Mesoporous silica particles as potential carriers for protein drug delivery: protein immobilization and the effect of displacer on γ-globulin release.

The adsorption of γ-globulin was evaluated with experiments with silica particles marketed as Syloid AL1-FP (SAL), XDP-3150 (SXDP), and 244FP (SFP). The influence of pH, pore sizes, and degree of surface porosity on the extent of γ-globulin immobilization was examined. Protein adsorption on these particles was largely related to their surface porosity and pore sizes. The adsorption capacity was established to be greater with mesoporous SFP and SXDP particles at 474 and 377 mg/g, respectively, when compared to significantly low-porosity SAL (16 mg/g). Additionally, γ-globulin immobilization was favored at pH closer to iso-electric point. A key aim of this work was to better understand and improve the limited reversibility of protein adsorption. Protein desorption was found to be lower in simulated intestinal fluid (SIF) in comparison to pH 7.4 phosphate buffer (PB). The use of displacer molecules (sodium dodecyl sulfate [SDS]/Tween 80/Pluronic F127 [PF127]) promoted protein desorption from the adsorbent surface by the exchange mechanism. The PF127 provided substantial release in both studied condition but the highest release of 83% of γ-globulin from SXDP was obtained with tween 80 in PB. The released protein was analyzed with circular dichroism (CD) spectroscopy which indicated that the secondary structure of desorbed γ-globulin was dependent on the pH and displacer molecule. The conformation largely remained unchanged when desorption was carried out in SIF but changed markedly in PB specially in the presence of SDS.

A study on reducing the absorption of lidocaine from the airway in cats.

PURPOSE:: To determine if the combination of lidocaine with epinephrine or gamma globulin would decrease the rate or reduce the amount of local absorption of lidocaine through the airway. METHODS:: Twenty adult male cats were randomly and evenly distributed into four groups: 1) Group LG: lidocaine administered with gamma globulin; 2) Group LS: lidocaine administered with physiological saline); 3) Group LE: lidocaine administered with epinephrine; 4) Group C: control group. Invasive blood pressure, heart rate, and concentration of lidocaine were recorded before and after administration. RESULTS:: The peak of plasma concentrations appeared difference (Group LG: 1.39 ± 0.23 mg/L; Group LS: 1.47 ± 0.29 mg/L and Group LE: 0.99 ± 0.08 mg/L). Compared to Group C, there were significant differences in the average heart rate of Groups LG, LS, and LE (P < 0.05). The average systolic blood pressures were significantly different when each group was compared to Group C (P < 0.05). The biological half-life, AUC0-120, peak time, and half-life of absorption among the three groups have not presented statistically significant differences (P > 0.05). CONCLUSION:: Administering lidocaine in combination with gamma globulin through airway causes significant decrease the rate and reduce the amount of local absorption of lidocaine in cats.

A study on reducing the absorption of lidocaine from the airway in cats

Abstract Purpose: To determine if the combination of lidocaine with epinephrine or gamma globulin would decrease the rate or reduce the amount of local absorption of lidocaine through the airway. Methods: Twenty adult male cats were randomly and evenly distributed into four groups: 1) Group LG: lidocaine administered with gamma globulin; 2) Group LS: lidocaine administered with physiological saline); 3) Group LE: lidocaine administered with epinephrine; 4) Group C: control group. Invasive blood pressure, heart rate, and concentration of lidocaine were recorded before and after administration. Results: The peak of plasma concentrations appeared difference (Group LG: 1.39 ± 0.23 mg/L; Group LS: 1.47 ± 0.29 mg/L and Group LE: 0.99 ± 0.08 mg/L). Compared to Group C, there were significant differences in the average heart rate of Groups LG, LS, and LE (P < 0.05). The average systolic blood pressures were significantly different when each group was compared to Group C (P < 0.05). The biological half-life, AUC0-120, peak time, and half-life of absorption among the three groups have not presented statistically significant differences (P > 0.05). Conclusion: Administering lidocaine in combination with gamma globulin through airway causes significant decrease the rate and reduce the amount of local absorption of lidocaine in cats.

Progress in gammaglobulin therapy for immunodeficiency: from subcutaneous to intravenous infusions and back again.

The year 1952 marked the first use of subcutaneous immunoglobulin therapy to treat primary immunodeficiency disease. Subsequently, intramuscular and then intravenous administration became the norm in the United States and most of Europe. Intravenous immunoglobulin therapy, however, can be burdensome and often causes systemic side effects. To overcome obstacles presented by the intravenous route of administration, subcutaneous preparations were developed. To further enhance patient satisfaction, adherence, and quality of life, enzyme-enhanced subcutaneous immunoglobulin administration using hyaluronidase, an enzyme spreading agent, was studied. The dose and flow rate of traditional subcutaneous immunoglobulin infusion is limited by the inhibition of bulk fluid flow by the extracellular matrix. Recombinant human hyaluronidase, administered with or immediately prior to infusate, increases the absorption and dispersion of infused fluids and drugs. Results from a phase III clinical trial indicate that subcutaneous immunoglobulin infusion, facilitated by recombinant human hyaluronidase, is well tolerated, and delivers infusion volumes at treatment intervals and rates equivalent to intravenous administration. This review surveys the state of the art of immunoglobulin replacement therapy.

Protein coverage on silicon surfaces modified with amino-organic films: a study by AFM and angle-resolved XPS.

An approach to determine structural features, such as surface fractional coverage F and thickness d of protein layers immobilized on silicon substrates coated with amino-organic films is presented. To demonstrate the proposed approach rabbit gamma globulins (RgG) are adsorbed from a 0.66muM solution onto SiO(2) and Si(3)N(4) modified with (3-aminopropyl)triethoxysilane (APTES). Atomic force microscopy data are analyzed by applying an integral geometry approach to yield average coverage values for silanized Si(3)N(4) and SiO(2) coated with RgG, F=0.99+/-0.01 and 0.76+/-0.08, respectively. To determine the RgG thickness d from angle-resolved X-ray photoelectron spectroscopy (ARXPS), a model of amino-organic bilayer with non-homogeneous top lamellae is introduced. For an APTES layer thickness of 1.0+/-0.1nm, calculated from independent ARXPS measurements, and for fractional surface RgG coverage determined from AFM analysis, this model yields d=1.0+/-0.2nm for the proteins on both silanized substrates. This value, confirmed by an evaluation (1.0+/-0.2nm) from integral geometry analysis of AFM images, is lower than the RgG thickness expected for monomolecular film ( approximately 4nm). Structures visible in phase contrast AFM micrographs support the suggested sparse molecular packing in the studied RgG layers. XPS data, compared for bulk and adsorbed RgG, suggest preferential localization of oxygen- and nitrogen-containing carbon groups at silanized silicon substrates. These results demonstrate the potential of the developed AFM/ARXPS approach as a method for the evaluation of surface-protein coverage homogeneity and estimation of adsorbed proteins conformation on silane-modified silicon substrates used in bioanalytical applications.

Biodistribution of liposome-entrapped human gamma-globulin.

The present study was aimed at the preparation and performance evaluation of Intacglobin-loaded liposomes for selective drug presentation to the lungs. Egg phosphatidylcholine- and cholesterol-based liposomes (1:1 and 1:0.25 mol/mol) were prepared by a dehydration-rehydration procedure. A tissue distribution study after single intranasal administration of 0.5 microCi 125I-Intacglobin-loaded liposomes was conducted in Balb/c mice. The efficiencies of drug entrapment (30%) and the average diameters did not differ significantly between the two liposome formulations. However, liposomes composed of an increased cholesterol amount showed a lower in vitro drug release rate. The airway penetration efficiency of the liposomal formulation was determined by the cumulative percentage of the dose reaching the lungs (AUC) and its sojourn time therein, and were 1.7- and 2.2-times higher compared with the plain 125I- Intacglobin solution-based formulation, respectively. A significantly greater (p<0.001) drug localization index after 24 h was found at the lungs in comparison with the other tissues (p<0.01), although similar values were detected between groups following administration of either liposomes or control solutions, despite the formulations attributes. In conclusion, it is suggested that longer Intacglobin exposure at the pulmonary region is observed after administration of the liposomal formulation. The results open future perspectives in assessing local passive immunization for the treatment of respiratory infectious diseases.

The effect of the administration of human gamma globulins in a model of BCG infection in mice.

The effect of the administration of a commercial preparation of human gamma globulins has been evaluated in a mouse model of intranasal infection with BCG. First, we demonstrated the passage of specific antibodies to saliva and lung lavage following the intranasal or intraperitoneal administration to mice of human gamma globulins. This treatment of mice inhibited BCG colonization of the lungs (p < 0.01). A similar inhibitory effect was observed after infection of mice with gamma globulin opsonized BCG organisms (p < 0.01). These results are relevant for the development of new strategies for the control and treatment of tuberculosis.

Sustained release of human growth hormone from in situ forming hydrogels using self-assembly of fluoroalkyl-ended poly(ethylene glycol).

Poly(ethylene glycol)s modified with fluorocarbon end groups are capable of in situ transition from an injectable liquid to a viscoelastic hydrogel by hydrophobic interaction of the end groups; this class of materials is useful for a variety of biomedical applications, including sustained protein release. The hydrogel state can be transformed into an injectable state by the addition of a toxicologically acceptable organic solvent, such as N-methyl pyrrolidone; after injection, this solution quickly returns to a gel state by diffusion of the water-miscible organic solvent into the surrounding environment. In vitro characterization of sustained release of human growth hormone (hGH) using this injectable depot shows that hGH remains stable inside the hydrogel formed, and demonstrates more than 2 weeks of prolonged release of hGH complexed with Zn(2+) ions without protein aggregation or initial burst.

Protein adsorption and monocyte activation on germanium nanopyramids.

Germanium can form defect-free pyramidal islands on Si(1 0 0)-2 x 1 with a height of 15 nm and a width of 60 nm. Using chemical vapor deposition we have prepared substrates with different nanopyramid densities to study the impact on contact angles, protein adsorption and cell behavior. The advancing contact angle of a water droplet of millimeter size significantly raises with nanopyramid density. The dynamic contact angle measurements reveal that the substrate surface is highly hydrophilic. On such a surface the adsorption of hydrophilic proteins, i.e. albumin and globulin, is drastically increased by the presence of nanopyramids. More important, however, the globulin is inactive after adsorption on nanopyramid edges. This observation is supported by the cytokine release of IL-1beta and TNF-alpha of monocyte-like cell line U937. Consequently, the presence of nanopyramidal structures gives rise to less inflammatory reactions.

Diffusion of gamma globulin into the arterial wall identifies localized entry of lipid and cells in atherosclerosis.

BACKGROUND: The localization of atheromatous lesions in vulnerable arteries and their relatively rare occurrence in other arteries of the same subject cannot be explained by current theories of the aetiology of atherosclerosis. OBJECTIVE: To determine whether abnormal diffusion of gamma globulin into the arterial wall from the lumen will identify defects of barrier function allowing localized entry of lipid and cells in atherosclerosis. METHODS: Paraffin sections of left anterior descending coronary arteries and corresponding internal thoracic arteries from 80 human subjects aged 1-65 years were stained for gamma globulin by the immunoperoxidase technique. Duplicate sections were stained with orcein to demonstrate the elastin structure. RESULTS: The barrier function of the luminal surface of the thickened intima was associated with the presence of an elastin lamina beneath the endothelial cells. With advancing age, the coronary arteries exhibited breakdown of this barrier function in localized areas with entry into the arterial wall of gamma globulin, lipid and cells. This was rare in the internal thoracic artery. CONCLUSION: Lack of continuity or incomplete formation of this sub-endothelial lamina, which was seen particularly in the coronary artery, was associated with localized entry into the arterial wall of gamma globulin, lipid and cells from the circulating blood and with the development of atheromatous lesions.

Oral immunization of rainbow trout with antigen microencapsulated in poly(DL-lactide-co-glycolide) microparticles.

The model protein antigen, human gamma globulin (HGG) was microencapsulated in poly(DL-lactide-co-glycolide) microparticles and administered orally to rainbow trout. Oncorhynchus mykiss (Walbaum, 1792). Using a Western blotting technique it was demonstrated that the dynamics of passage through the gut were different for the microencapsulated and soluble antigen. The association of HGG with microparticles increased the retention time of the antigen in the stomach and delayed its entry into the intestinal region. After the delivery of microencapsulated HGG, antigen was detected in gut contents in fragmented form which suggested that some of the antigen was present at the particle surface and therefore susceptible to proteolysis. However, a greater amount of intact antigen was detected in the posterior intestine and in the bloodstream of fish, which were administered with microparticle-associated than soluble antigen, indicating that the antigen was partially protected. Immunization with microencapsulated HGG resulted in the detection of specific antibody in the serum but levels were not significantly greater than after the delivery of soluble antigen. However, specific antibody was detected in the intestinal mucus of fish which were administered with the microencapsulated antigen after boosting with soluble HGG but not in fish which were primed with the soluble antigen.

Effect of chronic ethanol administration and dietary protein regimens on intestinal absorption of macromolecules in rats.

The effect of feeding ethanol daily for 40 days has been studied on intestinal absorption of bovine serum albumin (BSA) and gamma-globulin (IgG) in rats fed a low (8%) protein (LP) or a high (30%) protein (HP) diet. Feeding the LP diet enhanced the tissue uptake of BSA (p < 0.05) and absorption of BSA and IgG into serum (p < 0.001) as compared with controls. Feeding the HP diet also augmented the uptake of IgG (p < 0.001) by the intestinal tissue and significantly enhanced serum levels of BSA and IgG. Ethanol feeding to rats for 40 days enhanced the uptake of BSA and IgG (24-84%) and their absorption into serum (p < 0.001) as compared with the controls. Ethanol administration to rats fed LP or HP diets did not alter the uptake of these proteins as compared with their respective controls. Luminal degradation of BSA and IgG was higher in ethanol-administered (48-50% and 36-39%, respectively) and LP-fed rats (50 and 38%, respectively). It was reduced by 17-21% in HP-fed rats as compared with the control group. This indicated that the observed increase in protein absorption is not related to the luminal degradation of the proteins under these conditions. These findings suggest that the absorption of macromolecules from intestine in response to ethanol feeding is influenced by the dietary status of the animals.

The influence of hypoxia on transvascular leakage in the isolated rat heart: quantitative and ultrastructural studies.

1. The multiple indicator dilution method was used to study the transvascular movement of gamma-globulin, bovine serum albumin, insulin and cyanocobalamin in the isolated rat heart. 2. Perfusion of the heart with well-oxygenated solution for 75 min (constant flow) did not produce a significant change either in the total area under the dilution curve or the 'leakage index' (an arbitrary measure of transvascular flux) for all the tracers. 3. Perfusion of the heart with hypoxic solution produced a significant increase in leakage of gamma-globulin of 38.6 +/- 18, 48.5 +/- 17.6, 60.5 +/- 24 and 58 +/- 20% after 15, 30, 45 and 60 min, respectively, compared with the well-oxygenated equilibration period. Permeability- surface area products (PS) for the smaller diffusible solutes, therefore, could not be estimated. 4. The flux of albumin, insulin and cyanocobalamin in response to hypoxia was similar to that of gamma-globulin. 5. Ultrastructural examination of well-oxygenated hearts revealed that Monastral Blue-labelled albumin remained within the lumen and that endothelial integrity remained intact. 6. Conversely biopsies from hypoxic hearts showed that the labelled albumin had passed to the interstitium through gaps (approximately 3 microns) in venular endothelium. 7. The results showed that, in intact hearts, hypoxia produced gaps in the endothelium of venules and that these gaps could be the possible route for transvascular leakage of macromolecules.

Fluorinated proteins as potential 19F magnetic resonance imaging and spectroscopy agents.

Fluorinated proteins have been synthesized and characterized as potential in vivo 19F magnetic resonance imaging (MRI) and spectroscopy (MRS) agents. Proteins labeled with fluorine include bovine serum albumin, gamma-globulin, and purified immunoglobulin (IgG). The amino groups in proteins were selectively trifluoroacetylated using S-ethyl trifluorothioacetate to synthesize fluorinated proteins (TFA-protein; 1-3). In another approach, trifluoroacetamidosuccinic anhydride has been used to prepare corresponding fluorinated derivatives of proteins (TFASA-protein; 4-6). The fluorinated proteins have been purified by exhaustive dialysis and isolated in good yields (55-76%). The fluorinated proteins exhibit useful NMR characteristics and the biocompatibility for in vivo studies. The initial investigations demonstrate the potential of these new fluorinated proteins as in vivo MRI/MRS probes.

Effect of polycations on permeability of glomerular epithelial cell monolayers to albumin.

Polycations can interact with the surface negative charges of the glomerular epithelial cells in the kidney and give rise to metabolic alterations. This study examined whether charge neutralization can affect intercellular junctions and increase macromolecular permeability across epithelial monolayers. We examined this question by studying the effect of polycations on the leakage of albumin across monolayers of glomerular epithelial cells. Cells were grown to confluency on filter-lined cups. They were treated apically with cationic bovine gamma globulin or protamine (100 micrograms/ml) for 2 hours at 37 degrees C. After washing the cells, the monolayers were tested for leakage of albumin by the addition of radioactive bovine serum albumin on the apical side and determining the time course of its appearance on the basal side. Polycation treatment caused significant leakage of albumin in the absence of any toxic effect on viability or lactate dehydrogenase release. The leakage was shown to be through the tight junctions of the monolayer. Permeability alterations were compared at 4 degrees C and 37 degrees C to determine whether impairment was due to the membrane ruffling effect of charge neutralization or due to intracellular metabolic changes. Despite equal bindings of polycations at 4 degrees C and 37 degrees C, significant leak occurred only at 37 degrees C, suggesting the role of active processes in the maintenance of permselectivity. This was consistent with the rapid interiorization of the polycation at 37 degrees C after membrane binding. Further substantiation of this point was obtained by studying the protective effect of removing bound polycation with heparin. Removal of polycation after initial binding failed to protect the monolayer from albumin leakage. The conclusion was that neutralization of glomerular epithelial cell surface charges results in subtle impairment of tight junction control of permeability to macromolecules across the monolayer. The impairment was due to active intracellular processes that ensue after polycation binding to cell surface charges and their subsequent internalization.

Bioavailability of gamma-globulin after subcutaneous infusions in patients with common variable immunodeficiency.

Replacement therapy, using subcutaneous infusions of gamma-globulin, is being applied increasingly for antibody-deficient patients, as this form of treatment has been found to be related to a very low frequency of adverse systemic reactions. However, the uptake of IgG from subcutaneous tissue may be low, owing to degradation locally, especially for the IgG3 molecule. Therefore, the kinetics of IgG and IgG-subclass concentrations in the sera of 23 patients with common variable immunodeficiency was investigated during 18 months of subcutaneous infusions of gamma-globulin (100 mg/kg/week). Seventeen patients were previously treated with intramuscular injections or intravenous infusions. The mean serum IgG level increased twice in the previously treated patients and four times in the previously untreated patients. A steady state was reached after 6 months if the subcutaneous infusions were given weekly and after 1 week if the patients were given daily infusions for 5 consecutive days and, thereafter, weekly infusions. The fractional catabolic rate of IgG (4.1-5.9% per day) was found to be at the lower limit reported for normal controls, if 100% bioavailability of the infused IgG was assumed. The fractional contents of IgG subclasses in the patients' serum IgG resembled the physiological pattern, with the exception of IgG4, which was not present in the gamma-globulin preparations used. Significantly increased levels of IgG1 and -2 were seen in both previously treated and untreated patients during the treatment.

Intestinal absorption of immunologically intact macromolecules in germfree colostrum-deprived piglets maintained on total parenteral nutrition.

We have compared the neonatal absorption of anti-bovine gamma-globulin (BGG) antibody supplied in colostrum or saline in three groups of piglets born and maintained under different environmental conditions to determine the effect of these conditions on the cessation of intestinal absorption of macromolecules (anti-BGG antibody), termed "closure." An enzyme-linked immunosorbent assay was used to estimate the concentration of anti-BGG antibody in sera from each group of piglets. Three stages of macromolecular absorption through the piglet's intestine could be detected. The first stage is a nonselective massive absorption of macromolecules (in milligram levels) that lasts up to 3 days in germfree (GF) colostrum-deprived or conventional colostrum-fed piglets but up to 5 days in GF piglets maintained on total parenteral nutrition. In this stage, absorption was significantly (r = .05) higher in piglets fed anti-BGG serum with colostrum than in piglets fed anti-BGG serum without colostrum on GF day 0 (31.28% vs 15.59%) and GF-total parenteral nutrition day 3 (3.08% vs 0.11%). Thus, whenever there was the ability to absorb a massive amount of macromolecules, the sow colostrum had an enhancing affect. Although there was a minor effect of environmental or orally received stimuli in delaying closure, absorption of macromolecules decreased in all piglets maintained either parenterally or enterally after day 3. Thus, intestinal closure to massive absorption of macromolecules in piglets is primarily time (age)-dependent. The second stage is a selective absorption of immunoglobulins in much smaller quantities (microgram levels), inasmuch as absorption of 0.02% to 0.1% was determined in all 5-day-old piglets.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of intravenous gamma globulin to passively immunize high-risk children against respiratory syncytial virus: safety and pharmacokinetics. The RSVIG Study Group.

Infants with cardiopulmonary disease develop severe illness from respiratory syncytial virus (RSV) infection. Safety, feasibility, and pharmacokinetics of intravenous gamma globulin (IVIG) to prevent RSV illness were studied in 23 high-risk infants in a phase I trial. IVIG with an RSV neutralizing antibody titer of 1:1,100 in 5% solution was given monthly over a 2- to 4-h period in a clinical setting during the RSV season. The first group (n = 7) received 500 mg/kg of body weight, the second group (n = 9) received 600 mg/kg, and the third group (n =7) received 750 mg/kg. Serum was drawn prior to infusion and 2, 14, and 30 days after infusion. Total immunoglobulin G and RSV A2 and RSV B neutralizing antibody levels were obtained after the first IVIG infusion. Two children developed mild reversible pulmonary edema (group receiving 600 mg/kg per dose), and one developed hives and wheezing during one infusion (group receiving 500 mg/kg per dose). Twelve children developed subsequent RSV infection during two RSV seasons (November to April) over a 2-year follow-up period; 9 of 12 developed infection during the infusion year. Eleven illnesses were mild; one child died of progressive RSV illness (group receiving 500 mg/kg per dose). A cumulative infusion effect was not observed. IVIG appears safe and feasible in an outpatient setting, and at 750 mg/kg per dose, a target RSV antibody level of greater than or equal to 1:100 was achieved.

Comparative study of reaction kinetics in membrane and agarose bead affinity systems.

Compared to conventional agarose bead affinity supports, a microporous nylon membrane exhibits greatly improved reaction kinetics as quantified in the reaction between gamma-globulin and immobilised protein A. The improvement is only observed when the solution of gamma-globulin is forced through the membrane pores. In the absence of flow in the pores, it is possible to relate approximately the rate of uptake onto either type of affinity support to independently determined diffusion coefficients. In the presence of flow, the reaction rate is similar for membranes having 0.45 and 3.0 microns diameter pores, and considerably smaller than predicted by the Smoluchowski formula.