Characterization and Biocompatibility Studies of Novel Humic Acids

Multilayered films of humic acids (HAs) (naturally occurring biopolymers) were investigated as a potential semipermeable membrane for implantable gluc...
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Biomacromolecules 2001, 2, 1249-1255

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Characterization and Biocompatibility Studies of Novel Humic Acids Based Films as Membrane Material for an Implantable Glucose Sensor Izabela Galeska,† Tammy Hickey,‡ Francis Moussy,‡ Donald Kreutzer,‡ and Fotios Papadimitrakopoulos*,† Nanomaterials Optoelectronic Laboratory, Department of Chemistry, Polymer Program, Institute of Materials Science, University of Connecticut, Storrs, Connecticut 06269; and Center for Biomaterials & Surgical Research Center, University of Connecticut Health Center, Farmington, CT 06030-1615. Received June 29, 2001

Multilayered films of humic acids (HAs) (naturally occurring biopolymers) were investigated as a potential semipermeable membrane for implantable glucose sensors. These films were grown using a layer-by-layer self-assembly process of HAs and oppositely charged ferric ions. The growth of these assemblies exhibited strong dependence on the pH and ionic strength of HAs solutions, which correlated with the degree of ionization of the carboxyl groups and neutralization-induced surface spreading. Quartz crystal microbalance (QCM) and ellipsometric studies have shown repeatable, stepwise increase in mass (as high as 5.63 µg/ cm2) and in film thickness (ca. 24.3 nm per layer) for these assemblies. The permeability of glucose through these membranes can be regulated by varying the number of self-assembled HAs/Fe3+ layers. Moreover, a 200 nm thick HAs/Fe3+ film (in its hydrated state) had a shear modulus of about 80 MPa, implying stability upon implantation. These films were determined to be biocompatible since in vivo studies indicated only mild tissue reaction along with some neovascularization. Introduction In vivo membrane degradation, fouling, inflammation, and fibrosis (i.e., fibrous encapsulation) have so far prevented the widespread use of implantable biosensors.1,2 Nafion has been successfully used in nonbiological sensors either as a semipermeable or protective membrane.3-6 However, in implantable devices Nafion has been associated with (1) calcification and (2) fibrosis of the surrounding tissue, leading to loss of vascularization and restricted analyte flow, and together resulting in premature sensor failure.7 Hence there is an urgent need for inert, bioacceptable materials capable of withstanding the strong oxidative environment in vivo. Humic acids (HAs) are biopolymers found in soil, sediments, water, and some plants such as tobacco.8-11 Although their origin is ambiguous, it is speculated that they may have evolved from the auto-oxidation and condensation of lignin and polyphenols during the humification process12 or are produced during in vivo biosynthesis in plants.9 Humic acids are deemed as the final product of the biodegradative and oxidative route, with a mean residence time up to 1200 years in soil,13 and probably will not undergo any further breakdown unless they are exposed to agents they have not previously encountered.14 HAs are very heterogeneous in nature and contain species with molecular weight ranging from a few thousand daltons to hundreds of kilodaltons. Their structures,15,16 although still under investigation, are domi* To whom correspondence should be addressed: Telephone: (860)486-3447. Fax: (860)-486-4745. E-mail: [email protected]. † University of Connecticut. ‡ University of Connecticut Health Center.

nated by aromatic moieties containing carboxylic, carbonyl, phenol, catechol, and quinone, along with a few amine groups.17 The physiological and toxicological effects of humic acids on humans and animals have been explored to some degree. HAs are known to exist in the gastrointestinal tract of humans and animals,18 have been found in the blood19 and also are known to be metabolized in the liver.20 Oral doses of HAs reduce heavy metal adsorption in animals19 and also decrease pesticide toxicity.21 Antiviral, antiinflammatory and profibrinolytic activity of HAs have been previously reported.19 HAs also have the ability to interact with metals22 and absorb to a variety of surfaces such as minerals,23-27 cellulose,28 and chitin and chitosan29 as well as to bacteria.25 Although ingestion of large quantities of HAs via drinking water have been reported to be one of the causative factors of KahinBeck disease30 (an endemic osteoarthritic disorder) and of Black Foot disease31 (a peripheral vascular disease), the physiological effects associated with these diseases require the HAs to be available in solution. On the basis of the above-mentioned attributes we have investigated multilayered films of HAs and ferric ions, as a possible membrane material for an implantable glucose sensor. The present paper examines the effect of experimental conditions on the HAs/ Fe3+ electrostatic layer-by-layer assembly, glucose permeability and biocompatibility of the resulting films. The self-assembly involving oppositely charged moieties32 has the potential to address membrane mineralization, often experienced in anionic biomaterials.7,33-34 Along the lines of what was demonstrated in our previous work for Nafion/

10.1021/bm010112y CCC: $20.00 © 2001 American Chemical Society Published on Web 10/12/2001

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Fe3+ system,35 the carboxylic groups in HAs will be saturated with iron(III) during the self-assembly process, thus potentially preventing calcification and fouling on the sensor’s surface.36 Furthermore, the rich chemical environment in the HAs structure and the water-based self-assembly process may be easily employed for the attachment or coupling biologically active molecules such as growth factors, antifouling agents and drug delivery systems, which would further minimize the adverse tissue reactions to the implanted sensor. Experimental Section A. Electrostatic Layer-by-Layer Film Growth and Characterization. Materials and Reagents. Humic acid, lot no. 11909LR, was purchased from Aldrich and was used without any further purification. The HAs sample used in our studies was characterized by elemental and trace metal analysis, as well as molecular weight and functional groups determination. In particular elemental analysis (as supplied by Aldrich Chemical Co., Inc. and verified within 1% by Galbrith Laboratory) for C, N, and H indicated (%): C, 43.7; O, 41.8; H, 4.02; N, 0.94; Na, 6.2; Ca, 0.9; S, 0.7; Fe, 0.8; Al, 0.6; Mg, 0.2. Trace metal analysis (in ppm) (as supplied by Aldrich Chemical Co.) detected: K, 535; Si, 260; Mn, 175; Ti, 164; Sr, 139; Ba, 90; V, 10; Zr, 9; Cu, 6; Li, 4; Cr, 2. Gel permeation chromatography (GPC) in N-methyl pyrolidone/water (9:10) mixture indicated a weight-average molecular weight of 169 kDa with polydispersity index Mw/ Mn ) 2.58. Volumetric titration (from pH 2.5 to 12) showed a cumulative acidic functionality content on the order of 820 mequiv/100 g of HAs. The intermediate deflection points in the titration curve ((I) pH 3.2; (II) pH 5.8 and 6.8; (III) pH 10.3) indicated presence of different types of acidic groups ((I) aromatic carboxylic groups ortho to phenolic substitutions; (II) weak aromatic and aliphatic carboxylic acids; (III) phenolic and catecholic-OH). A 1 mg/mL HAs solution in deionized water was used for all experiments. pH of the HAs solutions used for the assembly was adjusted with diluted hydrochloric acid (J. T. Baker) and the ionic strength of these solutions were modified by the addition of ACS Certified potassium chloride (Fischer). A 5 mg/mL solution of iron(III) chloride hexahydrate (FeCl3‚6H2O) (Aldrich) was used for the self-assembly with HAs. Millipore quality deionized water with resistivity >18 MΩ was utilized in all experiments. The self-assembled films were deposited either on silicon wafers or quartz crystal resonators. Silicon wafers were cleaned in piranha solution (H2SO4/H2O2 (7:3)), rinsed with deionized water and methanol, kept in deionized water overnight, and used for the self-assembly without further surface modification. For all QCM experiments, 9 MHz AT cut quartz crystal resonators with an electrode surface area of 0.32 cm2 (USI, Japan) were used. The resonators with gold electrodes were used for the shear modulus experiments and those with silver electrodes for all other experiments. Prior to film deposition, the resonators were immersed for ca. 20 s. in ethanol/water/potassium hydroxide mixture (59/ 40/1), rinsed in deionized water and used without further surface modification.

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Techniques and Instrumentation. An HMS series programmable slide stainer from Carl Zeiss, Inc., was used for the layer-by-layer assembly of humic acids with Fe3+. The sample holder in the HMS Series Slide Stainer was covered to reduce solvent evaporation thereby improving film quality. A Hewlett-Packard Network analyzer (HP-1005A, 10 kHz - 300 MHz) was utilized to detect changes in both the fundamental frequency and in the higher harmonics after every dip cycle during the self-assembly. The Sauerbrey equation37 was used to quantify the change in frequency with the apparent mass deposited on the resonator (eq 1), where ∆f ) -2fo2∆m/A (µQFQ)0.5

(1)

fo is the resonant frequency of the quartz resonator, ∆m is the mass change, A is the active surface area, µQ is the shear modulus of quartz, and FQ is the density of quartz. The higher frequency harmonics measured for an empty resonator and after every dip cycle during the HA/Fe3+ assembly were utilized for the shear modulus calculation. The shear compliance (J′) can be determined by plotting the equivalent mass (meq) (eq 2), derived from different harmonics, vs the square of the frequency ω2 ) (2πf)2.38 The true equivalent mass, deposited on the resonator, can be obtained from the intercept of this plot and the shear compliance and shear modulus can be further calculated from the slope, utilizing eq 3.38 The equivalent mass obtained by this procedure gives much better representation of the true mass deposited on the resonator by taking into account the viscoelastic losses of the self-assembled film. meq ) -(Zq/2f0)(δfn/fn)

(2)

slope ) J′(meq)2d/3

(3)

where Zq ) 8.8 × 106 kg m-2 s-1 is the piezoelectric stiffened acoustic impedance, f0 is the fundamental frequency of an empty quartz, δf/f ) (fin - fn)/fn; fin is the resonant frequency of the “ith” layer for the “nth” harmonic and fn is the resonant frequency of the empty resonator for the “nth” harmonic. J′ is the shear compliance, and d is the thickness of the film, determined by ellipsometry. The thickness of self-assembled films was independently determined by ellipsometry. Ellipsometric data was acquired using a variable-angle WVASE32 spectroscopic ellipsometer from J. A. Woolman Co., Inc. Films were scanned in the range 250-1000 nm at the incident angles of 65, 70, and 75°. Data were collected every 10 nm at 10 revolutions per measurement. A standard Cauchy model was used to fit for thickness and optical constants in the entire optical range. A dialysis chamber was used to determine permeability of 1 µm glass fiber membranes (Gelman Sciences) coated with self-assembled HA/Fe3+ layers. The interior of the dialysis chamber was filled with 1 mL of 1 M glucose in 0.9% NaCl solution. Subsequently, the concentration of glucose that diffused out from the dialysis chamber to the dialysis buffer (50 mL of 0.9% NaCl solution, no glucose) was determined by a Beckman glucose analyzer. The detailed procedure has been described elsewhere.39 The apparent diffusion coefficients (DApp) of glucose through the self-

Novel Humic Acids Based Films

assembled membranes were estimated based on the model described previously.35 The following assumptions were taken into account: (i) the glass fiber membrane was considered extremely porous and did not contribute to the resistance; hence the flux was assumed to be through the self-assembled layer; (ii) the flow rate did not effect the flux of glucose through the membrane; (iii) rapid diffusion was assumed through the boundary layers. Fourier transform infrared (FTIR) spectroscopy data was obtained from a Nicolet Magna 560 using TeGeSe detector with 4 cm-1 resolution. HAs/Fe3+ films were assembled on double polished Si wafer and held at an angle of 45° with respect to the incident IR beam. This increases the sampling path length and eliminates the interference fringes from the double polished silicon wafer. A minimum of 128 scans was signal averaged and the background, consisting of doublepolished Si wafers and air was carefully subtracted from the sample’s spectra. The stability of the HAs/Fe3+ films used for the in vivo experiments was evaluated by depositing them on glass slides and monitoring change in UV absorption as a function of immersion time in 100 mL of PBS buffer (phosphate buffer saline, Sigma Diagnostics) in a thermostated bath at 37 °C. A Perkin-Elmer Lambda 6 UV/vis spectrophotometer was used for these experiments. B. In Vivo Biocompatibility Studies. Humic acids solution at pH 5, with and without 0.01 M KCl, was used to coat medical grade silastic tubing, which is a well-known biocompatible material.40 Nine layers of HA/Fe3+ were selfassembled. The 1 cm long silastic tubing samples were sterilized in ethylene oxide (3M steri-gas at 55 °C for 2.5 h and 12 h of aeration cycle) before implantation. Three groups of three Sprague-Dawley male rats were used for this study. Both types of humic acids coatings on silastic tubing, prepared with and without salt, as well plain medical-grade silastic tubing as a control, were implanted into each rat. The first group of rats was euthanized after 1 day, the second after 1 week, and the last after 1 month. Samples were inserted subcutaneously using sterile 18 gauge iv catheters. The samples were slid into the rats through the lumen of the catheter. The backs of the rats had been shaved the day before, to minimize the inflammatory response due to the irritation of the shaving procedure. During implantation and shaving procedures the rats were anesthetized with isofluorane. After the rats were euthanized with carbon dioxide, an approximately 1 cm by 2 cm rectangle of tissue containing the implanted tubing was removed. It was fixed in formalin and later cut into several sections across its length, so that the tubing was cut in cross section. These tissue samples were prepared for the histology by embedding them in paraffin wax and staining by hematoxylin and eosin (H&E). Hematoxylin is known to dye the nucleus dark purple-blue,42 while the eosin stains other cell structures pink, especially the cytoplasm.42 Gomori’s one-step trichrome was also used to stain fibrous tissue (i.e., collagen fibers).42 These staining procedures help in elucidating the inflammatory response induced by the foreign implant.

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Figure 1. Ellipsometrically determined thickness vs dip cycle as a function of pH and ionic strength of HAs solutions for HAs/Fe3+ assemblies: (A) pH 5, 0.01 M KCl; (B) pH 5, no salt; (C) pH 9.5, 0.01 M KCl; (D) pH 9.5, no salt.

Results and Discussion A. Characterization. The technique of electrostatic layerby-layer self-assembly32 was used to deposit films of HAs with oppositely charged ferric ions. The HAs/Fe3+ selfassembly dip cycle consists of consecutive dipping in HAs and ferric chloride solution followed in each case by washing in deionized water. The wash step between the dipping steps helps to remove the loosely bounded moieties on the surface. The acidic functional groups in humic acids, primarily carboxylic (pKa 4-5)43 and cateholic (pKa 8-11)43 provide active sites for the positively charged ferric ions, which result in electrostatic and complexation driven interactions.44,45 The excess charge of the trivalent iron ions reverses the charge on the adsorbed HAs layer rendering it positive and thus allowing the next layer of HAs to assemble. Thus, multilayered HAs/Fe3+ films can be deposited both as a function of the pH and ionic strength of the humic acids solution. A detailed experimental description and discussion on the selfassembly process is provided in a previous study on Nafion/ Fe3+ assemblies.35 Figure 1 shows the ellipsometrically determined thickness of HAs/Fe3+ films vs the number of HAs/Fe3+ layers (i.e. dip cycles) as a function of the pH and ionic strength of humic acids solutions. The ellipsometric studies indicate a stepwise increase in film thickness. However, in some instances, particularly at a higher pH (which yields lesser ionization of the Si/SiOx surface), steady state is reached only after 3-5 dip cycles (see curve C in Figure 1). The growth characteristics of these assemblies exhibit strong dependence on the pH and ionic strength of the HAs solution, which can be correlated to the HAs conformation in solution and also that attained during surface deposition, as described below. The net charge on HAs is governed by the degree of ionization of its carboxylic and phenolic groups, which in turn is a function of solution pH.46 At a high pH these weak acid functionalities in HAs are ionized and attain a negative charge resulting in both inter- and intramolecular electrostatic repulsion. Therefore, in solutions of high pH, HAs are expected to attain a more expanded conformation. pH plays a very important role on the nature of the substrate surface that HAs have to assemble on due to iron(III) precipitation in its hydroxide form (Ksp ∼ 6 × 10-39).47 Above pH 4.3, the formation of iron hydroxides transforms the iron domi-

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Galeska et al. Table 1. Summary of Experimentally Determined HAs/Fe3+ Film Thickness and Apparent Mass Deposited per Dip Cycle on QCM Resonators as a Function of pH and Ionic Strength of HAs Solution pH 9 9 5 5 3 a

KCl [M] 0.01 0.01

deposited mass

layer thickness per dip cycle, nm

µga

µg/cm2

1.3 9.2 19.4 24.3 22.1

0.34 0.83 1.34 1.80 2.04

1.06 2.59 4.20 5.63 6.36

Mass deposited on QCM resonator in one dip cycle.

Figure 2. Frequency change as determined by QCM (left axes) upon self-assembly on the resonator and the calculated apparent mass (right axes) vs dip cycle as a function of the pH of HAs solutions for HAs/Fe3+ assemblies: (A) pH 3; (B) pH 5; (C) pH 7; (D) pH 9.5.

Figure 4. Changes in shear modulus obtained from QCM (right axis (b)) and equivalent mass (left axis (0)) of a HAs (pH 5.0, no salt)/ Fe3+ film as a function of self-assembled layers.

Figure 3. Frequency change as determined by QCM in (left axes) upon self-assembly on the resonator and the calculated apparent mass (right axes) vs dip cycle as a function of pH and ionic strength of HAs solutions for HAs/Fe3+ assemblies: (A) pH 5, 0.01 M KCl; (B) pH 5, no salt; (C) pH 9.5, 0.01 M KCl; (D) pH 9.5, no salt.

nated surface into a strong base that induces greater surface spreading of HAs due to acid-base interactions and affects the number of available complexation sites.35 Thus, the film growth is considerably slower, (i.e., less than 2 nm per dip cycle at pH 9.5) as compared to that at pH 5, where the growth rate is on the order of 19.4 nm per dip cycle. Similar acid-base neutralization-induced surface spreading leading to a lower growth rate was also observed in our previously reported Nafion/Fe3+ assemblies.35 Thus, neutralizationinduced surface spreading of HAs on the iron(III) surface is believed to be one of the governing factors in these assemblies. Another important factor could be the conformation dependence of HAs on pH. At a lower pH, the degree of ionization on HAs is lesser, which causes them to attain a tighter, globular conformation and simultaneously aggregate in solution.48 Upon adsorption on the surface, the hydrophobic interactions that are responsible for the globularization of the HAs, compete with neutralization-induced surface spreading leading to an enhanced growth rate i.e., 19.4 nm per dip cycle. The notable acceleration in film growth is observed upon increasing the ionic strength of HAs solution by adding potassium chloride (i.e. 24.3 nm per HA/ Fe3+ layer at pH 5 and 9.2 nm at pH 9.5). This faster film

Figure 5. Effect of film thickness on glucose permeability through the self-assembled HAs films (pH 5/Fe3+) supported on 1 µm glass fiber membranes. (-) Empty membrane (curve A); (9) 1 layer (curve B); (b) 2 layers; (1) 4 layers; (+) 6 layers; (/) 8 layers; (|) 20 layers (curve C).

growth is attributed to salt induced charge screening49 that leads to more compact HAs conformation capable of further resisting neutralization-induced surface spreading.35 Along with ellipsometry, QCM was utilized to obtain better understanding of the growth of HA/Fe3+ assemblies. QCM is an analytical method for the in situ probing of interfacial processes and has attracted considerable interest in the scientific community, with applications in various areas.38,50-52 For the HA/Fe3+ assembly, change in the resonant frequency (f0) of the QCM resonator was monitored after each dip cycle, where the decrease in f0 signifies the apparent mass adsorption. Because of the well-defined quartz resonant frequency, high precision measurements allow the detection of minute amounts of deposited material. The measured frequency changes can be correlated with mass

Novel Humic Acids Based Films

Figure 6. UV/vis spectra of 10 layers HAs/Fe3+ film immersed in PBS buffer (pH 7.4) for one to 4 weeks at 37 °C.

deposition occurring at the QCM surface via the Sauerbrey equation (eq 1).37 However, we presently use the term apparent to signify the fact that mass values represent true mass only if the films are rigid and not viscoelastic.53 The deposition of the self-assembled HA/Fe3+ film on the QCM resonator as a function of pH and ionic strength of the HAs solution are depicted in Figures 2 and 3, respectively. The left ordinate axis shows change in frequency, whereas the right axis corresponds to deposited apparent mass of the self-assembled film. The similarity in film growth on the silicon substrates, as determined by ellipsometry (Figure 1) and by QCM (Figures 2 and 3), suggests that HAs/

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Fe3+ self-assembly is substrate independent. The average apparent mass deposited per dip cycle varies from 1.06 µg/ cm2 for pH 9.5 to 6.36 µg/cm2 for pH 3 in the presence of salt (Table 1). The viscoelastic properties of layer-by-layer assembled HAs/Fe3+ films are of profound importance. The viscoelastic processes occurring during film deposition were characterized using QCM impedance spectroscopy to obtain shear modulus of these assemblies. This provides insight into film stability and resilience, which are important factors in the design of an implantable sensor. The equivalent mass and the shear modulus were calculated as described in the Experimental Section and are shown in Figure 4 on the left and right y-axis, respectively. The shear modulus increased linearly with the number of layers deposited. This is presently attributed to improved HAs interlayer penetration that along with additionally formed Fe3+ interactions yields stiffer assemblies (ca. 80 MPa for a 200 nm thick film in its hydrated form) (Figure 4). This value is ca. 2-3 orders of magnitude larger than that of most soft tissues.54-56 Subsequent drying of the film for 12 h in a vacuum oven resulted in an increase in the shear modulus to ca. 120 MPa. Upon implantation these assemblies will soften since the in vivo environment will approaches their glass transition temperature (ca. 42 °C).57 The control of glucose diffusion through the sensor’s membrane is one of the most important characteristics or the biosensor membrane. Figure 5 illustrates glucose perme-

Figure 7. Histology slides: (i) silastic tubing alone, implanted for (a) 1 day, (d) 1 week, and (g) 1 month; (ii) HAs (pH 5, 0.01 M KCl)/Fe3+coated silastic tubing, implanted for (b) 1 day, (e) 1 week, and (h) 1 month and (iii) HAs (pH 5, no salt)/Fe3+-coated silastic tubing, implanted for (c) 1 day, (f) 1 week, and (i) 1 month.

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ability through HAs/Fe3+ films (pH 5, in the absence of salt, supported on 1 µm glass fiber membranes) as a function of self-assembled HAs/Fe3+ layers. A comparison of curves A (porous supporting membrane) and B (membrane after the assembly of one HAs/Fe3+ layer) implies that the permeability of glucose through the membrane is dramatically altered after the very first dip cycle. Additional film deposition gradually decreases glucose permeability as the film grows on the glass fibers filling the interstitial pores of the supporting membrane. This trend suggests that glucose permeability can be selectively tuned as a function of pH and ionic strength of the HAs solution. The apparent diffusion coefficient, derived based on our previously reported model35 for a ca. 400 nm film (20 dip cycles) on the glass fiber membrane (curve C), was on the order of 5 × 10-8 cm2/s. This is about 2 orders of magnitude larger than for the Nafion/Fe3+ system,35 suggesting a relatively porous structure for the hydrated HA/Fe3+ membranes. The long-term stability of HA/Fe3+ films is of interest for their in vivo applications. Our in vitro studies (Figure 6) indicate a relatively high stability for these assemblies with a partial leaching of the HAs/Fe3+ membrane (ca. 15% loss of UV-active species i.e., HAs and Fe3+). The in vitro studies were performed by immersing the substrates in PBS buffer (pH 7.4) for a period of 4 weeks. However, extensive in vitro and in vivo experiments are necessary to confirm our initial observation and fully understand this process. B. In Vivo Evaluation in Rats. Since HAs present in soil have been shown to interact with ethylene oxide during sterilization (primarily due to hydroxyalkylation of labile hydrogen atoms)41 the extent of chemical modification of HAs/Fe3+ membrane has been evaluated by FTIR. Unlike the HAs from the soil samples, in the self-assembled films most of the functional groups except for those on the surface are satiated with Fe3+ and thus not available for esterification. This hypothesis was corroborated by FTIR results, where an increase in intensity of aliphatic band (2850-2950 cm-1) and the appearance of a very weak ester band (1725 cm-1) was observed upon sterilization. Significantly there was no detectable change in the optical thickness of these films after sterilization as measured by ellipsometry, suggesting the formation of a monolayer of ethylene oxide and not its polymerization. This is further corroborated by the lack of the typical polyether bands in the FTIR spectra. However, there was a slight decrease in hydrophilicity of these assemblies after sterilization (contact angle change from 49 to 67°) presumably due to some esterification of the functional groups on the film surface. Figure 7 shows some examples of the hematoxylin and eosin (H&E) stained tissue for each type of implanted sample: silastic tubing (a, d, and g), HAs/Fe3+-coated silastic tubing in the presence of salt (b, e, and h) and HAs/Fe3+coated silastic tubing without salt (c, f, and i). The implants, removed during histology processing, were located on the left of each picture. The arc bordering the clear area was the implant/tissue interface. At day one (a-c), monocytes and some polymorphonuclear leukocytes (PMN’s), which are associated with the acute inflammatory response are clearly

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seen. The nucleus of these cells is stained dark purple by hematoxylin. After 1 week (d-f), the acute inflammatory response has dissipated leaving only residual PMN’s and monocytes and the chronic and/or fibrotic responses have begun. The band of darker pink material at the tissue/implant interface is a fibrous encapsulation of the implants. This is seen more clearly in the samples implanted for 1 month (g-i). The three types of samples used in this study [(i) silastic, (ii) silastic + HAs (pH ) 5)/Fe3+/no salt and (iii) silastic + HAs (pH ) 5)/Fe3+/salt] have induced the same types of tissue reaction following subcutaneous implantation in rats. After 1 day of implantation (Figure 7, parts a-c), all samples have shown a moderate to fairly intense acute inflammation. This reaction appears to have been mostly caused by the tissue damage created during implantation since there was no significant difference between the silastic tubing (control) and the HAs-coated samples. The tissue reaction was characterized by many monocytes and a few PMNs. One week after implantation (Figure 7, parts d-f), the tissue reaction in each sample was characterized by very little residual acute inflammation, a low-grade fibroblast encapsulation and a few focal points of blood vessels. Four weeks after implantation (Figure 7, parts g-i), the tissue reaction showed almost no inflammation, light to moderate fibrosis with some neovascularization and no giant cells. Since there was no difference in the intensity and nature of the tissue reaction observed among these samples and silastic tubing controls, the HAs/Fe3+ assemblies appear to be well-tolerated by the tissue. Conclusions Novel humic acids (HAs) based films grown via layerby-layer self-assembly are presently reported as a plausible membrane material for implantable glucose sensors. The HAs/Fe3+ film growth shows strong dependence on the pH and ionic strength of the HAs solution. The underlying reasons of this behavior appears to be related to pHdependent conformational rearrangement of HAs molecules in solution as well as neutralization-induced spreading on the surface. Film growth can be significantly altered by the incorporation of salt, where the charge-screening effect contributes to thicker film deposition, irrespective of pH. With a shear modulus in the range of 80 MPa, HAs-based assemblies are soft and pliable, qualities that should potentially minimize tissue damage. The glucose permeability through these films can be tuned by varying the number of self-assembled layers. These films can be considered biocompatible since in vivo studies in rats indicate mild tissue reaction along with some neovascularization around the HAs/ Fe3+-coated implants. Moreover, the absence of significant fibrosis around the implant is a considerable advantage over the Nafion based membranes for biosensors that rely on glucose diffusion.58-60 Acknowledgment. The authors wish to thank our collaborators: Drs. D. J. Burgess, S. Huang, J. T. Koberstein, T. K. Kim, and D. Chattopadhyay for stimulating discussions.

Novel Humic Acids Based Films

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