Drug delivery into the brain using poly(lactide-co-glycolide) microspheres

Among the strategies developed for drug delivery into the CNS, locally controlled drug release by the way of an implantable polymeric device has been developed in recent years. The first polymeric devices developed were macroscopic implants needing open surgery for implantation. Over the last few years, poly(lactide-co-glycolide) microspheres have been shown to be safe and promising for drug delivery into the brain. Poly(lactide-co-glycolide) is biodegradable and biocompatible with brain tissue. Due to their size, these microspheres can be easily implanted by stereotaxy in discrete, precise and functional areas of the brain without causing damage to the surrounding -tissue. Brain tumour treatments have been developed using this approach and clinical trials have been performed. Potential applications in neurodegenerative diseases have also been explored, particularly neurotrophic factor delivery and cell therapy.     

Drug delivery into the brain using poly(lactide-co-glycolide) microspheres

Among the strategies developed for drug delivery into the CNS, locally controlled drug release by the way of an implantable polymeric device has been developed in recent years. The first polymeric devices developed were macroscopic implants needing open surgery for implantation. Over the last few years, poly(lactide-co-glycolide) microspheres have been shown to be safe and promising for drug delivery into the brain. Poly(lactide-co-glycolide) is biodegradable and biocompatible with brain tissue. Due to their size, these microspheres can be easily implanted by stereotaxy in discrete, precise and functional areas of the brain without causing damage to the surrounding -tissue. Brain tumour treatments have been developed using this approach and clinical trials have been performed. Potential applications in neurodegenerative diseases have also been explored, particularly neurotrophic factor delivery and cell therapy.     

Chitosan-modified poly(D,L-lactide-co-glycolide) nanospheres for plasmid DNA delivery and HBV gene-silencing

Gene silencing using small interfering RNA (siRNA) has several potential therapeutic applications. In the present study, we investigated nanoparticles (NS) formulated using the biodegradable polymer, poly(D,L-lactide-co-glycolide) (PLGA) for plasmid DNA (pDNA) delivery. A cationic polymer, Chitosan (CHS), was incorporated in the PLGA matrix to improve pDNA loading efficiency and cellular uptake ability. PLGA-CHS NS were prepared by a spontaneous emulsion diffusion (SED) method, and various formulation factors were investigated. Spherical nanoparticles with particle size of around 60 nm were obtained under optimum formulation condition. The effectiveness of pDNA-loaded PLGA-CHS nanoparticles in expressing the indicative enhanced Green Fluorescent Protein (eGFP) and in slicing Hepatitis B virus (HBV) gene were examined in HepG2.2.15 cells. CHS-modified PLGA NS exhibited much higher loading efficiency than unmodified PLGA NS. CHS-PLGA NS showed a positive zeta potential, while plain-PLGA NS were negatively charged. EGFP expression studies by observation with confocal leaser scanning microscopy (CLSM) indicated that pDNA-loaded CHS-PLGA NS were more effectively taken up by the cells than plain-PLGA NS. The corresponding results showed that the HBV gene-silencing efficiency of CHS-PLGA NS was higher than those of plain-PLGA NS and naked pDNA. Thus, CHS-PLGA NS containing pDNA could provide an effective pDNA delivery system in vitro, showing that such an approach could be useful in the treatment of viral diseases in vivo.     

Versatility of biodegradable poly(D,L-lactic-co-glycolic acid) microspheres for plasmid DNA delivery.

In this study, we have optimized different formulations of DNA encapsulated into PLGA microspheres by correlating the protocol of preparation and the molecular weight and composition of the polymer, with the main characteristics of these systems in order to design an efficient non-viral gene delivery vector. For that, we prepared poly(D,L-lactic-co-glycolic acid) (PLGA) microparticles with an optimized water-oil-water double emulsion process, by using several types of polymers (RG502, RG503, RG504, RG502H and RG752), and characterized in terms of size, zeta potential, encapsulation efficiency (EE%), morphology, DNA conformation, release kinetics, plasmid integrity and erosion. The size of the particles ranged between 0.7 and 5.7 microm depending on the protocol of formulation and the molecular mass of the polymer used. The microspheres prepared by using in their formulation polymers of high molecular weight (RG503 and RG504) were bigger in size than in the case of using a lower molecular weight polymer (RG502). The EE (%) of plasmid DNA increased with increasing the molecular mass of the polymer and by using the most hydrophilic polymer RG502H, which contains terminal acidic groups in its structure. The plasmid could be encapsulated without compromising its structural and functional integrity. Also a protective effect of PLGA on endonuclease digestion is observed. Plasmid DNA release from microspheres composed of low molecular weight or hydrophilic polymers, like RG502H, was faster than from particles containing high molecular weight or hydrophobic polymers. These PLGA microspheres could be an alternative to the viral vectors used in gene therapy, given that may be used to deliver genes and other bioactive molecules, either very rapidly or in a controlled manner.     

Pegylated poly(lactide) and poly(lactide-co-glycolide) nanoparticles: preparation, properties and possible applications in drug delivery

The preparation, properties and potential applications in drug delivery of biocompatible and biodegradable PLA-PEG and PLGA-PEG nanoparticles are discussed. PLA-PEG and PLGA-PEG nanoparticles have been produced by emulsification-solvent evaporation, solvent displacement and salting out methods. The nanoparticles can be stored as freeze-dried powders, but an adequate amount of a suitable lyoprotectant should be added prior lyophilisation to prevent nanoparticle aggregation and retain nanoparticle redispersibility. The nanoparticles have a core-shell structure with a PLA core and a PEG coating. Their basic colloidal properties and degradation depend on copolymer composition. The PLA-PEG and PLGA-PEG nanoparticles exhibit prolonged blood circulation following intravenous administration to animals. The composition of the nanoparticles determine their biodistribution properties, probably through its effects on the effectiveness of the PEG steric barrier and the size of the nanoparticles. The ability of the PLA-PEG and PLGA-PEG nanoparticles to evade rapid phagocytocis has extended the range of sites within the body that the nanoparticles can reach, which has significant implications with regard to their application in controlled drug delivery and targeting. The PLA-PEG and PLGA-PEG nanoparticles can be loaded with a variety of bioactive agents achieving satisfactory loading, especially in the case of hydrophobic drugs. The nanoparticles have been investigated for the treatment of infectious diseases and cancer, the intravenous and mucosal delivery of proteins, and oligonucleotide and gene delivery. The results have been encouraging and PLA-PEG and PLGA-PEG nanoparticle formulations, improving the therapeutic potential of both established and new drugs, may be expected to be available in the near future.     

Novel approach for delivery of insulin loaded poly(lactide-co-glycolide) nanoparticles using a combination of stabilizers

Insulin stability during microencapsulation and subsequent release is essential for retaining its biological activity. The successful delivery of insulin relies on the proper selection of stabilizers in addition to other parameters. Attempts were made to address the problem with a few combination of stabilizers for maintaining the integrity of insulin during formulation and delivery. Insulin loaded nanoparticles with different stabilizers such as pluronic F68, trehalose, and sodium bicarbonate were prepared by the double emulsion evaporation method using two different copolymer ratios of poly(DL-lactide-co-glycolide) (50:50 and 85:15). The presence of stabilizers in the nanoparticles resulted in an increase in the particle size but a reduction of encapsulation efficiency. Insulin release rate was comparatively higher for the batches containing stabilizers when compared with controls for both the copolymer ratios. Also the presence of stabilizers resulted in sustained release of insulin resulting in prolonged reduction of blood glucose levels in streptozotocin induced diabetic rats. From the in vitro and in vivo studies, we concluded that a combination of stabilizers results in beneficial effects without compromising the advantages of delivery systems.     

Preparation, characterization, cytotoxicity and transfection efficiency of poly(DL-lactide-co-glycolide) and poly(DL-lactic acid) cationic nanoparticles for controlled delivery of plasmid DNA

The objective of this study was to investigate the effect of formulation parameters (i.e. polymer molecular weight and homogenization speed) on various physicochemical and biological properties of cationic nanoparticles. Cationic nanoparticles were prepared using different molecular weights of poly(DL-lactide-co-glycolide) (PLGA) and poly(DL-lactic acid) (PLA) by double emulsion solvent evaporation at two different homogenization speeds, and were characterized in terms of size, surface charge, morphology, loading efficiency, plasmid release, plasmid integrity, cytotoxicity, and transfection efficiency. Cationic surfactant, cetyltrimethylammonium bromide (CTAB), was used to provide positive charge on the surface of nanoparticles. Reporter plasmid gWIZ Beta-gal was loaded on the surface of nanoparticles by incubation. Use of higher homogenization speed and lower molecular weight polymer led to a decrease in mean particle size, increase in zeta potential, increase in plasmid loading efficiency, and a decrease in burst release. The nanoparticles displayed good morphology as evident from scanning electron micrographs. In vitro cytotoxicity study by MTT assay showed a low toxicity. Structural integrity of the pDNA released from nanoparticles was maintained. Transfecting human embryonic kidney (HEK293) cells with nanoparticles prepared from low molecular weight PLGA and PLA resulted in an increased expression of beta-galactosidase as compared to those prepared from high molecular weight polymer. Our results demonstrate that the PLGA and PLA cationic nanoparticles can be used to achieve prolonged release of pDNA, and the plasmid release rate and transfection efficiency are dependent on the formulation variables.     

Vancomycin release from poly(D,L-lactide) and poly(lactide-co-glycolide) disks

A biodegradable and biocompatible polymeric system was developed for the controlled release of vancomycin for the treatment of brain abscesses. Poly(D,L-lactic acid) (PLA) and its copolymers poly(lactide-co-glycolide) PLGA 90:10 and PLGA 70:30, were prepared. Polymer disks containing vancomycin (VN) were prepared by solvent casting from methylene chloride solutions. Degradation of the polymer disk was studied by scanning electron microscopy, NMR and GPC. SEM revealed an increasing degree of degradation with time with both PLGAs, the effect being more distinct in the PLGA with the higher glycolide content (PLGA 70:30), which was confirmed with GPC, which showed both a decrease in the molecular weights of PLGA and a decrease in the heterogeneity index (chain length distribution) upon incubation in isotonic phosphate buffer at 37#176;C for up to 5 weeks. NMR showed a decrease in the CH 2 contents of the copolymers, implying that the glycolide component of the copolymers is being preferentially degraded. In situ, vancomycin release behaviour of the disks in pH 7.4 phosphate buffer saline (PBS) was followed for ~2 months in a static system. It was observed that release was according to Higuchi kinetics (Q vs. t 1/2) , and introduction of low molecular weight PLA or hydrophilic compounds like PEG increased the release rate.     

Cationic lipid vectors for plasmid DNA delivery

Successful gene therapy depends on efficient gene transfer vectors. Viral vectors and non-viral vectors have been investigated extensively. Cationic lipids are non-viral vectors, which resemble traditional pharmaceuticals, display little immunogenicity, and have no potential for viral infection. However, toxicity and low transfection efficiency are two barriers limiting the clinical applications of cationic lipids. Over the last decade, hundreds of cationic lipids have been synthesized to address these problems. In this brief review, we summarized recent research results concerning the structures of DNA/liposomes complexes, some important strategies used to design different classes of cationic lipids, and use of disulfide cationic lipids in plasmid DNA delivery.     

Vancomycin release from poly(D,L-lactide) and poly(lactide-co-glycolide) disks.

A biodegradable and biocompatible polymeric system was developed for the controlled release of vancomycin for the treatment of brain abscesses. Poly(D,L-lactic acid) (PLA) and its copolymers poly(lactide-co-glycolide) PLGA 90:10 and PLGA 70:30, were prepared. Polymer disks containing vancomycin (VN) were prepared by solvent casting from methylene chloride solutions. Degradation of the polymer disk was studied by scanning electron microscopy, NMR and GPC. SEM revealed an increasing degree of degradation with time with both PLGAs, the effect being more distinct in the PLGA with the higher glycolide content (PLGA 70:30), which was confirmed with GPC, which showed both a decrease in the molecular weights of PLGA and a decrease in the heterogeneity index (chain length distribution) upon incubation in isotonic phosphate buffer at 37 degrees C for up to 5 weeks. NMR showed a decrease in the CH2 contents of the copolymers, implying that the glycolide component of the copolymers is being preferentially degraded. In situ, vancomycin release behaviour of the disks in pH 7.4 phosphate buffer saline (PBS) was followed for approximately 2 months in a static system. It was observed that release was according to Higuchi kinetics (Q vs. t(1/2)), and introduction of low molecular weight PLA or hydrophilic compounds like PEG increased the release rate.     

Determination of water-soluble acid distribution in poly(lactide-co-glycolide)

Determination of the kinetics of water-soluble degradation products inside poly(lactide-co-glycolide) (PLGA) delivery systems during polymer degradation is important to evaluate the polymer microclimate conditions, particularly microclimate pH changes for optimization of encapsulated drug stability. A pre-derivatization high-performance liquid chromatography (HPLC) method was developed for separation and quantification of water-soluble acid impurities and degradation products in PLGA. Thin PLGA films (approximately 200 microm) were incubated in PBS/0.02% Tween 80, pH 7.4, for 6 weeks. Water-soluble monomers and oligomers were obtained from polymer films after repeated CHCl(3)/H(2)O extraction and then derivatized into bromophenacyl esters. With the common chromophore, the esters were separated and quantified by HPLC with increased ultraviolet (UV) sensitivity at 254 nm. The total amount of water-soluble acids in the extract was validated by potentiometric titration with tetrabutyl ammonium hydroxide. During the first 3 weeks of incubation of PLGA 50:50 (inherent viscosity = 0.63 dL/g), the principal water-soluble acids in the polymer were glycolic, lactic, and lactoyllactic acids, and an unknown oligomer. After 4 weeks of incubation, a large fraction of higher molecular weight oligomers was observed. Pre-derivatization HPLC can be used to accurately measure water-soluble acid distribution, and may be invaluable to examine the degradation behavior of PLGAs, including the underlying mechanism of polymer microclimate pH development.     

Sustained release of ganciclovir from poly(lactide-co-glycolide) microspheres

Biodegradable poly(lactide-co-glycolide) microspheres loaded with ganciclovir were produced using the emulsification/solvent evaporation technique. The effects of drug-to-polymer ratio and dispersion time on the drug content in the microspheres were investigated. The release rate of the drug was studied for 20 weeks in a phosphate buffered solution of pH 7 at 37°C. Data revealed that lower drug content was obtained with increasing drug-to-polymer ratio and decreasing dispersion time. The release of the drug followed a triphasic release pattern, i.e. an initial burst, a diffusive phase and a second burst. The initial burst occurred within the first 2 days of immersion. After the burst, the release was by diffusion for up to 13 weeks, followed by another burst release, which signals the onset of bulk degradation of the polymer. Gel permeation chromatography (GPC), differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and ultraviolet spectroscopy (UV) were used to follow the hydrolytic degradation and drug release rate of the microspheres.     

Analysis of residual solvents in poly(lactide-co-glycolide) nanoparticles

Biodegradable nanoparticles formulated from poly(lactide-co-glycolide) (PLGA) have been extensively investigated for drug delivery. The emulsification-solvent evaporation method for manufacturing PLGA nanoparticles is simple and reproducible, but the organic solvent, dichloromethane, has undesirable environmental and safety risks. Dichloromethane is also a suspected carcinogen and mutagen, so has to be completely removed from the nanoparticles. The International Committee for Harmonization suggests a limit of residual dichloromethane of no more than 600?ppm. Here, we evaluate the residual quantity of volatile dichloromethane after the manufacturing process of nanoparticles. We developed a simple, fast, and reliable method by gas chromatography with a flame ionization detector to determine dichloromethane levels. This method was validated according to regulatory requirements and produced acceptable results with respect to specificity, linearity, sensitivity, precision, and accuracy. We also tested the effects of various parameters in the preparation of the nanoparticles such as surfactant concentration, organic phase volume, and washing frequency. This study provides a useful method for manufacturing PLGA nanoparticles with minimal residual solvent by decreasing surfactant concentration or increasing washing frequency.     

Sustained release of ganciclovir from poly(lactide-co-glycolide) microspheres

Biodegradable poly(lactide-co-glycolide) microspheres loaded with ganciclovir were produced using the emulsification/solvent evaporation technique. The effects of drug-to-polymer ratio and dispersion time on the drug content in the microspheres were investigated. The release rate of the drug was studied for 20 weeks in a phosphate buffered solution of pH 7 at 37 degrees C. Data revealed that lower drug content was obtained with increasing drug-to-polymer ratio and decreasing dispersion time. The release of the drug followed a triphasic release pattern, i.e. an initial burst, a diffusive phase and a second burst. The initial burst occurred within the first 2 days of immersion. After the burst, the release was by diffusion for up to 13 weeks, followed by another burst release, which signals the onset of bulk degradation of the polymer. Gel permeation chromatography (GPC), differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and ultraviolet spectroscopy (UV) were used to follow the hydrolytic degradation and drug release rate of the microspheres.     

Topical delivery of plasmid DNA using biphasic lipid vesicles (Biphasix)

The development of non-invasive methods for the delivery of vaccines through the skin will greatly improve the safety and the administration of human and veterinary vaccines. In this study we examined the efficiency of topical delivery of plasmids by assessing the localization of gene expression using luciferase as a reporter gene and induction of immune responses using a plasmid encoding for the bovine herpesvirus type-1 glycoprotein D (pgD). Topical administration of plasmids in a lipid-based delivery system (biphasic lipid vesicles--Biphasix) resulted in gene expression in the lymph node, whereas with intradermal injection, antigen expression was found in the skin. Following administration of plasmid with the gene gun, antigen expression was observed in both the skin as well as in the draining lymph nodes. Transcutaneous immunization with pgD formulated in biphasic lipid vesicles elicited gD-specific antibody responses and a Th2-type cellular response. In contrast, immunization by the intradermal route resulted in the stimulation of a Th1-type response. These findings have implications for both vaccine design and tailoring of specific immune responses.     

Oral plasmid DNA delivery systems for genetic immunisation

The use and optimisation of plasmid DNA delivery systems for the purposes of eliciting transgene specific immune responses to orally administered DNA encoded antigen represents a significant challenge. Here, we have outlined a multicomponent polymer modified liposomal delivery system that offers potential for oral administration of plasmid DNA. It is shown that the polymer/liposome formulated DNA is able to elicit markedly enhanced transgene specific cytokine production following in vitro restimulation of splenocytes with recombinant antigen. This is discussed with reference to recent publications and the potential of plasmid DNA delivery systems for the purposes of genetic immunisation, as reported in selected literature, is assessed.     

Oral tolerance induced by poly (lactide-co-glycolide) containing B lactoglobulin

Allergies to milk proteins are frequently encountered in the new born population. In order to prevent this allergy by inducing oral tolerance, one of the major allergenic milk protein, B lactoglobulin (BLG) was entrapped into biodegradable Poly (lactide-co-glycolide) microspheres and was then orally given to mice. Microspheres are able to protect proteins against degradation by intestinal proteolytic enzymes and to target the Peyers patches which are one important priming site of the mucosal immune system. Microspheres were prepared by the multiple emulsion solvent evaporation method. The goal of the formulation study was to associate large amounts of proteins to the smallest amount of polymer so that a minimal quantity of microspheres would be administered. It was shown that introducing tween 20 in the formulation was able to increase the encapsulation efficiency and to better control protein release reducing the burst release effect. Moreover, Oral administration of microspheres containing BLG reduced significantly (by 10.000) the amount of protein necessary to decrease both specific anti BLG IgE and DTH response. In conclusion, microspheres appear to be optimal systems to induce oral tolerance.     

A one-step process in preparation of cationic nanoparticles with poly(lactide-co-glycolide)-containing polyethylenimine gives efficient gene delivery

A one-step preparation of nanoparticles with poly(lactide-co-glycolide) (PLGA) pre-modified with polyethylenimine (PEI) is better in requirements for DNA delivery compared to those prepared in a two-step process (preformed PLGA nanoparticles and subsequently coated with PEI). The particles were prepared by emulsification of PLGA/ethyl acetate in an aqueous solution of PVA and PEI. DLS, AFM and SEM were used for the size characteristics. The cytotoxicity of PLGA/PEI nanoparticles was detected by MTT assay. The transfection activity of the particles was measured using pEGFP and pβ-gal plasmid DNA. Results showed that the PLGA/PEI nanoparticles were spherical and non-porous with a size of about 0.2 μm and a small size distribution. These particles had a positive zeta potential demonstrating that PEI was attached. Interestingly, the zeta potential of the particles (from one-step procedure) was substantially higher than that of two-step process and is ascribed to the conjugation of PEI to PLGA via aminolysis. The PLGA/PEI nanoparticles were able to bind DNA and the formed complexes had a substantially lower cytotoxicity and a higher transfection activity than PEI polyplexes. In conclusion, given their small size, stability, low cytotoxicity and good transfection activity, PLGA/PEI-DNA complexes are attractive gene delivery systems.     

The initial release of cisplatin from poly(lactide-co-glycolide) microspheres

PLGA microspheres loaded with cisplatin were produced using a single emulsion method. A semi-empirical model, with bi-exponential terms, was found to give a better fit to the drug release profiles compared to a mono-exponential model. This model suggests that there are two separate fractions of drug present in the depot. A fraction of the drug is located near/at the surface of the depot, and is readily released during immersion in buffer. A second fraction of drug is entrapped deeper within the depot and is subsequently released. It was also found that the initial release of cisplatin from PLGA microsphere is highly diffusion-controlled and the classical Higuchi model provides a good fit. From studies of water diffusion using PFG-NMR, results suggested that 50:50 PLGA microsphere was most susceptible to swelling and this might have promoted the faster initial drug release. Results from NMR cryoporometry also indicated that the developed PLGA microspheres could have “ink-bottle” pores.     

Development of bioadhesive amino-pegylated poly(anhydride) nanoparticles designed for oral DNA delivery

Amino-pegylated poly(methyl vinyl ether-co-maleic anhydride) nanoparticles were prepared applying a solvent displacement method. The surface charge of the resulting pegylated particles was considerably higher (?2.7 mV) than that of the non-pegylated (?33.5 mV). After oral administration to rats the amino-pegylated nanoparticles exhibited great ability for bioadhesive interactions with the gastrointestinal mucosa. Furthermore, fluorescence microscopy revealed that the amino-pegylated were able to cross the cellular membrane of the absorptive enterocytes. Genomic salmon testes DNA was associated to the amino-pegylated poly(anhydride) particles by applying two procedures: (i) incubation of aqueous DNA solution with the freshly formed amino-pegylated particles; and (ii) initial incubation of DNA and DAP-PEG simultaneously, followed by blending with preformed non-pegylated particles. Gel electrophoresis showed that both methods were safe and DNA integrity was not affected. Based on the results describing their adhesive properties and intracellular transport, the amino-pegylated nanoparticles were considered as a suitable carrier for DNA.