs was 11-fold above baseline. In addition, there was an increase in Treg activation markers at doses ≥12.0 µg/kg. In contrast to effects on Tregs, no changes in percentages or numbers of conventional CD4+ or CD8+ T cells were observed at any dose tested. At the highest dose, there were low-level increases in the percentages and numbers of NK cells. Overall, NKTR-358 selectively induced Tregs, evidenced by a 12-fold increase in the mean peak Treg:CD8 ratio over baseline in the 24.0 µg/kg group.Conclusion:NKTR-358, an IL-2 conjugate Treg stimulator, was well tolerated when repeatedly administered (q2w) at doses up to 24 µg/kg. Its administration led to marked, selective, prolonged, and dose-dependent increases in circulating CD25brightTregs. This clinical study in SLE patients extends the previous results in healthy volunteers and provides strong support for continued testing of NKTR-358 as a new therapeutic in SLE and other inflammatory diseases.Disclosure of Interests:Suresh Siddhanti Shareholder of: Nektar Therapeutics, Employee of: Nektar Therapeutics, Christie Fanton Shareholder of: Nektar Therapeutics, Employee of: Nektar Therapeutics, Neha Dixit Shareholder of: Nektar Therapeutics, Employee of: Nektar Therapeutics, Lin Lu Shareholder of: Nektar Therapeutics, Employee of: Nektar Therapeutics, Vishala Chindalore Grant/research support from: Nektar Therapeutics for conducted studies, Speakers bureau: > 5 years ago, Robert Levin Grant/research support from: Payments for clinical research for industry-sponsored trials, Consultant of: Gilead, Exagen, Myriad Rheumatology, Speakers bureau: Sanofi/Genzyme, Regeneron, Bristol-Myers Squibb, AbbVie, Isam Diab: None declared, Richard Furie Grant/research support from: Nektar Therapeutics to Northwell Rheumatology to conduct this study, Consultant of: Nektar Therapeutics, Jonathan Zalevsky Shareholder of: Nektar Therapeutics, Employee of: Nektar Therapeutics, Brian Kotzin Shareholder of: Nektar Therapeutics, Employee of: Nektar Therapeutics

Calcium-modified carbon dots derived from polyethylene glycol: fluorescence-based detection of Trifluralin herbicide

In this work, fluorescent carbon dots were synthesized from polyethylene glycol. The carbon dots are of sizes below 10 nm as confirmed through dynamic light scattering studies and transmission electron microscope images. Carbon dot was further conjugated with Ca2+ to form metal-ion-conjugated polyethylene glycol carbon dot. The metal-ion-conjugated polyethylene glycol carbon dot exhibits enhanced photoluminescence properties. Fluorescence of the Ca-modified carbon dots was quenched in the presence of Trifluralin. The practical utility of the system was demonstrated by using it to detect Trifluralin contaminated soil sample. Also, an array of sensor plates prepared using the drop-casting method on glass plates showed visual changes in fluorescence in the presence of Trifluralin. Development of such fluorescent-based detection system will lead to visual detection kits for detecting herbicides in general. We developed a fluorescent-based metal conjugated polyethylene glycol carbon dot (Ca-PEG-CD) that was used to detect Trifluralin herbicide.

Sulfoxide-Containing Polymer-Coated Nanoparticles Demonstrate Minimal Protein Fouling and Improved Blood Circulation.

Minimizing the interaction of nanomedicines with the mononuclear phagocytic system (MPS) is a critical challenge for their clinical translation. Conjugating polyethylene glycol (PEG) to nanomedicines is regarded as an effective approach to reducing the sequestration of nanomedicines by the MPS. However, recent concerns about the immunogenicity of PEG highlight the demand of alternative low‐fouling polymers as innovative coating materials for nanoparticles. Herein, a highly hydrophilic sulfoxide‐containing polymer—poly(2‐(methylsulfinyl)ethyl acrylate) (PMSEA)—is used for the surface coating of iron oxide nanoparticles (IONPs). It is found that the PMSEA polymer coated IONPs have a more hydrophilic surface than their PEGylated counterparts, and demonstrate remarkably reduced macrophage cellular uptake and much less association with human plasma proteins. In vivo study of biodistribution and pharmacokinetics further reveals a much‐extended blood circulation (≈2.5 times longer in terms of elimination half‐life t 1/2) and reduced accumulation (approximately two times less) in the organs such as the liver and spleen for IONPs coated by PMSEA than those by PEG. It is envisaged that the highly hydrophilic sulfoxide‐containing polymers have huge potential to be employed as an advantageous alternative to PEG for the surface functionalization of a variety of nanoparticles for long circulation and improved delivery.

SAT-283 Generation of a Long-Acting Human Growth Hormone Receptor Antagonist by Site-Specific Pegylation

Growth hormone (GH) is a peptide hormone that mediates actions through binding to a cell surface GH receptor (GHR), activating key signalling pathways including the JAK/STAT pathway. Excess GH secretion leads to acromegaly and tumoral expression has been implicated in cancer progression, suggesting that GH is also a potential target for anticancer therapy. Pegvisomant is the only GHR antagonist approved for clinical use. This antagonist is a PEGylated form of a mutated GH (B2036) that binds and blocks the receptor. Conjugation to polyethylene glycol (PEG) at multiple amine residues reduces in vitro bioactivity but extends the serum half-life resulting in improved in vivo bioactivity. We investigated whether we could generate a long-acting PEGylated GHR antagonist through site-specific conjugation of PEG. A codon optimised GHR antagonist, with an introduced free cysteine residue at amino acid site 144 (S144C), was generated by gene synthesis and recombinantly engineered by gene fusion with thioredoxin. Recombinant protein was expressed in E. coli and purified using a series of chromatographic methods. Antagonists were PEGylated using cysteine-specific conjugation chemistry. In vitro activity was determined using a Ba/F3-GHR viability assay, and in vivo pharmacokinetic and bioactivity was determined in mice. Fusion to thioredoxin was found to improve soluble protein expression at 30℃, resulting in dramatically increased yield. After a series of purification steps, including Ni-NTA, 3C protease cleavage and ion-exchange chromatography, a single band with a molecular mass of 22 kDa was observed by SDS-PAGE analysis. The recombinant antagonist was conjugated to 20 kDa or 30 kDa-PEG at amino acid site S144C. After purification, a single band with an effective molecular size of approximately 60 kDa (PEG-20kDa conjugate) or 70 kDa (PEG-30kDa conjugate) was observed by SDS-PAGE analysis. The unconjugated antagonist inhibited the proliferation of Ba/F3-GHR cells in a dose-dependent manner with a half maximal inhibitory concentration (IC50) of 10.1 ± 2.5 nM. Following PEGylation and purification, the PEG-20kDa and PEG-30kDa conjugates retained high in vitro bioactivity with an IC50 of 66.2 ± 3.8 nM and 106.1 ± 7.1 nM, respectively. Pharmacokinetic analysis demonstrated that PEGylation increased the serum half-life to approximately 15 hours in mice. Subcutaneous administration of the PEG-30kDa conjugate (10 mg/kg/day) reduced serum IGF-I levels in mice. In conclusion, we have generated a novel long-acting human GHR antagonist conjugate by introducing a free cysteine at a non-essential site of the antagonist and targeted attachment of PEG.

Reduction sensitive nanocarriers mPEG-g-γ-PGA/SSBPEI@siRNA for effective targeted delivery of survivin siRNA against NSCLC

Poly γ-glutamic acid (γ-PGA) is attractive due to its desirable biological properties such as nontoxicity, excellent biocompatibility, and minimal immunogenicity. Additionally, γ-PGA could be recognized by γ-glutamyl transpeptidase, which is regarded as a potential biomarker for many tumors. In this study, we have developed a new biodegradable, reduction sensitive, and tumor-specific gene nano-delivery platform consisting of a cationic carrier (SSBPEI) for siRNA condensation, mPEG shell for nanoparticle stabilization, and γ-PGA for accelerated cellular uptake. Disulfide bonds (-SS-) could be reduced specifically in the tumor environment, which is full of reductants such as glutathione reductase. Conjugating polyethylene glycol (PEG) to the γ-PGA led to the formation of mPEG-g-γ-PGA, with a decreased positive charge on the surface of SSBPEI@siRNA and substantially higher stability in an aqueous medium. As a result, mPEG-g-γ-PGA/SSBPEI@siRNA nanoparticles could protect siRNAs from RNase A degradation and release siRNAs in a reduction sensitive way. The multifunctional delivery system was shown to silence the Survivin gene and further promote chemotherapeutic drug-induced apoptosis in the A549 NSCLC cell line efficiently, thereby representing a novel promising platform for the delivery of siRNAs.

Self-Therapeutic Nanoparticle That Alters Tau Protein and Ameliorates Tauopathy Toward a Functional Nanomedicine to Tackle Alzheimer's.

Tauopathy is a complex disorder associated at the junction of several other pathologies. Intrinsically disordered tau protein remains therapeutically challenging due to its undruggable nature and is a possible reason for monumental failure of several tau-based therapies. Herein, nanogold remodeled tau is reported as a pseudo-nanochaperon and shows therapeutic benefit by passive targeting in transgenic tau P301L mutant mice. Treatment with nanogold polyethylene glycol (Au-PEG) conjugate moderately improves the learning ability of the tau P301L mice that corroborates with diminished phosphorylated tau burden. Circulating total tau level that acts in a prion fashion is significantly reduced upon Au-PEG treatment. Similarly, a high level of tau is found in macaque monkey serum and Au-PEG inhibits amyloidosis of Alzheimer's patients and primate's serum samples ex vivo. Addtionally, brain MRI of an old aged macaque monkey shows the decrease of grey matter, which correlates with mutual loss of grey matter upon progressive dementia as reported. Au-PEG tunes tau and other circulating pro-dementia factors that are present in human AD serum, by remodeling the protein and repairing aberrant proteostasis. Alteration of proteotoxic tau function by nanogold as a kinetic stablizer holds translational potential to combat socially challenging dementia.

Plasma Polyethylene Glycol (PEG) Levels Reach Steady State Following Repeated Treatment with N8-GP (Turoctocog Alfa Pegol; Esperoct®).

BackgroundExtended half-life (EHL) factor VIII (FVIII)-replacement therapies enable patients with haemophilia A to maintain higher activity levels with fewer injections. N8-GP (turoctocog alfa pegol; Esperoct®) is an EHL product derived from conjugation of polyethylene glycol (PEG) to a recombinant FVIII protein. Upon activation, PEG is released from the active protein and excreted in urine and faeces. While PEG levels are expected to reach steady state with repeated dosing, there has been some discussion regarding whether abnormal accumulation of PEG in plasma and tissues may occur.ObjectiveOur objective was to examine plasma PEG concentrations in rats and humans repeatedly treated with N8-GP for periods of up to 5 years.MethodsPEG levels were measured using liquid chromatography-tandem mass spectrometry in plasma samples from rats treated with N8-GP as part of a 52-week toxicity study. Human plasma samples from children, adolescents and adults treated with N8-GP as part of the pathfinder programme were also examined (NCT01731600; NCT01480180). These data were compared with steady-state PEG levels predicted by pharmacokinetic modelling of single-dose rat data.ResultsPEG levels reached steady state in plasma in both rats and humans after repeated dosing. The timing and degree of PEG increase to steady state were in line with or below model predictions, confirming the utility of the pharmacokinetic model and indicating that rat data can be used to estimate human plasma PEG levels.ConclusionSteady-state PEG levels were reached in plasma from rats and humans repeatedly treated with N8-GP. No unexpected increase in PEG was observed.

Antibodies against polyethylene glycol in human blood: A literature review

Polyethylene glycol (PEG) conjugation, i.e. PEGylation, is a successful strategy to improve the pharmacokinetics and pharmacodynamics of biopharmaceuticals. In the past few decades, PEGylation technology has developed tremendously, and >15 PEGylated therapeutics have been brought to market, with more in development. However, the widely accepted assumption that PEG would have no antigenicity or immunogenicity is increasingly challenged with popularization of PEGylation technique. Although PEGylation indeed reduces the immunogenicities of the modified molecules, and even appears to completely eliminate their immunogenicities, yet emerging clinical evidence of anti-PEG antibodies (including both pre-existing and PEGylated therapeutics-treatment induced anti-PEG antibodies) have been attracted more and more attention. Anti-PEG antibodies were detected in not only patients treated with PEGylated therapeutics but also PEGylated drugs treatment-naïve individuals with a prevalence from <1% to 72%. In patients, the existing anti-PEG antibodies may attenuate therapeutic efficacy of PEGylated drugs and increase adverse effects. Although there is no golden standard avenue, several types of methods, including passive hemagglutination, Western Blot, enzyme linked immunosorbent assay, flow cytometry, Meso Scale Discovery technology, Acoustic Membrane Microparticle assay, and surface plasmon resonace technique, were established and used to screen, confirm and quantitatively detect anti-PEG antibodies. Herein, we focused on reviewing the prevalence of anti-PEG antibodies in healthy and PEGylated therapeutics-treated patients, and highlighting the detection methods for pre-screening and quantitative detection of anti-PEG antibodies.

A Tumor Microenvironment-Responsive Biodegradable Mesoporous Nanosystem for Anti-Inflammation and Cancer Theranostics.

A nanoplatform that integrates diagnostic and therapeutic functions with intrinsic tumor microenvironment-responsive biodegradability is highly desired. Herein, a biodegradable nanotheranostic agent based on hollow mesoporous organosilica nanoparticles (HMONs), followed by encapsulating of heat shock protein 90 (Hsp 90) inhibitor is described. Then, the pore-engineering including gating with bovine serum albumin-iridium oxide nanoparticles (BSA-IrO2 ) and conjugation of polyethylene glycol (PEG) is conducted to yield 17AAG@HMONs-BSA-IrO2 -PEG (AHBIP) nanotheranostics for multimode computed tomography (CT)/photoacoustic (PA) imaging-guided photodynamic therapy (PDT) and low-temperature photothermal therapy (PTT). Such nanoplatforms show extraordinary photothermal conversion efficiency, high cargo loading (35.4% for 17AAG), and stimuli-responsive release of 17AAG for inhibition of Hsp90, which induces cell apoptosis at low-temperatures (≈41 °C). Also, the IrO2 simultaneously endows the nanotheranostics with catalytic activity in triggering the decomposition of H2 O2 into O2 and thus reducing the tumor hypoxia, as well as protecting normal tissues against H2 O2 -induced inflammation. AHBIP shows good photocatalysis activity for PDT as a result of the generation of superoxide anion by laser irradiation. The resulting AHBIP-mediated synergistic PTT/PDT offers an outstanding therapeutic outcome both in vitro and in vivo. Overall, the incorporation of the BSA-IrO2 and biodegradable HMONs into one nanoplatform has great potential for clinical applications.

Preparation of Polyethylene Glycol-Ginsenoside Rh1 and Rh2 Conjugates and Their Efficacy against Lung Cancer and Inflammation.

Low solubility and tumor-targeted delivery of ginsenosides to avoid off-target cytotoxicity are challenges for clinical trials. In the present study, we report on a methodology for the synthesis of polyethylene glycol (PEG)-ginsenoside conjugates through a hydrolysable ester bond using the hydrophilic polymer polyethylene glycol with the hydrophobic ginsenosides Rh1 and Rh2 to enhance water solubility and passive targeted delivery. The resulting conjugates were characterized by 1H nuclear magnetic resonance (1H NMR) and Fourier-transform infrared spectroscopy (FT-IR). 1H NMR revealed that the C-6 and C-3 sugar hydroxyl groups of Rh1 and Rh2 were esterified. The conjugates showed spherical shapes that were monitored by field-emission transmission electron microscopy (FE-TEM), and the average sizes of the particles were 62 ± 5.72 nm and 134 ± 8.75 nm for PEG-Rh1and PEG-Rh2, respectively (measured using a particle size analyzer). Owing to the hydrophilic enhancing properties of PEG, PEG-Rh1 and PEG-Rh2 solubility was greatly enhanced compared to Rh1 and Rh2 alone. The release rates of Rh1 and Rh2 were increased in lower pH conditions (pH 5.0), that for pathophysiological sites as well as for intracellular endosomes and lysosomes, compared to normal-cell pH conditions (pH 7.4). In vitro cytotoxicity assays showed that the PEG-Rh1conjugates had greater anticancer activity in a human non-small cell lung cancer cell line (A549) compared to Rh1 alone, whereas PEG-Rh2 showed lower cytotoxicity in lung cancer cells. On the other hand, both PEG-Rh1 and PEG-Rh2 showed non-cytotoxicity in a nondiseased murine macrophage cell line (RAW 264.7) compared to free Rh1 and Rh2, but PEG-Rh2 exhibited increased efficacy against inflammation by greatly inhibiting nitric oxide production. Thus, the overall conclusion of our study is that PEG conjugation promotes the properties of Rh1 for anticancer and Rh2 for inflammation treatments. Depends on the disease models, they could be potential drug candidates for further studies.

Metronomic chemotherapy of carboplatin-loaded PEGylated MWCNTs: synthesis, characterization and in vitro toxicity in human breast cancer

Our objective of this study is to design and develop a polyethylene glycol (PEG2000)-modified multiwall carbon nanotube (PEGylated MWCNT) formulation for oral controlled metronomic chemotherapeutic drug delivery. Multiwall carbon nanotubes undergo various chemical modifications including oxidation with strong acids, conjugation of polyethylene glycol, and coating with cellulose acetate phthalate which resulted in the formation of aqueous dispersion and prevention of drug degradation in acidic environment. Advanced analytical procedure such as Fourier transform infra-red, X-ray diffraction, differential scanning calorimetry, thermal gravimetric analysis, transmission electron microscopy, and dynamic light scattering techniques were used to evaluate physicochemical characterization. We also performed in vitro cytotoxic study by MTT assay and results revealed that carboplatin-loaded PEGylated MWCNTs did not show significant detrimental effect on the viability of MDA-MB-231 (human breast cancer) cells. The maximum encapsulation and drug-loading capacity were determined to be 71.58 ± 0.04 and 39.62 ± 0.07%, respectively. The release of carboplatin from PEGylated MWCNTs was investigated at simulated intestinal fluid (SIF), pH 6.8, after optimizing at simulated gastric fluid (SGF), pH 1.2, by enteric coating. Enteric-coated PEGylated MWCNTs exhibit pH-responsive drug activity in a sustained manner especially at pH 6.8. This surface modification strongly suggests that PEGylated MWCNTs could be a potential carrier for metronomic chemotherapeutic agent for high drug resistance, drug with maximum adverse effect and poorly oral bioavailable drugs.

Current Considerations on Characterization of Immune Response to Multi-Domain Biotherapeutics.

Compounds containing two or more structural domains with a distinct mode of action relevant to functionality have been defined as multi-domain biotherapeutics (MDBs). Several modalities, including endogenous protein fusions with an antibody Fc fragment or another polypeptide, bispecific antibodies, antibody-drug conjugates, as well as polyethylene glycol conjugates have been viewed as examples of MDBs. Similar to other biotherapeutics, MDBs have the potential to induce a host immune response, commonly detected in the form of anti-drug antibodies (ADAs). The need to characterize ADA specificity to a particular domain of the MDB has been identified as a potential regulatory requirement based on the compound nature of the drug and associated immunogenicity risk factors. MDB-related immunogenicity risk factors are discussed herein. The relative risk level of each of the immunogenicity factors was analyzed based on publicly available information. It is proposed that MDB-related immunogenicity risk factors can be divided into major and minor categories. Major risk category factors include (a) presence of immunogenic structural or linear epitopes of either non-human or human sequence origin and (b) significant homology of an MDB domain to an endogenous protein with a specific and unique function. Proposed minor risk category factors include (a) epitope spread, (b) repetitive antigenic structure of MDB, and (c) hapten-like effect due to chemical conjugation or fusion with a larger protein. Detailed modality-based information on several examples of MDBs is presented to support this proposal.

A generic method for the detection of polyethylene glycol specific IgG and IgM antibodies in human serum

Detection of anti-drug antibodies is a critical step in the development of large molecule biopharmaceuticals. In the case of multicomponent/multifunctional molecules, such as fusion proteins and protein conjugates such as covalent polyethylene glycol (PEG)~protein conjugates, it is useful to further characterize anti-drug antibody (ADA) binding to key domains of the drug. The detection of anti-PEG antibodies poses special challenges that if overlooked can result in underreporting antibody responses. Here we describe the development and characterization of a novel ELISA to detect anti-PEG antibodies that provides a more complete interpretation of anti-PEG than other published methods. Being specific to the PEG moiety alone, this method is intended to detect anti-PEG antibodies independent of the protein to which PEG is conjugated.Based upon early indications that our assay could detect anti-PEG antibodies at a surprisingly high frequency in the general population, our emphasis throughout method development and validation was to ensure that non-specific signals and unintended interactions were not falsely contributing to detection of anti-PEG antibodies. Techniques, including orthogonal methods used to ensure that this ELISA detected antibodies specific to PEG included competition, immunodepletion, immunoprecipitation/western blot and an Octet kinetic binding analysis. The validated ELISA can detect 100 ng/mL of an anti-PEG IgG positive control and 800 ng/mL of an anti-PEG IgM positive control in the presence of 7.5 μg/mL of the PEGylated therapeutic (MW 64 kDa). The intra-assay percent co-efficient of variation (CV) and inter-assay CV of the low positive control samples in the screening method were 4.1 to 7.2% and 16.7 to 17.7%, respectively. Additional assay performance parameters that were validated are also described. When the validated assay was applied to a population of 200 healthy blood donors with no known exposure to biopharmaceutical PEG conjugates it indicated a pre-existing anti-PEG antibody prevalence of 97.5%. We suggest this surprising result is a consequence of exposure to PEG additives in everyday products, such as cosmetics, processed foods and over-the-counter (OTC) pharmaceuticals.

Impact of Site-Specific Bioconjugation on the Interfacial Activity of a Protein Biosurfactant.

Biosurfactants are surface active molecules that can be produced by renewable, industrially scalable biologic processes. DAMP4, a designer biosurfactant, enables the modification of interfaces via genetic or chemical fusion to functional moieties. However, bioconjugation of addressable amines introduces heterogeneity that limits the precision of functionalization as well as the resolution of interfacial characterization. Here, we designed DAMP4 variants with cysteine point mutations to allow for site-specific bioconjugation. The DAMP4 variants were shown to retain the structural stability and interfacial activity characteristic of the parent molecule, while permitting efficient and specific conjugation of polyethylene glycol (PEG). PEGylation results in a considerable reduction on the interfacial activity of both single and double mutants. Comparison of conjugates with one or two conjugation sites shows that both the number of conjugates as well as the mass of conjugated material impact the interfacial activity of DAMP4. As a result, the ability of DAMP4 variants with multiple PEG conjugates to impart colloidal stability on peptide-stabilized emulsions is reduced. We suggest that this is due to steric constraints on the structures of amphiphilic helices at the interface. Specific and efficient bioconjugation permits the exploration and investigation of the interfacial properties of designer protein biosurfactants with molecular precision. Our findings should therefore inform the design and modification of biosurfactants for their increasing use in industrial processes and nutritional and pharmaceutical formulations.

Renal-clearable ultrasmall covalent organic framework nanodots as photodynamic agents for effective cancer therapy

Covalent organic frameworks (COFs) and their derivatives represent an emerging class of crystalline porous materials with broad potential applications. However, the biomedical applications of them were limited by the large size, low dispersivity, poor bioavailability within cells and metabolic problems. Herein, renal-clearable ultrasmall COF nanodots have been synthesized and utilized as efficient cancer therapy agents. A simple liquid exfoliation strategy was used to prepare COF nanodots. After polyethylene glycol (PEG) conjugation, the PEG coated COF nanodots (COF nanodots-PEG) showed improved physiological stability and biocompatibility. In addition, the well isolated porphyrin molecules endowed COF nanodots-PEG good light-triggered reactive oxygen species production ability, which showed excellent photodynamic therapy efficiency with good tumor accumulation ability. In particular, due to the ultrasmall size, COF nanodots-PEG could be cleared from the body through the renal filtration with no appreciable in vivo toxicity. Our study highlights the potential of COFs-based nanoparticles for biomedical applications.

Chemistry Routes for Copolymer Synthesis Containing PEG for Targeting, Imaging, and Drug Delivery Purposes.

Polyethylene glycol (PEG) is one of the most frequently used polymers for coating nanocarriers to enhance their biocompatibility, hydrophilicity, stability, and biodegradability. PEG is now considered to be among the best biocompatible polymers. It offers sterical hindrance against other nanoparticles and blood components such as opsonin, preventing their macrophage phagocytosis and resulting in a prolonged circulation time in blood stream, consequently a ‘stealth character’ in vivo. Therefore, PEG has a very promising future for the development of current therapeutics and biomedical applications. Moreover, the vast number of molecules that PEG can conjugate with might enhance its ability to have an optimistic perspective for the future. This review will present an update on the chemistry used in the modern conjugation methods for a variety of PEG conjugates, such methods include, but are not limited to, the synthesis of targeting PEG conjugates (i.e., Peptides, Folate, Biotin, Mannose etc.), imaging PEG conjugates (i.e., Coumarin, Near Infrared dyes etc.) and delivery PEG conjugates (i.e., doxorubicin, paclitaxel, and other hydrophobic low molecular weight drugs). Furthermore, the type of nanoparticles carrying those conjugates, along with their biomedical uses, will be briefly discussed.

Influence of PEGylation on the Strength of Protein Surface Salt Bridges.

Conjugation of polyethylene glycol (PEGylation) is a well-known strategy for extending the serum half-life of protein drugs and for increasing their resistance to proteolysis and aggregation. We previously showed that PEGylation can increase protein conformational stability; the extent of PEG-based stabilization depends on the PEGylation site, the structure of the PEG-protein linker, and the ability of PEG to release water molecules from the surrounding protein surface to the bulk solvent. The strength of a noncovalent interaction within a protein depends strongly on its microenvironment, with salt-bridge and hydrogen-bond strength increasing in nonpolar versus aqueous environments. Accordingly, we wondered whether partial desolvation by PEG of the surrounding protein surface might result in measurable increases in the strength of a salt bridge near a PEGylation site. Here we explore this possibility using triple-mutant box analysis to assess the impact of PEGylation on the strength of nearby salt bridges at specific locations within three peptide model systems. The results indicate that PEG can increase the nearby salt-bridge strength, though this effect is not universal, and its precise structural prerequisites are not a simple function of secondary structural context, of the orientation and distance between the PEGylation site and salt bridge, or of salt-bridge residue identity. We obtained high-resolution X-ray diffraction data for a PEGylated peptide in which PEG enhances the strength of a nearby salt bridge. Comparing the electron density map of this PEGylated peptide versus that of its non-PEGylated counterpart provides evidence of localized protein surface desolvation as a mechanism for PEG-based salt-bridge stabilization.