Jet injection into polyacrylamide gels: investigation of jet injection mechanics

Jet injectors employ high-velocity liquid jets that penetrate into human skin and deposit drugs in the dermal or subdermal region. Although jet injectors have been marketed for a number of years, relatively little is known about the interactions of high-speed jets with soft materials such as skin. Using polyacrylamide gels as a model system, the mechanics of jet penetration, including the dependence of jet penetration on mechanical properties, was studied. Jets employed in a typical commercial injector, (orifice diameter: 152 microm, velocity: 170-180 m/s) were used to inject fluid into polyacrylamide gels possessing Young's moduli in the range of 0.06-0.77 MPa and hardness values in the range of 4-70 H(OO). Motion analysis of jet entry into polyacrylamide gels revealed that jet penetration can be divided into three distinct events: erosion, stagnation, and dispersion. During the erosion phase, the jet removed the gel at the impact site and led to the formation of a distinct cylindrical hole. Cessation of erosion induced a period of jet stagnation ( approximately 600 micros) characterized by constant penetration depth. This stage was followed by dispersion of the liquid into the gel. The dispersion took place by crack propagation and was nearly symmetrical with the exception of injections into 10% acrylamide (Young's modulus of 0.06 MPa). The penetration depth of the jets as well as the rate of erosion decreased with increasing Young's modulus. The mechanics of jet penetration into polyacrylamide gels provides an important tool for understanding jet injection into skin.     

Dependence of invadopodia function on collagen fiber spacing and cross-linking: computational modeling and experimental evidence

Invadopodia are subcellular organelles thought to be critical for extracellular matrix (ECM) degradation and the movement of cells through tissues. Here we examine invadopodia generation, turnover, and function in relation to two structural aspects of the ECM substrates they degrade: cross-linking and fiber density. We set up a cellular automaton computational model that simulates ECM penetration and degradation by invadopodia. Experiments with denatured collagen (gelatin) were used to calibrate the model and demonstrate the inhibitory effect of ECM cross-linking on invadopodia degradation and penetration. Incorporation of dynamic invadopodia behavior into the model amplified the effect of cross-linking on ECM degradation, and was used to model feedback from the ECM. When the model was parameterized with spatial fibrillar dimensions that closely matched the organization, in real life, of native ECM collagen into triple-helical monomers, microfibrils, and macrofibrils, little or no inhibition of invadopodia penetration was observed in simulations of sparse collagen gels, no matter how high the degree of cross-linking. Experimental validation, using live-cell imaging of invadopodia in cells plated on cross-linked gelatin, was consistent with simulations in which ECM cross-linking led to higher rates of both invadopodia retraction and formation. Analyses of invadopodia function from cells plated on cross-linked gelatin and collagen gels under standard concentrations were consistent with simulation results in which sparse collagen gels provided a weak barrier to invadopodia. These results suggest that the organization of collagen, as it may occur in stroma or in vitro collagen gels, forms gaps large enough so as to have little impact on invadopodia penetration/degradation. By contrast, dense ECM, such as gelatin or possibly basement membranes, is an effective obstacle to invadopodia penetration and degradation, particularly when cross-linked. These results provide a novel framework for further studies on ECM structure and modifications that affect invadopodia and tissue invasion by cells.     

Characterization of kappa-carrageenan gels used for immobilization of Bacillus firmus.

In this study, aimed at a biochemical and physical characterization of kappa-carrageenan gels used for entrapment of Bacillus firmus NRS 783 (a superior producer of an alkaline protease), effects of carrageenan concentration, gelation temperature, initial cell loading, and strength of the curing agent (KCl) on the properties of cell-free and cell-laden gels were examined. The physical properties of the differently prepared gels that were examined included density, free volume fraction, mechanical strength, and change in gel volume during gel curing. The biochemical characteristics studied included viability of gel-entrapped cells, cell leakage from cell-laden gels, and cell penetration into cell-free gels. For the range of carrageenan contents investigated [between 2% and 5% (w/v)], the mechanical strength of the gels with/without KCl curing was observed to increase with an increase in carrageenan content of gels. The mechanical strength of each gel increased substantially upon extensive curing. Free volume fractions in excess of 0.8 were observed for all gels. Of cells that were viable prior to immobilization, 90-92% remained viable after formation and extensive curing of gels for cell-gel mixtures prepared at 45 degrees C. Attempts at prolonged storage of cell-laden gel beads at 0 degrees C as stock cultures of immobilized B. firmus were unsuccessful due to a significant decline in cell viability during such storage. On the basis of the cell leakage studies, the average pore sizes of 2%, 3%, 4%, and 5% gels were deduced to increase in the following order of carrageenan content (w/v): 4%, 3%, 2%, and 5%. Commensurate with the decrease in the average pore size (or the increased tightness of the gels) with increasing carrageenan content, both the extent of cell leakage and the extent of net cell penetration decreased with increasing carrageenan content for the first three gels. Owing to non-uniform distribution of free space and much larger pores, the extent of net cell penetration in 5% carrageenan gels was considerably low, while the extent of cell leakage in 5% carrageenan gels was an order of magnitude greater than the extents of cell leakage in the other three gels.     

Bifunctional reagents as vapour- and liquid-phase fixatives for immunohistochemistry

Synopsis Out of a total of eleven bifunctional reagents tested as fixatives for light microscopic immunohistochemistry, four were found satisfactory when applied in the vapour phase to freeze-dried blocks. These were diethylpyrocarbonate, as observed in carlier studies, dimethyladipimidate,p-benzoquinone, and diacetyl. Adequate but less satisfactory liquid-phase fixation was provided by three reagents (dimethyladipimidate, dimethylsuberimidate,p-benzoquinone).None of the eleven reagents gave optimal preservation of antigenicity and structure when tested at the ultrastructural level. Encouraging results were obtained withp-benzoquinone, however.     

Purification and properties of Escherichia coli protein i, a prepriming protein in phi X174 DNA replication

Protein i, one of seven Escherichia coli proteins essential for primosome initiation of DNA chains in the in vitro conversion of single-stranded phi X174 DNA to duplex replicative form, has been purified approximately 15,000-fold to more than 98% purity. The protein is an oligomer of 22,000-dalton subunits migrating as a single electrophoretic band on native, as well as on denaturing polyacrylamide gels. Estimates of a Stokes radius of 41 A, a sedimentation coefficient of 3.5 S, a Mr = 61,000, and a frictional coefficient of 1.57 suggest that native protein i is a highly asymmetric oligomer composed of three identical subunits. About 50 such molecules are present/cell. Cross-linking the protein with dimethylsuberimidate or dimethyladipimidate produced three major bands corresponding to the monomer, dimer, and trimer, as well as two minor bands corresponding to the tetramer and pentamer. Incorporation of 3H-labeled "trimeric" protein i into the prepriming replication intermediate (primosome) occurs at a stage requiring participation of dnaB and dnaC proteins, and follows the actions of proteins n, n', and n". After extension of primers by DNA polymerase III holoenzyme, protein i is not retained in the isolated primosome complex. Thus, protein i is essential in the assembly of a functional primosome, but its precise physiologic role and genetic locus are still unknown.     

Effect of saponins and apigenin mixtures on feeding behavior of the pea aphid,Acyrthosiphon pisum Harris

Electrical penetration graphs (DC EPG) were used to monitor the feeding behavior of the pea aphid Acyrthosiphon pisum Harris (Hemiptera: Aphididae) exposed to mixtures of saponins: 3GlcA, 28AraRhaXyl medicagenic acid glycoside (MAG) and zanhic acid tridesmoside (ZAD) with flavonoid apigenin, on agarose-sucrose gels. In general, mixtures of saponins with apigenin incorporated into agarose-sucrose gels resulted in a reduction in both the number of aphid probes and delayed their duration. Aphid feeding on gels impregnated with mixtures of the tested chemicals also showed a reduction in salivation into the gels and elongation of passive ingestion from the gels (EPG patterns g-E1 and g-E2, respectively). There were significant differences among the feeding behaviors of the pea aphids on gels containing mixtures of either MAG or ZAD with apigenin. These differences were also depended on the concentration and proportions of these compounds.     

Effect of PEGylation on the diffusion and stability of chitosan-DNA polyplexes in collagen gels

Diffusion through the extracellular matrix (ECM) is a critical step for the delivery of nanoparticles and genes. Gene delivery requires a carrier that protects the nucleic acid from degradation and facilitates transport. Chitosan is a promising carrier. To increase the circulation time, PEGylation of the carrier is performed. However, the effect of PEGylation on the transport and stability of gene delivery systems in the ECM has only been studied in solutions containing ECM components. We used polymerized collagen and collagen-hyaluronic acid (HA) gels to study the effects of PEGylation on the diffusion and stability of chitosan-DNA polyplexes. We found that PEGylation of the polyplexes was required for diffusion to occur, and PEGylation increased the dissociation between DNA and chitosan to some extent. The presence of HA had a contradictory role: it decreased the penetration depth of PEGylated polyplexes into the gels and increased the diffusion of the polyplexes being mixed into the gels.     

Structure of beef heart mitochondrial F1-ATPase. Arrangement of subunits as disclosed by cross-linking reagents and selective labeling by radioactive ligands.

1. The following bifunctional reagents, dimethylsuberimidiate, dimethyladipimidate, methylmercaptobutyrimidate have been used to produce dimers between the neighboring subunits of beef heart F1-ATPase. 2. Treatment of beef heart F1-ATPase with dimethylsuberimidate or dimethyladipimidate resulted in the formation of four cross-linked products. Their molecular weights determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 11 500, 105 000, 95 000 and 80 000, respectively. The products of molecular weight 115 000 and 105 000 were predominant and could be detected at the early stage of the cross-linking reaction. Treatment of beef heart F1-ATPase with methylmercaptobutyrimidate resulted in the accumulation of the product of molecular weight 115 000 and in traces of products of lower molecular weight. When the cross-linked products obtained with methylmercaptobutyrimidate were cleaved by beta-mercaptoethanol, the original gel electrophoresis pattern was restored. 3. Cross-linking of beef heart F1-ATPase by dimethylsuberimidate, dimethyladipimidate and methylmercaptobutyrimidate was accompanied by a loss of the ATPase activity. Cleavage of the cross-linked products obtained with methylmercaptobutyrimidate did not restore the original ATPase activity. 4. Identification of subunits A and B in the products of molecular weight 115 000 and 105 000 was achieved by specific labeling of subunit A with N-[14C]ethylmaleimide and of subunit B by chloronitro [14C]benzooxodiazole. Both products were able to bind N-[14C]ethylmaleimide; only the 105 000 dalton product was able to bind chloronitro [14C]benzooxodiazole. 5. The product of molecular weight 115 000 obtained by treatment of beef heart ATPase with methylmercaptobutyrimidate could bind N-[14C]ethylmaleimide. Its cleavage, following N-[14C]ethylmaleimide binding, yielded one labeled peptide identified with subunit A by polyacrylamide gel electrophoresis. 6. The above results indicate that the product of molecular weight 115 000 is a dimer containing two subunits A and that the product of molecular weight 105 000 is a dimer containing one subunit A and one subunit B. It can therefore be concluded that, in beef heart F1-ATPase, the A subunits are close to each other and that subunit A is close to subunit B. In contrast the B sublnits are probably too far from each other to be cross-linked by dimethylsuberimidate, dimethyladipimidate or methylmercaptobutyrimidate.     

Human hypoxanthine-guanine phosphoribosyltransferase. Evidence for tetrameric structure

Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) has been purified 23,000-fold from normal human erythrocytes. The purification includes affinity chromatography on a GMP column. The subunit molecular weight of the enzyme obtained from this purification is 24,000. The finding of four protein species after cross-linkage of the highly purified enzyme with dimethylsuberimidate, dimethyladipimidate, and glutaraldehyde suggests that the enzyme may exist in the native state as a tetramer.     

Investigating the penetrating potential of nanocomposite β-cycloethosomes: development using central composite design,in vitro and ex vivo characterization

Context: Atopic dermatitis (AD) is a chronic skin disease characterized by inflammation of the skin and has exhibited remarkable repercussions on human life across the globe. Fluocinolone acetonide (FA), a topical corticosteroid is employed in the treatment of atopic dermatitis, but suffers from limited penetration into deeper epidermis of atopic skin.

Objective: The present investigation was focused to explore the utility of β-cylcoethosomes in improvising the penetration deep into the skin.

Materials and methods: β-Cylcoethosomes developed using β-cycloamylose by injection method were evaluated for vesicle size, entrapment efficiency and in vitro release. Central Composite design employed for the preparation depicted FA8 as an optimized formulation which was then formulated as dermatological gel using carbomer 934P as a gel base. The gels were characterized for pH, viscosity, drug content and in vitro permeability.

Results and discussion: Optimized formulation (FA8) showed maximum desirability (0.795) with vesicle size of 228.33?±?1.23?nm), EE (82.49?±?1.21%) and CDR (90.90?±?0.29%). FA8-loaded gels showed maximum in vitro permeability as found in BG and BGP (83.22?±?0.72% and 84.02?±?0.87). BG was selected as an optimized gel and compared with optimized reference ethosomal gel and control gel. CLSM studies depicted deeper uniform penetration of fluorescent dye deep into the epidermis via BG. Improved penetration was observed due to the synergistic effect exerted by ethanol and β-cycloamylose.

Conclusion: β-cylcoethosomes proved to be a promising carrier for improvised penetration of fluocinolone acetonide via topical gel.     

Collagen cross-linking produced by dimethyladipimidate. Its comparison with dimethylsuberimidate

The cross-linking produced by dimethyladipimidate (DMA) was studied using reconstituted fibrils of type I calf skin collagen. The results show that DMA cross-links collagen to a lesser degree than dimethylsuberimidate. Other evidence suggests that DMA does not produce any morphological alterations in the positive staining pattern.     

Lymphocyte migration into three-dimensional collagen matrices: a quantitative study

Lymphocytes have been plated onto the surface of three-dimensional gels of native collagen fibers, and their distribution throughout the three- dimensional collagen matrix has been determined in a quantitative fashion at various times thereafter. Information regarding the total number of applied cells may be obtained by this means. Lymphocyte penetration into the collagen gel does not appear to involve the expression of collagenolytic activity, nor does it require the presence of serum. Analysis of the kinetics of lymphocyte penetration into the gel matrix indicates that lymphocytes are migrating in a "random-walk" fashion. Our objective has been to establish a model system for studying the cell-matrix and cell-cell interactions which influence the pattern of lymphocyte recirculation in vivo and the results presented here are discussed in this context.     

Fibroblast behavior on gels of type I, III, and IV human placental collagens

Various collagens were extracted and purified from human placenta after partial pepsin digestion. We prepared type III + I (57:43), enriched type I, type III, and type IV collagens on an industrial level, and studied their biological properties with MRC5 fibroblast cells. Using the process of contraction of a hydrated collagen lattice described by Bell, we found tha the contraction rate was dependent on collagen type composition. The contraction was faster and more pronounced with pepsinized type I collagen than with pepsinized type III + I (57:43) collagen; the lowest rate was obtained with the pepsinized type III collagen. Using a new technique of collagen cross-linking, a gel was made with type IV collagen. This cross-linking procedure, based on partial oxidation of sugar residues and hydroxylysine by periodic acid, followed by neutralization, resulted in an increased number of natural cross-link bridges between oxidized and nonoxidized collagen molecules, without internal toxic residues. The fibroblasts were unable to contract type IV/IVox collagen gels. The type IV/IVox collagen gel was transparent and its amorphous ultrastructure lacked any visible striated fibrils. Fibroblast cells exhibited atypical behavior in these type IV/IVox collagen gels as evidenced by optical and electron microscopy. The penetration of fibroblasts could be measured. Fibroblasts penetrated faster in type IV/IVox collagen gels than in untreated type III + I collagen gels. The lowest rate of penetration was obtained with cross-linked type III + I gels. Fibroblast proliferation was similar on untreated or cross-linked type III + I collagen gels and slightly increased on type IV/IVox collagen gels, suggesting that this cross-linking procedure was not toxic.     

Development and evaluation of topical formulation containing solid lipid nanoparticles of vitamin A

The purpose of this research was to investigate novel particulate carrier system such as solid lipid nanoparticles (SLN) for topical application of vitamin A palmitate and to study its beneficial effects on skin. Topical gels enriched with SLN of vitamin A were prepared. The solid lipid nanoparticulate dispersion was prepared using high-pressure homogenization technique and was incorporated into polymeric gels of Carbopol, Pemulen, Lutrol, and Xanthan gum for convenient application. The nanoparticulate dispersion and its gels were evaluated for various parameters such as particle size, in vitro drug release, in vitro penetration, in vivo skin hydration, and skin irritation. The solid lipid nanoparticulate dispersion showed mean particle size of 350 nm. Differential scanning calorimetry studies revealed no drugexcipient incompatibility. In vitro release profile of vitamin A palmitate from nanoparticulate dispersion and its gel showed prolonged drug release up to 24 hours, which could be owing to embedment of drug in the solid lipid core. In vitro penetration studies showed almost 2 times higher drug concentration in the skin with lipid nanoparticle-enriched gel as compared with conventional gel, thus indicating better localization of the drug in the skin. In vivo skin hydration studies in albino rats revealed increase in the thickness of the stratum corneum with improved skin hydration. The developed formulation was nonirritant to the skin with no erythema or edema and had primary irritation index of 0.00. Thus it can be concluded that SLN represents a promising particulate carrier having controlled drug release, improved skin hydration, and potential to localize the drug in the skin with no skin irritation.     

Erythrocyte membrane cholesterol levels and their effects on membrane proteins

The susceptibility of the band 3 protein of the erythrocyte membrane to proteolytic digestion at either surface of the membrane was not altered when the membrane cholesterol level was increased by 65–103%. Cross-linking of the major membrane proteins by o-phenanthroline · Cu, glutaraldehyde, dimethylsuberimidate and dimethyladipimidate was also unaffected.     

Hydrophilic Ionic Liquids as Ingredients of Gel-Based Dermal Formulations

Ionic liquids (ILs) have several properties that offer many advantages in dermal drug delivery systems. Depending on the chemical structure, ILs can be used for protection against microorganisms, to enhance skin penetration, and as a solvent. In the present work, SEPINEO? P 600 formulations and hydroxyethylcellulose gels containing the hydrophilic ILs hexylpyridinium chloride, choline dihydrogen phosphate, and 1-ethyl-3-methylimidazolium ethyl sulfate were prepared, and the influence of the ILs on the formulation properties was evaluated. ILs were successfully incorporated into the emulsion structure, resulting in stable formulations. The antimicrobial activity of the ILs was estimated. The minimal inhibitory concentration values for hexylpyridinium chloride are about 2.5 mg/mL. The other two ILs have no antimicrobial activity. Skin penetration enhancement of caffeine, a hydrophilic model substance, was observed in the presence of hexylpyridinium chloride.     

Development of triptolide-nanoemulsion gels for percutaneous administration: physicochemical,transport, pharmacokinetic and pharmacodynamic characteristics

Background

This work aimed to provide useful information on the use of nanoemulsions for the percutaneous administration of triptolide. Lipid nanosystems have great potential for transdermal drug delivery. Nanoemulsions and nanoemulsion gels were prepared to enhance percutaneous permeation. Microstructure and in vitro/in vivo percutaneous delivery characteristics of triptolide (TPL)-nanoemulsions and TPL-nanoemulsion gels were compared. The integrity of the nanoemulsions and nanoemulsion gels during transdermal delivery and its effects on the surface of skin were also investigated. The penetration mechanisms of nanoemulsions and nanoemulsion gels were investigated by differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FTIR). The transport characteristics of fluorescence-labelled nanoemulsions were probed using laser scanning confocal microscopy. A chronic dermatitis/eczema model in mice ears and the pharmacodynamic of the TPL-nanoemulsion gels were also investigated.

Results

Compared to TPL gels, significantly greater cumulative amounts of TPL-nanoemulsion gels and TPL-nanoemulsions penetrated rat skin in vitro. The in vivo microdialysis showed the concentration–time curve AUC_0–t for TPL-NPs is bigger than the TPL-gels. At the same time, TPL-NPs had a larger effect on the surface of skin. By hydrating keratin and changing the structure of both the stratum corneum lipids and keratin, nanoemulsions and nanoemulsion gels influence skin to promote percutaneous drug penetration. Both hairfollicles and the stratum corneum are also important in this transdermal drug delivery system. Moderate and high dosages of the TPL-nanoemulsion gels can significantly improve the symptoms of dermatitis/eczema inflammation and edema erythematic in mice ears and can reduce the expression of IFN-γ and IL-4. Moreover, the TPL-nanoemulsion gels cause less gastrointestinal damage than that of the Tripterygium wilfordii oral tablet does.

Conclusions

Nanoemulsions could be suitable for transdermal stably releasing drugs and maintaining the effective drug concentration. The TPL-nanoemulsion gels provided higher percutaneous amounts than other carriers did. These findings suggest that nanoemulsion gels could be promising percutaneous carriers for TPL. The TPL-nanoemulsion gels have a significant treatment effect on dermatitis/eczema in the mice model and is expected to provide a new, low-toxicity and long-term preparation for the clinical treatment of dermatitis/eczema in transdermal drug delivery systems.

     

Histone propinquity using imidoesters.

Native chromatin was reacted with bifunctional protein cross-linking reagents of varying molecular sizes. Short cross-linkers do not produce significant yields of polymeric histones whereas intermediate-sized materials such as dimethyladipimidate produce extensive interaction between histones. Dimethylsuberimidate reacts with all histone fractions but only generates polymers of F₁ and of F_2b and F_2a2.     

A Sandwich Cup Method for the Penetration Assay of Antimicrobial Agents through Pseudomonas Exopolysaccharides

We developed new sandwich cup method to assay the penetration of various antimicrobial agents through Pseudomonas exopolysaccharides. Using alginate extracted from mucoid-type Pseudomonas aeruginosa and gellan gum from Pseudomonas elodea, the role of exopolysaccharides as a barrier against drug penetration was examined. The penetration of positively charged hydrophilic drugs such as aminoglycosides and polypeptides was markedly inhibited by the gels tested, but that of β-lactams, quinolones, and macrolides was not inhibited. The penetration of gentamicin was strongly influenced by the gel concentration, the solution to be used, and the presence of Ca²⁺. These results suggest that the microenvironment at the infection site could greatly influence drug penetration through biofilms in vivo.     

Down-regulation of NOSII gene expression by iontophoresis of anti-sense oligonucleotide in endotoxin-induced uveitis

Transcorneoscleral iontophoresis was used to enhance ocular penetration of a 21-bp NH(2) protected anti-NOSII oligonucleotides (ODNs) (fluorescein or infrared-41 labeled) in Lewis rats. Both histochemical localization and acrylamide sequencing gels were used. To evaluate the potential to down-regulate NOSII expression in the rat model of endotoxin-induced uveitis (EIU), anti-sense NOSII ODN, scrambled ODN or saline were iontophorezed into these animals' eyes. Iontophoresis facilitated the penetration of intact ODNs into the intraocular tissues of the rat eye and only the eyes receiving ODNs and electrical current demonstrated intact ODNs within the ocular tissues of both segments of the eye. Iontophoresis of anti-NOSII ODN significantly down-regulated the expression of NOSII expression in iris/ciliary body compared to the saline or scrambled ODN treated eyes. Nitrite production was also significantly reduced in the anti-NOSII applied eyes compared to those treated with saline. Using this system, intraocular delivery of ODNs can be significantly enhanced increasing the potential for successful gene therapy for human eye diseases.