Surface PEGylation Research Articles

Insulin loaded mucus permeating nanoparticles: Addressing the surface characteristics as feature to improve mucus permeation

AimIt was the aim of this study to combine two strategies – namely the virus-mimicking strategy and the surface PEGylation strategy – in order to generate highly mucus permeating nanocarriers for oral insulin delivery. MethodsChondroitin sulphate was covalently conjugated with poly(ethylene glycol) 5kDa at different degree of modification and with the functionalized polymers NPs were formulated via complexation with chitosan. NPs were characterized by particle size, zeta potential, surface hydrophilicity and permeation ability in porcine mucus and on excised mucosa. ResultsThe NPs presented a size between 510 and 670nm and a zeta potential between −1 and −5mV when dispersed in simulated intestinal fluid. The mucus permeation test revealed a correlation between the NPs hydrophilicity and their ability to move through mucus. A 5-fold higher amount of NPs with the higher degree of PEGylation could permeate through fresh mucus compared to non-PEGylated NPs. Moreover, highly PEGylated NPs showed a 3.7-fold greater ability to be retained in intestinal mucosa against buffer flow compared to unmodified NPs. Finally, insulin was incorporated with a payload of 2.18% and the release profile showed a 65% release within 4h. ConclusionsResults of this study provide strong evidence for the potential of combining different surface modification strategies in order to improve the mucus permeating properties of NPs for oral peptide delivery.

Methods of protein surface PEGylation under structure preservation for the emulsion-based formation of stable nanoparticles

We evaluated different methods for a high surface PEGylation of lysozyme. The resulting lipophilic enzymes can be used for the formation of stable nanoparticles.

Mussel-inspired PEGylated carbon nanotubes: biocompatibility evaluation and drug delivery applications.

Carbon nanotubes (CNTs) have been widely examined for biomedical applications. However, surface functionalization of CNTs with polymers is often required to improve their application performance. To obtain these CNT-based polymer nanocomposites, surface-initiated polymerization strategies are generally adopted. However, all of these methods rely on the surface oxidation of CNTs, which is a rather complex and time-consuming procedure, and involves hazardous reagents. In this work, a facile and efficient bio-inspired strategy was developed for surface PEGylation of CNTs via a combination of mussel-inspired chemistry and the Michael addition reaction. The potential biomedical applications of these PEGylated CNTs were evaluated for intracellular delivery of a normally used anticancer drug (Doxorubicin hydrochloride). Two steps were involved in this strategy, which included the surface coating of CNTs with polydopamine (PDA) through self-polymerization of dopamine, and a Michael addition reaction between the PDA-coated CNTs (CNT-PDA) and amino-functionalized polymers, which were obtained by free radical polymerization using poly(ethylene glycol) methyl methacrylate and N-(3-aminopropyl) methacrylamide as monomers. Results suggested that these PEGylated CNTs are well dispersed in aqueous solution and showed improved biocompatibility toward cancer cells. On the other hand, we also demonstrated that DOX can be effectively loaded on these PEGylated CNTs and delivered into cells for cancer treatment. More importantly, this strategy can also be utilized for surface modification of many other materials with different polymers due to the strong and universal adhesion of PDA and designability of polymerization. Therefore, this method should be of great interest for the fabrication of multifunctional nanocomposites for biomedical applications.

Tumor Acidity-Sensitive Polymeric Vector for Active Targeted siRNA Delivery.

Although surface PEGylation of siRNA vectors is effective for preventing protein adsorption and thereby helps these vectors to evade the reticuloendothelial system (RES) in vivo, it also suppresses the cellular uptake of these vectors by target cells. This dilemma could be overcome by employing stimuli-responsive shell-detachable nanovectors to achieve enhanced cellular internalization while maintaining prolonged blood circulation. Among the possible stimuli, dysregulated pH in tumor (pHe) is the most universal and practical. However, the design of pHe-sensitive system is problematic because of the subtle differences between the pHe and pH in other tissues. Here, a simple acid-sensitive bridged copolymer is developed and used for tumor-targeted systemic delivery of siRNA. After forming the micelleplex delivery system, the corresponding nanoparticles (Dm-NP) might undergo several modifications as follows: (i) a poly(ethylene glycol) (PEG) corona, which is stable in the circulatory system and protects nanovectors from RES clearance; (ii) a pHe responsive linkage breakage, which induces PEG detachment at tumor sites and thereby facilitates cell targeting; and (iii) a cell-penetration peptide, which is exposed upon the removal of PEG and further enhances cellular uptake. Thus, Dm-NP achieved both prolonged circulation and effective accumulation in tumor cells and resulted in the safe and enhanced inhibition of non-small cell lung cancer growth.

Direct surface PEGylation of nanodiamond via RAFT polymerization

Nanodiamond (ND) is a novel class of carbon nanomaterials, which has been extensively investigated for biomedical applications because of its small size, high surface area and excellent biocompatibility. However, the biomedical applications of unmodified ND are still largely restricted because of their poor dispersibility in both aqueous and organic medium. In this work, we reported a novel strategy for the surface modification of ND via reversible addition fragmentation chain transfer (RAFT) polymerization. For preparation of the PEGylated ND (pPEGMA-ND), chain transfer agent (CTA) was immobilized onto ND through reaction between the hydroxyl group of ND and the carboxyl group of CTA, which was used as the initiator for surface-initiated RAFT polymerization. The successful preparation of pPEGMA-ND was characterized by nuclear magnetic resonance spectroscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectra and thermal gravimetric analysis in detail. Results demonstrated that pPEGMA-ND exhibited enhanced water dispersibility and desirable biocompatibility, making it promising for biomedical applications.

Human placental cell and tissue uptake of doxorubicin and its liposomal formulations.

The anticancer drug doxorubicin and its liposomal formulations are in clinical use, doxorubicin also during pregnancy. However, little is known about how doxorubicin and its liposomal formulations are taken up by placental cells and whether they can cross human placenta. We therefore investigated quantitative cellular uptake and toxicity of doxorubicin and its two liposomal formulations, pH-sensitive liposomal doxorubicin (L-DOX) and commercially available pegylated liposomal doxorubicin (PL-DOX), in human placental choriocarcinoma (BeWo) cells. PL-DOX showed significantly lower cellular uptake and toxicity compared with doxorubicin and L-DOX. In preliminary studies with human placental perfusion, PL-DOX did not cross the placenta at all in 4h, whereas doxorubicin and L-DOX crossed the placenta at low levels (max 12% of the dose). Furthermore, PL-DOX did not accumulate in placental tissue while doxorubicin did (up to 70% of the dose). Surface pegylation probably explains the low placental cell and tissue uptake of PL-DOX. Formulation of doxorubicin thus seems to enable a decrease of fetal exposure.

Binding and conformation of dendrimer-based drug delivery systems: a molecular dynamics study

All atomistic molecular dynamics simulations were performed on poly (amidoamine) (PAMAM) dendrimers that compound non-covalently with anticancer drug molecules including DOX, MTX, CE6, and SN38. The binding energies as well as their associated interaction energies and deformation energies were combined to evaluate the relative binding strength among drug, PAMAM, and PEG chains. We find that the deformation of dendrimers due to drug loading plays a crucial role in the drug binding. It is energetically favorable for the drug molecules to bind with PAMAM while the drugs bind with PEG metastable chains via kinetic confinement. Surface PEGylation helps dendrimers to accommodate more drug molecules with greater strength without inducing too much expansion. This work indicates that tuning the functionalized terminal groups of dendrimers is critical to design efficient dendrimer-based drug delivery systems.

Bifunctionalized dextrans for surface PEGylation via multivalent host–guest interactions

The main goal of this work was to develop a supramolecular chemistry strategy to decorate interfaces with polyethylene glycol (PEG) grafts. A series of novel bifunctionalized dextrans, bearing 40–60 PEG pending chains and 12–24 hydrophobic adamantyl groups, have been prepared by copper(I)-catalyzed azide-alkyne cycloaddition. Their binding properties toward native βCD and βCD polymers were characterized both in solution and at interface using isothermal titration microcalorimetry and surface plasmon resonance. The polymers were found to form stable layers onto neutral and positively charged βCD-polymer films pre-adsorbed on gold surfaces, due to multivalent interpolymer host–guest interactions. The thickness and stability of the layers could be tuned by varying the ratio between the degrees of substitution by PEG and adamantyl groups.

Organic coating of 1–2-nm-size silicon nanoparticles: Effect on particle properties

Photoluminescent silicon nanoparticles 1–2 nm in size were synthesized by a wet chemical procedure and derivatized with propylamine (NH2SiNP). Surface NH2 groups were used as linkers for additional poly(ethylene glycol) (PEG) and folic acid (Fo) attachment (PEG-NHSiNP and Fo-NHSiNP, respectively) to enable efficient targeting of the particles to tumors and inflammatory sites. The particles were characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, ζ potential, dynamic light scattering, and time-resolved anisotropy.The photophysical properties and photosensitizing capacity of the particles and their interaction with proteins was dependent on the nature of the attached molecules. While PEG attachment did not alter the photophysical behavior of NH2SiNP, the attachment of Fo diminished particle photoluminescence. Particles retained the capacity for 1O2 generation; however, efficient 1O2 quenching by the attached surface groups may be a drawback when using these particles as 1O2 photosensitizers. In addition, Fo attachment provided particles with the capacity to generate the superoxide anion radical (O −2 ).The particles were able to bind tryptophan residues of bovine serum albumin (BSA) within quenching distances. NH2SiNP and PEG–NHSiNP ground state complexes with BSA showed binding constants of (3.1 ± 0.3) × 104 and (1.3 ± 0.4) × 103 M−1, respectively. The lower value observed for PEG-NHSiNP complexes indicates that surface PEGylation leads to a reduction in protein adsorption, which is required to prevent opsonization. An increase in particle luminescence upon BSA binding was attributed to the hydrophobic environment generated by the protein. NH2SiNP-BSA complexes were also capable of resonance energy transfer.

Versatile, tannic acid-mediated surface PEGylation for marine antifouling applications.

In this study, we report a facile and versatile approach to the formation of marine antifouling surface coatings. The approach consists of a combined coating of polydopamine (pDA) and tannic acid (TA) and subsequent immobilization of polyethylene glycol (PEG) on solid substrates. TA coating of a pDA-coated surface was carried out using iron(III) coordination chemistry, and PEG was immobilized on the TA-coated surface via hydrogen bond formation. Stainless steel and nylon were successfully modified by this approach, and the resulting substrates were used for marine antifouling applications, in which diatom adhesion was significantly inhibited. Advantageously, this approach allowed marine antifouling coatings to be prepared by a simple immersion process under environmentally friendly conditions.

Surface PEGylation on PLA membranes via micro-swelling and crosslinking for improved biocompatibility/hemocompatibility

A feasible and efficient strategy was developed to enable persistent PEGylation on a PLA membrane surface via micro-swelling and subsequent UV-initiated crosslinking of poly(ethylene glycol) diacrylate.

Biocompatible PEGylated MoS2 nanosheets: Controllable bottom-up synthesis and highly efficient photothermal regression of tumor

Two-dimensional transition metal dichalcogenides, particularly MoS2 nanosheets, have been deemed as a novel category of NIR photothermal transducing agent. Herein, an efficient and versatile one-pot solvothermal synthesis based on “bottom-up” strategy has been, for the first time, proposed for the controlled synthesis of PEGylated MoS2 nanosheets by using a novel “integrated” precursor containing both Mo and S elements. This facile but unique PEG-mediated solvothermal procedure endowed MoS2 nanosheets with controlled size, increased crystallinity and excellent colloidal stability. The photothermal performance of nanosheets was optimized via modulating the particulate size and surface PEGylation. PEGylated MoS2 nanosheets with desired photothermal conversion performance and excellent colloidal and photothermal stability were further utilized for highly efficient photothermal therapy of cancer in a tumor-bearing mouse xenograft. Without showing observable in vitro and in vivo hemolysis, coagulation and toxicity, the optimized MoS2-PEG nanosheets showed promising in vitro and in vivo anti-cancer efficacy.

Tuning PEGylation of mixed micelles to overcome intracellular and systemic siRNA delivery barriers

A series of endosomolytic mixed micelles was synthesized from two diblock polymers, poly[ethylene glycol-b-(dimethylaminoethyl methacrylate-co-propylacrylic acid-co-butyl methacrylate)] (PEG-b-pDPB) and poly[dimethylaminoethyl methacrylate-b-(dimethylaminoethyl methacrylate-co-propylacrylic acid-co-butyl methacrylate)] (pD-b-pDPB), and used to determine the impact of both surface PEG density and PEG molecular weight on overcoming both intracellular and systemic siRNA delivery barriers. As expected, the percent PEG composition and PEG molecular weight in the corona had an inverse relationship with mixed micelle zeta potential and rate of cellular internalization. Although mixed micelles were internalized more slowly, they generally produced similar gene silencing bioactivity (∼80% or greater) in MDA-MB-231 breast cancer cells as the micelles containing no PEG (100D/no PEG). The mechanistic explanation for the potent bioactivity of the promising 50 mol% PEG-b-DPB/50 mol% pD-b-pDPB (50D) mixed micelle formulation, despite its relatively low rate of cellular internalization, was further investigated as a function of PEG molecular weight (5 k, 10 k, or 20 k PEG). Results indicated that, although larger molecular weight PEG decreased cellular internalization, it improved cytoplasmic bioavailability due to increased intracellular unpackaging (quantitatively measured via FRET) and endosomal release. When delivered intravenously in vivo, 50D mixed micelles with a larger molecular weight PEG in the corona also demonstrated significantly improved blood circulation half-life (17.8 min for 20 k PEG micelles vs. 4.6 min for 5 kDa PEG micelles) and a 4-fold decrease in lung accumulation. These studies provide new mechanistic insights into the functional effects of mixed micelle-based approaches to nanocarrier surface PEGylation. Furthermore, the ideal mixed micelle formulation identified (50D/20 k PEG) demonstrated desirable intracellular and systemic pharmacokinetics and thus has strong potential for in vivo therapeutic use.

Magneto-fluorescent core-shell supernanoparticles.

Magneto-fluorescent particles have been recognized as an emerging class of materials that exhibit great potential in advanced applications. However, synthesizing such magneto-fluorescent nanomaterials that simultaneously exhibit uniform and tunable sizes, high magnetic content loading, maximized fluorophore coverage at the surface, and a versatile surface functionality has proven challenging. Here we report a simple approach for co-assembling magnetic nanoparticles with fluorescent quantum dots to form colloidal magneto-fluorescent supernanoparticles. Importantly, these supernanoparticles exhibit a superstructure consisting of a close packed magnetic nanoparticle “core” which is fully surrounded by a “shell” of fluorescent quantum dots. A thin layer of silica-coating provides high colloidal stability and biocompatiblity and a versatile surface functionality. We demonstrate that after surface pegylation, these silica-coated magneto-fluorescent supernanoparticles can be magnetically manipulated inside living cells while being optically tracked. Moreover, our silica-coated magneto-fluorescent supernanoparticles can also serve as an in vivo multi-photon and magnetic resonance dual-modal imaging probe.

Combined effects of PEGylation and particle size on uptake of PLGA particles by macrophage cells

Objective: At the present study, relationship between phagocytosis of PLGA particles and combined effects of particle size and surface PEGylation was investigated. Materials and Methods: Microspheres and nanospheres (3500 nm and 700 nm) were prepared from three types of PLGA polymers (non-PEGylated and PEGylation percents of 9% and 15%). These particles were prepared by solvent evaporation method. All particles were labeled with FITC-Albumin. Interaction of particles with J744.A.1 mouse macrophage cells, was evaluated in the absence or presence of 7% of the serum by flowcytometry method. Results: The study revealed more phagocytosis of nanospheres. In the presence of the serum, PEGylated particles were phagocytosed less than non-PEGylated particles. For nanospheres, this difference was significant (P<0/05) and their uptake was affected by PEGylation degree. In the case of microsphere formulation, PEGylation did not affect the cell uptake. In the serum-free medium, the bigger particles had more cell uptake rate than smaller ones but the cell uptake rate was not influenced by PEGylation. Conclusion: The results indicated that in nanosized particles both size and PEgylation degree could affect the phagocytosis, but in micron sized particles just size, and not the PEGylation degree, could affect this.

Features of complement activation-related pseudoallergy to liposomes with different surface charge and PEGylation: Comparison of the porcine and rat responses

Pigs are known to provide a sensitive model for studying complement (C) activation-related pseudoallergy (CARPA), a hypersensitivity reaction to liposomal and many other nanomedicines that limits their clinical use. The utility of rats as a CARPA model has, however, not been analyzed to date in detail. The present study compared the two models by inducing CARPA with i.v. bolus injections of two reactogenic liposomes that differed from each other in surface properties: one was AmBisome, a strong anionic, free-surface small unilamellar liposome (SUV), while the other was neutral, polyethylene glycol (PEG)-grafted SUV wherein the 2kDa-PEG was anchored to the membrane via cholesterol (Chol-PEG). Both in pigs and rats AmBisome caused significant consumption of C3, indicating C activation, along with paralleling massive changes in blood pressure, white blood cell, platelet counts and in plasma thromboxane B2 levels, indicating CARPA. These processes were similar in the two species in terms of kinetics, but significantly differed in the doses that caused major hemodynamic changes (~0.01 and ~22mg phospholipid (PL)/kg in pigs and rats, respectively). Pigs responded to AmBisome with pulmonary hypertension and systemic hypotension, and the reaction was not tachyphylactic. The major response of rats was systemic hypotension, leukopenia followed by leukocytosis, and thrombocytopenia. Chol-PEG liposomes caused severe reaction in pigs at 0.1mg/kg, while the reaction they caused in rats was mild even at 300mgPL/kg. Importantly, the reaction to Chol-PEG in pigs was partly tachyphylactic. These observations highlight fundamental differences in the immune mechanisms of porcine and rat CARPA, and also show a major impact of liposome surface characteristics, determining the presence or absence of tachyphylaxis. The data suggest that rats are 2–3 orders of magnitude less sensitive to liposomal CARPA than pigs; however, the causes of these differences, the PEG-dependent tachyphylaxis and the massive reactivity of Chol-PEG liposomes remain unclear.

Adjustable bioadhesive control of PEGylated hyperbranch brushes on polystyrene microplate interface for the improved sensitivity of human blood typing.

A PEGylated 96-well polystyrene (PS) microplate was first introduced for applications in high-throughput screening for selective blood typing to minimize the risks in blood transfusions. Herein, we present a hemocompatible PS 96-well microplate with adjustable PEGylated hyperbranch brush coverage prepared by ozone pretreated activation and thermally induced surface PEGylation. The grafting properties, hydration capacity, and blood compatibility of the PEGylated hyperbrush immobilized PS surfaces in human blood were illustrated by the combined chemical and physical properties of the surface, and the dependence of the specific absorption of human plasma fibrinogen onto the PEGylated surfaces on the grafting density was analyzed by monoclonal antibodies. The surface coverage of PEGylated brushes plays a major role in the bioadhesive properties of modified PS microplates, which in turn control the level of agglutination sensitivity in blood typing. The bioadhesive resistance toward proteins, platelets, and erythrocytes in human whole blood showed a correlation to the controlled hydration properties of the PEGylated hyperbrush-modified surfaces. Therefore, we suggested that the surface coverage of PEGylated hyperbrushes on PS surfaces can increase the sensitivity of cross-matching blood agglutination by up to 16-fold compared to that of the conventional 96-well virgin PS due to the regulated biorecognition of hematocrit and antibodies of the PEGylated hyperbrush-modified surfaces.

PEGylation of microbead surfaces reduces unspecific antibody binding in glycan-based suspension array

Glycan-based suspension array (SGA) is an “in-house” developed multi-target immunoassay, employing commercially available fluorescent microbeads as a solid support for unique chemically synthesized glycopolymers which capture naturally occurring human anti-glycan antibodies. SGA is a sensitive and reliable tool for the high-throughput screening of anti-glycan antibody alterations characteristic for a vast number of human diseases including cancer. However, unspecific background binding, for instance binding of non-target antibodies, is a common obstacle in such immunoassays. In an attempt to reduce unspecific background binding of serum (or plasma) antibodies, we prepared glycosylated microbeads modified with linear poly(ethylene glycols) (PEGs) of different lengths. We compared several kinds of PEG modifications: (a) partial side-chain substitution of glycopolymers by PEGs of different lengths, (b) end-point addition of biotin-linked PEGs to glycopolymer-coupled beads, and (c) linking of heterobifunctional PEGs to the bead surface prior to glycopolymer immobilization. Among the various modifications investigated, the direct modification of the bead surface with linear heterobifunctional PEGs, consisting of 23- and 60PEG-units significantly reduced the background binding. The end-point addition of biotin-linked PEGs, especially in the case of PEG consisting from 50PEG-units, helped to repel non-target binding caused by endogenous biotin. We observed unspecific binding predominantly for antibodies of IgG but of IgM class. The novel design of fluorescent microbeads allows the detection of human anti-glycan antibodies with increased specificity and opens new horizons for practical application of SGA as a diagnostic tool.

Monofunctional Stealth Nanoparticle for Unbiased Single Molecule Tracking Inside Living Cells

On the basis of a protein cage scaffold, we have systematically explored intracellular application of nanoparticles for single molecule studies and discovered that recognition by the autophagy machinery plays a key role for rapid metabolism in the cytosol. Intracellular stealth nanoparticles were achieved by heavy surface PEGylation. By combination with a generic approach for nanoparticle monofunctionalization, efficient labeling of intracellular proteins with high fidelity was accomplished, allowing unbiased long-term tracking of proteins in the outer mitochondrial membrane.

Enhanced Performance of Plasmid DNA Polyplexes Stabilized by a Combination of Core Hydrophobicity and Surface PEGylation.

Nonviral gene therapy has high potential for safely promoting tissue restoration and for treating various genetic diseases. One current limitation is that conventional transfection reagents such as polyethylenimine (PEI) form electrostatically stabilized plasmid DNA (pDNA) polyplexes with poor colloidal stability. In this study, a library of poly(ethylene glycol-b-(dimethylaminoethyl methacrylate-co-butyl methacrylate)) [poly(EG-b-(DMAEMA-co-BMA))] polymers were synthesized and screened for improved colloidal stability and nucleic acid transfection following lyophilization. When added to pDNA in the appropriate pH buffer, the DMAEMA moieties initiate formation of electrostatic polyplexes that are internally stabilized by hydrophobic interactions of the core BMA blocks and sterically stabilized against aggregation by a PEG corona. The BMA content was varied from 0% to 60% in the second polymer block in order to optimally tune the balance of electrostatic and hydrophobic interactions in the polyplex core, and polymers with 40 and 50 mol% BMA achieved the highest transfection efficiency. Diblock copolymers were more stable than PEI in physiologic buffers. Consequently, diblock copolymer polyplexes aggregated more slowly and followed a reaction-limited colloidal aggregation model, while fast aggregation of PEI polyplexes was governed by a diffusion-limited model. Polymers with 40% BMA did not aggregate significantly after lyophilization and produced up to 20-fold higher transfection efficiency than PEI polyplexes both before and after lyophilization. Furthermore, poly(EG-b-(DMAEMA-co-BMA)) polyplexes exhibited pH-dependent membrane disruption in a red blood cell hemolysis assay and endosomal escape as observed by confocal microscopy.Lyophilized polyplexes made with the lead candidate diblock copolymer (40% BMA) also successfully transfected cells in vitro following incorporation into gas-foamed polymeric scaffolds. In summary, the enhanced colloidal stability, endosomal escape, and resultant high transfection efficiency of poly(EG-b-(DMAEMA-co-BMA))-pDNA polyplexes underscores their potential utility both for local delivery from scaffolds as well as systemic, intravenous delivery.