AVS2016 Session BI+PB-TuP: Biomaterial Interfaces Poster Session (preceded by Oral Flash Presentations)

We present an investigation of gelatin film response to an array of pancreas-specific enzymes using a Quartz Crystal Microbalance (QCM) system. In fluids secreted from the pancreas into the upper small intestine, highly concentrated enzymes (α-amylase, trypsin, and lipase) are mixed to complete digestion of partially broken-down materials entering from the stomach. Sensors, such as that illustrated in Fig 1, are utilizing biomaterials such as gelatin, as it can be used to enter the body due to its biocompatibility and tailorability both chemically and structurally. Gelatin is known to be highly sensitive to degradation by trypsin, one of the aforementioned enzymatic pancreas biomarkers, thereby offering potential to indirectly monitor trypsin levels [1]. The composition of pancreatic fluid is a biomarker in testing exocrine function of the pancreas, a process often involving invasive procedures toward quantifying losses in enzyme levels [2]. However, its interactions with other interfering enzymes such as lipase or α-amylase have not been studied. While these enzymes are believed to cleave specific to bonds not found in gelatin, it is critical to be able to determine how these non-specific enzymes impact the signals produced when gelatin interacts with specific enzymes, i.e. trypsin, when they are each in the media.

In this work, we utilize a QCM system in sensing the mass change of gelatin films deposited onto standard crystals with gold electrodes. Films are subjected to a constant flow rate of buffer before introducing pancreatic enzymes in the setup illustrated in Fig 2. After system stabilization under buffer flow, material loss is quantified from the surface of the crystal. For trypsin, as expected, we observe degradation in a concentration-dependent manner, shown in Fig. 3. With either lipase or α-amylase, however, we observe no change, as illustrated in the top of Fig 4. After rinsing with buffer, we reintroduce trypsin in combination with each enzyme to determine if the presence of nonspecific enzymes affected the sensitivity of the gelatin to proteolytic activity, shown in the bottom of Fig 4. Digestion rates are found to decrease by ­­­­­83% and 77% with exposure to lipase or α-amylase, respectively, indicating a decrease in gelatin sensitivity to trypsin in the presence of these enzymes. The next phase in this work will be to combine all three enzymes to further model pancreatic juices. This work emphasizes the necessity in characterizing gelatin’s response to other enzymes in understanding its sensitivity and specificity in the digestive environment, leading the avenue for devices designed to monitor gastrointestinal health.

Bacteriorhodopsin (bR) is the integral protein of the purple membrane of Halobacterium salinarum and is the most studied proton pump. It is a chiral system composed of seven parallel, upright-oriented alpha-helices. Recent photoemission and electrochemical studies have shown that it can act as a natural electron spin filter as a result of the chiral-induced spin selectivity effect.[1] Previous structural studies using hard x-ray synchrotron radiation (SR) have shown that such radiation can significantly impact the structural integrity of bR,[2] while earlier work has demonstrated that X-ray-induced, low energy secondary electrons can play a major role in surface chemical reactions of adsorbed biological molecules.[3] In the present study we use SR x-ray absorption and photoelectron spectroscopy (XPS) to characterize the initial state of the adsorbed bR. Time-dependent changes in the core-level XPS spectra are utilized to follow the dynamics of the X-ray/secondary electron induced reaction. The results will be discussed in terms of previous studies of x-ray induced reactions in bR and other biological molecules.

The work performed at the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-06CH11357.

REFERENCES

1 D. Mishra, T. Z. Markus, R. Naaman et al., Proc. Nat. Acad. Sci. 110, 14872 (2013).

2 V. I. Borshchevskiy, E. S. Round, A. N. Popov et al., J. Mol. Biol. 409, 813 (2011).

3 B. Boudaïffa, P. Cloutier, D. Hunting et al., Science 287, 1658 (2000); R. A. Rosenberg, D. Mishra, and R. Naaman, Angew. Chem. Int. Ed. 54, 7295 (2015); R. A. Rosenberg, J. M. Symonds, K. Vijayalakshmi et al., Phys. Chem. Chem. Phys. 16, 15319 (2014).

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a powerful technique for the nanoanalysis of biological samples, but improvements in sensitivity are needed in order to detect large biomolecules, such as peptides, on the individual cell level at physiological concentrations. An increase in the detection efficiency for larger molecules and reduced fragmentation rates could be obtained by a) the use of cluster ion beams such as Au_n⁺ , Bi_n⁺ , C₆₀⁺ , or even large Ar_n⁺ clusters in order to maximize the energy deposited close to the surface or b) by modifying the surface by organic matrices in the so-called matrix-enhanced SIMS (ME-SIMS). This approach is based on embedding analyte molecules in low weight organic matrices, like common MALDI matrices, prior to ion bombardment

We used dual beam ToF-SIMS to image the incorporation of three peptides with different hydrophobicities, bradykinin, substance P, and vasopressin, into two classical MALDI matrices, 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (HCCA) prepared with dried droplet sample preparation method. For depth profiling, an Ar cluster ion beam was used to gradually sputter through the matrix crystals without causing significant degradation of matrix or biomolecules. A pulsed Bi₃⁺ ion cluster beam was used to image the lateral analyte distribution in the center of the sputter crater. Using this dual beam technique, the 3D distribution of the analytes and spatial segregation effects within the matrix crystals were imaged with sub-µm resolution.

This presentation reports on the use of Zr-based (Zr₅₃Cu₃₃Al₉Ta₅) thin film metallic glass (TFMG) for the coating of various medical devices and compares the results with those obtained using conventional titanium nitride and pure titanium coatings . TFMG was selected as the coating material for its unique properties such as good biocompatibility and antibacterial property due to its amorphous atomic structure, revealing a great potential for biomedical applications. The TFMG coating was shown to reduce insertion forces and retraction forces by up to over seventy percent when tested using polyurethane rubber block. The benefits of TFMG-coated needles were also seen when tested using pig muscle tissues. Based on the nano-scratch test, the TFMG coatings achieved a low coefficient of friction (COF), about one order of magnitude lower than those of bare surface and other coatings. Furthermore, the adhesions of cancer cells and platelets to coatings are also examined. TFMG coating is shown to appreciably minimize the attachment of cancer cells and platelets by more than eighty percent in relative to those of Ti coating and bare surface. The low COF and non-stick coated surfaces by TFMG can be attributed to the absence of grain boundaries in the TFMG coating, smooth surface and low surface free energy.

Two hydrolases secreted by Pseudomonas protegens Pf-5 bacteria, PueA and PueB, have been demonstrated to be active towards polyester - polyurethane (PU) hydrolysis. In this work, the impact of these enzymes towards PU degradation was directly compared at biofilm / PU interfaces through deletion of PueA and PueB genes. Unsaturated biofilm assays were used where biofilm growth took place on solid, hydrated PU discs in air. PU degradation was analyzed using confocal Raman microscopy of intact samples. Additionally, cross-sectional analysis of microtomed sections was done using FTIR microscopy and combined atomic force microscopy – infrared spectroscopy (AFM-IR). Results showed varying degrees of biofilm related permeation and polymer degradation within the ~300 um thick discs. Degradation took place through a pitting process involving preferential loss of the ester component. Wild type and PueB knockout mutants showed the highest levels of hydrolysis, measured by loss of carbonyl intensity in vibrational spectra, while the PueA and double knockout mutants showed lower hydrolysis levels. The apparent higher level of PueA activity is consistent with higher enzymatic activity for the hydrophobic lipase substrate, p-nitrophenyl palmitate. Relationships between biofilm morphology and PU degradation were also observed for the wild type and mutant biofilms.

Until recently, biodegradable implants have exclusively been used in non-load bearing applications such as stents and sutures. Mg-Al-Zn alloys like AZ31B are currently being considered as biodegradable materials because of their similar mechanical properties to that of bone. In addition, the corrosion product resulting from Mg alloys in body fluid contains Mg and Ca-phosphates (hydroxyapatite) and has been shown to stimulate bone regeneration. The degradation of Mg alloys is also considered non-toxic when corroding in the human body. However, the structural integrity is poor due to rapid corrosion caused by microstructural heterogeneities in the form of electrochemically noble secondary phases leading to micro-galvanic couples and preferential dissolution of the α-Mg matrix in physiological media. Laser surface processing of the Mg-Al-Zn alloy, AZ31B, reduced the corrosion rate in simulated body fluid (SBF) experiments by minimizing the impact of secondary phases.

Experiments utilized a pulsed excimer laser (λ = 248 nm and FWHM = 25 ns) in combination with a novel surface modification chamber. Samples of AZ31B in the as-received and laser processed condition were submerged in a 27 mM HCO₃^- Tris ((HOCH₂)₃CNH₂) variant of SBF. The corrosion resistance was investigated through optical microscopy, scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), gravimetric mass loss, and polarization measurements.

No major corrosion product variations were observed for the as-received / laser processed specimens by SEM and EDS, both showing a similar amount of Ca and P. The laser processed alloy exhibited a reduction in anodic kinetics compared to the as-received material, suggesting the corrosion product is more compact and passivating. Furthermore, the laser processed surface exhibited a 50% reduction in mass loss after 24 hours immersion in SBF in comparison to the as-received samples. Optical micrographs of samples immersed in SBF reveal a reduction in the H₂ evolution rate of the laser processed versus as-received material. In addition, the laser treated specimens exhibited a significant increase in wettability with a 10^o contact angle compared to the 45^o angle of the as-received materials. The increased wettability of the laser processed samples may decrease the time required for osseointegration through allowing cells to more readily bind to the surface of an implant.

Bacterial cellulose and chitosan are renewable natural polymers and have many favorable properties such as biocompatibility, biodegradability and low toxicity. So, they have been extensively used in drug delivery systems, gene therapy, tissue engineering, and biosensor applications. Silver nanoparticles have attracted much attention for their unusual chemical and physical properties and have been widely applied in sensors, antibacterial and photocatalytic areas. The synthesis of nanomaterials with different chemical composition, size distribution, and controlled monodispersity has become an important research area in nanotechnology. Many kinds of methods such as vapor deposition, solvent-thermal, sol-gel, electrochemistry and microwave have been developed to fabricate nanomaterials. So far, plasma technology has become an important approach to prepare and reinforce materials and surfaces. This work seeks to fabricate nanoparticles/natural polymer functional composites via an atmospheric pressure plasma method. Comparing many traditional methods, atmospheric pressure plasma jet can induce chemical reactions in mild conditions, which can guarantee the purity of system and will not destroy the structure of natural polymers.

In this work, silver/bacterial cellulose/chitosan functional composites are fabricated via an atmospheric pressure plasma method. Plasma can produce many active radicals including reduction species and oxygen species, which can trigger chemical reaction. Ag+ in the reaction system can attach to the surface of bacterial cellulose and chitosan via bonding. By controlling the reaction condition, Ag+ can be reduced to Ag(0) and form Ag nanoparticles under plasma treatment. The existence of bacterial cellulose and chitosan can limit the growth and prevent the aggregation of particles, which is very critical to form nanostructure. SEM, XRD, XPS, FT-IR are employed to examine the morphology and structures of as prepared nano-composites. The biocompatibility and antibacterial properties are also studied. All results reveal that Ag nanoparticles are successfully formed and well dispersed in bacterial cellulose/chitosan. The as prepared silver/bacterial cellulose/chitosan nano-composites have excellent biocompatibility and antibacterial abilities, which can be used in biomedical areas. This convenient synthesis strategy based on atmospheric pressure plasma could be extended to fabricate other nanoparticles/bacterial cellulose/chitosan composites.

Laparoscopes, arthroscopes, and laryngoscopes lenses are hydrophobic and fog during closed body surgery, due to bodily fluids and differences between body and operating room temperatures [1,2]. Surgeons must repeatedly remove, clean, and reinsert scopes obscured by fog. Hencesurgery duration, infection risks, and scarring from air exposure increase. Methods to address fogging introduce other complications [3]. Alcohol-based coatings scar tissue and quickly evaporate, heated lenses require reheating every 5 to 20 minutes. A non-toxic, super-hydrophilic, anti-fog coating that is pH neutral (7.2-7.4), long-lasting has been developed VitreOx™ . [4] VitreOx™ can be used wet or dry, without alcohol, heat, or fluid evacuation. When applied in liquid form, it easily espouses a lens’s surface and edges, and dries within seconds to form a permanently super-hydrophilic surface on silica and most polymer surfaces . VitreOx™ avoids current shortfalls by foregoing frequent reapplications.

VitreOx™'s anti - fog properties can be explained by nucleation and growth theory, and the three mechanisms for condensation: 1) 3-D droplets, resulting in fogging; 2) 2-D sheets resulting in a flat transparent film . ; or 3) mixed, resulting in optical distortion. On hydrophobic surfaces (e.g. optical lenses ), condensation occurs with fogging via spherical 3-D droplets (Volmer-Weber model). 3 - D droplets scatter light in all directions through refraction yielding opaque or translucent films, or fog. VitreOx™ applied to hydrophobic lenses renders them super-hydrophilic. Similar to the 2-D Frank Van-der-Merwe growth mode, condensation with uniform wetting yields transparent 2-D films that don’t distort light.

In-vitro and in-vivo animal studies of VitreOx™ were conducted to measure performance and duration of anti-fog effectiveness and bio-compatibility. In-vitro testing spanned from 3 to 72 hours over a 3-year range. Side-by-side in-vivo gastro-endoscopies were conducted using a lens coated with VitreOx^TM and a Covidien Clearify ™ warmer with anti-fog, on Yucatan™ swine for 90 minutes . The VitreOx™ coating lasted without fogging nor reapplication, while Covidien Clearify™ only l ast ed at for 38 minutes without fogging, and required retreatment and reapplication . No adverse reaction was observed on swine in surgery, and in the 18 months that follows.

[1] Knuth A et al “ Anaesthesist. 2012 Dec;61(12):1036-44.

[2] Lawrentschuk N, et al “Laparoscopic Lens Fogging” (2010). Journal of Endourology. 24(6):905-913

[3] US Pat. 5,518,502 (1994) and its 129 cites.

[4] Herbots N, Watson CF, patents pending