Herein we firstly reported a simple, environment-friendly, controllable synthetic method of CuSe nanosnakes at room temperature using copper salts and sodium selenosulfate as the reactants, and bovine serum albumin (BSA) as foaming agent. As the amounts of selenide ions (Se2−) released from Na2SeSO3 in the solution increased, the cubic and snake-like CuSe nanostructures were formed gradually, the cubic nanostructures were captured by the CuSe nanosnakes, the CuSe nanosnakes grew wider and longer as the reaction time increased. Finally, the cubic CuSe nanostructures were completely replaced by BSA–CuSe nanosnakes. The prepared BSA–CuSe nanosnakes exhibited enhanced biocompatibility than the CuSe nanocrystals, which highly suggest that as-prepared BSA–CuSe nanosnakes have great potentials in applications such as biomedical engineering.
Copper selenide; Nanosnakes; Bovine serum albumin; Synthesis; Characterization; Mechanism; Biocompatibility
Thallium(I) copper(I) thorium(IV) triselenide, TlCuThSe3, crystallizes with four formula units in the space group Cmcm in the KCuZrS3 structure type. There is one crystallographically independent Th, Tl, and Cu atom at a site of symmetry 2/m.., m2m, and m2m, respectively. There are two crystallographically independent Se atoms at sites of symmetry m.. and m2m. The structure consists of sheets of edge-sharing ThSe6 octahedra and CuSe4 tetrahedra stacked parallel to the (010) face, separated by layers filled with chains of Tl running parallel to . Each Tl is coordinated by a trigonal prism of Se atoms.
Spectroscopic ellipsometry was used to characterize the optical properties of thin (<5 nm) films of nanostructured titanium dioxide (TiO2). These films were then used to investigate the dynamic adsorption of bovine serum albumin (BSA, a model protein), as a function of protein concentration, pH, and ionic strength. Experimental results were analyzed by an optical model and revealed that hydrophobic interactions were the main driving force behind the adsorption process, resulting in up to 3.5 mg/m2 of albumin adsorbed to nanostructured TiO2. The measured thickness of the adsorbed BSA layer (less than 4 nm) supports the possibility that spreading of the protein molecules on the material surface occurred. Conformational changes of adsorbed proteins are important because they may subsequently lead to either accessibility or inaccessibility of bioactive sites which are ligands for cell interaction and function relevant to physiology and pathology.
Nanomaterials; protein adsorption; spectroscopic ellipsometry; titanium dioxide; bovine serum albumin
The interaction between Avelox and bovine serum albumin (BSA) was investigated at different temperatures by fluorescence spectroscopy. Results showed that Avelox could quench the intrinsic fluorescence of BSA strongly, and the quenching mechanism was a static quenching process with Förester spectroscopy energy transfer. The electrostatic force played an important role on the conjugation reaction between BSA and Avelox. The order of magnitude of binding constants (Ka) was 104, and the number of binding site (n) in the binary system was approximately equal to 1. The binding distance (r) was less than 3 nm and the primary binding site for Avelox was located in subdomain IIA of BSA. Synchronous fluorescence spectra clearly revealed that the microenvironment of amino acid residues and the conformation of BSA were changed during the binding reaction. In addition, the effect of some antibiotics on the binding constant of Avelox with BSA was also studied.
Nanocrystalline lead selenide (PbSe) thin films were prepared on glass substrates by a chemical bath deposition method, using sodium selenosulfate (Na2SeSO3) as a source of Se2− ions, and lead acetate as a source of Pb2+ ions. Trisodium citrate (TSC) was used as a complexing agent. PbSe films were prepared at various deposition temperatures while the pH value was kept fixed at 11, and the effect on the resulting film properties was studied by X-ray diffraction (XRD), X-ray fluorescence (XRF), scanning electron microscopy (SEM) and optical absorption studies. The structural parameters, such as the lattice constant (a), crystallite size (D), dislocation density (ρ) and microstrain (ε) were evaluated from the XRD spectra. It was found that average crystallite size, as calculated from Scherrer’s formula, increased from 23 to 33 nm as the deposition temperature was varied from 303 to 343 K. The dislocation density and microstrain were found to vary inversely with the crystallite size, whereas the lattice constant was found to increase with an increase in crystallite size. The optical absorption spectra of the nanocrystalline PbSe films showed a blue shift, and the optical band gap (E
) was found to increase from 1.96 to 2.10 eV with the decrease in crystallite size.
chemical bath deposition; lattice parameter; lead selenide; Nelson–Riley plot; optical absorption
Assessment of behavioral skills remains critical to the evaluation of HIV prevention interventions; however, investigators often rely upon participant reports of self-efficacy to estimate such skills. We evaluated the relationship between self-efficacy beliefs for condom use and behavioral performance. Forty-three men completed the Condom Use Self-Efficacy Scale (CUSES) and participated in two behavioral assessments. Regression analyses indicated that the CUSES subscales relevant to negotiation of condom use did not account for a significant amount of variability in interpersonal skills; similarly, the CUSES subscale relevant to technical condom use skill did not account for variability in the condom application scores. We caution investigators against the assumption that higher self-efficacy reflects behavioral competence for HIV-risk reduction.
Self-efficacy; HIV; AIDS; assessment
By combination of isoelectric focusing and immunoelectrophoresis of fresh bovine plasma it is shown that 10% of the albumin in plasma has isoionic points equal to the intramolecular SS-interchanged isomers of bovine serum albumin (BSA). It is also shown that (a) albumin with the isoionic point of SS-interchanged BSA is produced in the cow from radioiodinated BSA depleted from SS-interchanged albumin before injection and (b) purified radiolabeled SS-interchanged BSA can be converted in vivo to albumin with the native isoionic point. On this basis, it is proposed that SS-interchanged albumin in vivo is in postsynthetic equilibrium with the "native" albumin conformation. The SS-interchanged isomers purified either from commerical BSA or from BSA submitted to SH-SS interchange was, after radioiodination with 125I, compared metabolically with "native" albumin labeled with 131I in the same calf. Both species of SS-interchanged albumins have fast initial turnover rates but obtain a normal rate of degradation after the reversion to native albumin. If the isomers formed in vivo have the same properties as the ones present in commercial BSA, at least 50% of the physiological degradation of albumin can be accounted for by the 6-7 times faster catabolic rates of these isomers.
In this study, we applied electrical polarization technique to increase adsorption and control protein release from biphasic calcium phosphate (BCP). Three different biphasic calcium phosphate (BCP) composites, with hydroxyapatite (HAp) and β-tricalcium phosphate (β-TCP), were processed and electrically polarized. Our study showed that stored charge was increased in the composites with the increase in HAp percentage. Adsorption of bovine serum albumin (BSA), as a model protein, on the poled, as well as unpoled surfaces of the composites, was studied. The highest amount of BSA adsorption was obtained on positively poled surfaces of each composite. Adsorption isotherm study suggested a multilayer adsorption of BSA on the BCP composites. The effect of electrical polarization on BSA release kinetics from positively charged BCP surfaces was studied. A gradual increase in percent BSA release from positively charged BCP surfaces with decreasing stored charge was observed. Our study showed that the BCP based composites have the potential to be used as drug or growth factor delivery vehicle.
The purpose of the present study was to evaluate the effects of bovine serum albumin (BSA) and essentially fatty acid-free BSA (BSA-FAF) on the biliary clearance of compounds in sandwich-cultured rat hepatocytes. Unbound fraction (fu), biliary excretion index (BEI), and unbound intrinsic biliary clearance (intrinsic Cl’biliary) were determined for digoxin, pravastatin, and taurocholate in the absence or presence of BSA or BSA-FAF. BSA had little effect on the BEI or intrinsic Cl’biliary of these compounds. Surprisingly, BSA-FAF decreased both BEI and intrinsic Cl’biliary for digoxin and pravastatin, which represent low and moderately-bound compounds, respectively. The BEI and intrinsic Cl’biliary of taurocholate, a highly-bound compound, were not altered significantly by BSA-FAF. Neither BSA nor BSA-FAF had a discernable effect on the bile canalicular networks, based on carboxydichlorofluorescein (CDF) retention. The addition of physiological concentrations of calcium, or the addition of fatty acids to BSA-FAF, was unable to restore the BEI or intrinsic Cl’biliary of the model compounds to similar values in the absence or presence of BSA. Careful consideration is warranted when selecting the type of BSA for addition to in vitro systems such as sandwich-cultured rat hepatocytes.
Microchip capillary electrophoresis of proteins labeled either off- or on-chip with the “chameleon” CE dye 503 using poly(methyl methacrylate) microchips is presented. A simple dynamic coating using the cationic surfactant cetyltrimethyl ammonium bromide prevented nonspecific adsorption of protein and dye to the channel walls. The labeling reactions for both off- and on-chip labeling proceeded at room temperature without requiring heating steps. In off-chip labeling, a 9 ng/mL concentration detection limit for bovine serum albumin (BSA), corresponding to a ~7 fg (100 zmol) mass detection limit, was obtained. In on-chip tagging, the free dye and protein were placed in different reservoirs of the microchip, and an extra incubation step was not needed. A 1 μg/mL concentration detection limit for BSA, corresponding to a ~700 fg (10 amol) mass detection limit, was obtained from this protocol. The earlier elution time of the BSA peak in on-chip labeling resulted from fewer total labels on each protein molecule. Our on-chip labeling method is an important part of automation in miniaturized devices.
Dye conjugation is a common strategy improving the surface enhanced Raman detection sensitivity of biomolecules. Reported is a proof-of-concept study of a novel surface enhanced Raman spectroscopic tagging strategy termed as acid-cleavable SERS tag (ACST) method. Using Rhodamine B as the starting material, we prepared the first ACST prototype that consisted of, from the distal end, a SERS tag moiety (STM), an acid-cleavable linker, and a protein reactive moiety. Complete acid cleavage of the ACST tags was achieved at a very mild condition that is 1.5% trifluoroacetic acid (TFA) aqueous solution at room temperature. SERS detection of this ACST tagged protein was demonstrated using bovine serum albumin (BSA) as the model protein. While the SERS spectrum of intact ACST-BSA was entirely dominated by the fluorescent signal of STM, quality SERS spectra can be readily obtained with the acid cleaved ACST-BSA conjugates. Separation of the acid cleaved STM from protein further enhances the SERS sensitivity. Current SERS detection sensitivity, achieved with the acid cleaved ACST-BSA conjugate is ~5 nM in terms of the BSA concentration and ~1.5 nM in ACST content. The linear dynamic range of the cleaved ACST-BSA conjugate spans four orders of magnitudes from ~10 nM to ~100 μM in protein concentrations. Further improvement in the SERS sensitivity can be achieved with resonance Raman acquisition. This cleavable tagging strategy may also be used for elimination of protein interference in fluorescence based biomolecule detection.
Tumor necrosis factor alpha (TNFα) is a cytokine that regulates immune and inflammatory overactivation in various pathological states. Protein therapeutics may antagonize this cytokine, but may also have systemic toxicities. Small molecule natural products are also efficacious, but can suffer from poor oral bioavailability. A drug delivery vehicle is needed to sustain release of active therapeutics and address localized inflammation.
Chitosan is a biocompatible aminopolysaccharide that undergoes thermally-initiated gelation in cosolutions with glycerophosphate (GP), and may entrap and sustain release of additive therapeutics. Gelation time and temperature of chitosan/GP were evaluated by turbidity (OD350), as was the kinetic effect of bovine serum albumin (BSA) entrapment. We investigated in vitro release of BSA and various anti-TNF agents (curcumin, sTNFRII, anti-TNF antibody) and confirmed in vitro activity of the released drugs using an established bioassay.
Turbidity results show that chitosan/GP thermogel achieves gelation at 37°C within 10 minutes, even with significant protein loading. Sustained BSA release occurred with 50% retained at 7 days. All anti-TNF therapeutics exhibited sustained release, with 10% of sTNFRII and anti-TNF antibody remaining after 7 days and 10% of curcumin remaining after 20 days. After release, each compound antagonized TNFα-cytotoxicity in murine fibrosarcoma cells.
This study demonstrates that thermogelling chitosan/GP entraps and sustains release of a broad range of anti-TNF agents. Such delivery of disease-modifying therapy could establish a drug depot to treat local inflammation. The breadth of molecular sizes demonstrates significant versatility, and slow release could protect against toxicities of systemic delivery.
chitosan; drug delivery; drug depot; tumor necrosis factor alpha; inflammation; curcumin
We investigated fluorescence enhancements and lifetime reductions of Cy5 probe molecules at various distances from the deposited silver island film surface using single molecule spectroscopic methods. The proximity of fluorophore molecules to the surface was controlled by alternating layers of biotinylated bovine serum albumin (BSA-biotin) and avidin, followed by binding of Cy5-labeled oligonucleotides to the top of a BSA-biotin layer structure. We observed dramatically varied brightness of fluorophores with distances from metal structures as well with reduced blinking in the presence of silver island films. In addition, distributions of fluorescence lifetimes and apparent emission intensities from individual molecules indicate an inhomogeneous nature of local matrix surface near metallic nanostructures. These studies illustrate the exclusive information that is otherwise hidden in ensemble measurements.
Our laboratory has pioneered the use of bovine serum albumin (BSA) linked to different positions of the ring structure of progesterone to investigate steroid-membrane interactions. The complex can be radioiodinated to demonstrate the existence of specific membrane progesterone binding sites in the rat brain. Not only are these progesterone complexes specific ligands, but they also elicit functional responses in the central nervous system (CNS), particularly in the corpus striatum (CS) where progesterone-BSA conjugates linked at C-3 and C-11 positions (P-3-BSA and P-11-BSA) alter amphetamine-evoked dopamine release. In this communication we will report our current studies that use radioiodinated progesterone-BSA conjugates (P-3-125I-BSA, P-6-125I-BSA, and P-11-125I-BSA) and estradiol-BSA conjugates linked at C-6 position (17 beta-E-6-125I-BSA and 17-E-6-BSA) to demonstrate the existence of specific membrane binding sites for progesterone and estrogen in several regions of the rat brain. In addition, initial studies to isolate and purify these membrane binding sites from digitonin-solubilized P2-membrane fractions by affinity chromatography are reported. The data indicate that these sites are part of a complex membrane receptor for either estrogen or progesterone, the so-called membrane estrogen receptor (mER) and the membrane progesterone receptor (mPR), respectively.
Immune cells induced by bovine serum albumin (BSA) and its methylated derivative (MBSA) have been compared in a cooperative cell transfer system for their content of BSA-specific antibody-forming cell precursors (AFCP, B) and BSA-specific helper (T) cells. When MBSA immune cells were transferred together with hapten-primed cells into recipient mice which were stimulated by a hapten-BSA conjugate, their cooperative secondary anti-hapten response was as good as in case of transferred BSA immune cells. Their secondary anti-BSA response, however, was markedly reduced (reduction factor > 30). Hapten-MBSA conjugates had the same capacity to react with BSA-specific helper cells in the cooperative secondary anti-hapten response as hapten-BSA conjugates but had a reduced ability to react with BSA-specific AFCP cells. In spite of the pronounced reduction of the B cell response, MBSA had the same threshold dose as BSA for activating BSA-specific T cells. These data suggest that B and T cells recognize different epitopes on the BSA molecule, only those recognized by B cells being affected by the methylation procedure.
The binding of coomassie brilliant blue G (CBB) to bovine serum albumin (BSA) was investigated under simulative physiological conditions employing different optical spectroscopic techniques viz., fluorescence emission, UV–visible absorption and FTIR. Fluorescence quenching data obtained at different temperatures suggested the presence of dynamic type of quenching mechanism. The binding constant of CBB-BSA and the number of binding sites (n) for CBB in BSA were calculated and found to be 4.20 × 104 M−1 and 0.96 respectively, at 302 K. The value of n close to unity indicated that the protein has a single class of binding sites for CBB. The thermodynamic parameters revealed that the hydrophobic forces played a major role in the interaction of CBB to BSA. The distance between the CBB and protein was calculated using the theory of Föster’s Resonance Energy Transfer (FRET). The conformational change in the secondary structure of BSA upon interaction with dye was investigated by synchronous fluorescence and FTIR techniques. Competitive binding studies were also carried out to know the location of binding of CBB on BSA.
Coomassie brilliant blue G; Physiological condition; Optical spectroscopy; Binding parameter; Thermodynamic parameter
Chemical coupling of polyethylene glycol (PEG) to proteins or particles (PEGylation), prolongs their circulation half-life by greater than 50-fold, reduces their immunogenicity, and also promotes their accumulation in tumors due to enhanced permeability and retention effect. Herein, phase separation method was used to prepare bovine serum albumin (BSA) nanoparticles. PEGylation of BSA nanoparticles was performed by SPA activated mPEG through their free amino groups. Effect of process variables on PEGylation efficiency of BSA nanoparticles was investigated and optimized through response surface methodology with the amount of free amino groups as response. Optimum conditions was found to be 32.5 g/l of PEG concentration, PEG-nanoparticle incubation time of 10 min, incubation temperature of 27°C, and pH of 7 for 5 mg of BSA nanoparticles in 1 mL phosphate buffer. Analysis of data showed that PEG concentration had the most noticeable effect on the amount of PEGylated amino groups, but pH had the least. Mean diameter and zeta potential of PEGylated nanoparticles under these conditions were 217 nm and −14 mV, respectively. In conclusion, PEGylated nanoparticles demonstrated reduction of the negative surface charge compared to the non modified particles with the zeta potential of −31.7 mV. Drug release from PEGylated nanoparticles was almost slower than non-PEGylated ones, probably due to existence of a PEG layer around PEGylated particles which makes an extra resistance in opposition to drug diffusion.
5-fluorouracil; BSA nanoparticle; PEGylation optimization; response surface method (RSM)
Background—Although many studies
have investigated macromolecular uptake in the stomach and small
intestine, little is known about macromolecular uptake in the colon.
Aims—To investigate the mechanisms
involved in the transport of large antigenically intact macromolecules
across the proximal and distal colonic epithelium in the rabbit.
Methods—The mucosal to serosal
movement of bovine serum albumin (BSA) was examined in modified Ussing
chambers under short circuited conditions. The mucosal surface was
exposed to varying concentrations of BSA, and after a 50 minute
equilibration period, the mucosal to serosal flux of immunologically
intact BSA was determined by ELISA. Total BSA flux was determined by the transport of radiolabelled 125I-BSA.
Results—Intact BSA transport in
proximal and distal colonic tissue showed saturable kinetics. Intact
BSA transport in the proximal and distal segment was 7% and 2% of the
total 125I-BSA flux respectively. Immunologically intact
BSA transport in the distal segment was significantly less than that in
the proximal segment. Intact BSA transport in the proximal colon was significantly reduced following treatment with sodium fluoride, colchicine, and tetrodotoxin. Cholinergic blockade had no effect on the
uptake of intact BSA.
Conclusion—The findings indicate
that the transport of intact macromolecules across the proximal and
distal large intestine is a saturable process. Further, intact BSA
transport in the proximal colon is an energy dependent process that
utilises microtubules and is regulated by the enteric nervous system.
colon; transport; protein
Bovine serum albumin (BSA) protein incorporated with hydroxyapatite (HA) nanoparticles (NPs) were synthesized by in situ precipitation process. 2 mol% Zn2+ and Mg2+ were used as dopants to synthesize Zn2+/Mg2+ doped HA-BSA NPs by in situ synthesis route. In our study we used BSA as a model protein. The amount of BSA uptake by doped and undoped HA NPs and subsequent release of BSA from NPs were investigated. Zn doped HA NPs showed the highest amount of BSA uptake, whereas the amount of BSA loaded in undoped HA NPs was the lowest. A two-stage BSA release profile from doped and undoped HA NPs was observed in phosphate buffer solution (PBS) at pH 7.2 ± 0.2. Initial burst release was due to the desorption of BSA from the HA surface. The later stage of slow release was controlled by the dissolution of BSA incorporated HA NPs. BSA release rate from Zn doped HA NPs was found to be the highest, whereas undoped HA NPs released BSA at the slowest rate. Our study showed that the protein release rate from HA NPs can be controlled by the addition of suitable dopants and doped HA based NP systems can be used in bone growth factor and drug release study.
Nanoparticles; Hydroxyapatite; Dopant; Bovine serum albumin; Protein release
Aldehyde- and NHS-activated magnetic microspheres were used to immobilize trypsin (CHO-trypsin and NHS-trypsin), and their performance for protein digestion was evaluated by reversed phase liquid chromatography-electrospray ionization-tandem mass spectrometry using an LTQ Orbitrap Velos instrument. NHS-trypsin provided greater sequence coverage and identified more peptides for the digestion of bovine serum albumin. A one-minute digestion at room temperature using the immobilized trypsin also identified more peptides (96 ± 6 vs. 48 ± 1) and produced higher sequence coverage (90 ± 2% vs. 75 ± 2%) than traditional free trypsin digestion for 12 hours at 37 °C. Analysis of 15 nM (0.001 mg/mL) BSA digested by NHS-trypsin in 1 min. at room temperature consistently yielded one detected peptide; 150 nM BSA generated 22 peptides. Peptide intensity and protein spectral count were used to evaluate the run-to-run digestion reproducibility of NHS-trypsin with a three-protein-mixture. Three high intensity peptides for each protein generated intensity ratios from 0.70 to 1.09 and spectral count ratios from 0.78 to 1.18. Finally, RAW 264.7 cell lysates were digested by NHS-trypsin for 10 min. and 30 min. at room temperature; 604 and 697 protein groups, respectively, were identified by RPLC-ESI-MS/MS, with a peptide false discovery rate of less than 1%. Digestion by solution phase trypsin for 12 hours at 37 °C resulted in identification of 878 protein groups.
protein digestion; trypsin-immobilized magnetic microspheres; reproducibility; RPLC-ESI-MS/MS
A specific antibody for 2,3-dihydro-2,3-dihydroxyaflatoxin B1 (AFB1-diol) was prepared, and its reactivity was characterized for the major aflatoxin (AF) B1 (AFB1) metabolites. Reductive alkylation was used to conjugate AFB1-diol to ethylenediamine-modified bovine serum albumin (EDA-BSA) and horseradish peroxidase for use as an immunogen and an enzyme-linked immunosorbent assay (ELISA) marker, respectively. High reactant ratios, 1:5 and 1:10, for AFB1-diol-EDA-BSA (wt/wt) resulted in precipitated conjugates which were poorly immunogenic. However, a soluble conjugate obtained by using a 1:25 ratio of AFB1-diol to EDA-BSA could be used for obtaining high-titer AFB1-diol rabbit antibody within 10 weeks. Competitive ELISAs revealed that the AFB1-diol antibody detected as little as 1 pmol of AFB1-diol per assay. Cross-reactivity of AFB1-diol antibody in the competitive ELISA with AF analogs was as follows: AFB1-diol, 100%; AFB1, 200%; AFM1, 130%; AFB2a, 100%; AFG1, 6%; AFG2, 4%; aflatoxicol, 20%; AFQ1, 2%; AFB1-modified DNA, 32%; and 2,3-dihydro-2-(N7-guanyl)-3-hydroxy AFB1, 0.6%. These data indicated that the cyclopentanone and methoxy moieties of the AF molecule were the primary epitopes for the AFB1-diol antibody. The AFB1-diol competitive ELISA was subject to substantial interference by human, rat, and mouse serum albumins but not by BSA, Tris, human immunoglobulin G, or lysozyme. By using a noncompetitive, indirect ELISA with an AFB1-modified DNA solid phase, a modification level of one AFB1 residue for 200,000 nucleotides could be determined.
This document reports a novel method of measuring association rate constant (ka) for antibody-antigen interaction using evanescent wave-based combination tapered fiber-optic biosensor (CTFOB) dip-probes. The method was demonstrated by measuring association rate constant for bovine serum albumin (BSA) and anti-BSA antibody interaction. “Direct method” was used for detection; goat anti-BSA “capture” antibodies were immobilized on the probe surfaces while the antigen (BSA) was directly labeled with Alexa 488 dye. The probes were subsequently submerged in 3 nM Labeled BSA in egg albumin (1 mg/ml). The fluorescence signal recorded was proportional to BSA anti-BSA conjugates and continuous signal was acquired suing a fiber optic spectrometer (Ocean Optics, Inc.). A 476 nm diode laser was use as an excitation source. Association constant was estimated from a plot of signal as a function of time. Measured association rate constant ka for the binding of BSA with anti-BSA at room temperature is (8.33 ± 0.01) ×104 M−1 s−1.
association constant; BSA; anti-BSA; fiber-optic; biosensor; real-time
Polycaprolactone (PCL) was coated on porous tricalcium phosphate (TCP) scaffolds to achieve controlled protein delivery. Porous TCP scaffolds were fabricated using reticulated polyurethane foam as sacrificial scaffold with a porosity of 70–90 vol %. PCL was coated on sintered porous TCP scaffolds by dipping-drying process. The compressive strength of TCP scaffolds increased significantly after PCL coating. The highest strength of 2.41 MPa at a porosity of 70% was obtained for the TCP scaffold coated with 5% PCL solution. Model protein bovine serum albumin (BSA) was encapsulated efficiently within the PCL coating. The amount of BSA encapsulation was controlled by varying proteins’ composition in the PCL coating. The FTIR analysis confirmed that BSA retained its structural conformation and did not show significant denaturization during PCL coating. The release kinetics in phosphate buffer solution indicated that the protein release was controlled and sustained, and primarily dependant on protein concentration encapsulated in the PCL coating.
porous tricalcium phosphate; polycaprolactone; coating; protein delivery
Thymus cells from 5- to 6-week-old normal (unimmunized) BALB/c mice showed an increased incorporation of [3H]thymidine in the presence of 2,4-dinitrophenyl-bovine serum albumin, fluorescein-bovine serum albumin, and bovine serum albumin (BSA) in tissue culture. The concentrations of antigen (BSA and haptenated proteins) required for stimulation were approximately 25- to 50-fold higher than those of the nonspecific mitogen, concanavalin A. In contrast to the stimulation by concanavalin A, which was maximal at 24 to 72 h, the stimulation by antigen was most marked earlier in the culture period (6 to 24 h). The BSA response was diminished to a statistically significant degree (especially at low BSA concentrations) in thymocytes from animals injected 72 h previously with BSA, indicating that the stimulation is immunologically specific.
To characterize thermoresponsive hydrogels (liquids at room temperature, gels at body temperature) as a novel drug delivery platform to the posterior segment.
Thermoresponsive hydrogels were synthesized using poly(N-isopropylacrylamide) (PNIPAAm), cross-linked with poly(ethylene glycol) diacrylate (PEG-DA). Proteins were then encapsulated into the hydrogels, including bovine serum albumin (BSA), immunoglobulin G (IgG), and, finally, bevacizumab and ranibiumab. By varying the degree of cross-linker density, the rate of protein release could be adjusted. The rate of release was assessed at various time points with Bradford assay, and the bioactivity of the released anti–vascular endothelial growth factor agents was studied in an in vitro cell culture assay.
Cross-linked PNIPAAm hydrogel exhibited a fast and reversible phase change with alteration in temperature. The rate of protein release was examined as a function of cross-link density. Release profiles of the proteins showed that there was an initial burst of release within 48 hours, and then a steady state was reached, which was sustained for approximately 3 weeks. Hydrogels with less cross-linking showed faster release and yielded a more pliable gel for intravitreal injection via small-gauge needles. Examination of the gels after the release experiment revealed significant residual entrapped protein.
Thermoresponsive hydrogels were successfully synthesized and exhibited fast and reversible phase changes. The gel was able to encapsulate and release various proteins. Current formulation of the gel will be modified to extend the release time and to be made fully biodegradable. Thermoresponsive hydrogels appear to be a promising, minimally invasive platform for extended drug delivery to the posterior segment.