Self-Hardening Hydrogel for Bone Tissue Engineering

Summary: We have developed a self-reticulating polymer based on silanized hydroxypropylmethylcellulose (Si-HPMC). The aim of this study was to determine whether this Si-HPMC hydrogel with or without calcium phosphate granules could represent a potential scaffold for bone tissue engineering. This study showed that Si-HPMC hydrogel didn't affect SaOS-2 and rat bone marrow cells viability. In addition, SaOS-2 cells are able to proliferate within Si-HPMC hydrogel containing or not calcium phosphate granules whereas Rat bone marrow cells proliferate only at the surface of calcium phosphate granules contained within Si-HPMC hydrogel. Finally, SaOS-2 cells seeded at the surface of reticulated Si-HPMC were not able to penetrate the hydrogel, while J774, a macrophage cells line, were able to move into the Si-HPMC hydrogel. These data indicate that Si-HPMC is a promissing scaffold for tissue engineering.     

A new drug delivery system using plasma-irradiated pharmaceutical aids. IX. Controlled-release of theophylline from double-compressed tablet composed of cellulose derivatives as wall material.

The rapid release from a double-compressed tablet containing theophylline with the water-soluble polymer, hydroxypropylmethylcellulose (HPMC) or hydroxypropylmethylcellulose phthalate (HPMCP), used as a wall material can be suppressed by argon plasma-irradiation and changed into the sustained-release system due to a decrease in solubility of the outer layer. It was shown that the release profiles can be varied so as to cause theophylline release at different rates, depending on the set of conditions chosen for tablet manufacture and for plasma operation.     

Quantitative measurement of collagen methylation by capillary electrophoresis

Collagen methylation has been exploited in various applications involving living cells. We have observed correlation between the collagen methylation with the rate of cell proliferation in three-dimensional (3-D) microenvironment. To quantify the degree of collagen methylation, we have developed a capillary zone electrophoresis method. Using a polyvinyl alcohol-coated fused-silica capillary and UV detection at 200 nm, we have optimized pH and separated the native collagen into three major bands in phosphate buffer (50 mM, pH 2.5) with 0.05% hydroxypropylmethylcellulose. Under these conditions, the methylated collagens were separated into four major bands, which changed with different methylation reaction conditions. We propose an index to quantify the degree of collagen methylation that also correlates with their effects on cell proliferation.     

Determination of phosphate in seawater by CZE with on-line transient ITP

We developed CZE with indirect UV detection for the determination of phosphate in seawater using transient ITP as an on-line concentration procedure. The following optimum conditions were established: BGE, 5 mM 2,6-pyridinedicarboxylic acid (PDC) containing 0.01% hydroxypropylmethylcellulose (HPMC) adjusted to pH 3.5; detection wavelength, 200 nm; vacuum injection period of sample, 3 s (45 nL); terminating ion solution, 500 mM MES adjusted to pH 4.0; vacuum injection period of the terminating ion solution, 30 s (450 nL); applied voltage, 30 kV with the sample inlet side as the cathode. The LOD for phosphate was 16 microg/L (PO(3-)(4) -P) at S/N of 3. The respective values of the RSD of the peak area, peak height, and migration time for phosphate were 2.6, 2.3, and 0.34%. The proposed method was applied to the determination of phosphate in a seawater certified reference material for nutrients, MOOS-1, distributed by the National Research Council of Canada (NRC). The results were very similar to certified values. The method was also applied to the determination of phosphate in coastal seawaters. The results agreed with those obtained using a conventional spectrophotometric method.     

Protein Fractionation of Cowpea (Vigna unguiculata (L.) Walp) Leaf, Flower and Seed by Capillary Electrophoresis and Its Potential for Variety Identification

The proteins of different faction of cowpea [Vigna unguiculata (L.) Walp] were fractionated by capillary electrophoresis (CE). The extracting solvent system was one of the most critical factors in the optimization exercise. To improve reproducibility, seed samples needed to be defatted with chloroform/methanol (V:V=2:1) as preferred prior to protein extraction. Proteins were extracted from seeds, leaves and flowers with 50% aqueous 1‐propanol and separated on a 50 (m×20 cm fused silica capillary column using a UV detector at 200 nm. Separation was conducted at constant voltage (10 kV, 40°C), using iminodiacetic acid (pH 2.5) containing 0.05% hydroxypropylmethylcellulose (HPMC) and 20% acetonitile. The results showed that proteins extracted from all fraction of cowpea were successfully separated by CE in less than 20 min. Seed extracts provided the greatest number of eluted protein peaks, followed by flower and leaf, respectively. The seed‐protein extracts provided unique CE patterns for different varieties; hence the seed was the tissue chosen as being most suitable for variety identification. As a result, an optimized procedure was developed to provide rapid identification of cowpea varieties, based on capillary electrophoregram patterns.     

Stability and solution concentration enhancement of resveratrol by solid dispersion in cellulose derivative matrices

Resveratrol is a highly biologically active phytoalexin, found in many plant materials that are common elements of the human diet, such as grapes, nuts, and red wine. The therapeutic or disease preventative potential of this natural polyphenolic antioxidant has been limited in part due to its poor aqueous solubility and low oral bioavailability. We hypothesized that solid dispersion of resveratrol (Res) in cellulose derivative matrices might afford amorphous dispersions, from which supersaturated Res solutions would be produced in the human gastrointestinal (GI) tract, resulting in higher Res bioavailability. We carried out structure–property studies employing cellulose esters with a range of physical characteristics but possessing features suitable for use in amorphous solid dispersions: carboxymethylcellulose acetate butyrate (CMCAB), hydroxypropylmethylcellulose acetate succinate (HPMCAS) and cellulose acetate adipate propionate (CAAdP). The cellulose derivative results were compared with those of a negative control, pure crystalline Res, and a positive control, Res/poly(vinylpyrrolidinone) (PVP). Solid dispersions were characterized by powder X-ray diffraction (XRPD), modulated differential scanning calorimetry (MDSC), nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FT-IR) of solid dispersions. HPMCAS and PVP solid dispersions afforded faster and more complete Res release at pH 6.8; however Res is also released from PVP matrices at pH 1.2. The carboxyl-containing cellulose derivatives release Res to only a small extent at pH 1.2. This combination of solution and solid phase stabilization against crystallization, and pH-triggered drug release makes these cellulose esters attractive candidates for Res bioavailability enhancement.     

Varietal identification of rice prolamins by capillary zone electrophoresis

Summary Capillary electrophoretic methods were developed to investigate the possibility of prolamin identification of four rice varieties i.e. Thai white fragrant rice KD105, Korkhor15, Thai white rice A, and a Chinese white rice H20. Two extraction methods were compared and varietal identification of rice prolamins was successfully accomplished by capillary electrophoresis in pH 2.5, sodium phosphate buffer containing 20% acetonitrile and 0.05% hydroxypropylmethylcellulose.     

Automated instrumentation for miniaturized displacement electrophoresis with on-column photometric detection

This paper describes an automated equipment for capillary displacement electrophoresis (isotachophoresis). The equipment employs the advantages of a separation channel with a nonuniform cross-section. The system works in a closed mode, the analytes are determined as anions in the examined arrangement. The pH gradient is within the range 4.4–11.0, the total voltage over the separation channel is from 150 V to 900 V at a constant current of 20 μA or 10 μA, respectively. Cetyltrimethylammoniumbromide and either hydroxypropylcellulose or hydroxypropylmethylcellulose are used in the leading electrolyte to control the electroosmotic flow.

The reproducibility and the minimum detectable concentrations are determined and calibration curves are measured using a model mixture of p1 markers and myoglobin as analytes and either low-molecular-mass ampholytec buffers or synthetic carrier ampholytes as spacers. Both Gaussian peaks and the square-wave zones were evaluated.     

Collection of alpha1-acid glycoprotein molecular species by capillary electrophoresis and the analysis of their molecular masses and carbohydrate chains. Basic studies on the analysis of glycoprotein glycoforms

A highly heterogeneous glycoprotein, alpha1-acid glycoprotein, was resolved into their glycoforms by capillary electrophoresis using a surface-modified capillary in 20 mM acetate buffer (pH 4.2) containing 0.5% (w/v) hydroxypropylmethylcellulose. We collected the fractions containing each glycoform as nearly pure state by capillary electrophoresis, and examined the molecular masses of these glycoforms by matrix assisted laser desorption time-of-flight mass spectrometry. We also analyzed carbohydrate chains after releasing them with N-glycosidase F followed by fluorescent labeling with 8-aminopyrene-1,3,6-trisulfonate. We found that the separation of glycoforms was mostly due to the presence of multiantennary carbohydrate chains. We propose that the present technique is useful for the analysis of post translational modification of proteins with carbohydrate chains.     

The zeta potential of cyclo-olefin polymer microchannels and its effects on insulative (electrodeless) dielectrophoresis particle trapping devices

While cyclo-olefin polymer microchannels have the potential to improve both the optical detection sensitivity and the chemical resistance of polymer microanalytical systems, their surface properties are to date not thoroughly characterized. These surface properties dictate, among other things, electrokinetic effects when electric fields are present. Here, we report the measurement of the zeta potential of cyclo-olefin polymers (injection-molded and hot-embossed Zeonor 1060R and 1020R) microchannels as a function of pH, counter-ion concentration, storage conditions, and chemical treatment in aqueous solutions both with and without EOF-suppressing additives. In contrast with previous reports, significant surface charge is measured, consistent with titration of charged sites with pK(a) = 4.8. Storage in air, acetonitrile, or aqueous solutions has relatively minor effects. While the source of the surface charge is unclear, chemical functionalization has shown that carboxylic acid groups are not present at the surface, consistent with the chemical structure of Zeonor. EOF-suppressing additives (hydroxypropylmethylcellulose) and conditioning in perchloric acid allow the surface charge to be suppressed. We demonstrate dielectrophoretic particle trapping devices in Zeonor 1060R substrates that show reduced trapping voltage thresholds as compared to previous implementations in glass.     

Electrophoretic migration behavior of DNA fragments in polymer solution

A newly developed polymer coil shrinking theory is described and compared with the existing entangled solution theory to explain electrophoretic migration behaviour of DNA in hydroxypropylmethylcellulose (HPMC) polymer solution in buffer containing 100 mM tris(hydroxymethyl)aminomethane 100 mM boric acid, 2 mM ethylenediaminetetraacetic acid at pH 8.3. The polymer coil shrinking theory gave a better model to explain the results obtained. The polymer coil shrinking concentration, Cs, was found to be 0.305% and the uniform entangled concentration, C+, 0.806%. The existence of three regions (the dilute, semidilute, and concentrated solution) at different polymer concentrations enables a better understanding of the system to guide the selection of the best conditions to separate DNA fragments. For separating large fragments (700/ 800 bp), dilute solutions (HPMC < 0.3%) should be used to achieve a short migration time (10 min). For small fragments (200/300 bp), concentrated solutions are preferred to obtain constant resolution and uniform separation. The best resolution is 0.6% HPMC due to a combined interaction of the polymer coils and the entangled structure. The possibility of DNA separation in semidilute solution is often neglected and the present results indicate that this region has a promising potential for analytical separation of DNA fragments.     

Fabrication,characterization and evaluation of bacterial cellulose-based capsule shells for oral drug delivery

Bacterial cellulose (BC) was investigated for the first time for the preparation of capsule shells for immediate and sustained release of drugs. The prepared capsule shells were characterized using X-ray diffraction, scanning electron microscopy and Fourier transform infrared spectroscopy. The BC capsule shells were studied for drug release using an USP type-I dissolution apparatus. Irrespective of the drying method and the thickness of the BC sheet, the capsule shells displayed an immediate drug release profile. Moreover, the addition of release-retardant cellulosic polymers sustained the drug release having first-order kinetics for hydroxypropylmethylcellulose and carboxymethyl cellulose sodium with R ² values of 0.9995 and 0.9954, respectively. Furthermore, these capsules shells remained buoyant in 0.1 N HCl (pH 1.2) solution up to 12 h. This study showed that BC is a promising alternative to gelatin capsules with both immediate and sustained drug release properties depending upon the compositions of the encapsulated materials.     

Click synthesis by Diels-Alder reaction and characterisation of hydroxypropyl methylcellulose-based hydrogels

The Diels-Alder reaction was used to fabricate hydroxypropyl methylcellulose-based hydrogels. First, hydroxypropylmethylcellulose (HPMC) was modified by a carboxyl-containing diene molecule (SFA) which was synthesised from furfurylamine and succinic anhydride. Second, dienophile groups were introduced into HPMC by the coupling reaction with N-maleoyl alanine (AMI) using N,N′-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP). Subsequently, the asprepared furan- and maleimide-modified HPMC were dissolved in water and gelation was observed at a pre-determined temperature after a period of time. The samples thus obtained were characterised by FTIR, NMR, SEM, etc. The gelation time changing with temperature, concentration of the solution, and solvent was measured. It was found that gelation time decreased with increasing temperature and concentration of the solution, and that water had a rate-accelerating effect on Diels-Alder reaction. The swelling behaviour indicates that the hydrogels have a high swelling ratio in water and the swelling ratio increases with the increasing temperature. Taking into consideration that the HPMC-based hydrogels are prepared under mild reaction conditions with an adjustable gelation time and thermal stability, the method described here has a potential application in biomaterials, especially in the areas of tissue-engineering and drug-controlled release carriers.     

Design of a poly(L‐histidine)‐carbohydrate conjugate for a new pH‐sensitive drug carrier

A poly(L ‐histidine) (PLH)‐carbohydrate conjugate has been synthesized as a new macromolecule extracting pH‐dependent properties of PLH with imidazole groups. Because of poor water solubility at physiological pH, the application of PLH with a pK_a around 6.0 has been limited in spite of the native possession of the pH‐dependent property change at endosomal pH. Although the PLH modified with aliphatic primary amino groups suddenly precipitated out of the aqueous medium above pH 6.0 as a result of the deprotonation of the imidazole groups, the water solubility of PLH was improved at physiological pH by the conjugation of the aminated PLH with hydrophilic maltopentaose. The resulting PLH‐maltopentaose conjugates and metalloporphyrins formed the complexes which varied their assembling structure below pH 6.0. The PLH‐maltopentaose would be the fundamental compound for designing various drug carriers with the pH sensitivity at endosomal pH. Copyright © 2004 John Wiley & Sons, Ltd.     

A double-blind placebo-controlled study of 5-fluorouracil:cyclodextrin complex loaded thermosensitive gel for the treatment of HPV induced condyloma

Objective of this double-blind placebo-controlled study was to determine the efficacy of thermosensitive mucoadhesive gel loaded with 5-fluorouracil (5-FU):hydroxypropyl-ß-cyclodextrin (HP-ß-CD) complex via topical administration or intralesional injection for the treatment of human papilloma virus induced condyloma in 44 women. The diagnosis of human papilloma virus was established with clinical, histopathological and polymerase chain reaction techniques. Subjects were randomized into four parallel groups to evaluate topical or intralesional administration of drug-loaded or blank gel. The formulation used in the study consisted of 20% Pluronic PF 127 and 0.2% hydroxypropylmethylcellulose (HPMC) to render thermosensitive and mucoadhesive properties to the blank and drug-loaded gels. 5-FU was complexed to hydroxypropyl-ß-cyclodextrin to improve its solubility and this complex was loaded into thermosensitive gel to obtain controlled release of the cytotoxic drug in administration site over a two-week period cure regimen aiming therapeutic efficacy with lower 5-FU doses. Complete response was achieved in 61% of patients through intralesional administration while topical administration resulted in only 29% complete cure. Relapse rates of all therapy groups were significantly low in the 6-month follow-up time.     

pH Sensitive supramolecular assembling system between a new linear water soluble β-cyclodextrin terpolymer and an amphiphilic poly(ethylene oxide)

A linear water soluble β-cyclodextrin terpolymer (P(MVE-AM)-g-βCD) was synthesized by grafting different amounts of βCD on the copolymer P(MVE-AM). The resulting terpolymer showed a pH sensitive behaviour in aqueous solution, due to the presence of carboxylic acid groups. Whatever the substitution degree in βCD, the terpolymer behaved like a polyacid at pH 7, and had neutral properties at pH 2. The mixture of P(MVE-AM)-g-βCD with an amphiphilic poly(ethylene oxide) (PEO-Ad₃) showed an associative phase separation influenced by the pH and concentration of the medium. In the dilute range and intermediate range, associative phase separations were induced reversibly by decreasing the pH from 7 to 2. The results in the semi-dilute range showed that networks were elaborated with much stronger dynamic modulus at low pH (pH 2) than at higher pH (pH 7). Coupled inclusion complex interactions (between the adamantyl groups of PEO-Ad₃ and the cyclodextrin cavities of P(MVE-AM)-g-βCD) with hydrogen bonding (between carboxylic acid groups and PEO backbone) could be a way to modulate the strength of the interactions in supramolecular assemblies. These results suggest that the mixture of P(MVE-AM)-g-βCD with PEO-Ad₃ could be a potential pH sensitive system.     

On the pH dependence of Cu(II) binding to insulin: ESR and electronic studies

The ESR and electronic absorption spectra have been used to investigate co-ordination of Cu(II) to insulin in aqueous solutions at different pH. Two series of complexes with low- and high copper content were examined and the values of the magnetic tensor components and the shape of the diffuse reflectance transitions suggested that these copper-insulin derivatives have tetragonal symmetry with Cu(II) in a (N_xO_y) ligand field, where oxygen donor groups are predominant at low pH and nitrogenous ligands at high pH. Such a trend was further supported by the presence of superhyperfine structure at pH = 13. Oxygen of the carboxylato groups, nitrogen of α- and ε-amino groups and of imidazoles, all contribute to the coordinations field. At very high pH only, a preferential binding site for Cu(II) is found, which probably involves deprotonated peptide nitrogens.     

Sustained-release phenylpropanolamine hydrochloride bilayer caplets containing the hydroxypropylmethylcellulose 2208 matrix. I. Formulation and dissolution characteristics

The purpose of this study was to develop a new sustained-release phenylpropanolamine hydrochloride (PPA) bilayer caplets that consists of an immediate-release portion and a prolonged-release portion containing a hydroxypropylmethylcellulose 2208 (HPMC2208) matrix. Since PPA is a highly water-soluble drug, incorporation of 60% HPMC2208 level in the matrix was required for giving the product a PPA-slow releasing property. Difference in the viscosity grade of HPMC2208 in the matrices did not greatly influence the PPA dissolution characteristics from the matrices. Therefore, we formulated the prolonged-release portion consisting of 10% PPA, 30% excipients, and 60% HPMC2208 (Metolose 90SH4000) into the sustained-release PPA bilayer caplets. The PPA dissolution characteristics from the formulated bilayer caplets showed the prolonged dissolution profile after rapid dissolution and was close to the targeted profile calculated from PPA pharmacokinetics study. The manufacturing methods of the prolonged-release portion and the filling order of the prolonged-release portion in bilayer compression did not significantly affect the PPA dissolution characteristics from the bilayer caplets. The PPA dissolution characteristics from the bilayer caplets was pH independent. Moreover, the PPA dissolution characteristics from the bilayer caplets was not affected by mechanical shear. The sustained-release PPA bilayer caplets is expected to present constant prolonged-release of PPA after rapid dissolution in vivo without dissolution change due to pH and mechanical shear.     

Enhancement of naringenin solution concentration by solid dispersion in cellulose derivative matrices

Naringenin (Nar) is an important bioactive flavonoid with poor organic solubility and oral bioavailability. It is highly promising for treatment of conditions including diabetes, hyperlipidemia, and hepatitis C infection. Amorphous solid dispersion (ASD) of Nar is an appealing way to enhance its solubility, and carboxylated cellulose esters are attractive polymers for this purpose because of their ability to stabilize drugs against crystallization in both solid and solution phases, while restricting drug release to the pH of the small intestine (ca. 6.8). We demonstrate that ASDs of Nar can be formed using such carboxylated cellulose derivatives as cellulose acetate adipate propionate (CAAdP), carboxymethylcellulose acetate butyrate (CMCAB) and hydroxypropylmethylcellulose acetate succinate (HPMCAS). We compare Nar solution concentrations and release profiles from these cellulosic ASDs to those from pure crystalline Nar, and to Nar ASD in poly(vinylpyrrolidinone) (PVP). We show that all polymers in this study form ASDs with Nar, that the PVP ASDs release Nar at both gastric (1.2) and small intestine (6.8) pH, and that the cellulosic polymers release Nar selectively at neutral pH. Solution concentrations of Nar are significantly enhanced from these ASDs. These preliminary studies indicate that HPMCAS, CAAdP, and CMCAB are practical ASD polymers for Nar due to their ability to generate and stabilize high solution concentrations, and their pH-triggered drug release.     

Improved dissolution of an insoluble drug using a 4-fluid nozzle spray-drying technique

A solid dispersion of the drug can be made using a polymer carrier to improve solubility. Generally, drugs become amorphized when solid dispersion is formed using a polymer carrier. In such high energy conditions, the solubility of the drug molecule is increased. We previously prepared solid dispersion using a spray-drying technique and reported its solubility and crystallinity. In this study, hydroxypropylmethylcellulose (HPMC) was used as the carrier, and tolubutamide was the model drug, which is water-insoluble. Solubility was evaluated by preparing a solid dispersion using a newly developed 4-fluid nozzle spray dryer. Observation of particle morphology by scanning electron microscopy (SEM) revealed that the particles from the spray drying were atomized to several microns, and they had also become spherical. Assessment of the crystallinity of the spray-dried particles by powder X-ray diffraction and differential scanning calorimetry demonstrated that the tolbutamide had been amorphized, forming a solid dispersion. The apparent release rate constant K of the drug from the spray-dried particles was 4 to 6 times faster than the original drug in pH 1.2, and it was also 1.5 to 1.9 times faster than the original drug in pH 6.8. The 70% release time (T(70)) of the drug from the spray-dried particles was 20 to 30 times faster than the original drug in pH 1.2 solution as well as 2 to 3 times faster than the original drug in pH 6.8 solution. Pharmaceutical preparations prepared in this way using the 4-fluid nozzle system spray dryer formed composite particles, resulting in a remarkably improved dissolution rates of the drug.