Simulated Physiological Fluids Research Articles

Composite Nanoparticles Based on Hyaluronic Acid Chemically Cross-Linked with α,β-Polyaspartylhydrazide

In this paper, new composite nanoparticles based on hyaluronic acid (HA) chemically cross-linked with alpha,beta-polyaspartylhydrazide (PAHy) were prepared by the use of a reversed-phase microemulsion technique. HA-PAHy nanoparticles were characterized by FT-IR spectroscopy, confirming the occurrence of the chemical cross-linking, dimensional analysis, and transmission electron micrography, showing a sub-micrometer size and spherical shape. Zeta potential measurements demonstrated the presence of HA on the nanoparticle surface. A remarkable affinity of the obtained nanoparticles toward aqueous media that simulate some biological fluids was found. Stability studies showed the absence of chemical degradation in various media, while in the presence of hyaluronidase, a partial degradation occurred. Cell compatibility was evaluated by performing in vitro assays on human chronic myelogenous leukaemia cells (K-562) chosen as a model cell line and a haemolytic test. HA-PAHy nanoparticles were also able to entrap 5-fluorouracil, chosen as a model drug, and release it in a simulated physiological fluid and in human plasma with a mechanism essentially controlled by a Fickian diffusion.

Elucidation of the physicomechanical and ab initio quantum energy transitions of a crosslinked PLGA scaffold

This study elucidated the in vitro physicomechanical transitions of a crosslinked polylactic- co-glycolic acid (PLGA) scaffold, utilizing quantum mechanics to compute the ab initio energy requirements of a salted-out and subsequently crosslinked PLGA scaffold interacting with simulated physiological fluid, phosphate buffered saline (PBS) (pH 7.4, 37 °C) at a molecular level. Twenty-six salted-out PLGA scaffolds were formulated using a four factor, two centerpoint quadratic Face-Centered Central Composite Design (FCCD). PLGA molecular mass, PLGA concentration, water volume and salting-out reaction time were the dependant formulation variables. Subsequent to PLGA solubilization in dimethyl formamide (DMF), protonated water was added to induce salting-out of PLGA into a scaffolds that were immersed in PBS, oscillated at 100 rpm, and analyzed at pre-determined time intervals for their physicomechanical and ab initio quantum energy transitions. Results indicated that the matrix resilience (MR) decreased with longer incubation periods (MR=35–45%) at day 30. Scaffolds salted-out using higher PLGA concentrations exhibited minimal changes in MR and the matrix ability to absorb energy was found to closely correlate with the scaffold residence time in PBS. Spartan-based ab initio quantum energy predictions elucidated the potential scaffold stability from a molecular viewpoint and its suitability for use in rate-modulated drug delivery.

Biotribological performance of NCD coated Si 3N 4–bioglass composites

A nanocrystalline diamond (NCD) coated Si 3N 4–bioglass composite, with potential use for hip and knee joint implants, was tribologically tested in simulated physiological fluids. NCD was deposited using a hot-filament chemical vapour deposition (HFCVD) apparatus in an Ar–H 2–CH 4 gas mixture. Self-mated reciprocating experiments were performed using a pin-on-flat geometry in Hanks' balanced salt solution (HBSS) and dilute fetal bovine serum (FBS). A nominal contact pressure of 25 MPa was applied for up to 500,000 cycles. Very low friction coefficients of 0.01–0.02 were measured using HBSS, while for FBS lubricated tests the values are slightly higher (0.06–0.09), due to a protein attaching effect. AFM assessed wear rates by an approach using the bearing function for volume loss quantification, yielding wear rates of k ∼ 10 − 10 mm 3 N − 1 m − 1 in HBSS and k ∼ 10 − 9 –10 − 8 mm 3 N − 1 m − 1 for FBS, characteristic of very mild wear regimes.

Nucleation and growth of calcium phosphate on amine-, carboxyl- and hydroxyl-silane self-assembled monolayers

Upon implantation, calcium phosphate (Ca–P) surfaces form on materials that are bone bioactive. In this study, the evolving surface characteristics associated with calcium phosphate precipitation are modeled using self-assembled monolayers (SAMs), in a one-step nucleation process. SAMs were used to create amine (–NH 2), carboxyl (–COOH) and hydroxyl (–OH) functionalized surfaces by grafting 3-aminopropyltriethoxysilane, 3-triethoxysilylpropyl succinic anhydride and glycidoxypropyl tri-methoxysilane, respectively, onto oxidized silicon wafers. The SAM surfaces were characterized using ellipsometry to establish the presence of grafted molecules. On the surfaces incubated in simulated physiological fluids for 7 days, the thickness of Ca–P layer grew slowly over the first few hours, increasing strongly between 1 and 5 days and then slowed down again. FTIR showed the dependence of calcium phosphate morphology on the type of surface groups, with stronger P–O bands seen on the OH-terminated surface. SEM analysis showed dispersed Ca–P precipitates on the –COOH and –OH terminated surfaces after 1 day immersion. After 7 days, all SAM surfaces were covered with uniformly dispersed and denser Ca–P precipitates. The underlying Ca–P layer showed cracks on the –NH 2-terminated surface. Rutherford backscattering spectrometry (RBS) data analysis confirmed that Ca/P ratio is in excellent agreement with the theoretical value of 1.67 for hydroxyapatite. X-ray diffraction (XRD) analysis also showed evidence of apatite formation on all the surfaces, with stronger evidence on the –OH-terminated surface. Highly porous Ca–P precipitates were observed on the SAM surfaces portrayed by the AFM scans with nanoscale RMS roughness. Thus, using highly controlled surface chemistry, under physiological conditions, in vitro, this study demonstrates that a hydroxylated surface enhances Ca–P nucleation and growth relative to other surfaces, thereby supporting the concept of its beneficial effect on bone tissue formation and growth.

Nucleation and Growth of Bone-Like Apatite on Surfaces of Metals, Ceramics and Polymers in Simulated Body Fluids

The biomimetic approach of mineralization in vitro is adopted to investigate systematically the nucleation and growth of bone-like apatite on the surface of biomaterials such as bioceramics, metals and polymers, and those chemically surface-treated. The simulated environment is kept isothermic at the human body temperature of 36.5C with three kinds of simulated physiological fluids. The experimental results show that (1) inherent properties of biomaterials determine their bioactivity and the different crystalline structure of same materials results in the difference in bioactivity; (2) the bioactivity can effectively be improved by the surface treatment of biomaterials via chemical methods and by the addition of bioactive particles in a polymer matrix; (3) the bone-like apatite, nucleated and grown in the simulated body fluid with the same ion concentrations to that of the human plasma, possesses the same composition, structure and morphology despite of matrixes; (4) the difference in bioactivity with biomaterials is indicated by the different time for bone-like apatite to nucleate and to grow on their surfaces.

Preparation of Polymeric Nanoparticles by Photo‐Crosslinking of an Acryloylated Polyaspartamide in w/o Microemulsion

AbstractSummary: Biodegradable polymeric nanoparticles have been prepared by UV irradiation of an acryloylated water soluble polymer by an inverse microemulsion. The starting polymer was a α,β‐poly(N‐2‐hydroxyethyl)‐D,L‐aspartamide (PHEA) partially functionalized with glycidyl methacrylate (GMA) in order to introduce reactive vinyl groups in the side chain. The PHEA‐GMA copolymer obtained (PHG) was crosslinked by UV irradiation of the inverse microemulsion prepared by mixing an aqueous solution of PHG with propylene carbonate (PC)/ethyl acetate (EtOAc) in the presence of sorbitan trioleate (SPAN 85) as surfactant. Nanoparticles obtained were characterized by FTIR spectrophotometry, transmission electron microscopy, size distribution analysis and zeta potential measurements. Nanoparticles investigated revealed spherical and homogeneous shading, the particle size having a mean diameter of 88 ± 13 nm (PDI = 0.21) and a negative surface charge in several aqueous media. Moreover, in vitro chemical and enzymatic hydrolysis studies evidenced the partial biodegradability of PHG nanoparticles, which is more evident after incubation with enzymes such as esterases. PHG nanoparticles were loaded during UV irradiation process with Cytarabine, chosen as a model drug, and Cyt‐loaded PHG nanoparticles were able to release it in a simulated physiological fluid (phosphate buffer at pH 7.4) and in blood plasma.Transmission electron micrograph of photo‐crosslinked PHG.magnified imageTransmission electron micrograph of photo‐crosslinked PHG.

Immersion characteristics of electrolytic calcium phosphate coated Ti in simulated physiological fluid

The electrolytic calcium phosphate coatings have been deposited on titanium at room temperature and presented several differences with respect to other documented reports. The present study aimed to investigate the immersion behaviors of the uncoated titanium, the hydroxyapatite (HA) coated and the dicalcium phosphate dihydrate (DCPD) coated in the simulated physiological fluid (Hank's solution) at 37°C for 1, 7, 14, and 30 days. For the evaluation in dissolution or precipitation, morphology, composition, and crystal structure, the specimens were characterized by weight variation, X-ray diffraction, scanning electron microscopy, and energy dispersive spectroscopy before and after immersion. The plate-like DCPD structure was not stable in Hank's solution. However, its dissolution revealed the enhancing effect on the precipitation of newly formed hydroxyapatite. The calcium deficient hydroxyapatite (CDHA) can be conducted directly by the HA as-coated or the HA500 (annealed at 500°C, Ca/P = 1.63), or indirectly by the DCPD coated (Ca/P = 1.58), but not by the HA700 (annealed at 700°C, Ca/P = 1.80). The newly formed granular hydroxyapatite showed the more crystallization than the as-coated. Besides, the annealing enhanced the crystallization of the as-coated hydroxyapatite. Generally, the electrolytic CaP coating including the DCPD coated, HA as-coated, HA500, and HA700 specimens played an important role on the precipitation of newly formed hydroxyapatite in Hank's solution, but the uncoated Ti did not.

In vivo biocompatibility of radiation crosslinked acrylamide copolymers

In vitro swelling and in vivo biocompatibility of radiation crosslinked acrylamide copolymers such as acrylamide/crotonic acid (AAm/CA) and acrylamide/itaconic acid (AAm/IA) were studied. The swelling kinetics of acrylamide copolymers were performed in distilled water, human serum and some simulated physiological fluids such as phosphate buffer, pH 7.4, glycine–HCl buffer, pH 1.1, physiological saline solution, and some swelling and diffusion parameters have been calculated. AAm/CA and AAm/IA hydrogels were subcutaneously implanted in rats for up to 10 weeks and the immediate short- and long-term tissue response to these implants were investigated. Histological analysis indicated that tissue reaction at the implant site progressed from an initial acute inflammatory response. No necrosis, tumorigenesis or infection was observed at the implant site up to 10 weeks. The radiation crosslinked AAm/CA and AAm/IA copolymers were found well tolerated, non-toxic and highly biocompatible. However, AAm/IA copolymer was not found to be compatible biomaterials, because one of the AAm/IA samples was disintegrated into small pieces in the rat.

Bioactive, degradable composite microspheres: effect of filler material on surface reactivity.

Composite microspheres with two different fillers were developed using a solid-in-oil-in-water (s/o/w) emulsion solvent removal method. Two types of bioactive ceramic powders, specifically calcium hydroxyapatite (HA) and modified bioactive glass (MBG), were incorporated into degradable poly(lactic acid) (PLA) polymer matrix to form composite microspheres. For each filler material, microspheres with three different weight ratios of filler material to polymer, namely, 1: 1, 1: 3, and 1: 9, were synthesized. In vitro immersion using simulated physiological fluid (SPF) was employed to evaluate the surface reactivity of the microspheres. SEM analysis revealed that after a 14-day immersion the surface of the microspheres containing 50% MBG was fully transformed into a bone-like apatite. In contrast, a limited number of mineral nodules were present on the surface of microspheres containing HA. The solution chemical analyses performed to determine changes of Ca, P, and Si concentrations as a function of the immersion time showed that the ion concentration profiles were similar for all microspheres, except the [Si] profile. A higher Si concentration was detected in the SPF immersed with MBG-containing microspheres. These data show that the MBG filler significantly enhances the surface reactivity of the composite microspheres. This observation enables us to conclude that the composite MBG-containing microspheres are the preferable microspheres for three-dimensional bone tissue engineering.

Design of biodegradable particles for protein delivery

Major research issues in protein delivery include the stabilization of proteins in delivery devices and the design of appropriate protein carriers in order to overcome mucosal barriers. We have attempted to combine both issues through the conception of new biodegradable polymer nanoparticles: (i) poly(ethylene glycol) (PEG)-coated poly(lactic acid) (PLA) nanoparticles, chitosan (CS)-coated poly(lactic acid–glycolic acid (PLGA) nanoparticles and chitosan (CS) nanoparticles. These nanoparticles have been tested for their ability to load proteins, to deliver them in an active form, and to transport them across the nasal and intestinal mucosae. Additionally, the stability of some of these nanoparticles in simulated physiological fluids has been studied. Results showed that the PEG coating improves the stability of PLA nanoparticles in the gastrointestinal fluids and helps the transport of the encapsulated protein, tetanus toxoid, across the intestinal and nasal mucosae. Furthermore, intranasal administration of these nanoparticles provided high and long-lasting immune responses. On the other hand, the coating of PLGA nanoparticles with the mucoadhesive polymer CS improved the stability of the particles in the presence of lysozyme and enhanced the nasal transport of the encapsulated tetanus toxoid. Finally, nanoparticles made solely of CS were also stable upon incubation with lysozyme. Moreover, these particles were very efficient in improving the nasal absorption of insulin as well as the local and systemic immune responses to tetanus toxoid, following intranasal administration. In summary, these results show that a rational modification in the composition and structure of the nanoparticles, using safe materials, increases the prospects of their usefulness for mucosal protein delivery and transport.

In vitro dynamic swelling behaviors of radiation synthesized polyacrylamide with crosslinkers in the simulated physiological body fluids

Acrylamide hydrogels, containing different amounts and types of crosslinkers, were synthesized via γ-irradiation technique. Their swellings in simulated body fluids, such as physiological saline (0.89% NaCl) isoosmotic phosphate buffer at pH 7.4, gastric fluid at pH 1.1 (glycine–HCl), protein (aqueous solution of bovine serum albumin), urine (aqueous solution of urea), glucose and distilled water, were studied. Equilibrium swellings of the hydrogels were changed in the range 27–85 depending upon the fluids, type and amount of crosslinkers. The diffusion exponents were found over half for all hydrogels.

In vitro dynamic swelling behaviors of polyhydroxamic acid hydrogels in the simulated physiological body fluids

Influence of some simulated physiological body fluids on the dynamic swelling behaviour of polyelectrolytic hydroxamic acid hydrogels (PHA) was investigated at 37 °C in vitro. The simulated physiological body fluids are distilled water, human sera, physiological saline (0.89 % NaCl), isoosmotic phosphate buffer at pH 7.4, gastric fluid at pH 1. 1, (gylicine-HCl buffer), urea (0.3 mol L−1), and the aquatic solutions of K2HPO4 and KNO3 (the sources of K+). The values of equilibrium swelling of PHA hydrogels varied in the range of 130–4625%, while the values of equilibrium fluid content of the hydrogels varied in the range of 57–97%. The initial rate of swelling, diffusional exponent, and, diffusion coefficient were calculated using swelling kinetics data. Diffusion of the fluids into the hydrogel was found to be non-Fickian character. The diffusion coefficients of the hydrogel varied between 0.6×10−6– 8.1×10−6 cm2 s−1.

In vivo biocompatibility of radiation induced acrylamide and acrylamide/maleic acid hydrogels

In vitro swelling and in vivo biocompatibility of radiation induced acrylamide (AAm) and acrylamide/maleic acid (AAm/MA) hydrogels were investigated. The swelling kinetics of AAm and AAm/MA hydrogels of are investigated in distilled water, human serum and some simulated physiological fluids such as phosphate buffer at pH 7.4, glycine-HCl buffer at pH 1.1, physiological saline solution and, some swelling and diffusion parameters have been calculated. AAm and AAm/MA hydrogels were subcutaneously implanted in rats for up to 10 weeks and the tissue response to these implants was studied. Histological analysis indicated that tissue reaction at the implant site progressed from an initial acute inflammatory response characterized. No necrosis, tumorigenesis or infection was observed at the implant site up to 10 week. In vivo studies indicated that the radiation induced acrylamide and acrylamide/maleic acid hydrogels were found to be well-tolerated, non-toxic and highly biocompatible.

3D bone tissue engineered with bioactive microspheres in simulated microgravity.

Three-dimensional (3D) osteoblast cell cultures were obtained in rotating-wall vessels (RWV), simulating microgravity. Three types of bioactive microcarriers, specifically modified bioactive glass particles, bioceramic hollow microspheres, and biodegradable bioactive glass-polymer composite microspheres, were developed and used with osteoblasts. The surfaces of composite microspheres fully transformed into bone apatite after 2-wk immersion in simulated physiological fluid, which demonstrated their bone-bonding ability. The motion of microcarriers in RWVs was photographically recorded and numerically analyzed. The trajectories of hollow microspheres showed that they migrated and eventually stayed around at the central region of the RWV. At their surfaces, shear stresses were low. In contrast, solid glass or polymer particles moved toward and finally bounced off the outer wall of the RWVs. Cell culture studies in the RWV using bone marrow stromal cells showed that the cells attached to and formed 3D aggregates with the hollow microspheres. Extracellular matrix and mineralization were observed in the aggregates. Cell culture studies also confirmed the ability of the composite microspheres to support 3D bone-like tissue formation. These data suggest that the new hollow bioceramic microspheres and degradable composite microspheres can be used as microcarriers for 3D bone tissue engineering in microgravity. They also have potential applications as drug delivery systems.

Biomimetic Mineralisation on Plasma Sprayed Apatite Particles and Coatings in Different Simulated Physiological Fluids

Biomimetic Mineralisation on Plasma Sprayed Apatite Particles and Coatings in Different Simulated Physiological Fluids

New bioactive, degradable composite microspheres as tissue engineering substrates.

Novel bioactive, degradable polymer/glass/ceramic composite microspheres were developed using a solid-in-oil-in-water (s/o/w) emulsion solvent removal method. Modified bioactive glass (MBG) powders were encapsulated into the polylactic acid (PLA) matrix. Scanning electron microscopy and energy-dispersive X-ray analyses revealed that the MBG powders were mostly embedded in the polymer matrix, and submicron-size pores were present at the surface. Immersion in simulated physiological fluid (SPF) was used to evaluate the surface reactivity of the microspheres. The polymeric surface was fully transformed into carbonated calcium hydroxyapatite (c-HA) after 3 weeks of immersion. In contrast, PLA microspheres showed no evidence of any calcium phosphate deposition. Ion concentration analyses revealed a decrease in Ca and P concentrations and an increase in Si concentration in the SPF immersed with composite microspheres during the 3-week period. The Ca and P uptake rates decreased after 2 days of incubation. This coincided with the decrease of the Si release rate. These data lend support to the suggestion that the Si released from the MBG powders present in the polymer matrix is involved in the formation of the Ca-P layer. Our results support the concept that these new bioactive, degradable composite microspheres may serve as microcarriers for synthesis of bone and other tissues in vitro and in vivo.

Rebuttal on Garlic Chemistry: Stability of S-(2-Propenyl) 2-Propene-1-sulfinothioate (Allicin) in Blood, Solvents, and Simulated Physiological Fluids

Rebuttal on Garlic Chemistry: Stability of <i>S</i>-(2-Propenyl) 2-Propene-1-sulfinothioate (Allicin) in Blood, Solvents, and Simulated Physiological Fluids

Comments on Garlic Chemistry: Stability of S-(2-Propenyl) 2-Propene-1-sulfinothioate (Allicin) in Blood, Solvents, and Simulated Physiological Fluids

Comments on Garlic Chemistry: Stability of <i>S</i>-(2-Propenyl) 2-Propene-1-sulfinothioate (Allicin) in Blood, Solvents, and Simulated Physiological Fluids

In vitro dynamic solubility test: influence of various parameters.

This article discusses the dissolution of mineral fibers in simulated physiological fluids (SPF), and the parameters that affect the solubility measurement in a dynamic test where an SPF runs through a cell containing fibers (Scholze and Conradt test). Solutions simulate either the extracellular fluid (pH 7.6) or the intracellular fluid (pH 4.5). The fibers have various chemical compositions and are either continuously drawn or processed as wool. The fiber solubility is determined by the amount of SiO2 (and occasionally other ions) released in the solution. Results are stated as percentage of the initial silica content released or as dissolution rate v in nm/day. The reproducibility of the test is higher with the less soluble fibers (10% solubility), than with highly soluble fibers (20% solubility). The influence of test parameters, including SPF, test duration, and surface area/volume (SA/V), has been studied. The pH and the inorganic buffer salts have a major influence: industrial glasswool composition is soluble at pH 7.6 but not at pH 4.5. The opposite is true for rock- (basalt) wool composition. For slightly soluble fibers, the dissolution rate v remains constant with time, whereas for highly soluble fibers, the dissolution rate decreases rapidly. The dissolution rates believed to occur are v1, initial dissolution rate, and v2, dissolution rate of the residual fibers. The SA of fibers varies with the mass of the fibers tested, or with the fiber diameter at equal mass. Volume, V, is the chosen flow rate. An increase in the SA/V ratio leads to a decrease in the dissolution rate.(ABSTRACT TRUNCATED AT 250 WORDS)

SEM-EDS analysis of glass fibers corroded in physiological solutions by dynamic tests with variable flow rates.

The dissolution of mineral fibers has been studied in simulated physiological fluids using a dynamic testing procedure. Fibers of different chemical composition and obtained by different processes with a mean diameter of about 1 micron, have been characterized with respect to their solubility under various test conditions of flow-rate. The surfaces were analyzed using scanning electron microscopy (SEM), energy dispersive spectrometry (EDS) and X-ray diffraction techniques. SEM examinations show the formation of various corrosion patterns: porous, gel-like outer layers; precipitation zones and even, in some cases, no modification of the surface aspect. EDS analyses performed on the fibers, on the fiber surface layers, or on the deposits show three types of chemical composition: areas enriched in Al, in Ca and P, or in Al, Ca, and P. These surface compositions can be found for the same type of fiber tested, depending on the flow rate of the solution. Surface changes depend strongly on the initial composition of the glass and on the test conditions, particularly the flow rate. It is of particular interest to characterize the remaining surfaces (if any) obtained at the end of the in vitro test run and to compare them with surface analysis of the recovered fibers from the in vivo tests to assess the validity of the in vitro tests.