Gentamicin-loaded microspheres for treatment of experimental Brucella abortus infection in mice

Objectives: To evaluate the efficacy of gentamicin-loaded poly (lactide-co-glycolide) 50:50H (PLGA 50:50H) microspheres for the treatment of mice experimentally infected with Brucella abortus 2308. Methods: The microspheres were dispersed in either 2% (w/v) poloxamer 188 saline solution, or deionized water with the help of a cell homogenizer to break up particle aggregates, and were administered intravenously or intraperitoneally to B. abortus-infected mice 7 days post-infection. Results: Neither a single intravenous or intraperitoneal dose of 67 μg of gentamicin per mouse, nor three intraperitoneal doses of 100 μg of gentamicin per mouse, reduced the Brucella infection in the spleen compared with untreated mice 1 and 3 weeks post-treatment. Histological examination revealed granulation and tissue reaction in the periphery of spleen and liver of animals given three doses of the gentamicin-loaded microspheres. Conclusions: The lack of therapeutic activity of the gentamicin-loaded microspheres might be related to inappropriate microsphere size and aggregation, resulting also in a poor distribution of the microspheres in the spleen. The results might provide an example of practical problems related to particle size and aggregation for in vivo therapy with PLGA microsphere

Gentamicin-loaded microspheres for reducing the intracellular Brucella abortus load in infected monocytes

Objectives: The intracellular antibiotic efficiency of gentamicin-loaded microspheres in the context of Brucella-infected murine monocytes was examined in vitro with a view to developing improved therapies for the treatment of brucellosis. Methods: Biodegradable microspheres made of end-group capped and uncapped poly(lactide-co-glycolide) 50:50 (PLGA 50:50 and PLGA 50:50H) and containing gentamicin sulphate were used to target Brucella abortus-infected J774 monocyte-macrophages. The infected cells were treated with 15 µg of free or microencapsulated gentamicin and the efficacy of the treatments was measured after 24 h. Results: The particle sizes were below 8 µm and in vitro release of gentamicin from the microspheres followed a continuous (PLGA 50:50H) or a multiphasic (PLGA 50:50) pattern over 50 days. Treatment with gentamicin microencapsulated into the end-group uncapped PLGA 50:50H microspheres, decreased significantly the number of intracellular bacteria (typically by 2 log10) in comparison with untreated infected cells. Addition of 2% poloxamer 188 to the microsphere dispersion medium further reduced the infection (3.5 log10). Opsonization of the particles with non-immune mouse serum had no effect on the antibacterial efficacy of the microspheres. End-group capped PLGA 50:50 type microspheres containing the antibiotic were less effective at reducing intracellular bacteria (∼1 log10 reduction), although addition of poloxamer 188 to the dispersion medium again enhanced their intracellular antibacterial activity. Placebo PLGA 50:50 and PLGA 50:50H microspheres had no bactericidal activity. Conclusions: The results indicate that PLGA 50:50-microencapsulated gentamicin sulphate may be suitable for efficient drug targeting and delivery to reduce intracellular Brucella infection

Effect of Oil-in-Water Emulsions on 5-Aminolevulinic Acid Uptake and Metabolism to PpIX in Cultured MCF-7 Cells

No Heading: Purpose.: To identify the optimal vehicle for fast and efficient cellular production of the photosensitizer, protoporphyrin IX (PpIX), upon administration of 5-aminolevulinic acid (ALA). Methods.: ALA in various oil/water o/w emulsions was applied to the human mammary epithelial cell line (MCF-7) cultured in microplates. Upon incubation for 1-4 h, the accumulated amount of PpIX was determined by fluorescence spectroscopy. Variables such as the pH and concentration of the emulsions, the temperature and duration of incubation were examined along with the importance of ALA concentration and the presence of endocytosis inhibitors. Results.: An increase in the amount of produced PpIX was observed with an increase in extracellular pH, incubation temperature, and ALA concentration. A saturable mechanism of PpIX accumulation was evident, mainly as a result of the uptake mechanism for ALA. Some of the o/w emulsions increased the amount of intracellular PpIX, and the results indicated that this was not due to an increased km of the extracellular ALA to intracellular PpIX conversion, but to the increased endocytotic uptake in the presence of the emulsions. In general, the increase in PpIX in the presence of emulsions relative to the control was more pronounced after 1 h as compared to after 2-4 h. Conclusions.: The formation of PpIX in MCF-7 cells exposed to ALA is improved by the presence of certain o/w emulsions, which could be explained by endocytosi

Insulin-like Growth Factor I—Releasing Alginate-Tricalciumphosphate Composites for Bone Regeneration

Purpose: Development and characterization of an in situ-forming, osteoconductive, and growth factor-releasing bone implant. Methods: Injectable in situ-forming scaffolds were prepared from a 2% (m/v) alginate solution, tricalciumphosphate (TCP) granules, and poly(lactide-co-glycolide) microspheres (MS), loaded with the osteoinductive growth factor insulin-like growth factor I (IGF-I). Scaffolds were prepared by mixing the components followed by hydrogel formation through calcium carbonate-induced physical cross-linking of the alginate at slightly acidic pH. Physical-chemical properties and cell biocompatibility using osteoblast-like cells (MG-63 and Saos-2) of these scaffolds were investigated. Results: The addition of TCP to the alginate resulted in reduced swelling and gelation time and an increase in stiffness. Osteoblast-like cells (MG-63 and Saos-2) did not show toxic reactions and adhered circumferentially to the TCP granules surface. The addition of the IGF-I MS resulted in an up to sevenfold increased proliferation rate of MG-63 cells as compared to scaffold preparations without IGF-I MS. The alkaline phosphate (ALP) activity—a parameter for osteblastic activity—increased with increasing amounts of TCP in Saos-2 loaded composite scaffolds. Conclusions: A prototype in situ-hardening composite system for conformal filling of bone defects supporting osteoblastic activity for further clinical testing in relevant fracture models was developed and characterize

A Protective Allergy Vaccine Based on CpG- and Protamine-Containing PLGA Microparticles

Purpose: Allergen-specific immunotherapy (SIT) requires dozens of subcutaneous injections over 3 to 5years in order to control IgE-mediated hypersensitivity, which is a T-helper 2 (Th2)-associated pathology. This study investigates the use of poly(lactide-co-glycolide) (PLGA) microparticles combined with immunostimulatory oligodeoxynucleotide (CpG), as well as protamine in SIT. Materials and Methods: We prepared microparticle formulations with the major allergen of bee venom, phospholipase A2 (PLA2), and analyzed the effect of co-encapsulated or admixed CpG in both naïve and bee venom allergic mice. Results: Mice immunized with microparticles containing only PLA2 induced weak antibody responses. In contrast, the combination with CpG resulted in strong PLA2-specific antibody responses. The presence of CpG was required for the induction of the Th1-associated isotype IgG2a, and the titers of IgG2a in sensitized mice correlated with a better protection against an allergen challenge. The effect of CpG was further strengthened when protamine was co-encapsulated for complexation of CpG. Conclusions: This study shows that allergen-specific immunotherapy with a PLGA-based allergen-delivery system in combination with CpG enhanced the induction of protective IgG2a immune responses. This may improve SIT compliance and shorten its duratio

Gentamicin-loaded microspheres for reducing the intracellular Brucella abortus load in infected monocytes

Objectives: The intracellular antibiotic efficiency of gentamicin-loaded microspheres in the context of Brucella-infected murine monocytes was examined in vitro with a view to developing improved therapies for the treatment of brucellosis. Methods: Biodegradable microspheres made of end-group capped and uncapped poly(lactide-co-glycolide) 50:50 (PLGA 50:50 and PLGA 50:50H) and containing gentamicin sulphate were used to target Brucella abortus-infected J774 monocyte-macrophages. The infected cells were treated with 15 µg of free or microencapsulated gentamicin and the efficacy of the treatments was measured after 24 h. Results: The particle sizes were below 8 µm and in vitro release of gentamicin from the microspheres followed a continuous (PLGA 50:50H) or a multiphasic (PLGA 50:50) pattern over 50 days. Treatment with gentamicin microencapsulated into the end-group uncapped PLGA 50:50H microspheres, decreased significantly the number of intracellular bacteria (typically by 2 log10) in comparison with untreated infected cells. Addition of 2% poloxamer 188 to the microsphere dispersion medium further reduced the infection (3.5 log10). Opsonization of the particles with non-immune mouse serum had no effect on the antibacterial efficacy of the microspheres. End-group capped PLGA 50:50 type microspheres containing the antibiotic were less effective at reducing intracellular bacteria (∼1 log10 reduction), although addition of poloxamer 188 to the dispersion medium again enhanced their intracellular antibacterial activity. Placebo PLGA 50:50 and PLGA 50:50H microspheres had no bactericidal activity. Conclusions: The results indicate that PLGA 50:50-microencapsulated gentamicin sulphate may be suitable for efficient drug targeting and delivery to reduce intracellular Brucella infections

Importance of single or blended polymer types for controlled in vitro release and plasma levels of a somatostatin analogue entrapped in PLA/PLGA microspheres.

The aim of the work was to develop biodegradable microspheres for controlled delivery of the somatostatin analogue vapreotide and maintenance of sustained plasma levels over 2–4 weeks after a single injection in rats. Vapreotide was microencapsulated into end-group capped and uncapped low molecular weight poly(lactide) (PLA) and poly(lactide-co-glycolide) (PLGA) by spray-drying and coacervation. Microspheres were prepared from single and blended (1:1) polymer types. The microparticles were characterized for peptide loading, in vitro release and pharmocokinetics in rats. Spray-drying and coacervation produced microspheres in the size range of 1–15 and 10–70 μm, respectively, and with encapsulation efficiencies varying between 46% and 87%. In vitro release of vapreotide followed a regular pattern and lasted more than 4 weeks, time at which 40–80% of the total dose were released. Microspheres made of 14-kDa end-group uncapped PLGA50:50 or 1:1 blends of this polymer with 35 kDa end-group uncapped PLGA50:50 gave the best release profiles and yielded the most sustained plasma levels above a pre-defined 1 ng/ml over approximately 14 days. In vitro/in vivo correlation analyses showed for several microsphere formulations a linear correlation between the mean residence time in vivo and the mean dissolution time (r=0.958) and also between the amount released between 6 h and 14 days and the AUC6h–14d (r=0.932). For several other parameters or time periods, no in vitro/in vivo correlation was found. This study demonstrates that controlled release of the vapreotide is possible in vivo for a duration of a least 2 weeks when administered i.m. to rats. These results constitute a step forward towards a twice-a-month or once-a-month microsphere-formulation for the treatment of acromegaly and neuroendocrine tumors

On Technological and Immunological Benefits of Multivalent Single-Injection Microsphere Vaccines

Purpose. With the aim of developing multivalent vaccines for single-injection, we examined the feasibility of combining antigens in biodegradable microspheres. Such vaccines are expected to improve vaccination coverage by reducing the number of vaccination sessions required to generate immunity. Methods. Mono- and multivalent vaccines of Haemophilus influenzae type b (Hib) conjugate, diphtheria toxoid (DT), tetanus toxoid (TT), and pertussis toxin (PT) in poly (lactic acid) and poly(lactic-coglycolic acid) microspheres were prepared by spray drying, and the influence of coencapsulated antigens and excipients on antigen loading, release, and stability was examined. Two tetravalent formulations were tested in guinea pigs. Results. Monovalent Hib and PT vaccines showed loading efficiencies of 10% (Hib) and 30% (PT) in both polymers. The loading efficiencies increased upon addition of trehalose and, even more, when the antigens were coencapsulated in di- and trivalent combinations. Highest loading efficiencies (>80%) were achieved with trivalent formulations (DT + PT + Hib) that also contained coencapsulated albumin. The percentage of antigen released during 24 h of incubation was typically 10-40% and decreased as loading efficiency increased. Enzyme-linked immunosorbent assay (ELISA) data revealed that TT, DT, and PT remained antigenic throughout the encapsulation and subsequent release processes. Finally, all antigens maintained their immunogenicity, since strong and sustained antibody responses were elicited after a single injection of tetravalent microsphere vaccines (DT + TT + PT + Hib) in guinea pigs. Conclusions. This study reveals technologic benefit as well as an immunological potential of multivalent single-injection microsphere vaccines. The results support our hypothesis that coencapsulation of several antigens may intrinsically improve entrapment of antigenic and immunogenic antigen probably by virtue of increased protein concentration during microencapsulation leading to mutual stabilization of the component

Traffic Prediction Based on a Local Exchange of Information

We propose a decentralized method for traffic monitoring, fully distributed over the vehicles. An algorithm is provided, specifying which information should be tracked to reconstruct an instantaneous map of traffic flow. We test the accuracy of our method in a simple cellular automata traffic simulation model, for which the traffic condition can be controlled and analyzed theoretically. We show how local communication parameters affect the method accuracy

In vitro and in vivo evaluation of a somatostatin analogue released from PLGA microspheres

The purpose of this study was to design poly(lactide-co-glycolide) (PLGA) microspheres for the continuous delivery of the somatostatin analogue, vapreotide, over 2–4 weeks. The microspheres were produced by spray-drying and the desired characteristics, i.e. high encapsulation efficiency and controlled release over 2–4 weeks, achieved through optimizing the type of polymer, processing solvent, and co-encapsulated additive. The in vitro release was tested in fetal bovine serum preserved with 0.02% of thiomersal. Furthermore, formulations were injected intramuscularly into rats to obtain pharmacokinetic profiles. Encapsulation efficiency was between 34 and 91%, depending on the particular formulation. The initial peptide release (within 6 h) was lowest, i.e. 1 ng/ml) over 21–28 days in rats was the one made with end-group uncapped PLGA 50:50, the solvent acetic acid and the additive polyethyleneglycol. In conclusion, the optimization of formulation parameters allowed us to produce vapreotide-loaded PLGA microspheres of suitable characteristics for therapeutic use