Protein-resistant Properties Research Articles

Effects of Long-Term Water-Aging on Novel Anti-Biofilm and Protein-Repellent Dental Composite.

The aims of this study were to: (1) synthesize an anti-biofilm and protein-repellent dental composite by combining 2-methacryloyloxyethyl phosphorylcholine (MPC) with quaternary ammonium dimethylaminohexadecyl methacrylate (DMAHDM); and (2) evaluate the effects of water-aging for 180 days on protein resistance, bacteria-killing ability, and mechanical properties of MPC-DMAHDM composite. MPC and DMAHDM were added into a resin composite. Specimens were stored in distilled water at 37 °C for 1, 30, 90, and 180 days. Mechanical properties were measured in three-point flexure. Protein attachment onto the composite was evaluated by a micro bicinchoninic acid approach. An oral plaque microcosm biofilm model was employed to evaluate oral biofilm viability vs. water-aging time. Mechanical properties of the MPC-DMAHDM composite after 180-day immersion matched those of the commercial control composite. The composite with 3% MPC + 1.5% DMAHDM had much stronger resistance to protein adhesion than control (p < 0.05). MPC + DMAHDM achieved much stronger biofilm-eradicating effects than MPC or DMAHDM alone (p < 0.05). Biofilm colony-forming units on the 3% MPC + 1.5% DMAHDM composite were three orders of magnitude lower than commercial control. The protein-repellent and antibacterial effects were durable and showed no loss in water-aging from 1 to 180 days. The novel MPC-DMAHDM composite possessed strong and durable resistance to protein adhesion and potent bacteria-eradicating function, while matching the load-bearing ability of a commercial dental composite. The novel MPC-DMAHDM composite represents a promising means of suppressing oral plaque growth, acid production, and secondary caries.

Polydimethylsiloxane Substrates with Surfaces Decorated by Immobilized Hyaluronic Acids of Different Molecular Weight for Biomedical Applications

Polydimethylsiloxane elastomers are a promising class of materials for biomedical applications. However, the surface hydrophobicity of the elastomer causes non-specific adsorption of proteins from the surrounding biological environment, which is undesirable in any biomaterial application. Therefore, this study aims to develop non-degradable and stable hydrophilic surface to minimize protein adsorption and enhance cell interaction. A polydimethylsiloxane surface was prepared from Sylgard 184 PDMS pre-polymer using a crosslinking catalyst, which was then modified by the incorporation of a Si–H bond, followed by the incorporation of amine, to prepare amino functionally modified silicon elastomers. Following the introduction of amino groups, hyaluronic acid was incorporated into the PDMS surfaces. ATR-FTIR and water contact angle measurements confirmed that HA was grafted to the PDMS surfaces. Atomic force microscopy suggested that the PDMS surface was covered by HA. All modified surfaces exhibited very low water contact angles, suggesting that the HA-modified surfaces had increased the hydrophilicity under aqueous conditions relative to the PDMS control. The surface stability, protein-resistant properties and biocompatibility of the different molecular weight of HA-modified PDMS surfaces were explored. All the HA-modified surfaces are found to be very stable and durable. HA 3-modified PDMS surfaces markedly decrease the fibrinogen and albumin adsorption as much as 98% from a buffer. The biocompatibility of the HA 3-modified PDMS surface to the RPE cells was evaluated. These highly protein-resistant hydrophilic transparent surfaces, particularly the HA 3-modified PDMS surface, can potentially be used as an extraordinary biomaterial for biomedical applications.

Crosslinked Platform Coatings Incorporating Bioactive Signals for the Control of Biointerfacial Interactions.

Control over biointerfacial interactions on material surfaces is of significant interest in many biomedical applications and extends from the modulation of protein adsorption and cellular responses to the inhibition of bacterial attachment and biofilm formation. Effective control over biointerfaces is best achieved by reducing nonspecific interactions on the surface while also displaying specific bioactive signals. A poly(ethylene glycol) (PEG)-based multifunctional coating has been developed that provides effective reduction of protein fouling while enabling covalent immobilization of peptides in a one or two-step manner. The highly protein resistant properties of the coating, synthesized via the crosslinking of PEG diepoxide and diaminopropane, are confirmed via europium-labeled fibronectin adsorption and cell attachment assays. The ability to covalently incorporate bioactive signals is demonstrated using the cyclic peptides cRGDfK and cRADfK. L929 cells show enhanced attachment on the biologically active cRGDfK containing surfaces, while the surface remains nonadhesive when the nonbiologically active cRADfK peptide is immobilized. The crosslinked PEG-based coating also demonstrates excellent resistance toward Staphylococcus aureus attachment in a 48 h biofilm assay, achieving a >96% reduction compared to the control surface. Additionally, incorporation of the antimicrobial peptide melimine during coating formation further significantly decreases biofilm formation (>99%).

Physical Cross-Linking Starch-Based Zwitterionic Hydrogel Exhibiting Excellent Biocompatibility, Protein Resistance, and Biodegradability

In this work, a novel starch-based zwitterionic copolymer, starch-graft-poly(sulfobetaine methacrylate) (ST-g-PSBMA), was synthesized via Atom Transfer Radical Polymerization. Starch, which formed the main chain, can be degraded completely in vivo, and the pendent segments of PSBMA endowed the copolymer with excellent protein resistance properties. This ST-g-PSBMA copolymer could self-assemble into a physical hydrogel in normal saline, and studies of the formation mechanism indicated that the generation of the physical hydrogel was driven by electrostatic interactions between PSBMA segments. The obtained hydrogels were subjected to detailed analysis by scanning electron microscopy, swelling ratio, protein resistance, and rheology tests. Toxicity and hemolysis analysis demonstrated that the ST-g-PSBMA hydrogels possess excellent biocompatibility and hemocompatibility. Moreover, the cytokine secretion assays (IL-6, TNF-α, and NO) confirmed that ST-g-PSBMA hydrogels had low potential to trigger the activation of macrophages and were suitable for in vivo biomedical applications. On the basis of these in vitro results, the ST-g-PSBMA hydrogels were implanted in SD rats. The tissue responses to hydrogel implantation and the hydrogel degradation in vivo were determined by histological analysis (Hematoxylin and eosin, Van Gieson, and Masson's Trichrome stains). The results presented in this study demonstrate that the physical cross-linking, starch-based zwitterionic hydrogels possess excellent protein resistance, low macrophage-activation properties, and good biocompatibility, and they are a promising candidate for an in vivo biomedical application platform.

Reversible Interactions of Proteins with Mixed Shell Polymeric Micelles: Tuning the Surface Hydrophobic/Hydrophilic Balance toward Efficient Artificial Chaperones.

Molecular chaperones can elegantly fine-tune its hydrophobic/hydrophilic balance to assist a broad spectrum of nascent polypeptide chains to fold properly. Such precious property is difficult to be achieved by chaperone mimicking materials due to limited control of their surface characteristics that dictate interactions with unfolded protein intermediates. Mixed shell polymeric micelles (MSPMs), which consist of two kinds of dissimilar polymeric chains in the micellar shell, offer a convenient way to fine-tune surface properties of polymeric nanoparticles. In the current work, we have fabricated ca. 30 kinds of MSPMs with finely tunable hydrophilic/hydrophobic surface properties. We investigated the respective roles of thermosensitive and hydrophilic polymeric chains in the thermodenaturation protection of proteins down to the molecular structure. Although the three kinds of thermosensitive polymers investigated herein can form collapsed hydrophobic domains on the micellar surface, we found distinct capability to capture and release unfolded protein intermediates, due to their respective affinity for proteins. Meanwhile, in terms of the hydrophilic polymeric chains in the micellar shell, poly(ethylene glycol) (PEG) excels in assisting unfolded protein intermediates to refold properly via interacting with the refolding intermediates, resulting in enhanced chaperone efficiency. However, another hydrophilic polymer-poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) severely deteriorates the chaperone efficiency of MSPMs, due to its protein-resistant properties. Judicious combination of thermosensitive and hydrophilic chains in the micellar shell lead to MSPM-based artificial chaperones with optimal efficacy.

Zwitterionic-Modified Starch-Based Stealth Micelles for Prolonging Circulation Time and Reducing Macrophage Response.

Over the last few decades, nanoparticles have been emerging as useful means to improve the therapeutic efficacy of drug delivery and medical diagnoses. However, the heterogeneity and complexity of blood as a medium is a fundamental problem; large amounts of protein can be adsorbed onto the surface of nanoparticles and cause their rapid clearance before reaching their target sites, resulting in the failure of drug delivery. To overcome this challenge, we present a rationally designed starch derivative (SB-ST-OC) with both a superhydrophilic moiety of zwitterionic sulfobetaine (SB) and a hydrophobic segment of octane (OC) as functional groups, which can self-assemble into "stealth" micelles (SSO micelles). The superhydrophilic SB kept the micelles stable against aggregation in complex media and imbued them with "stealth" properties, eventually extending their circulation time in blood. In stability and hemolysis tests the SSO micelles showed excellent protein resistance properties and hemocompatibility. Moreover, a phagocytosis test and cytokine secretion assay confirmed that the SSO micelles had less potential to trigger the activation of macrophages and were more suitable as a drug delivery candidate in vivo. On the basis of these results, doxorubicin (DOX), a hydrophobic drug, was used to investigate the potential application of this novel starch derivative in vivo. The results of the pharmacokinetic study showed that the values of the plasma area under the concentration curve (AUC) and elimination half-life (T1/2) of the SSO micelles were higher than those of micelles without SB modifications. In conclusion, the combination of excellent protein resistance, lower macrophage activation, and longer circulation time in vivo makes this synthesized novel starch derivative a promising candidate as a hydrophobic drug carrier for long-term circulation in vivo.

Protein-resistant properties of a chemical vapor deposited alkyl-functional carboxysilane coating characterized using quartz crystal microbalance

The protein-resistant properties of a chemical vapor deposited alkyl-functional carboxysilane coating (Dursan®) were compared to that of an amorphous fluoropolymer (AF1600) coating and bare 316L grade stainless steel by studying non-specific adsorption of various proteins onto these surfaces using quartz crystal microbalance with dissipation monitoring (QCM-D). A wash solution with nonionic surfactant, polyoxyethyleneglycol dodecyl ether (or Brij 35), facilitated 100% removal of the adsorbed bovine serum albumin (BSA), mouse immunoglobulin G (IgG), and normal human plasma proteins from the Dursan surface and of the adsorbed normal human plasma proteins from the AF1600 surface, whereas these proteins remained adsorbed on the bare stainless steel surface. Mechanical stress in the form of sonication demonstrated durability of the Dursan coating to mechanical wear and showed no negative impact on the coating's ability to prevent adsorption of plasma proteins. Surface delamination was observed in case of the sonicated AF1600 coating, which further led to adsorption of normal human plasma proteins.

Polypropylene microfiltration membranes modified with TiO2 nanoparticles for surface wettability and antifouling property

TiO2 nanoparticles have attracted much attention in the surface modification of polymer membranes for antifouling purpose due to their excellent hydrophilicity. In this work, a polydopamine/polyethyleneimine (PDA/PEI) intermediate layer was fabricated on the polypropylene microfiltration membrane via a co-deposition method and then TiO2 nanoparticles (NPs) were modified on the membrane surfaces through a sol–gel process. TiO2-modified membranes with various Ti/C ratios were prepared and their surface structures and properties are compared with those of the nascent and PDA/PEI-deposited ones. Results indicate that the TiO2 NPs modification increases both the surface wettability and the water permeation flux of the membranes remarkably. TiO2 NPs also endow the membranes with promoted protein resistance properties. The relative flux reduction and flux recovery ratio are 31±3% and 82±5% for bovine serum albumin solution, while 26±4% and 86±3% for lysozyme solution. The TiO2 NPs are also stable on the membrane surfaces under a wide range of pH value.

Surface functionalization of hydrogel by thiol-yne click chemistry for drug delivery

Abstract Although hydrogel has some outstanding performance, traditional hydrogel still has some drawbacks in the application of drug delivery field. Herein, we attempted to utilize the thiol-yne photopolymerization, which had advantages of high efficiency, mild reaction condition and low toxicity as a type of click chemistry, to realize β-cyclodextrin (β-CD) functionalized hydrogel in order to improve its performance for drug delivery. As a prerequisite, alkynylation was conducted on the per-formed hydrogel. The above-mentioned synthesis was verified by the results of atomic concentration from X-ray Photoelectron Spectroscopy (XPS) characterization and semiquantitative analysis of Fourier-transformed infrared spectroscopy (FTIR) spectra. Similarly, the optimum reaction condition including optimal reaction time and feeding molar ratio were also determined by semiquantitative analysis of FTIR spectra and XPS results. The alkynylation resulted in higher contact angle, lower equilibrium water and coarser surface. Then, β-CD functionalization was beneficial to the hydrophilicity and protein resistant property of hydrogel. With respect to drug delivery performance, an increasing drug concentration and β-CD molecules that grafted onto hydrogel benefited to aggregating orfloxacin molecules in hydrogels. The loading drug concentration as well as β-CD content on hydrogel exerted influences upon drug release behaviors.

Cellulose-based polymeric liquid crystals as a biomimetic modifier for suppressing protein adsorption

A novel biomimetic protein-resistant modifier based on cellulose-based polymeric liquid crystals was described (PLCs). Two types of PLCs of propyl hydroxypropyl cellulose ester (PPC) and octyl hydroxypropyl cellulose ester (OPC) were prepared by esterification from hydroxypropyl cellulose, and then were mixed with polyvinyl chloride and polyurethane to obtain composite films by solution casting, respectively. The surface morphology of PLCs and their composite films were characterized by polarized optical microscopy (POM) and scanning electron microscopy (SEM), suggesting the existence of microdomain separation with fingerprint texture in PLC composite films. Water contact angle measurement results indicated that hydrophilicity of PLC/polymer composite films was dependent on the type and content of PLC as well as the type of matrix due to their interaction. Using bovine serum albumin (BSA) as a model protein, protein adsorption results revealed that PLCs with protein-resistant property can obviously suppress protein adsorption on their composite films, probably due to their flexible LC state. Moreover, all PLCs and their composites exhibited non-toxicity by MTT assay, suggesting their safety for biomedical applications.

Surface modification of silk fibroin fabric using layer-by-layer polyelectrolyte deposition and heparin immobilization for small-diameter vascular prostheses.

There is an urgent need to develop a biologically active implantable small-diameter vascular prosthesis with long-term patency. Silk-fibroin-based small-diameter vascular prosthesis is a promising candidate having higher patency rate; however, the surface modification is indeed required to improve its further hemocompatibility. In this study, silk fibroin fabric was modified by a two-stage process. First, the surface of silk fibroin fabric was coated using a layer-by-layer polyelectrolyte deposition technique by stepwise dipping the silk fibroin fabric into a solution of cationic poly(allylamine hydrochloride) (PAH) and anionic poly(acrylic acid) (PAA) solution. The dipping procedure was repeated to obtain the PAH/PAA multilayers deposited on the silk fibroin fabrics. Second, the polyelectrolyte-deposited silk fibroin fabrics were treated in EDC/NHS-activated low-molecular-weight heparin (LMWH) solution at 4 °C for 24 h, resulting in immobilization of LMWH on the silk fibroin fabrics surface. Scanning electron microscopy, atomic force microscopy, and energy-dispersive X-ray data revealed the accomplishment of LMWH immobilization on the polyelectrolyte-deposited silk fibroin fabric surface. The higher the number of PAH/PAA coating layers on the silk fibroin fabric, the more surface hydrophilicity could be obtained, resulting in a higher fetal bovine serum protein and platelets adhesion resistance properties when tested in vitro. In addition, compared with untreated sample, the surface-modified silk fibroin fabrics showed negligible loss of bursting strength and thus reveal the acceptability of polyelectrolytes deposition and heparin immobilization approach for silk-fibroin-based small-diameter vascular prostheses modification.

Polyglycerol based coatings to reduce non-specific protein adsorption in sample vials and on SPR sensors

Coatings based on dendritic polyglycerol (dPG) were investigated for their use to control nonspecific protein adsorption in an assay targeted to analyze concentrations of a specific protein. We demonstrate that coating of the sample vial with dPG can significantly increase the recovery of an antibody after incubation. First, we determine the concentration dependent loss of an antibody due to nonspecific adsorption to glass via quartz crystal microbalance (QCM). Complementary to the QCM measurements, we applied the same antibody as analyte in an surface plasmon resonance (SPR) assay to determine the loss of analyte due to nonspecific adsorption to the sample vial. For this purpose, we used two different coatings based on dPG. For the first coating, which served as a matrix for the SPR sensor, carboxyl groups were incorporated into dPG as well as a dithiolane moiety enabling covalent immobilization to the gold sensor surface. This SPR-matrix exhibited excellent protein resistant properties and allowed the immobilization of amyloid peptides via amide bond formation. The second coating which was intended to prevent nonspecific adsorption to glass vials comprised a silyl moiety that allowed covalent grafting to glass. For demonstrating the impact of the vial coating on the accuracy of an SPR assay, we immobilized amyloid beta (Aβ) 1-40 and used an anti-Aβ 1-40 antibody as analyte. Alternate injection of analyte into the flow cell of the SPR device from uncoated and coated vials, respectively gave us the relative signal loss (1−RUuncoated/RUcoated) caused by the nonspecific adsorption. We found that the relative signal loss increases with decreasing analyte concentration. The SPR data correlate well with concentration dependent non-specific adsorption experiments of the analyte to glass surfaces performed with QCM. Our measurements show that rendering both the sample vial and the sensor surface is crucial for accurate results in protein assays.

A zwitterionic surface with general cell-adhesive and protein-resistant properties

A choline phosphate functionalized surface is proved to have general cell-adhesive and protein-resistant properties, which give it potential for biomaterials.

Antifouling property of monothiol-terminated bottle-brush poly(methylacrylic acid)-graft-poly(2-methyl-2-oxazoline) copolymer on gold surfaces.

In this study, a series of well-controlled bottle-brush polymers, poly(methylacrylic acid)-g-poly(2-methyl-2-oxazoline) with monothiol-terminated group ((PMAA-g-PMOXA)-SH) were synthesized by using reversible addition-fragmentation chain transfer (RAFT) polymerization and cationic ring-opening polymerization (CROP). (PMAA-g-PMOXA)-SH were grafted to the surface of gold sensors in in situ aminolysis reactions. Cyclic voltammetry (CV), X-ray photoelectron spectroscopy (XPS), variable angle spectroscopic ellipsometry (VASE), water contact angle (CA), and atomic force microscopy (AFM) were used to characterize the copolymer modified gold sensor. The protein-resistant properties were investigated by surface plasmon resonance (SPR), and platelet adhesion was observed by scanning electron microscopy (SEM). Compared with the bare gold sensor, the (PMAA20-g-PMOXA12)-SH modified gold sensor can reduce the levels of fibrinogen (Fg), bovine serum albumin (BSA), and lysozyme (Lyz) adsorption by 96.5 ± 3.1%, 85.8 ± 5.7%, and 49.4 ± 1.6%, respectively. Meanwhile, the (PMAA20-g-PMOXA12)-SH modified gold sensor also possesses excellent resistance to platelet adhesion. All these data demonstrate that this simple method is feasible, and that a bottle-brush (PMAA-g-PMOXA)-SH modified gold sensor has potential applications in biosensors and biomedical areas.

Choline phosphate functionalized surface: protein-resistant but cell-adhesive zwitterionic surface potential for tissue engineering.

A choline phosphate (CP) modified surface is designed to resist protein adsorption due to its zwitterionic properties and simultaneously promote cell adhesion though its universal interaction with phosphate choline (PC) headgroups of the cell membrane. This work provides a new approach to obtain a cell-adhesive surface with a non-biofouling 'background', which has a potential for tissue engineering.

Zwitterionic siloxane-polyurethane fouling-release coatings

An approach to the design of polyurethane coatings having amphiphilic/zwitterionic surfaces via a self-stratification approach has been explored for marine non-fouling applications. Zwitterionic materials such as poly(sulfobetaine methacrylate) poly(SBMA), are used as non-fouling materials due to their protein resistance properties. ABA-type triblock copolymers, poly(SBMA)-block-PDMS-block-poly(SBMA), having PDMS as the central block and poly(SBMA) as the flanking blocks with secondary amines at the junction points were synthesized using ring opening equilibration polymerization (ROEP), Michael addition and atom transfer radical polymerization (ATRP). A series of triblocks were synthesized and incorporated into a polyurethane coating system and were evaluated for water contact angle (WCA), surface energy (SE) and pseudobarnacle adhesion (PB). The fouling-release (FR) performance of the coatings was evaluated in the laboratory using a suite of representative marine organisms. The coatings showed excellent FR performance toward the bacterium Halomonas pacifica and the diatom (microalga) Navicula incerta while the bacterium Cellulophaga lytica and sporelings of the green macroalga Ulva linza showed a relatively higher affinity for amphiphilic coating surfaces when compared to a series of standard control coatings.

Fabrication of a photocontrolled surface with switchable wettability based on host-guest inclusion complexation and protein resistance.

A novel surface-modification strategy has been developed for the construction of a photocontrolled silicon wafer surface with switchable wettability based on host-guest inclusion complexation. The silicon wafer was first modified by guest molecule azobenzene (Azo) via a silanization reaction. Subsequently, a series of polymers with different polarities were attached to host molecule β-cyclodextrin (β-CD) to prepare β-CD-containing hemitelechelic polymers via click chemistry. Finally, a photocontrolled silicon wafer surface modified with polymers was fabricated by inclusion complexation between β-CD and Azo, and the surface properties of the substrate are dependent on the polymers we used. The elemental composition, surface morphology, and hydrophilic/hydrophobic property of the modified surfaces were characterized by X-ray photoelectron spectroscopy (XPS), atomic force microscope, and contact angle measurements, respectively. The antifouling property of the PEG-functionalized surface was evaluated by a protein adsorption assay using bovine serum albumin, which was also characterized by XPS. The results demonstrate that the surface modified with PEG possesses good protein-resistant properties.

Preparation and characterization of brush-like PEGMA-graft-PDA coating and its application for protein separation by CE

In this paper, a novel brush-like copolymer consisting of poly(ethylene glycol) methyl ether methacrylate and 2-aminoethyl methacrylate (AEMA) named as poly(PEGMA300-co-AEMA) was synthesized by atom transfer radical polymerization (ATRP), and then, poly(PEGMA300-co-AEMA) copolymer was immobilized onto material surfaces through polydopamine (PDA)-anchored coating. The defined copolymer structure was characterized by nuclear magnetic resonance hydrogen spectroscopy (1H NMR) and gel permeation chromatography (GPC). The chemical component and surface morphology of the brush-like copolymer-graft-PDA coating were studied by using X-ray photoelectron spectroscopy (XPS) and scanning electron microscope (SEM), respectively. The hydrophilicity of the brush-like copolymer-graft-PDA coating was investigated by using static water contact angle. The protein-resistant property of the brush-like copolymer-graft-PDA coating was investigated by using quartz crystal microbalance with dissipation (QCM-D), and finally the coating was applied to capillary inner surface for protein separation by capillary electrophoresis (CE).

One-step preparation of vinyl-functionalized material surfaces: a versatile platform for surface modification

A simple approach has been developed to functionalize various substrates, such as gold and polyvinylchloride, with dopamine methacrylamide—a molecule with adhesive properties that mimic those of mussels—to produce a versatile and general platform for subsequent surface modification. With active double bonds on the surface, various polymers, such as poly([2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl) ammonium hydroxide (PMEDSAH) and poly(N-vinylpyrrolidone) (PVP), can be grafted by conventional radical polymerization. Double bond surface functionalization and subsequent polymer grafting have been verified by static water contact angle, Fourier transform infrared-attenuated total reflectance (FTIR-ATR) spectroscopy and X-ray photoelectron spectroscopy (XPS) measurements. Protein adsorption assays showed that the polymer-modified substrates have good protein-resistant properties. Considering the advantages of facility, versatility and substrate-independence, this method should be useful in designing functional interfaces for bioengineering applications.

Layer-by-layer nanoencapsulation of camptothecin with improved activity.

160 nm nanocapsules containing up to 60% of camptothecin in the core and 7-8 polyelectrolyte bilayers in the shell were produced by washless layer-by-layer assembly of heparin and block-copolymer of poly-l-lysine and polyethylene glycol. The outer surface of the nanocapsules was additionally modified with polyethylene glycol of 5 kDa or 20 kDa molecular weight to attain protein resistant properties, colloidal stability in serum and prolonged release of the drug from the capsules. An advantage of the LbL coated capsules is the preservation of camptothecin lactone form with the shell assembly starting at acidic pH and improved chemical stability of encapsulated drug at neutral and basic pH, especially in the presence of albumin that makes such formulation more active than free camptothecin. LbL nanocapsules preserve the camptothecin lactone form at pH 7.4 resulting in triple activity of the drug toward CRL2303 glioblastoma cell.