A subunit vaccine based on biodegradable microspheres carrying rHsp65 protein and KLK protects BALB/c mice against tuberculosis infection

Of the hundreds of new tuberculosis ( TB) vaccine candidates some have therapeutic value in addition to their prophylactic properties. This is the case for the DNA vaccine encoding heat-shock protein 65 (DNAhsp65) from Mycobacterium leprae. However, there are concerns about the use of DNA vaccines in certain populations such as newborns and pregnant women. Thus, the optimization of vaccination strategies that circumvent this limitation is a priority. This study evaluated the efficacy of a single dose subunit vaccine based on recombinant Hsp65 protein against infection with M. tuberculosis H37Rv. The Hsp65 protein in this study was either associated or not with immunostimulants, and was encapsulated in biodegradable PLGA microspheres. Our results demonstrate that the protein was entrapped in microspheres of adequate diameter to be engulfed by phagocytes. Mice vaccinated with a single dose of Hsp65-microspheres or Hsp65 + CpG-microspheres developed both humoral and cellular-specific immune responses. However, they did not protect mice against challenge with M. tuberculosis. By contrast, Hsp65+KLK-microspheres induced specific immune responses that reduced bacilli loads and minimized lung parenchyma damage. These data suggest that a subunit vaccine based on recombinant protein Hsp65 is feasible.

Characterization and in vitro activities of cell-free antigens from Histoplasma capsulatum-loaded biodegradable microspheres

In the last decades, the incidence of histoplasmosis, a pulmonary fungal disease caused by Histoplasma capsulatum, has increased worldwide. In this context, vaccines for the prevention of this infection or therapies are necessary. Cell-free antigens (CFAgs) from H. capsulatum when administered for murine immunization purposes are able to confer protection and control of the infection, since they activate cellular immunity. However the most of vaccination procedures need several anti, gens administrations and immunoadjuvants, which are not approved for use in humans. The aim of this study was to develop and characterize a vaccination approach using biodegradable PLGA microspheres (MS) that could allow the controlled and/or sustained release of the encapsulated antigens from H. capsulatum. CFAgs-loaded MS presented a size less than 10 mu m, were marked engulfed by bone marrow-derived macrophages (BMDM phi) and induced the nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production by these cells. Our data show that CFAgs-loaded MS induce cell activation, suggesting an immunostimulant effect to be further investigated during immunization procedures. CFAgs-loaded MS present potential to be used as vaccine in order to confer protection against H. capsulatum infection. (C) 2009 Elsevier B.V. All rights reserved.

Childhood and malaria vaccines combined in biodegradable microspheres produce immunity with synergistic interactions.

Biodegradable microspheres may represent a potential tool for the delivery of combination vaccines. We demonstrate strong immunogenicity of five co-encapsulated antigens after a single subcutaneous inoculation in guinea pigs. Tetanus- and diphtheria-specific antibodies were not significantly affected by the presence of either antigen or by the presence of pertussis or Haemophilus influenzae type b (Hib) antigens. Microsphere formulations gave better protection against diphtheria toxin than did two injections of a licensed tetravalent vaccine. Finally, a synthetic malaria peptide antigen (PfCS) also encapsulated in PLGA microspheres increased diphtheria and tetanus-specific immunity and improved protection against diphtheria. These findings demonstrate the potential of microspheres as an alternative and promising strategy for combination vaccines with a further aptitude in reducing the number of inoculations required to gain functional immunity.

Enhanced in vivo angiogenesis within a model tissue engineered construct using biodegradable microspheres containing encapsulated VEGF

Introduction. Tissue engineering techniques offer a potential means to develop a tissue engineered construct (TEC) for the treatment of tissue and organ deficiencies. However, a lack of adequate vascularization is a limiting factor in the development of most viable engineered tissues. Vascular endothelial growth factor (VEGF) could aid in the development of a viable vascular network within TECs. The long-term goals of this research are to develop clinically relevant, appropriately vascularized TECs for use in humans. This project tested the hypothesis that the delivery of VEGF via controlled release from biodegradable microspheres would increase the vascular density and rate of angiogenesis within a model TEC. ^ Materials and methods. Biodegradable VEGF-encapsulated microspheres were manufactured using a novel method entitled the Solid Encapsulation/Single Emulsion/Solvent Extraction technique. Using a PLGA/PEG polymer blend, microspheres were manufactured and characterized in vitro. A model TEC using fibrin was designed for in vivo tissue engineering experimentation. At the appropriate timepoint, the TECs were explanted, and stained and quantified for CD31 using a novel semi-automated thresholding technique. ^ Results. In vitro results show the microspheres could be manufactured, stored, degrade, and release biologically active VEGF. The in vivo investigations revealed that skeletal muscle was the optimal implantation site as compared to dermis. In addition, the TECs containing fibrin with VEGF demonstrated significantly more angiogenesis than the controls. The TECs containing VEGF microspheres displayed a significant increase in vascular density by day 10. Furthermore, TECs containing VEGF microspheres had a significantly increased relative rate of angiogenesis from implantation day 5 to day 10. ^ Conclusions. A novel technique for producing microspheres loaded with biologically active proteins was developed. A defined concentration of microspheres can deliver a quantifiable level of VEGF with known release kinetics. A novel model TEC for in vivo tissue engineering investigations was developed. VEGF and VEGF microspheres stimulate angiogenesis within the model TEC. This investigation determined that biodegradable rhVEGF 165-encapsulated microspheres increased the vascular density and relative rate of angiogenesis within a model TEC. Future applications could include the incorporation of microvascular fragments into the model TEC and the incorporation of specific tissues, such as fat or bone. ^

CNS delivery of antisense oligodeoxynucleotides using biodegradable microspheres

Antisense oligodeoxynucleotides can selectively inhibit gene expression provided they are delivered to their target site successfully for a sufficient duration. Biodegradable microspheres have previously been developed for the potential systemic delivery of antisense oligodeoxynucleotides and offer an excellent strategy for central administration of antisense oligodeoxynucleotides, providing a sustained-release delivery system. Biodegradable microspheres were formulated to entrap antisense oligodeoxynucleotides for stereotaxic implantation into site-specific regions of the rat brain.Release profiles of antisense oligodeoxynucleotides from biodegradable microspheres over 56 days that were triphasic were observed with high molecular weight polymers. Antisense oligodeoxynucleotides loaded into microspheres (1-10μm) had a five-fold increase in cellular association with glial and neuronal cells compared to the naked molecule, which was partially due to a greater cellular accumulation as observed by a slower efflux profile. In vivo distribution studies of antisense oligodeoxynucleotides demonstrated that the use of microspheres provided a sustained-release over more than 2 days compared to 12 hours of the naked molecule. Efficacy of antisense oligodeoxynucleotides was demonstrated during locomotor activity investigations, which significantly reduced cocaine-induced locomotor activity, where no efficacy was demonstrated with microspheres, possibly attributed to antisense loading and measurements being taken during a lag phase of antisense oligodeoxynucleotide release. Biodegradable microspheres can be delivered site-specifically into the brain and provide sustained-release of antisense oligodeoxynucleotides, offering the potential of in vivo efficacy in these reagents in the brain.

The delivery of bioactive proteins using biodegradable microspheres

In this project, antigen-containing microspheres were produced using a range of biodegradable polymers by single and double emulsion solvent evaporation and spray drying techniques. The proteins used in this study were mainly BSA, tetanus toxoid, F1 and V, Y. pestis subunit vaccines and the cytokine, interferon-gamma. The polymer chosen for use in the vaccine preparation will directly determine the characteristics of the formulation. Full in vitro analysis of the preparations was carried out, including surface hydrophobicity and drug release profiles. The influence of the surfactants employed on microsphere surface hydrophobicity was demonstrated. Preparations produced with polyhydroxybutyrate and poly(DTH carbonate) polymers were also shown to be more hydrophobic than PLA microspheres, which may enhance particle uptake by antigen presenting cells and Peyer's patches. Systematic immunisation with microspheres with a range of properties showed differences in the time course and extent of the immune response generated, which would allow optimisation of the dosing schedule to provide maximal response in a single dose preparation. Both systematic and mucosal responses were induced following oral delivery of microencapsulated tetanus toxoid indicating that the encapsulation of the antigen into a microsphere preparation provides protection in the gut and allows targeting of the mucosal-associated lymphoid tissue. Co-encapsulation of adjuvants for further enhancement of immune response was also carried out and the effect on loading and release pattern assessed. Co-encapsulated F1 and interferon-gamma was administered i.p. and the immune responses compared with singly encapsulated and free subunit antigen.

Prostaglandin E(2)-loaded microspheres as strategy to inhibit phagocytosis and modulate inflammatory mediators release

PGE(2), an arachidonic acid metabolite produced by various type of cells regulates a broad range of physiological activities in the endocrine, cardiovascular, gastrointestinal, and immune systems, and is involved in maintaining the local homeostasis. In the immune system, PGE(2) is mainly produced by APCs and it can suppress the Th1-mediated immune responses. The aim of this study was to develop PGE(2)-loaded biodegradable MS that prolong and sustain the in vivo release of this mediator. An o/w emulsion solvent extraction-evaporation method was chosen to prepare the MS. We determined their diameters, evaluated the in vitro release of PGE(2), using enzyme immunoassay and MS uptake by peritoneal macrophages. To assess the preservation of biological activities of this mediator, we determined the effect of PGE(2) released from MS on LPS-induced TNF-alpha release by murine peritoneal macrophages. We also analyzed the effect of encapsulated PGE(2) on inflammatory mediators release from HUVECs. Finally, we studied the effect of PGE(2) released from biodegradable MS in sepsis animal model. The use of this formulation can provide an alternative strategy for treating infections, by modulating or inhibiting inflammatory responses, especially when they constitute an exacerbated profile. (C) 2008 Elsevier B.V. All rights reserved.

The effect of complexation on characteristics and drug release from PLGA microspheres loaded by cyclosporine-cyclodextrin complex

The purpose of this study was to evaluate the effect of cyclosporine (CyA)-cyclodextrin (CD) complex incorporated within PLGA inicrospheres on microsphere characteristics, with particular emphasis on drug release kinetics. For this purpose, microspheres encapsulated with CyA and those loaded by CyA-CD complex were prepared by solvent evaporation and multiple emulsification solvent evaporation methods, respectively. Morphology, size, encapsulation efficiency and drug release pattern from microspheres were evaluated. Also, physicochemical properties of drug inside microspheres were characterized by differential scanning calorimetry (DSC) and infrared spectroscopy (IR) studies. Scanning electron microscopy (SEM) studies showed that microspheres encapsulated with CyA had islands on the microsphere surface but the islands were not seen on the surface of microspheres loaded by complex. Size range varied from 1 to 25 mu m for CyA encapsulated microspheres and 1 to 50 mu m for complex loaded microspheres. The release of CyA was biphasic with an initial more rapid release phase followed by a slower phase but drug release was twice as fast for complex loaded microspheres. IR studies did not indicate any chemical interaction between the components of microspheres and DSC thermograms revealed that CyA was present either in its amorphous state in microspheres or the presence of CyA as an inclusion complex within microspheres loaded by complex. In conclusion, using CyA as an inclusion complex with CD within microspheres can affect microsphere characteristics and drug release and it is possible to modify microsphere properties like drug release by incorporating CDs as complexing agents.

Novel formulations for antigen delivery using biodegradable polymers: new approaches for the use of new and established adjuvants

The use of immunological adjuvants has been established since 1924 and ever since many candidates have been extensively researched in vaccine development. The controlled release of vaccine is another area of biotechnology research, which is advancing rapidly with great potential and success. Encapsulation of peptide and protein drugs within biodegradable microspheres has been amongst the most successful of approaches within the past decade. The present studies have focused on combining the advantages of microsphere delivery systems composed of biodegradable polylactide (PLLA) and polylactide-co-glycolide (PLGA) polymers with that of safe and effective adjuvants. The research efforts were directed to the development of single-dose delivery vehicles which, can be manufactured easily, safely, under mild and favourable conditions to the encapsulated antigens. In pursuing this objective non ionic block copolymers (NIBCs) (Pluronics@ LI01 and L121) were incorporated within poly-dl-lactide (PDLA) micorospheres prepared with emulsification-diffusion method. LI0I and L121 served both as adjuvants and stabilising agents within these vaccine delivery vehicles. These formulations encapsulating the model antigens lysozyme, ovalbumin (OVA) and diphtheria toxoid (DT) resulted in high entrapment efficiency (99%), yield (96.7%) and elicited high and sustained immune response (IgG titres up to 9427) after one single administration over nine months. The structural integrity of the antigens was preserved within these formulations. In evaluating new approaches for the use of well-established adjuvants such as alum, these particles were incorporated within PLLA and PLGA microspheres at much lesser quantities (5-10 times lower) than those contained within conventional alum-adsorbed vaccines. These studies focused on the incorporation of the clinically relevant tetanus toxoid (TT) antigen within biodegradable microspheres. The encapsulation of both alum particles and TT antigen within these micropheres resulted in preparations with high encapsulation efficiency (95%) and yield (91.2%). The immune response to these particles was also investigated to evaluate the secretion of serum IgG, IgG1, IgG2a and IgG2b after a single administration of these vaccines. The Splenic cells proliferation was also investigated as an indication for the induction of cell mediated immunity. These particles resulted in high and sustained immune response over a period of 14 months. The stability of TT within particles was also investigated under dry storage over a period of several months. NIBC microspheres were also investigated as potential DNA vaccine delivery systems using hepatitis B plasmid. These particles resulted in micro spheres of 3-5 μm diameter and were shown to preserve the integrity of the encapsulated (27.7% entrapment efficiency) hepatitis B plasmid.

A Single Intraoperative Sub-Tenon's Capsule Injection of Triamcinolone and Ciprofloxacin in a Controlled-Release System for Cataract Surgery

PURPOSE. To compare intraoperative injection of triamcinolone and ciprofloxacin in a controlled-release system (DuoCat) with prednisolone and ciprofloxacin eye drops after cataract surgery.METHODS. In this randomized, double-masked, controlled trial, a total of 135 patients undergoing cataract surgery were randomly allocated to two groups: 67 patients treated after surgery with prednisolone 1% and ciprofloxacin 3% eye drops four times daily (week 1), three times daily (week 2), twice daily (week 3), and once daily (week 4) and 0.3% ciprofloxacin drops four times daily (weeks 1 and 2), and 68 patients treated at the end of surgery with a sub-Tenon's injection of 25 mg triamcinolone and 2 mg ciprofloxacin in biodegradable microspheres. The patients were examined on postoperative days 1, 3, 7, 14, and 28. The main outcome measures were postoperative anterior chamber cell and flare, intraocular pressure (IOP), lack of anti-inflammatory response, and presence of infection.RESULTS. No significant differences were observed between the groups in anterior chamber cell (P > 0.14) and flare (P > 0.02) at any postoperative visits. The mean (99% confidence interval) differences in IOP between the prednisolone and triamcinolone groups on days 1, 3, 7, 14, and 28 were -0.4 mm Hg (-2.1 to 1.3), 0.0 mm Hg (-1.4 to 1.3), 0.0 mm Hg (-1.1 to 1.1), -0.2 mm Hg (-1.1 to 0.8), and -0.1 mm Hg (-1.1 to 0.9), respectively. No patient had a postoperative infection.CONCLUSIONS. One injection of DuoCat had a therapeutic response and ocular tolerance that were equivalent to conventional eye drops in controlling inflammation after cataract surgery. (Clinical-Trials. gov number, NCT00431028.) (Invest Ophthalmol Vis Sci. 2009; 50: 3041-3047) DOI: 10.1167/iovs.08-2920

Micropartículas de quitosana estruturadas com aerosil®: estabilidade, adsorção, encapsulação e liberação de substâncias ativas

Biodegradable microspheres used as controlled release systems are important in pharmaceutics. Chitosan biopolymer represents an attractive biomaterial alternative because of its physicochemical and biological characteristics. Chitosan microspheres are expected to become promising carrier systems for drug and vaccine delivery, especially for non-invasive ways oral, mucosal and transdermal routes. Controlling the swelling rate and swelling capacity of the hydrogel and improving the fragile nature of microspheres under acidic conditions are the key challenges that need to be overcomed in order to enable the exploration of the full pharmaceutical potential use of these microparticles. Many studies have focused on the modification of chitosan microsphere structures with cross-linkers, various polymers blends and new organic-inorganic hybrid systems in order to obtain improved properties. In this work, microspheres made of chitosan and nanosized hydrophobic silica (Aerosil R972) were produced by a method consisting of two steps. First, a preparation of a macroscopically homogeneous chitosan-hydrophobic silica dispersion was prepared followed by spray drying. FTIR spectroscopy, X-ray powder diffraction, differential scanning calorimetry, thermal gravimetric analysis, scanning electron microscopy (SEM) and high-resolution transmission electron microscopy (TEM) were used to characterize the microspheres. Also, the were conducted acid stability, moisture sorption capacity, release properties and biological assays. The chitosan-hydrophobic silica composite microspheres showed improved thermal degradation, lower water affinity, better acid stability and ability to retard rifampicin and propranolol hydrochloride (drug models) release under simulated physiological conditions. In vitro biocompatibility studies indicated low cytotoxicity and low capacity to activate cell production of the pro-inflammatory mediator nitric oxide. The results show here encourage further studies on the use of the new chitosan-hydrophobic silica composite microspheres as drug carrier systems via oral or nasal routes.

PHBHV/PCL microspheres as biodegradable drug delivery systems (DDS) for photodynamic therapy (PDT)

Unloaded microspheres were prepared from polyhydroxybutyrate-co-valerate (PHBHV) and poly(epsilon-caprolactone) (PCL) polymers using the emulsification-solvent evaporation method (EE). The study was conducted to determine the ideal polymeric composition and ideal molecular weight for the microspheres preparation to be used as a Drug Delivery System (DDS) for cancer therapy. In this work, NzPC, a new photosensitizer, has been investigated when incorporated into microspheres of PHBHV/PCL evaluating its application for Photodynamic Therapy (PDT) of neoplastic tissue. The biodegradation studies were conducted to analyze the effects of the incorporation of the NzPC and also to determine the release profiles in vitro condition. We also evaluated the dark toxicity and the photobiological effect of the PHBHV-PCL microspheres in cutaneous melanoma cell line (B-16-A1) used as a biological neoplastic medium.

Characterization of Atrazine-Loaded Biodegradable Poly(Hydroxybutyrate-Co-Hydroxyvalerate) Microspheres

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The suitability of biodegradable poly(dl-lactide-co-glycolide)75:25 microspheres for the sustained release of antibiotics

Post-operative infections resulting from total hip arthroplasty are caused by bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa entering the wound perioperatively or by haemetogenous spread from distant loci of infection. They can endanger patient health and require expensive surgical revision procedures. Gentamicin impregnated poly (methyl methacrylate) bone cement is traditionally used for treatment but is often removed due to harbouring bacterial growth, while bacterial resistance to gentamicin is increasing. The aim of this work was to encapsulate the antibiotics vancomycin, ciprofloxacin and rifampicin within sustained release microspheres composed of the biodegradable polymer poly (dl-lactide-co-glycolide) [PLCG] 75:25. Topical administration to the wound in hydroxypropylmethylcellulose gel should achieve high local antibiotic concentrations while the two week in vivo half life of PLCG 75:25 removes the need for expensive surgical retrieval operations. Unloaded and 20% w/w antibiotic loaded PLCG 75:25 microspheres were fabricated using a Water in Oil emulsification with solvent evaporation technique. Microspheres were spherical in shape with a honeycomb-like internal matrix and showed reproducible physical properties. The kinetics of in vitro antibiotic release into newborn calf serum (NCS) and Hank's balanced salt solution (HBSS) at 37°C were measured using a radial diffusion assay. Generally, the day to day concentration of each antibiotic released into NCS over a 30 day period was in excess of that required to kill St. aureus and Ps. auruginosa. Only limited microsphere biodegradation had occurred after 30 days of in vitro incubation in NCS and HBSS at 37°C. The moderate in vitro cytotoxicity of 20% w/w antibiotic loaded microspheres to cultured 3T3-L1 cells was antibiotic induced. In conclusion, generated data indicate the potential for 20% w/w antibiotic loaded microspheres to improve the present treatment regimens for infections occurring after total hip arthroplasty such that future work should focus on gaining industrial collaboration for commercial exploitation.

The sustained delivery of antisense oligodeoxynucleotides using biodegradable polymer microspheres

Antisense technology is a novel drug discovery method, which provides an essential tool for directly using gene sequence information to rationally design specific inhibitions of mRNA, to treat a wide range of diseases. The efficacy of naked oligodeoxynucleotides (ODNs) is relatively short lived due to rapid degradation in vivo. The entrapment of ODNs within biodegradable sustained-release delivery systems may improve ODN stability and reduce dose required for efficacy. Biodegradable polymer microspheres were evaluated as delivery devices for ODNs and ribozymes. Poly(lactide-co-glycolide) polymers were used due to their biocompatibility and non toxic degradation products. Microspheres were prepared using a double emulsion-deposition method and the formulations characterised. In vitro release profiles were characterised by an initial burst effect during the first 48 hours of release followed by a more sustained release. The release profiles were influenced by microsphere size, copolymer molecular weight, copolymer ratio, ODN loading, ODN length, and ODN chemistry. The serum stability of ODNs was significantly improved when entrapped within polymer microspheres. The cellular association of ODNs entrapped within small spheres (1-2μm) was improved by approximately 20-fold in A431 carcinoma cells compared with free ODNs. Fluorescence microscopy studies showed a more diffuse subcellular distribution when delivered as a microsphere formulation compared with free ODNs, which exhibited the characteristic punctate periplasmic distribution. For in vivo evaluation, polymer microspheres containing fluorescently-labelled ODNs were stereo-taxically administered to the neostriatum of the rat brain. Free ODN resulted in a punctate cellular distribution after 24 hours. In comparison ODN delivered using polymer microspheres were intensely visible in cells 48 hours post administration, and fluorescence appeared to be diffuse covering both cytosolic and nuclear regions. Whole-body autoradiography was also used to evaluate the biodistribution of free tritium labelled ODN and ODN entrapped microspheres, following subcutaneous administration to Balb-C mice. Polymer entrapped ODN gave a similar biodistribution to free ODN. Free ODN was distributed within 24 hours, whereas polymer released ODN was observed still presented in organs and at the site of administration seven days post administration.