Tetanus Toxoid Loaded Nanoparticles from Sulfobutylated Poly(Vinyl Alcohol)-Graft-Poly(Lactide-co-Glycolide): Evaluation of Antibody Response After Oral and Nasal Application in Mice

Purpose. Aim of the study was the evaluation of the potential of novel tetanus toxoid (TT) loaded nanoparticles (NP) for electing an immune response in mice against TT. Methods. Six week-old female Balb/c mice were immunized by oral (p.o.), nasal (i.n.) and intraperitoneal (i.p.) application of TT NP loaded by adsorption. As polymer a novel polyester, sulfobutylated poly(vinyl alcohol)-graft-poly(lactide-co-glycolide), SB(43)-PVAL-g-PLGA was used. Blood samples were collected 4 and 6 weeks after immunization and assayed for serum IgG- as well as IgA antibody titers by ELISA. NP formulations varying in size and loading were compared to alum adsorbates as well as to TT solutions. Results. Both, p.o. and i.n. administration of TT associated NP increased serum titers up to 3 × 10³ (IgG) and 2 × 10³ (IgA). While small NP induced significantly higher titers then larger ones after oral administration, intermediate NP induced antibodies after nasal application. Of the mucosal routes investigated, i.n. seems to be more promising compared to p.o. immunization. Conclusions. Antigen loaded NP prepared from surface modified polyesters combined with CT show considerable potential as a vaccine delivery system for mucosal immunization. The results warrant further experiments to explore in more detail the potential use of NP as mucosal vaccine delivery system.     

Poly(D,L-lactide-co-glycolide) encapsulated poly(vinyl alcohol) hydrogel as a drug delivery system

Purpose. The efficiency of encapsulation of water-soluble drugs in biodegradable polymer is often low and occasionally these microcapsules are associated with high burst effect. The primary objective of this study is to develop a novel microencapsulation technique with high efficiency of encapsulation and low burst effect. Method. Pentamidine was used as a model drug in this study. Pentamidine/polyvinyl alcohol (PVA) hydrogel was prepared by freeze-thaw technique. Pentamidine loaded hydrogel was later microencapsulated in poly(lactide-co-glycolide) (PLGA) using solvent evaporation technique. The microcapsules were evaluated for the efficiency of encapsulation, particle size, surface morphology, thermal characteristic, and drug release. Results. Scanning Electron Microscope (SEM) studies revealed that the microcapsules were porous. The microcapsules were uniform in size and shape with the median size of the microcapsules ranging between 27 and 94 m. The samples containing 10% PLGA showed nearly three times increase in drug loading (18-53%) by increasing the hydrogel content from 0-6%. The overall drug release from the microencapsulated hydrogel, containing 3% and 6% PVA, respectively, was significantly lower than the control batches. Conclusions. The use of a crosslinked hydrogel such as PVA can significantly increase the drug loading of highly water-soluble drugs. In addition, incorporation of the PVA hydrogel significantly reduced the burst effect and overall dissolution of pentamidine.     

A Heterogeneously Structured Composite Based on Poly(lactic-co-glycolic acid) Microspheres and Poly(vinyl alcohol) Hydrogel Nanoparticles for Long-Term Protein Drug Delivery

Purpose. To prepare a heterogeneously structured composite based on poly (lactic-co-glycolic acid) (PLGA) microspheres and poly(vinyl alcohol) (PVA) hydrogel nanoparticles for long-term protein drug delivery. Methods. A heterogeneously structured composite in the form of PLGA microspheres containing PVA nanoparticles was prepared and named as PLGA-PVA composite microspheres. A model protein drug, bovine serum albumin (BSA), was encapsulated in the PVA nanoparticles first. The BSA-containing PVA nanoparticles was then loaded in the PLGA microspheres by using a phase separation method. The protein-containing PLGA-PVA composite microspheres were characterized with regard to morphology, size and size distribution, BSA loading efficiency, in vitroBSA release, and BSA stability. Results. The protein-containing PLGA-PVA composite microspheres possessed spherical shape and nonporous surface. The PLGA-PVA composite microspheres had normal or Gaussian size distribution. The particle size ranged from 71.5 m to 282.7 m. The average diameter of the composite microspheres was 180 m. The PLGA-PVA composite microspheres could release the protein (BSA) for two months. The protein stability study showed that BSA was protected during the composite microsphere preparation and stabilized inside the PLGA-PVA composite microspheres. Conclusions. The protein-containing PLGA-PVA composite may be suitable for long-term protein drug delivery.     

Combined Poly(isobutylcyanoacrylate) and Cyclodextrins Nanoparticles for Enhancing the Encapsulation of Lipophilic Drugs

Purpose. The aim of this study was to prepare and characterize nanoparticulate systems constituted of poly(isobutylcyanoacrylate) and cyclodextrins and intended for increasing the loading of the particles with lipophilic substances. Progesterone was used as a model substance. Methods. Nanoparticles were prepared by polymerization of isobutyl-cyanoacrylate in presence of cyclodextrins or progesterone/ hydroxy-propyl--cyclodextrin complex. Particle size, zeta potential, cyclodextrin and progesterone loading of the particles were determined. Results. Nanoparticles could be easily prepared in presence of cyclodextrins. An increase in hydroxypropyl--cyclodextrin concentration resulted in small nanoparticles (less than 50 nm). It was found that large amounts of cyclodextrins remained associated to the particles, resulting in a 50 fold increase in progesterone loading compared to nanoparticles prepared in absence of cyclodextrins. Conclusions. The poly(isobutylcyanoacrylate)—cyclodextrin nanoparticles were characterized by the presence of many lipophilic sites belonging to the cyclodextrins which were firmly anchored to the structure of the particles. Therefore, this new type of nanoparticles offers probably an opportunity for increasing the loading of nanoparticles with various lipophilic drugs.     

Charged nanoparticles as protein delivery systems: A feasibility study using lysozyme as model protein

The aim of this study was to investigate the feasibility of negatively charged nano-carriers (nanoparticles), consisting of polymer blends of poly(lactide-co-glycolide) (PLGA) and poly(styrene-co-4-styrene-sulfonate) (PSS), to improve the loading capacity and release properties of a positively charged model protein, lysozyme, through an adsorption process. Nanoparticles were prepared by a solvent displacement method and characterized in terms of size, ζ-potential, morphology, as well as loading capacity of model protein lysozyme. Morphology of these particles was investigated using transmission electron microscopy (TEM), scanning electron microscopy (SEM) and atomic force microscopy (AFM). The loading capacity of lysozyme was evaluated as a function of polymer blend ratio, protein concentration, pH, and ionic strength; in vitro release profiles were also studied. The results show that negatively charged nanoparticles were obtained using polymer blends of PLGA and PSS, characterized by increased net negative surface charge with increasing ratios of PSS. Moreover, protein loading capacity increased as function of PSS/PLGA ratio. Increased pH facilitated the adsorption process and improved the loading capacity. Maximum loading efficiency was achieved at salt concentrations of 50 mM. In vitro release of lysozyme from the polymer blend nanoparticles was dependent on drug loading and full bioactivity of lysozyme was preserved throughout the process. These findings suggest that this is a feasible method to prepare nanoparticles with high surface charge density to efficiently adsorb oppositely charged protein through electrostatic interactions.     

Polyester-Poly(Ethylene Glycol) Nanoparticles Loaded with the Pure Antiestrogen RU 58668: Physicochemical and Opsonization Properties

Purpose. The pure antiestrogen RU58668 (RU) was encapsulated within nanospheres (NS) and nanocapsules (NC) prepared from different polyester copolymers with poly(ethylene glycol) (PEG) chains. The influence of their physicochemical properties on drug release in vitro and their susceptibility to opsonization were evaluated. Methods. RU-loaded PEG-bearing nanoparticles (NP) prepared by interfacial deposition of preformed polymer were characterized (size, zeta potential, percentage encapsulation and loading). In vitro release kinetics were studied in the presence of 10% fetal calf serum (FCS). Their opsonization in mouse serum was evaluated by silver staining of SDS-PAGE and Western blotting of desorbed proteins. Results. The NS were smaller than NC and had a zeta potential close to zero and a higher percentage of loading. RU release from NS in vitro was reduced as compared with the dissolution profile of free RU in a serum-containing medium. Decreased opsonin adsorption at the surface of pegylated NS was observed. Conclusion. Small nanoparticulate systems containing a high load of pure antiestrogen, showing reduced drug release, have been developed. Among the six nanosphere preparations containing RU, two show a size below 200 nm, and two others undergo reduced protein adsorption in the presence of serum, compatible with increased persistence in the blood.     

Toward the understanding of the photodynamic activity of m-THPP encapsulated in PLGA nanoparticles: correlation between nanoparticle properties and in vivo activity

Biodegradable polymeric nanoparticles (NP) are promising delivery systems for photosensitizers (PS). A hydrophobic PS, the meso-tetra(p-hydroxyphenyl)porphyrin (m-THPP) was encapsulated into NP made of poly(d,l-lactide-co-glycolide) using an emulsification-diffusion method, and poly(vinyl alcohol) (PVAL) as stabilizing agent during the emulsification step. Three batches of NP with mean diameters of 117, 285, and 593?nm, respectively, were prepared. NP of 117?nm exhibited the highest rate of reactive oxygen species production and the fastest m-THPP release determined by the transfer of m-THPP to serum proteins in vitro. Similarly, the 117?nm batch exhibited the highest in vivo photodynamic activity, as established in the chick embryo chorioallantoic membrane model. Our data suggest that m-THPP release before illumination is an important step for in vivo photodynamic activity especially for large NP. The NP size and the amount of residual PVAL at the NP surface were shown to control both the PS release and activity.     

Modified Paclitaxel-loaded Nanoparticles for Inhibition of Hyperplasia in a Rabbit Arterial Balloon Injury Model

Purpose This study tested the possibility of localized intravascular infusion of positive charged paclitaxel-loaded nanoparticles (NPs) to better prevent neointimal formation in a rabbit carotid artery injury model. Materials and Methods NPs were prepared by oil–water emulsion/solvent evaporation technique using biodegradable poly (lactide-co-glycolide) (PLGA). A cationic surfactant, didodecyldimethylammonium bromide (DMAB), was absorbed on the NP surface by electrostatic attraction between positive and negative charges. NPs were characterized in such aspects as size, surface morphology, surface charges as well as in vitro drug release profile. Balloon injured rabbit carotid arteries were treated with single infusion of paclitaxel-loaded NP suspension and observed for 28 days. The inhibitory effects of NPs on neointima formation were evaluated as end-point. Results NPs showed spherical shape with a diameter ranging from 200 to 500 nm. Negatively charged PLGA NPs shifted to positive after the DMAB modification. The in vitro drug release profile showed a biphasic release pattern. Morphometric analyses on the retrieved artery samples revealed that the inhibitory effect of intima proliferation was dose-dependent. At a concentration of 30 mg ml⁻¹, NP infusion completely inhibited intima proliferation in a rabbit vascular injury model. Conclusions Paclitaxel-loaded NPs with DMAB modification were proven an effective means of inhibiting proliferative response to vascular injury in a rabbit model.     

Ligand-Specific Targeting of Microspheres to Phagocytes by Surface Modification with Poly(L-Lysine)-Grafted Poly(Ethylene Glycol) Conjugate

Purpose. The purpose of this study was to demonstrate specific receptor-mediated targeting of phagocytes by functional surface coatings of microparticles, shielding from nonspecific phagocytosis and allowing ligand-specific interactions via molecular recognition. Methods. Coatings of the comb polymer poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) were investigated for potential to inhibit 1) nonspecific spreading of human blood-derived macrophages (MOs) and dendritic cells (DCs) on glass and 2) nonspecific phagocytosis of PLL-g-PEG-coated, carboxylated polystyrene (PS) or biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres. Coating was performed by adsorption of positively charged PLL-g-PEG on negatively charged microparticles or plasma-cleaned glass through electrostatic interaction. The feasibility of ligand-specific interactions was tested with a model ligand, RGD, conjugated to PEG chains of PLL-g-PEG to form PLL-g-PEG-RGD and compared with inactive ligand conjugate, PLL-g-PEG-RDG. Results. Coatings with PLL-g-PEG largely impaired the adherence and spreading of MOs and DCs on glass. The repellent character of PLL-g-PEG coatings drastically reduced phagocytosis of coated PS and PLGA microparticles to 10% in presence of serum. With both MOs and DCs, we observed ligand-specific interactions with PLL-g-PEG-RGD coatings on glass and PS and PLGA microspheres. Ligand specificity was abolished when using inactive ligand conjugate PLL-g-PEG-RDG, whereas repellency of coating was maintained. Conclusions. Coatings of PLL-g-PEG-ligand conjugates provide a novel technology for ligand specific targeting of microspheres to MOs and DCs while reducing nonspecific phagocytosis.     

Preparation and Characterization of Innovative Protein-coated Poly(Methylmethacrylate) Core-shell Nanoparticles for Vaccine Purposes

Purpose This study aims at developing novel core-shell poly(methylmethacrylate) (PMMA) nanoparticles as a delivery system for protein vaccine candidates. Materials and Methods Anionic nanoparticles consisting of a core of PMMA and a shell deriving from Eudragit L100/55 were prepared by an innovative synthetic method based on emulsion polymerization. The formed nanoparticles were characterized for size, surface charge and ability to reversibly bind two basic model proteins (Lysozyme, Trypsin) and a vaccine relevant antigen (HIV-1 Tat), by means of cell-free studies. Their in vitro toxicity and capability to preserve the biological activity of the HIV-1 Tat protein were studied in cell culture systems. Finally, their safety and immunogenicity were investigated in the mouse model. Results The nanoparticles had smooth surface, spherical shape and uniform size distribution with a mean diameter of 220 nm. The shell is characterized by covalently bound carboxyl groups negatively charged at physiological pH, able to reversibly adsorb large amounts (up to 20% w/w) of basic proteins (Lysozyme, Trypsin and HIV-1 Tat), mainly through specific electrostatic interactions. The nanoparticles were stable, not toxic to the cells, protected the HIV-1 Tat protein from oxidation, thus preserving its biological activity and increasing its shelf-life, and efficiently delivered and released it intracellularly. In vivo experiments showed that they are well tolerated and elicit strong immune responses against the delivered antigen in mice. Conclusions This study demonstrates that these new nanoparticles provide a versatile platform for protein surface adsorption and a promising delivery system particularly when the maintenance of the biologically active conformation is required for vaccine efficacy.     

Improving Protein Delivery from Microparticles Using Blends of Poly(DL lactide co-glycolide) and Poly(ethylene oxide)-poly(propylene oxide) Copolymers

Purpose. Microparticles containing ovalbumin as a model for protein drugs were formulated from blends of poly(DL lactide-co-glycolide) and poly(ethylene oxide)-poly(propylene oxide) copolymers (Pluronic). The objectives were to achieve uniform release characteristics and improved protein delivery capacity. Methods. The water- in oil -in oil emulsion/solvent extraction technique was used for microparticle production. Results. A protein loading level of over 40% (w/w) was attained in microparticles having a mean diameter of approximately 5 µm. Linear protein release profiles over 25 days in vitro were exhibited by certain blend formulations incorporating hydrophilic Pluronic F127. The release profile tended to plateau after 10 days when the more hydrophobic Pluronic L121 copolymer was used to prepare microparticles. A delivery capacity of 3 µg OVA/mg particles/ day was achieved by formulation of microparticles using a 1:2 blend of PLG:Pluronic F127. Conclusions. The w/o/o formulation approach in combination with PLG:Pluronic blends shows potential for improving the delivery of therapeutic proteins and peptides from microparticulate systems. Novel vaccine formulations are also feasible by incorporation of Pluronic L121 in the microparticles as a co-adjuvant.     

Stabilization and Controlled Release of Bovine Serum Albumin Encapsulated in Poly(D,L-lactide) and Poly(ethylene glycol) Microsphere Blends

Purpose. The acidic microclimate in poly(D, L-lactide-co-glycolide) 50/50 microspheres has been previously demonstrated by our group as the primary instability source of encapsulated bovine serum albumin (BSA). The objectives of this study were to stabilize the encapsulated model protein, BSA, and to achieve continuous protein release by using a blend of: slowly degrading poly(D, L-lactide) (PLA), to reduce the production of acidic species during BSA release; and pore-forming poly(ethylene glycol) (PEG), to increase diffusion of BSA and polymer degradation products out of the polymer. Methods. Microspheres were formulated from blends of PLA (Mw 145,000) and PEG (Mw 10,000 or 35,000) by using an anhydrous oil-in-oil emulsion and solvent extraction (O/O) method. The polymer blend composition and phase miscibility were examined by FT-IR and DSC, respectively. Microsphere surface morphology, water uptake, and BSA release kinetics were also investigated. The stability of BSA encapsulated in microspheres was examined by losses in protein solubility, SDS-PAGE, IEF, CD, and fluorescence spectroscopy. Results. PEG was successfully incorporated in PLA microspheres and shown to possess partial miscibility with PLA. A protein loading level of 5% (w/w) was attained in PLA/PEG microspheres with a mean diameter of approximately 100 m. When PEG content was less than 20% in the blend, incomplete release of BSA was observed with the formation of insoluble, and primarily non-covalent aggregates. When 20%-30% PEG was incorporated in the blend formulation, in vitro continuous protein release over 29 days was exhibited. Unreleased BSA in these formulations was water-soluble and structurally intact. Conclusions. Stabilization and controlled relaease of BSA from PLA/PEG microspheres was achieved due to low acid and high water content in the blend formulation.     

Preparation and Characterization of Poly (D,L-Lactide-Co-Glycolide) Microspheres for Controlled Release of Poly(L-Lysine) Complexed Plasmid DNA

Purpose. To produce and characterize controlled release formulations of plasmid DNA (pDNA) loaded in poly (D,L-lactide-co-glycolide) (PLGA) microspheres both in free form and as a complex with poly (L-lysine). Methods. Poly (L-lysine) (PLL) was used to form pDNA/PLL complexes with complexation ratio of 1:0.125 and 1:0.333 w/w to enhance the stability of pDNA during microsphere preparation and protect pDNA from nuclease attack. pDNA structure, particle size, zeta potential, drug loading, in vitro release properties, and protection from DNase I were studied. Results. The microspheres were found to be spherical with average particle size of 3.1-3.5 m. Drug loading of 0.6% was targeted. Incorporation efficiencies of 35.1% and 29.4-30.6% were obtained for pDNA and pDNA/PLL loaded microspheres respectively. Overall, pDNA release kinetics following the initial burst did not correlate with blank microsphere polymer degradation profile suggesting that pDNA release is convective diffusion controlled. The percentage of supercoiled pDNA in the pDNA and pDNA/PLL loaded microspheres was 16.6 % and 76.7-85.6% respectively. Unencapsulated pDNA and pDNA/PLL degraded completely within 30 minutes upon the addition of DNase I. Encapsulation of DNA/PLL in PLGA microspheres protected pDNA from enzymatic degradation. Conclusions. The results show that using a novel process, pDNA can be stabilized and encapsulated in PLGA microspheres to protect pDNA from enzymatic degradation.     

The in Vitro Cell Association of Invasin Coated Polylactide-Co-glycolide Nanoparticles

Purpose. To determine the effect of particle size and ligand surface density on the cellular association of poly lactide-co-glycolide nanoparticles covalently coated with bacterial invasin. Methods. Poly lactide-co-glycolide nanoparticles containing a flourescent probe were prepared at four diameters 155nm, 200nm, 375nm and 600nm using standard techniques. Bacterial invasin was covalently coupled to the particles surface at varying surface concentrations using a water soluble carbodiimide. The modified particle's cellular association with HEp2 2B cells in tissue culture was determined using flow cytometry. Results. Cellular association of modified nanoparticles was time dependent, abolished at low temperature, competitively inhibited by free invasin or the RGD peptide ligand and saturable. Increased cell association was produced by increasing the particle's surface invasin concentration however, this effect was size dependent. Small particles (155 nm and 200 nm) exhibiting a maximal association with increasing invasin concentration whilst the larger particles (375 nm and 600 nm) provide a minimum at low invasin concentrations. Conclusions. Modified particle cell association provided results commensurate with a receptor dependent uptake mechanism related to the presence of invasin. The size and surface concentration dependency however illustrate that application of these ligands to particulate drug delivery or targeting systems will be controlled by their natural cellular association properties.     

Effect of poly(ethylene glycol) content and formulation parameters on particulate properties and intraperitoneal delivery of insulin from PLGA nanoparticles prepared using the double-emulsion evaporation procedure

Abstract

Context: Size, encapsulation efficiency and stability affect the sustained release from nanoparticles containing protein-type drugs.

Objectives: Insulin was used to evaluate effects of formulation parameters on minimizing diameter, maximizing encapsulation efficiency and preserving blood glucose control following intraperitoneal (IP) administration.

Methods: Homogenization or sonication was used to incorporate insulin into poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles with increasing poly(ethylene glycol) (PEG) content. Effects of polymer type, insulin/polymer loading ratio and stabilizer in the internal aqueous phase on physicochemical characteristics of NP, in vitro release and stability of encapsulated insulin were investigated. Entrapment efficiency and release were assessed by radioimmunoassay and bicinconnic acid protein assay, and stability was evaluated using SDS-PAGE. Bioactivity of insulin was assessed in streptozotocin-induced, insulin-deficient Type I diabetic mice.

Results: Increasing polymeric PEG increased encapsulation efficiency, while the absence of internal stabilizer improved encapsulation and minimized burst release kinetics. Homogenization was shown to be superior to sonication, with NP fabricated from 10% PEG–PLGA having higher insulin encapsulation, lower burst release and better stability. Insulin-loaded NP maintained normoglycaemia for 24?h in diabetic mice following a single bolus, with no evidence of hypoglycemia.

Conclusions: Insulin-loaded NP prepared from 10% PEG–PLGA possessed therapeutically useful encapsulation and release kinetics when delivered by the IP route.     

Surface Modification of Poly(lactide-co-glycolide) Nanospheres by Biodegradable Poly(lactide)-Poly(ethylene glycol) Copolymers

The modification of surface properties of biodegradable poly(lactide-co-glycolide) (PLGA) and model polystyrene nanospheres by poly(lactide)-poly(ethlene glycol) (PLA:PEG) copolymers has been assessed using a range of in vitro characterization methods followed by in vivo studies of the nanospheres biodistribution after intravenous injection into rats. Coating polymers with PLA:PEG ratio of 2:5 and 3:4 (PEG chains of 5000 and 2000 Da, respectively) were studied. The results reveal the formation of a PLA: PEG coating layer on the particle surface resulting in an increase in the surface hydrophilicity and decrease in the surface charge of the nanospheres. The effects of addition of electrolyte and changes in pH on stability of the nanosphere dispersions confirm that uncoated particles are electrostatically stabilized, while in the presence of the copolymers, steric repulsions are responsible for the stability. The PLA:PEG coating also prevented albumin adsorption onto the colloid surface. The evidence that this effect was observed for the PLA:PEG 3:4 coated nanospheres may indicate that a poly(ethylene glycol) chain of 2000 Da can provide an effective repulsive barrier to albumin adsorption. The in vivo results reveal that coating of PLGA nanospheres with PLA:PEG copolymers can alter the biodistribution in comparison to uncoated PLGA nanospheres. Coating of the model polystyrene nanospheres with PLA:PEG copolymers resulted in an initial high circulation level, but after 3 hours the organ deposition data showed values similar to uncoated polystyrene spheres. The difference in the biological behaviour of coated PLGA and polystyrene nanospheres may suggest a different stability of the adsorbed layers on these two systems. A similar biodistribution pattern of PLA:PEG 3:4 to PEG 2:5 coated particles may indicate that poly(ethylene glycol) chains in the range of 2000 to 5000 can produce a comparable effect on in vivo behaviour.     

Indirect Evidence that Drug Brain Targeting Using Polysorbate 80-Coated Polybutylcyanoacrylate Nanoparticles Is Related to Toxicity

Purpose. To investigate the mechanism underlying the entry of the analgesic peptide dalargin into brain using biodegradable polybutylcyanoacrylate (PBCA) nanoparticles (NP) overcoated with polysorbate 80. Methods. The investigations were carried out with PBCA NP and with non biodegradable polystyrene (PS) NP (200 nm diameter). Dalargin adsorption was assessed by HPLC. Its entry into the CNS in mice was evaluated using the tail-flick procedure. Locomotor activity measurements were performed to compare NP toxicities. BBB permeabilization by PBCA NP was studied in vitro using a coculture of bovine brain capillary endothelial cells and rat astrocytes. Results. Dalargin loading was 11.7 µg/mg on PBCA NP and 16.5µg/ mg on PS NP. Adding polysorbate 80 to NP led to a complete desorption. Nevertheless, dalargin associated with PBCA NP and polysorbate 80 induced a potent and prolonged analgesia, which could not be obtained using PS NP in place of PBCA NP. Locomotor activity dramatically decreased in mice dosed with PBCA NP, but not with PS NP. PBCA NP also caused occasional mortality. In vitro, PBCA NP (10 µg/ml) induced a permeabilization of the BBB model. Conclusions. A non specific permeabilization of the BBB, probably related to the toxicity of the carrier, may account for the CNS penetration of dalargin associated with PBCA NP and polysorbate 80.     

Preparation and Characterization of a Composite PLGA and Poly(Acryloyl Hydroxyethyl Starch) Microsphere System for Protein Delivery

Purpose. To prepare and characterize a novel composite microsphere system based on poly(D,L-lactide-co-glycolide) (PLGA) and poly(acryloyl hydroxyethyl starch) (acHES) hydrogel for controlled protein delivery. Methods. Model proteins, bovine serum albumin, and horseradish peroxidase were encapsulated in the acHES hydrogel, and then the protein-containing acHES hydrogel particles were fabricated in the PLGA matrix by a solvent extraction or evaporation method. The protein-loaded PLGA-acHES composite microspheres were characterized for protein loading efficiency, particle size, and in vitro protein release. Protein stability was examined by size-exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and monitoring the enzymatic activity. Results. Scanning electron microscopy showed discrete PLGA microspheres containing many acHES particles. The composite microspheres were spherical and smooth in size range of 39-93 m. The drug loading efficiency ranged from 51 to 101%. The composite microspheres showed more favorable in vitro release than conventional PLGA microspheres. The composite microspheres showed 20% less initial with a gradual sustained release compared to high burst (60%) followed by a very slow release with the conventional PLGA microspheres. The composite microspheres also stabilized encapsulated proteins from the loss of activity during the microsphere preparation and release. Proteins extracted from the composite microspheres showed good stability without protein degradation products and structural integrity changes in the size-exclusion chromatography and SDS-PAGE analyses. Horseradish peroxidase extracted from microspheres retained more than 81% enzymatic activity. Conclusion. The PLGA-acHES composite microsphere system could be useful for the controlled delivery of protein drugs.     

Novel Polyester-Polysaccharide Nanoparticles

Purpose. The aim of the present study was to develop a new type of core-shell nanoparticles from a family of novel amphiphilic copolymers, based on dextran (DEX) grafted with poly(–caprolactone) (PCL) side chains (PCL-DEX). Methods. A family of PCL-DEX copolymers was synthesized in which both the molecular weight and the proportion by weight of DEX in the copolymer were varied. The nanoparticles were prepared by a technique derived from emulsion-solvent evaporation, during which emulsion stability was investigated using a Turbiscan. The nanoparticle size distribution, density, zeta potential, morphology, and suitability for freeze-drying were determined. Results. Because of their strongly amphiphilic properties, the PCL-DEX copolymers were able to stabilize o/w emulsions without the need of additional surfactants. Nanoparticles with a controlled mean diameter ranging from 100 to 250 nm were successfully prepared. A mechanism of formation of these nanoparticles was proposed. Zeta potential measurements confirmed the presence of a DEX coating. Conclusion. A new generation of polysaccharide-decorated nanoparticles has been successfully prepared from a family of PCL-DEX amphiphilic copolymers. They may have potential applications in drug encapsulation and targeting.     

Interaction of a Self-Emulsifying Lipid Drug Delivery System with the Everted Rat Intestinal Mucosa as a Function of Droplet Size and Surface Charge

Purpose. To investigate the interaction of positively charged self-emulsifying oil formulations (SEOF) following aqueous dilution as a function of resulting emulsion droplet charge and size with rat everted intestinal mucosa, adherent mucus layer and Peyer's patches, using cyclosporine A (CsA) as a lipophilic model drug. Methods. Droplet size determination (TEM technique) and -potential measurements were used to characterize the resulting emulsions. For the ex vivo interaction study, the well-known rat intestine everted sac technique was used in combination with confocal microscopy. Results. The positively charged oil droplets formed by SEOF dilutions at ratios of 1/50 and 1/10 elicited the stronger interaction with the mucosal surface. The positive charge of the smaller droplets was more readily neutralized, and even reversed in aqueous solutions containing moderate subphysiological mucin concentrations. Parameters such as droplet size, negativity of the epithelial mucosa potential and presence of the mucus layer on the epithelial surface affected drug mucosa uptake and the adhesion of the positively charged droplets to the rat intestinal mucosa. Conclusions. The enhanced electrostatic interactions of positively charged droplets with the mucosal surface are mostly responsible for the preferential uptake of CsA from the positively charged droplets as compared to negatively charged droplets irrespective of the experimental conditions used. The increased uptake of the CsA from the negatively charged oil droplets was consistent with the dilution extent, as expected, whereas in the positively charged droplets, an intermediate droplet size range was identified resulting in optimum drug uptake and clearly suggesting that drug uptake was not consistent with either dilution extent or droplet size.