Biotinylated Poly Ethylene Glycol Research Articles

Biotinylated polyaminoacid-based nanoparticles for the targeted delivery of lenvatinib towards hepatocarcinoma

In this work, we describe the development of targeted polymeric nanoparticles loaded with lenvatinib for the treatment of hepatocellular carcinoma (HCC). A synthetic brush copolymer (PHEA-g-BIB-pButMA-g-PEG-biotin) was synthesized from α-poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA) by a three-step reaction involving atom transfer radical polymerisation (ATRP) to graft hydrophobic polybutylmethacrylate pendant groups and further conjugation with biotinylated polyethylene glycol via carbonate ester. Subsequently, lenvatinib-loaded nanoparticles were obtained and characterized demonstrating colloidal size, negative zeta potential, biotin exposure on the surface and the ability to release lenvatinib in a sustained manner. Lenvatinib-loaded nanoparticles were tested in vitro on HCC cells to evaluate their anticancer efficacy compared to free drug. Furthermore, the enhanced in vivo efficacy of lenvatinib-loaded nanoparticles on nude mice HCC xenograft models was demonstrated by evaluating tumor burdens, apoptotic markers and histological scores after administration of lenvatinib-nanoparticles via intraperitoneal or oral route. Finally, in vivo biodistribution studies were performed, demonstrating the ability of the prepared drug delivery systems to significantly accumulate in the solid tumor by active targeting, due to the presence of biotin on the nanoparticle surface.

Biotin-modified acid-sensitive micelles for enhancing antitumor effect of paclitaxel

This study was aimed at fabricating tumor targeting and acid-responsive micelles as carriers for paclitaxel (PTX). An amphipathic polymer TOS-Ace-PEG-Bio was designed and synthesized by conjugating α-tocopherol succinate (TOS) with biotinylated polyethylene glycol (PEG-Bio) via acetal bond. The polymer could self-assemble into nanosized micelles and encapsulate PTX. The PTX-loaded micelles possessed appropriate particle size with the mean value of 129.3 ± 0.9 nm. Strong pH dependent PTX release behavior was verified by in vitro drug release study. At cellular level, the PTX-loaded micelles could be selectively internalized by MCF-7 cells. The in vivo study also confirmed their excellent antitumor activity and reduced side effects. Based on these features, the TOS-Ace-PEG-Bio micelles were considered to be promising carriers for delivery of PTX.

Hypoxia-alleviating hemoglobin nanoclusters for sensitizing chemo-photodynamic therapy of cervical cancer

Hypoxia-alleviating hemoglobin (Hb) nanoclusters (NCs) have been developed for sensitizing combined chemo-photodynamic therapy (chemo-PDT) of cervical cancer. Hb was conjugated with chlorin e6 and biotinylated polyethylene glycol and adsorbed with doxorubicin (DOX), resulting in the self-assembly of 220 nm diameter protein NCs. These oxygen-carrying NCs, namely DOX@HPBC, exhibited improved colloidal stability in the serum compared to that of native Hb and pH-responsive drug release favorable for cancer treatment. DOX@HPBC alleviated hypoxia by 64.8% in HeLa cells cultured under hypoxia and normalized the levels of related biomarkers, HIF-1α and MDR1, with enhanced cellular uptake via biotin–receptor interactions. Moreover, antitumor efficacy tests performed in HeLa monolayer and spheroid cultures revealed that DOX@HPBC possessed 3.8-fold improved therapeutic efficacy compared to DOX and chlorin e6 physical mixture, showing synergism between chemotherapy and PDT. DOX@HPBC-mediated chemo-PDT significantly suppressed tumor growth in heterotopic (96.6% reduction in tumor volume compared to no-intervention) and orthotopic (comparable uterus weight to the normal group) HeLa-xenografted mice with no significant toxicity. These results showed the hypoxia amelioration effect, which lowered HIF-1α and MDR1 levels in tumor tissues, and efficient DOX distribution to hypoxic regions of the tumor, suggesting that DOX@HPBC could be a promising chemo-PDT strategy for cervical cancer treatment.

Tuning gold-based surface functionalization for streptavidin detection: A combined simulative and experimental study.

A rationally designed gold-functionalized surface capable of capturing a target protein is presented using the biotin-streptavidin pair as a proof-of-concept. We carried out multiscale simulations to shed light on the binding mechanism of streptavidin on four differently biotinylated surfaces. Brownian Dynamics simulations were used to reveal the preferred initial orientation of streptavidin over the surfaces, whereas classical molecular dynamics was used to refine the binding poses and to investigate the fundamental forces involved in binding, and the binding kinetics. We assessed the binding events and the stability of the streptavidin attachment through a quartz crystal microbalance with dissipation monitoring (QCM-D). The sensing element comprises of biotinylated polyethylene glycol chains grafted on the sensor's gold surface via thiol-Au chemistry. Finally, we compared the results from experiments and simulations. We found that the confined biotin moieties can specifically capture streptavidin from the liquid phase and provide guidelines on how to exploit the microscopic parameters obtained from simulations to guide the design of further biosensors with enhanced sensitivity.

Functionalization of Biotinylated Polyethylene Glycol on Live Magnetotactic Bacteria Carriers for Improved Stealth Properties.

Simple SummaryThe development of new approaches in the field of drug delivery systems is primarily based on increasing the accuracy and precision of the targeted site and improving the stability of the drug by preventing the phagocytosis process inside the body. Among many other methods used to fulfill the above-mentioned requirements, the use of magnetotactic bacteria (MTB) is proven to be a promising solution, as it is self-propelling in nature, and can also be controlled by an external magnetic field. For the present work, we developed an MTB/PEG–biotin complex by exploiting the process of covalent bond formation between bacteria and a biotin–PEG–NHS polymer. In addition to this, attachment efficacy and stability were also determined. In biological applications, cytotoxicity assay of THP-1 cells was performed, showing the MTB/PEG–biotin complex to be less harmful to the cells; meanwhile, to explore the stealth properties of the complex, we performed a cell association assay. With these results, we provide a significant contribution to the field of potential drug delivery system development.The early removal of drug delivery agents before reaching the affected target remains an area of interest to researchers. Several magnetotactic bacteria (MTB) have been used as self-propelled drug delivery agents, and they can also be controlled by an external magnetic field. By attaching the PEG–biotin polymer, the bacteria are turned into a stealth material that can escape from the phagocytosis process and reach the area of interest with the drug load. In the study, we developed a potential drug carrier by attaching the PEG–biotin to the MTB-through-NHS crosslinker to form a MTB/PEG–biotin complex. The attachment stability, efficacy, and bacterial viability upon attachment of the PEG–biotin polymer were investigated. Biological applications were carried out using a cytotoxicity assay of THP-1 cells, and the results indicate that the MTB/PEG–biotin complex is less harmful to cell viability compared to MTB alone. Along with cytotoxicity, an assay for cell association was also evaluated to assess the complex as a potential stealth material. The development of these complexes focuses on an easy, time-saving, and stable technique of polymer attachment with the bacteria, without damaging the cell’s surface, so as to make it a strong and reliable delivery agent.

Abstract P-17: Photophysical Properties of Freely Diffusing and Immobilized Fluorescent Conjugate Based on Calcium-Binding Protein Recoverin and Alexa647 Dye

Background: Recoverin is a calcium sensor membrane-associated protein that inhibits rhodopsin kinase thereby participating in the regulation of visual transduction. Here we examined calcium-induced conformational changes in recoverin conjugated with fluorescent dye Alexa647. Methods: Photophysical properties of immobilized and freely diffusing recoverin were investigated using fluorescence lifetime imaging microscopy and fluorescence emission spectroscopy. In solution, the formation and dissociation of the Ca2+-recoverin complex manifested as changes in Alexa647 spectra and the lifetime. In contrast, immobilization of recoverin on the microscopy glass via biotin–NeutrAvidin–biotinylated polyethylene glycol (PEG) tether inhibited changes in fluorescent signal. That can be provided by PEG as it prevented the calcium-induced changes in spectrum and lifetime of recoverin-bound Alexa647 in solution. The use of another immobilization facilitator, bovine serum albumin (BSA), did not affect calcium-induced changes in fluorescence of the conjugate in solution but produced the matrix, which was ineffective in recoverin immobilization. Results: Microscale thermophoresis demonstrated that biotinylated recoverin interacted with NeutrAvidin in solution indicating that immobilization affinity depended mainly on the geometry of the glass coating surface. Conclusion: Our results highlight the challenge of specific protein immobilization that does not affect protein functionality. By the example of recoverin, we showed that the employment of two common immobilization facilitators, PEG and BSA, yielded surfaces with different space geometry, which differently affect NeutrAvidin-based immobilization affinity as well as Ca2+-dependent conformational changes of the biotinylated protein.

Photodegradable avidin-biotinylated polymer conjugate hydrogels for cell manipulation.

Protein-synthetic polymer hybrid hydrogels crosslinked via protein-ligand binding are promising materials for the three-dimensional culture of various cells, while photo-responsive hydrogels have been widely used for the spatio-temporal control of cell functions and patterning. Photo-responsive protein-polymer hybrid hydrogels are therefore attractive candidates for use in cell and artificial tissue fabrication; however, no examples combining these properties have been reported to date. Herein, a photodegradable hydrogel consisting of avidin and biotinylated polyethylene glycol (PEG) was developed as a multi-functional matrix for cell culture and sorting. A four-branched PEG with a biotinylated photocleavable group at the end of each chain was crosslinked with avidin to produce a photodegradable hydrogel. A cytokine-dependent immunocyte was successfully cultured in the hydrogel by supplying cytokine from a medium layered on the hydrogel. Additionally, the adhesion and survival of fibroblasts could be controlled by decorating the hydrogel with a biotinylated cell-adhesive peptide. Cells embedded in the hydrogels could be recovered without cell damage as a result of light-induced hydrogel degradation. Moreover, model target cells expressing red fluorescent protein were selectively liberated from a hydrogel containing cells of different colors by irradiating with a targeted light. Owing to both the selective biotin-binding ability of avidin and the photocleavable properties of the synthetic polymer, the hydrogels were easy to prepare and decorate with functional molecules; they provided an internal structure suitable for cell culture, and allowed light-guided cell manipulation. The hydrogels are therefore expected to contribute to various cell fabrication processes as useful cell engineering and sorting tools.

Whole-Cell Pseudomonas aeruginosa Localized Surface Plasmon Resonance Aptasensor.

The detection of whole-cell Pseudomonas aeruginosa presents an intriguing challenge with direct applications in health care and the prevention of nosocomial infection. To address this problem, a localized surface plasmon resonance (LSPR) based sensing platform was developed to detect whole-cell Pseudomonas aeruginosa strain PAO1 using a surface-confined aptamer as an affinity reagent. Nanosphere lithography (NSL) was used to fabricate a sensor surface containing a hexagonal array of Au nanotriangles. The sensor surface was subsequently modified with biotinylated polyethylene glycol (Bt-PEG) thiol/PEG thiol (1:3), neutravidin, and biotinylated aptamer in a sandwich format. The 1:3 (v/v) ratio of Bt-PEG thiol/PEG thiol was specifically chosen to maximize PAO1 binding while minimizing nonspecific adsorption and steric hindrance. In contrast to prior whole-cell LSPR work, the LSPR wavelength shift was shown to be linearly related to bacterial concentration over the range of 10-103 cfu mL-1. This LSPR sensing platform is rapid (∼3 h for detection), sensitive (down to the level of a single bacterium), selective for detection of Pseudomonas strain PAO1 over other strains, and exhibits a clinically relevant dynamic range and excellent shelf life (≥2 months) when stored at ambient conditions. This versatile LSPR sensing platform should be extendable to a wide range of supermolecular analytes, including both bacteria and viruses, by switching affinity reagents, and it has potential to be used in point-of-care and field-based applications.

Monitoring growth and antibiotic susceptibility of Escherichia coli with photoluminescence of GaAs/AlGaAs quantum well microstructures

Development of quick and reliable methods to investigate antibiotic susceptibility of bacteria is vital to prevent inappropriate and untargeted use of antibiotics and control the antibiotic resistance crisis. The authors have developed an innovative, low-cost and rapid approach to evaluate antibiotic susceptibility of bacteria by employing photoluminescence (PL) emission of photocorroding GaAs/AlGaAs quantum well (QW) biochips. The biochips were functionalized with self-assembled monolayers of biotinylated polyethylene glycol thiols, neutravidin and biotinylated antibodies to immobilize bacteria. The illumination of a QW biochip with the above bandgap radiation leads to formation of surface oxides and dissolution of a limited thickness GaAs cap material (≤10nm) that results in the appearance of a characteristic maximum in the PL plot collected over time. The position of the PL maximum depends on the photocorrosion rate which, in turn, depends on the electric charge immobilized on the surface of the GaAs/AlGaAs biochips. Bacteria captured on the surface of biochips retard the PL maximum, while growth of these bacteria further delays the PL maximum. For the bacteria affected by antibiotics a faster occurring PL maximum, compared with growing bacteria, is observed. By exposing bacteria to nutrient broth and penicillin or ciprofloxacin, the authors were able to distinguish in situ antibiotic-sensitive and resistant Escherichia coli bacteria within less than 3h, considerable more rapid than with culture-based methods. The PL emission of the heterostructures was monitored with an inexpensive reader. This rapid determination of bacterial sensitivity to different antibiotics could have clinical and research applications.

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide.

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

GaAs/AlGaAs heterostructure based photonic biosensor for rapid detection of Escherichia coli in phosphate buffered saline solution

We have investigated photonic biosensing of bacteria based on photoluminescence (PL) of GaAs/AlGaAs heterostructures. The method takes advantage of the GaAs PL sensitivity to the perturbation of the semiconductor near-surface electric field induced by the charge of bacteria immobilized in its vicinity. Maintaining the balance between device sensitivity and stability in the biosensing (aqueous) environment is one of the key parameters allowing successful biosensing. To immobilize bacteria, we have employed a network of biotinylated antibodies interfaced with biotinylated polyethylene glycol thiols through the link provided by neutravidin. Post-processing of thiolated samples in ammonium sulfide was applied to increase the stability of the biosensing architectures while allowing biosensing at an attractive level of detection. Escherichia coli was detected in phosphate buffered saline solutions at 103CFU/mL; however, it appears that even greater sensitivity levels are feasible with this technique.

Comparative study of the ToF‐SIMS, FT‐IR and XPS techniques for quantitative analyses of mixed self‐assembled monolayers

We present a comparative study of the time‐of‐flight‐secondary ion mass spectrometry (ToF‐SIMS), Fourier transform infrared (FT‐IR) spectroscopy and X‐ray photoelectron spectroscopy (XPS) techniques on a mixed self‐assembled monolayer (SAM) of biotinylated polyethylene glycol alkane thiol (BPT) and 11‐mercaptoundecanol (MUD) as a model system. This mixed SAM was employed to maximize specific binding between the biotin groups in the mixed SAM and the streptavidin, while minimizing nonspecific adsorption of the streptavidin. For a better quantitative analysis of our mixed SAM by using ToF‐SIMS, we performed a principal component analyses (PCA) with various peak sets and found that the negative ions of the MUD complementary peak set showed good correlation between the XPS and FT‐IR quantification results and the scores for principal component 1 from a PCA of the ToF‐SIMS data. These findings show that ToF‐SIMS analysis along with PCA would make a good surface analysis technique for identifying intact molecules of adsorbates and for quantifying at once each adsorbate in a mixed SAM. Copyright © 2014 John Wiley & Sons, Ltd.

Micro-patterning of streptavidin-biotin-ampicillin/heparin on poly(tetrafluoroethylene) (PTFE) surfaces: simultaneous antimicrobial and anticoagulant activity.

Formation of heterogeneous and controllable surface patterns on polymeric materials containing antimicrobial and anticoagulant components represent an attractive way of maintaining synthetic materials "clean" from adverse bio-activities. The primary surface "contaminants" are microbial films as well as blood coagulation, both affecting not only performance of internal or external devices, but often exhibiting detrimental effects on patients. In an effort to simultaneously inhibit formation of microbial films and surface blood coagulation multifunctional assemblies containing streptavidin (STR)-biotin bioconjugates were developed on poly(tetrafluoroethylene) (PTFE) surfaces. Using STR conjugated to a biotin-functionalized PTFE surface, spatially controlled micro-patterning was produced by grafting biotinylated polyethylene glycol (B-PEG) to COOH modified PTFE (MA-PTFE), followed by inkjet micro-printing of biotinylated ampicillin (B-AM) and biotinylated heparin (B-HP) molecules. These surfaces exhibit simultaneous antimicrobial and anticoagulant attributes manifested by "zone of inhibition" and anticoagulant measurements. Quantitative spectroscopic analysis revealed that the required surface density of COOH groups on PTFE is 2.94 × 10-7 g cm-2, and B-PEG and STR densities of 9.2 × 10-8 g cm-2 and 3.5 × 10-8 g cm-2, respectively, are sufficient to achieve simultaneous antimicrobial and anticoagulant responses. These studies also showed that the force required to remove STR-biotin conjugates attached to PTFE surfaces measured by atomic force microscopy is approximately 1090 pN, thus providing desirable surface mechanical stability.

A prodrug nanoassembly entrapping drugs as a tumor-targeted delivery system

We presented here a targeted drug delivery system, biotinylated polyethylene glycol prodrug loading with free drugs (doxorubicin or camptothecin), which showed high drug loading capacity and superior targeted therapeutic efficacy for cancerous cells.

A photoluminescence-based quantum semiconductor biosensor for rapid in situ detection of Escherichia coli

This work describes a novel method of detecting Escherichia coli using photoluminescence (PL) emission from III–V quantum semiconductor (QS) devices functionalized with two different antibody-based architectures. The first approach employed self-assembled monolayers of biotinylated polyethylene glycol thiols to immobilize biotinylated antibody via neutravidin. In the second approach, we used QS microstructures coated with a thin layer of Si 3N 4 allowing direct functionalization with E. coli antibodies through hydrofluoric acid etching and glutaraldehyde-based reticulation. Atomic force, optical and fluorescence microscopy measurements were used to assess the immobilization process. Depending on the biosensing architecture, density of the immobilized bacteria was observed in the range of 0.5–0.7 bacteria/100 μm 2. The detection of E. coli at 10 4 CFU/ml was achieved within less than 120 min of the bacteria exposure. It is expected that an even better sensitivity threshold could be achieved following further optimization of the method.

Developing microcolin A analogs as biological probes

Three microcolin A and B analogs have been synthesized. Their biological activity profiles were evaluated against several cell lines, revealing the existence of a structural determinant whose role in mediating the antiproliferative effect of the microcolins has heretofore gone unrecognized. While previously described as potent immunosuppressive natural products, we found that these microcolin analogs possessed no selective cytotoxicity when comparing immune cell versus nonimmune cell proliferation. In addition, the amino-terminus of microcolin A has been modified to incorporate a biotinylated polyethylene glycol linker. The biological activity of this biotinylated microcolin A analog was determined. This affinity reagent was shown to retain limited biological activity and thus can serve as a useful probe for elucidating the mechanism of action of microcolin A.