Microencapsulated drug delivery: a new approach to pro-inflammatory cytokine inhibition.

CONTEXT: This article reviews the use of albumin microcapsules 3-4 µm in size containing cytokine inhibiting drugs which include neutralizing antibodies to TNF and IL1, CNI-1493, antisense oligonucleotides to TNF and NF-kappaB, and the antioxidant catalase. OBJECTIVE: Describe the effects, cellular uptake and distribution of microencapsulated drugs and the effect in both a peritonitis model of infection and a model of adjuvant-induced arthritis. METHODS: The studies performed by our group are reviewed, the only such studies available. RESULTS: Microencapsulation of these compounds produced high intracellular drug concentrations due to rapid uptake by phagocytic cells, including endothelial cells, without toxicity. All compounds produced excellent inhibition of TNF and IL1 resulting in improved animal survival in a peritonitis model of septic shock and inflammation in an arthritis model. CONCLUSION: Albumin microencapsulated pro-inflammatory cytokine inhibiting compounds are superior to equivalent concentration of these compounds administered in solution form.

Potentiation of pro-inflammatory cytokine suppression and survival by microencapsulated dexamethasone in the treatment of experimental sepsis.

Cytokine inhibiting drugs are much more effective when delivered intracellularly to phagocytic cells in the microencapsulated form. Dexamethasone is a powerful inhibitor of TNF-α cytokine through inhibition of NF-κB which is a gene regulator of multiple pro-inflammatory cytokines. We have determined the effect of microencapsulated dexamethasone in pro-inflammatory cytokine release both in in vitro using whole blood model, and in vivo using peritonitis model of septic shock. Microspheres of 1-4 µm mean size were prepared by using albumin polymer matrix in a one-step spray drying method. Microencapsulated form of dexamethasone with concentration of 10(-1), 10(-2) and 10(-3) M was compared to an equivalent concentration of solution form of dexamethasone in the in vitro whole blood model. The results show microencapsulated dexamethasone inhibited tumor necrosis factor-alpha (TNF-α) and interleukin-beta (IL-1ß) significantly in comparison with the solution form of dexamethasone. The in vivo peritonitis model also demonstrated significant inhibition of TNF-α and IL-1ß cytokines in microencapsulated form in comparison with solution form of dexamethasone. In the in vivo study, the animal survival rate after 5 days was 90%, dexamethasone in solution with gentamicin was 40% and gentamicin alone was 30%. This study demonstrates significantly improved inhibition of TNF-α and IL-1ß both in vivo and in vitro when dexamethasone was used in microencapsulated form.

Synergism in survival to endotoxic shock in rats given microencapsulated CNI-1493 and antisense oligomers to NF-kappaB.

The synthesis of TNF may be inhibited at the transcriptional level by antisense to either TNF or NF-kappaB or at the post-transcriptional level by CNI-1493, a guanylhydrazone compound which inhibits p38 MAP kinase activity. Previous studies have demonstrated that targeting macrophages and other phagocytic cells by intracellular drug delivery using albumin microcapsules containing either antisense oligomers to NF-kappaB or CNI-1493 greatly enhances intracellular drug concentration and survival in both endotoxic shock and sepsis models. It is the purpose of this study to determine if microencapsulated drugs acting at different stages in the synthesis of TNF are synergistic. Four groups of 10 rats each were given 15 mg kg(-1) of E.coli endotoxin and treated with (1) CNI-1493 1 mg kg(-1), (2) antisense oligomers to NF-kappaB at 100 mcg, (3) CNI-1493 1 mg kg(-1) plus antisense kappa to NF- at 100 mg kg(-1) and (4) CNI-1493 200 mg kg(-1) plus antisense oligomer to NF-kappaB at 200 mg kg(-1). TNF and IL1 were measured by ELISA at 4, 8, 24 and 48 h. The rats were observed for 5 days. The combination of CNI-1493 and antisense oligomers to NF-kappaB inhibited TNF 41% greater that CNI alone and 51% greater than antisense oligomers to NF-kappaB alone at 4 h after endotoxin administration. Survival at 5 days with CNI alone was 0%, 20% with antisense oligomers to NF-kappaB and 60% with the combination. In conclusion, synergism in survival occurs using microencapsulated drugs acting at different points in the synthesis of TNF was demonstrated using an in-vivo model of endotoxic shock. Both the amount of TNF inhibition and the mortality were significantly improved with combination therapy. Multiple drugs acting at different sites in the synthesis of TNF may be useful in the treatment of disease states characterized by pro-inflammatory cytokine release.

Targeted delivery of antigens to the gut-associated lymphoid tissues: 2. Ex vivo evaluation of lectin-labelled albumin microspheres for targeted delivery of antigens to the M-cells of the Peyer's patches.

The purpose of this study was to evaluate the possibility of lectin-coupled microspheres to improve the targeted delivery of protein antigens to the lymphoid tissues of mucosal surfaces. Bovine serum albumin containing acid phosphatase model protein and polystyrene microspheres were coupled with mouse M-cell-specific Ulex europaeus lectin. The coupling efficiency, physical characteristics and the binding capabilities of the microspheres to the follicle associated epithelium of the Peyer's patches were evaluated in vitro and ex vivo in mice intestine. The results showed that coupling of lectin to albumin microspheres did not significantly affect the bioactivity of the encapsulated acid phosphatase model protein. It was also shown that there was preferential binding of the lectin-coupled microspheres to the follicle-associated epithelium. It was concluded from the results of the study that coupling of ligands such as lectin specific to cells of the follicle associated epithelium can increase the targeting of encapsulated candidate antigens for delivery to the Peyer's patches of the intestine for improved oral delivery.

The effect of intracellular delivery of catalase and antisense oligonucleotides to NF-kappaB using albumin microcapsules in the endotoxic shock model.

UNLABELLED: Microencapsulated (MC) catalase has been shown to inhibit H(2)O(2) and tumor necrosis factor (TNF) in vitro after endotoxin stimulation. It is the purpose of this study to determine whether MC catalase improves pro-inflammatory cytokine inhibition and mortality in an endotoxic shock model in vivo. We also examined whether MC catalase and antisense oligonucleotides (ASO) to nuclear factor kappaB (NF-kappaB) together improved survival by inhibiting pro-inflammatory cytokines using different mechanisms. METHODS: Albumin microcapsules containing catalase and ASO to NF-kappaB were prepared 2-7 microm in size by using a Büchi spray dryer. Progressively increasing doses of MC catalase, MC ASO to NF-kappaB, and the combination were given to rats before the administration of Escherichia coli endotoxin. Results demonstrated 60% survival in rats given 15 mg/kg MC catalase, 70% survival with 20 mg/kg MC ASO NF-kappaB, and 80% survival with the combination. TNF was inhibited by 53% in the MC catalase group 4 h after endotoxin administration, 43% in the ASO NF-kappaB group, and 78% in the combination group compared to controls. In conclusion, this study demonstrates the effectiveness of MC intracellular delivery of the naturally occurring antioxidant catalase in improving animal survival. The addition of ASO to NF-kappaB improved both cytokine inhibition and animal survival in endotoxic shock.

The effect of intracellular antioxidant delivery (catalase) on hydrogen peroxide and proinflammatory cytokine synthesis: a new therapeutic horizon.

UNLABELLED: Reactive oxygen species synthesized by endothelial cells may be responsible for cell damage and altered physiologic function. After endotoxin stimulation, free radicals including H(2)O(2) are produced. We have developed a method of intracellular drug delivery using albumin microcapsules. Catalase would be an excellent compound to alter H(2)O(2) production. However, the large molecular size of catalase limits cellular penetration. Endothelial cells have been previously shown to readily phagocytoze albumin microcapsules. METHODS: Catalase was added to an albumin solution to form a 10% solution of catalase. Microspheres from 2 to 7 microm in size were formed using a Bucchi spray dryer. Human endothelial cells were incubated with varying concentrations of microencapsulated catalase. The cells were then exposed to Escherichia coli endotoxin to determine if increased intracellular penetration of catalase would inhibit H(2)O(2), nitrate, and cytokine synthesis. RESULTS: There was a 7.2-fold increase in endothelial intracellular catalase after 48 h incubation. H(2)O(2) was inhibited by 72%, nitrate 96%, TNF 90%, IL1 21%, IL6 42%. CONCLUSIONS: These results demonstrate that inhibition of H(2)O(2) as a result of increased intracellular delivery of catalase inhibits proinflammatory cytokine synthesis after endotoxin exposure.

A methodology for quantitation and characterization of oligonucleotides in albumin microspheres.

A fluorescence assay was developed to quantify oligonucleotides (ODNs) encapsulated in bovine serum albumin (BSA) microspheres using antisense to Nuclear Factor-kappaB (NF-kappaB) as a model ODN and employing Oligreen as the fluorescent dye. Methodologies were optimized for the suspension of the microspheres as well as release of the encapsulated ODN using protease digestion. This was followed by the detection and quantitation of the ODN using the Oligreen dye. The Oligreen fluorescence assay gave a concentration-dependent fluorescent interaction with the ODN. Further characterization of the ODN with respect to their structural integrity in non-irradiated and gamma-irradiated antisense encapsulated in BSA microspheres was performed using HPLC, infrared spectroscopy and polyacrylamide gel electrophoresis. Results showed no structural modification of antisense in the BSA microspheres as determined by HPLC retention times for the pure antisense and microsphere-encapsulated ODN. The migration pattern of the antisense in polyacrylamide gels confirmed the absence of significant alterations as a result of the encapsulation process or due to gamma-irradiation. The infrared spectra of non-irradiated and gamma-irradiated antisense to NF-kappaB microspheres also displayed peaks characteristic of the functional groups. The fluorescence assay could also detect NF-kappaB antisense in the serum of rats administered with encapsulated antisense by oral and intravenous routes. This methodology should be valuable for the analysis of BSA-encapsulated antisense ODN and for pharmacokinetic studies during antisense therapy.

Formulation and characterization of catalase in albumin microspheres.

Catalase in albumin microspheres were formulated for intravenous administration to antagonize the effects of over-production of reactive oxygenated species (ROS) such as hydrogen peroxide (H(2)O(2)) in septic shock. The aim was to increase effective half-life of catalase and take advantage of the phagocytic uptake of the encapsulated catalase by the vascular endothelium. Catalase microspheres were prepared by spray-drying. The microspheres were evaluated for particle size, particle shape and surface morphology by scanning electron microscopy (SEM), drug encapsulation efficiency, chemical stability, thermal stability and in vitro drug release characteristics. The microspheres had a mean particle size of 4.7 +/- 2 microm, optimal for phagocytic uptake, as demonstrated by Makino et al. SEM revealed that microspheres were spherical with smooth surface morphology. An encapsulation efficiency of 91.5 +/- 3% was achieved and the encapsulated catalase was chemically and thermally stable. Application of in vitro drug release data to the Higuchi kinetic equation indicated matrix diffusion-controlled catalase release from albumin microspheres.

Treatment of adjuvant arthritis using microencapsulated antisense NF-κB oligonucleotides.

Antisense oligonucleotides are promising new therapeutic agents used to selectively inhibit target genes such as Nuclear Factor Kappa B (NF-κB), an important transcription factor in the pathogenesis of inflammatory disease. The purpose of the present study was to evaluate microencapsulated antisense oligonucleotides specific to NF-κB for in vitro efficacy and treatment of adjuvant-induced arthritis in rats. Oligonucleotide-loaded albumin microspheres were prepared and characterized in terms of size, zeta potential, morphology and release pattern. This study reports significant NF-κB inhibition in vitro after treatment with microencapsulated antisense oligonucleotides. Furthermore, microencapsulated antisense NF-κB oligonucleotides were found to inhibit paw inflammation associated with rat adjuvant-induced arthritis in a dose-dependent manner. Taken together, the results presented in this work described albumin microspheres to be effective delivery vehicles for antisense NF-κB oligonucleotides and a potential treatment for inflammatory diseases.

Reversal of LPS induced endothelial cell TNF synthesis and increased permeability with microencapsulated antisense oligomers to NF-kappaB.

Endothelial cells form the barrier between the circulation and interstitial space. Changes in permeability of endothelial cells allow penetration of inflammatory cells such as polymorphonuclear cells and macrophages to respond to infections and other inflammatory stimuli. Endothelial cells have also been shown to be phagocytic and produce pro-inflammatory cytokines such as TNF. It is the purpose of this study to evaluate endothelial cell phagocytosis of albumin microspheres containing antisense oligonucluetide to NF-kappaB (MASO), the effect of MASO on TNF synthesis after LPS stimulation and the effect of TNF inhibition on the permeability of endothelial cells in vitro. Results were (1) endothelial cells avidly phagocytozed albumin miocrospheres 1.0 and 1.7 microm in size, (2) phagocytosis of microspheres was potentiated by LPS, (3) TNF is synthesized by endothelial cells in cell culture with the peak concentrations occurring 4 h after stimulation with LPS, (4) MASO results in high intracellular concentration of oligomer, (5) MASO inhibits TNF synthesis to a greater extent than equivalent amounts of NF-kappaB antisense in solution and (6) the inhibition of TNF by MASO significantly decreases the permeability of albumin through endothelial cells in vitro.

Pro-inflammatory cytokine inhibition in the primate using microencapsulated antisense oligomers to NF-kappaB.

PRIMARY OBJECTIVE: Antisense oligomers to NF-kappaB (ASO) were incorporated into albumin microspheres to determine if microcapsules containing ASO inhibit pro-inflammatory cytokines to a greater extent than comparable doses of ASO in solution. Phagocytosis of microcapsules and intracellular release of ASO in macrophages was evaluated. RESEARCH DESIGN: Comparable doses of microencapsulated ASO and ASO in solution were evaluated in non-human primates. METHODS: Blood was sampled and stimulated with Escherichia coli endotoxin ex vivo. TNF, IL-1 and IL-6 concentrations were compared for 72 hrs. The intracellular concentration of ASO was measured in macrophages in vitro to evaluate the difference in intracellular penetration of microencapsulated ASO. RESULTS: Microencapsulated ASO produced significantly greater cytokine inhibition at all time points compared to ASO in solution. There were no side effects to ASO in the baboons. Intracellular ASO concentration was 10 fold greater in macrophages using microencapsulation. CONCLUSIONS: Microencapsulated ASO to NF-kappaB is more effective than ASO in solution in pro-inflammatory cytokine inhibition in non-human primates.

Formulation and testing of vancomycin loaded albumin microspheres prepared by spray-drying.

Microparticles are widely employed as carriers of biologically active compounds with many possible applications. For targeted drug delivery and sustained release purposes, biopolymers (i.e. polysaccharides and proteins) have been proposed. In this study, microsphere formulations of vancomycin were prepared by the spray-drying method. Bovine serum albumin (BSA) was used as a polymer matrix and was cross-linked with glutaraldehyde after microsphere preparation. The product yield obtained from the spray-drying method was approximately 75%. The mean particle size was 5 +/- 1.6 microm, with the majority of particles between 4 and 8 microm. The extent of cross-linking affected the release of vancomycin from microspheres. Moreover, both rate and extent of vancomycin release from microspheres decreased with increasing glutaraldehyde concentration. Encapsulation of vancomycin did not alter the bioactivity of the drug and it was more effective in killing Staphylococcus aureus than the solution form.

Treatment of experimental septic shock with microencapsulated antisense oligomers to NF-kappaB.

NF-kappaB is an ideal target for inhibition of proinflammatory cytokines. The purpose of this study was to determine if microencapsulated antisense oligomer to NF-kappaB can inhibit proinflammatory cytokine release in response to Escherichia coli endotoxin and bacteria. Microencapsulation takes advantage of the phagocytic function of the macrophage to deliver the oligomer intracellularly and enhance the effect. Albumin microcapsules 1 microm in size were prepared by a nebulization method containing antisense oligomers to NF-kappaB. E. coli endotoxin was incubated in 1 ml aliquots of whole blood. Microencapsulated antisense to NF-kappaB was given, and the inhibition of tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-6, and IL-8 was compared with similar amounts of oligomer in solution. Endotoxic shock was produced in rats using E. coli endotoxin (15 mg/kg). Peritonitis was induced by injecting 10(10) CFU E. coli. Cytokines were measured after simultaneous and delayed (4 h) administration of antisense to NF-kappaB in microcapsules and solution form. TNF was suppressed by 81% in whole blood, 56% in the endotoxic shock model, 89% in the peritonitis model (simultaneous treatment), and 56% in the delayed treatment group. Survival was 70% in the endotoxic shock group, 80% in the simultaneous peritonitis group, and 70% in the delayed treatment group. Microcapsule treatment using antisense to NF-kappaB suppressed TNF and IL-1 levels and mortality significantly better than all solution treatment groups in the whole blood model, endotoxic shock model, and peritonitis model.