The potency of the adjuvant, CpG oligos, is enhanced by encapsulation in PLG microparticles

The objective of this work was to evaluate the potency of the CpG containing oligonucleotide encapsulated within poly(lactide-co-glycolide), and coadministered with antigen adsorbed to poly(lactide-co-glycolide) microparticles (PLG particles). The formulations evaluated include, CpG added in soluble form, CpG adsorbed, and CpG encapsulated. The antigen from Neisseria meningitidis serotype B (Men B) was used in these studies. The immunogenicity of these formulations was evaluated in mice. Poly(lactide-co-glycolide) microparticles were synthesized by a w/o/w emulsification method in the presence of a charged surfactant for the formulations. Neisseria meningitidis B protein was adsorbed to the PLG microparticles, with binding efficiency and initial release measured. CpG was either added in the soluble or adsorbed or encapsulated form based on the type of formulation. The binding efficiency, loading, integrity and initial release of CpG and the antigen were measured from all the formulations. The formulations were then tested in mice for their ability to elicit antibodies, bactericidal activity and T cell responses. Encapsulating CpG within PLG microparticles induced statistically significant higher antibody, bactericidal activity and T cell responses when compared to the traditional method of delivering CpG in the soluble form.     

Polylactide-co-glycolide microparticles with surface adsorbed antigens as vaccine delivery systems

Several groups have shown that vaccine antigens can be encapsulated within polymeric microparticles and can serve as potent antigen delivery systems. We have recently shown that an alternative approach involving charged polylactide co-glycolide (PLG) microparticles with surface adsorbed antigen(s) can also be used to deliver antigen into antigen presenting cell (APC). We have described the preparation of cationic and anionic PLG microparticles which have been used to adsorb a variety of agents, which include plasmid DNA, recombinant proteins and adjuvant active oligonucleotides. These PLG microparticles were prepared using a w/o/w solvent evaporation process in the presence of the anionic surfactants, including DSS (dioctyl sodium sulfosuccinate) or cationic surfactants, including CTAB (hexadecyl trimethyl ammonium bromide). Antigen binding to the charged PLG microparticles was influenced by several factors including electrostatic and hydrophobic interactions. These microparticle based formulations resulted in the induction of significantly enhanced immune responses in comparison to alum. The surface adsorbed microparticle formulation offers an alternative and novel way of delivering antigens in a vaccine formulation.     

Anionic microparticles are a potent delivery system for recombinant antigens from Neisseria meningitidis serotype B

The adsorption behavior of model proteins onto anionic poly(lactide-co-glycolide) (PLG) microparticles was evaluated. PLG microparticles were prepared by a w/o/w solvent evaporation process in the presence of the anionic surfactant dioctyl sodium sulfosuccinate (DSS). The effect of surfactant concentration and adsorption conditions on the adsorption efficiency and release rates in vitro was also studied. Subsequently, the microparticle formulation was tested to evaluate the efficacy of anionic microparticles as delivery systems for recombinant antigens from Neisseria meningitides type B (Men B), with and without CpG adjuvant. Protein (antigen) binding to anionic PLG microparticles was influenced by both electrostatic interaction and by other mechanisms, including hydrophobic attraction. The Men B antigens adsorbed efficiently onto anionic PLG microparticles and, following immunization in mice, induced potent enzyme-linked immunosorbent assay (ELISA) and serum bactericidal activity in comparison to alum-adsorbed formulations. These Men B antigens represent an attractive approach for vaccine development.     

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.     

A Two-Stage Strategy for Sterilization of Poly(lactide-co-glycolide) Particles by γ-Irradiation Does Not Impair Their Potency for Vaccine Delivery

This study evaluated the feasibility of using γ-irradiation for preparing sterile poly(lactide-co-glycolide) (PLG) formulations for vaccines. PLG microparticles were prepared by water-in-oil-in-water double-emulsion technique and lyophilized. The vials were γ-irradiated for sterilization process. Antigens from Neisseria meningitidis were adsorbed onto the surface of the particles and were characterized for protein adsorption. Antigens adsorbed onto the surface of the irradiated particles within 30 min. Mice were immunized with these formulations, and vaccine potency was measured as serum bactericidal titers. The γ-irradiated PLG particles resulted in equivalent serum bactericidal titers against a panel of five N. meningitidis strains as the nonirradiated PLG particles. The use of PLG polymers with different molecular weights did not influence the vaccine potency. The PLG particles prepared by γ-irradiation of the lyophilized formulations replace the need for aseptic manufacturing of vaccine formulations. This approach may enable the use of PLG formulations with a variety of antigens and stockpiling for pandemics.     

Enhancing the therapeutic efficacy of CpG oligonucleotides using biodegradable microparticles

Oligonucleotides, with specific sequence surrounding CpG motifs, appear to be very effective for the induction of a potent Th1 responses. This molecule represents pathogen-associated molecular patterns (PAMPs) that allows the pathogen recognition receptors (PRRs) present on innate immune cells to recognize them and become activated. PAMPs and related compounds are often labelled as immunopotentiators, allowing a clear distinction between them and particulate delivery systems such as emulsions, liposomes, virus-like particles and microparticles.Microparticles prepared from biodegradable, biocompatible polyesters, and poly (lactide co-glycolide) (PLG). They have been proven to be a good particulate delivery system for the co-delivery of antigens and adjuvants. PLG has been used in humans for many years as a resorbable suture material and controlled-release drug delivery systems. It has been demonstrated that antigen presenting cells (APCs) efficiently uptake the PLG microparticles (∼ 1 μm) both in vivo and in vitro. After uptake, the PLG subsequently induces an antigen specific CTL response in rodents.Several groups, including our group, have evaluated CpG as an immunopotentiator in various formulations and delivery systems (i.e. emulsions and particulate systems). This review will discuss in detail the work conducted so far with CpG using PLG microparticles as a delivery system. We will also discuss the advantages and enhancement of immune properties of formulating CpG (soluble, adsorbed, and encapsulated forms) with PLG microparticles along with future directions for these microparticles with CpG.     

Adsorption of a Novel Recombinant Glycoprotein from HIV (Env gp120dV2 SF162) to Anionic PLG Microparticles Retains the Structural Integrity of the Protein,Whereas Encapsulation in PLG Microparticles Does Not

No HeadingPurpose. To evaluate the delivery of a novel HIV-1 antigen (gp120dV2 SF162) by surface adsorption or encapsulation within polylactide-co-glycolide microparticles and to compare both the formulations for their ability to preserve functional activity as measured by binding to soluble CD4.Methods. Poly(lactide-co-glycolide) microparticles were synthesized by a water-in-oil-in-water (w/o/w) emulsification method in the presence of the anionic surfactant dioctylsulfosuccinate (DSS) or polyvinyl alcohol. The HIV envelope glyocoprotein was adsorbed and encapsulated in the PLG particles. Binding efficiency and burst release measured to determine adsorption characteristics. The ability to bind CD4 was assayed to measure the functional integrity of gp120dV2 following different formulation processes.Results. Protein (antigen) binding to PLG microparticles was influenced by both electrostatic interaction and other mechanisms such as hydrophobic attraction and structural accommodation of the polymer and biomolecule. The functional activity as measured by the ability of gp120dV2 to bind CD4 was maintained by adsorption onto anionic microparticles but drastically reduced by encapsulation.Conclusions. The antigen on the adsorbed PLG formulation maintained its binding ability to soluble CD4 in comparison to encapsulation, demonstrating the feasibility of using these novel anionic microparticles as a potential vaccine delivery system.     

Inactive Vibrio cholerae whole-cell vaccine-loaded biodegradable microparticles: in vitro release and oral vaccination

An approach is proposed using Vibrio cholerae (VC)-loaded microparticles as oral vaccine delivery systems for improved vaccine bioavailability and increased therapeutic efficacy. The VC-loaded microparticles were prepared with 50:50 poly(DL-lactide-co-glycolide) (PLG), 75:25 poly(DL-lactide-co-glycolide) and poly(lactide acid) (PLA)/PEG blend copolymers by the solvent evaporation method. VC was successfully entrapped in three types of microparticles with loading efficiencies and loading levels as follows: 50:50 PLG systems: 97.8% and 55.4 ± 6.9 µg/mg; 75:25 PLG systems: 89.2% and 46.5 ± 4.4?µg/mg; PLA/PEG-blended systems: 82.6% and 53.7 ± 5.8?µg/mg. The different distributions of VC in the core region and on the surface were as follows: 50:50 PLG systems 25.7 ± 1.9 and 6.2 ± 0.9?µg/mg; 75:25 PLG systems: 25.8 ± 2.2 and 3.6 ± 0.4?µg/mg; PLA/PEG-blended systems: 32.4 ± 2.1 and 5.2 ± 1.0?µg/mg, respectively. In vitro active release of VC was affected mainly by matrix type and VC-loaded location in microparticles. The therapeutic immunogenic potential of VC loaded with 50:50 PLG, 75:25 PLG and PLA/PEG-blended microparticles was evaluated in adult mice by oral immunization. Significantly higher antibody responses and serum immunoglobin Ig G, IgA and IgM responses were obtained when sera from both VC-loaded 75:25 PLG and PLA/PEG-blended microparticles immunized mice were titrated against VC. The most immunogenicity in evoking serum IgG, IgA and IgM responses was immunized by VC-loaded PLA/PEG-blended microparticles, and with VC challenge in mice, the survival rate (91.7%).     

Monitoring Microviscosity and Microacidity of the Albumin Microenvironment Inside Degrading Microparticles from Poly(lactide-co-glycolide) (PLG) or ABA-triblock Polymers Containing Hydrophobic Poly(lactide-co-glycolide) A Blocks and Hydrophilic Poly(ethyleneoxide) B Blocks

Purpose. The purpose of this study was to monitor the microenvironment of an encapsulated model protein during the release from biodegradable microparticles (MP) made from three different polymers, namely poly(lactide-co-glycolide) (PLG) and ABA-triblock polymers containing hydrophobic poly(lactide-co-glycolide) A blocks and hydrophilic poly(ethyleneoxide) B blocks with an A:B ratio of 90:10 (ABA10) and 70:30 (ABA30). Methods. MP loaded with spin labeled albumin were prepared by a w/o/w technique. The particles were characterized by light scattering and electron microscopy. In vitro release of albumin was determined by size exclusion chromatography. Light microscopic experiments were conducted to visualize water penetration in the matrix. The protein microenvironment inside the degrading microparticles was characterized noninvasively by 2 GHz EPR spectroscopy. Results. Water penetrated rapidly into all MP in the range of few minutes. A burst release was observed for PLG. The release from ABA block-polymers continued for over 14 days despite the rapid solubilization of the protein inside the microparticles. The initial microviscosity of the protein environment inside the ABA particles after exposure to buffer was 2 mm²/s and increased with time. A gradual decrease of the pH to a value of 3.5 was observed within the MP. Conclusions. The data indicate that the microviscosity and microacidity inside protein loaded microparticles can be studied nondestructively by EPR spectroscopy. Our results clearly demonstrate that ABA-block polymers are superior to PLG allowing a controlled release of proteins from swollen microspheres.     

In vitro and in vivo evaluations of PLGA microsphere vaccine formulations containing pDNA coexpressing Hepatitis B surface antigen and Interleukin-2

Purpose. DNA-based vaccines encoding viral antigens have been shown to elicit immune responses in animal models. In this study, a plasmid DNA (pDNA) coexpressing the middle envelope protein of hepatitis B virus (HBV) and Interleukin-2 (IL-2) was incorporated into Poly (D,L-lactic-co-glycolic acid) (PLGA) microspheres and three different formulations were investigated for their potential as a vaccine delivery system.

Methods. Emulsion solvent evaporation methods of water-in-oil-in-water (w/o/w) and oil-in-water (o/w) were used to generate three different formulations in which PLGA microspheres contained pDNA either encapsulated within or adsorbed onto the microspheres.

Results. In vaccine formulation A2, prepared using the (w/o/w) method, pDNA was encapsulated within the microspheres. The other two formulations (B2 and B2a) were prepared using the (o/w) method and B2 contained pDNAs encapsulated within the microspheres while B2a contained pDNAs adsorbed onto the microspheres. pDNA loading efficiencies of A2, B2 and B2a were determined to be 15%, 25% and 45%, respectively. In vitro release of pDNAs from microspheres was evaluated for a 45-day period with no conformational changes and A2 displayed slower release than that of the B2 and B2a. When mice were immunized from anterior tibialis muscle using A2, B2 and B2a formulations containing 100 µg pDNA, antibody responses were detected for 6 months in mice sera.

Conclusions. Although all PLGA microsphere formulations containing pDNA elicited antibody responses by the end of the 6th month, the antibody titers were found to be higher with B2 and B2a formulations in comparison to A2 formulation and the naked pDNA in saline.     

An investigation of the factors controlling the adsorption of protein antigens to anionic PLG microparticles

This work examines physico-chemical properties influencing protein adsorption to anionic PLG microparticles and demonstrates the ability to bind and release vaccine antigens over a range of loads, pH values, and ionic strengths. Poly(lactide-co-glycolide) microparticles were synthesized by a w/o/w emulsification method in the presence of the anionic surfactant DSS (dioctyl sodium sulfosuccinate). Ovalbumin (OVA), carbonic anhydrase (CAN), lysozyme (LYZ), lactic acid dehydrogenase, bovine serum albumin (BSA), an HIV envelope glyocoprotein, and a Neisseria meningitidis B protein were adsorbed to the PLG microparticles, with binding efficiency, initial release and zeta potentials measured. Protein (antigen) binding to PLG microparticles was influenced by both electrostatic interaction and other mechanisms such as van der Waals forces. The protein binding capacity was directly proportional to the available surface area and may have a practical upper limit imposed by the formation of a complete protein monolayer as suggested by AFM images. The protein affinity for the PLG surface depended strongly on the isoelectric point (pI) and electrostatic forces, but also showed contributions from nonCoulombic interactions. Protein antigens were adsorbed on anionic PLG microparticles with varying degrees of efficiency under different conditions such as pH and ionic strength. Observable changes in zeta potentials and morphology suggest the formation of a surface monolayer. Antigen binding and release occur through a combination of electrostatic and van der Waals interactions occurring at the polymer-solution interface.     

In Vitro Myotoxicity of Selected Cationic Macromolecules Used in Non-Viral Gene Delivery

Purpose. Cationic lipid/DNA complexes have been proposed as a method of in vivo gene delivery via intravenous or intramuscular injection. A concern with using these polycationic molecules is whether they are associated with tissue toxicity at the injection site. Therefore, the objective of these studies was to investigate the myotoxic potential of selected non-viral gene delivery macromolecules (e.g., cationic lipids and polymers) with and without plasmid DNA (pDNA) in vitro. Methods. Myotoxicity was assessed by the cumulative release of creatine kinase (CK) over 90 minutes from the isolated rodent extensor digitorum longus muscle into a carbogenated balanced salt solution (BBS, pH 7.4, 37°C) following a 15 L injection of the test formulation. Phenytoin (Dilantin^®) and normal saline served as positive and negative controls, respectively. Results. The myotoxicity of plasmid DNA (pDNA, ~5000bp, 1 mg/ ml) was not statistically different from normal saline. However, the myotoxicity of Dilantin^® was 16-times higher than either normal saline or pDNA (p < 0.05). Cationic liposomes were found to be less myotoxic than polylysine and PAMAM dendrimers. Polylysine's myotoxicity was found to be dependent upon concentration and molecular weight. The myotoxicity of formulations of cationic liposomes(s), lower molecular weight polylysine (25,000) and higher concentration of PAMAM dendrimers with pDNA were found to be statistically less significant than those formulations without pDNA. Conclusions. The cationic liposomes were less myotoxic compared to the dendrimers and polylysine. Myotoxicity was dependent upon the type of cationic lipid macromolecule, concentration, molecular weight and the presence of pDNA. A possible explanation for this reduced tissue damage in cationic lipids complexed with pDNA is that the formation of complex reduces the overall positive charge of the injectable system resulting in less damage.     

Design of a pectin-based microparticle formulation using zinc ions as the cross-linking agent and glutaraldehyde as the hardening agent for colonic-specific delivery of resveratrol: In vitro and in vivo evaluations

The aim of this study was to develop a colon-specific microparticle formulation based on pectin. Resveratrol was used as a model drug due to its potential therapeutic efficacy on colitis and colon cancer. Microparticles were produced by cross-linking pectin molecules with zinc ions and with glutaraldehyde as hardening agent for pectins. Different microparticles were prepared by varying the formulation variables. Effect of these formulation variables were investigated on particle shape and size, moisture content and weight-loss during drying, encapsulation efficiency, swelling–erosion ratio, and drug release pattern of the formulated microparticles. Formulation conditions were optimized based on the in vitro drug release study. Morphology, Fourier transform infrared spectroscopy, stability, and in vivo pharmacokinetic study of the microparticles prepared at the optimized formulation conditions were performed. Microparticles were spherical with <1?mm diameter and encapsulation efficiencies of >94%. The glutaraldehyde-modified microparticles prepared at optimized formulation conditions revealed colon specific in vitro and in vivo drug release. Plasma appearance of drug was delayed for 4–5?h after their administration directly into stomach, but displayed comparable area under the curve to other controls in the experiment, indicating the potential of the developed formulation as a colon-specific drug delivery system.     

Cationic stearylamine-containing biodegradable microparticles for DNA delivery

Technical aspects for preparing a new type of cationic stearylamine (SA)-containing microparticle as a potential drug delivery system for negatively charged therapeutics were investigated. Cationic biodegradable microparticles based on poly(lactide) and poly(lactide-co-glycolide) were prepared upon incorporation of SA either by solvent evaporation or by spray-drying. Water-insoluble SA offers the advantage over other water-soluble cationic compounds that it can be dissolved directly in the organic solution together with the polymers. This facilitated the subsequent preparation of the microparticle formulations. Particle size was controlled by the respective process parameters, resulting in either large polymer aggregates within the range 50-100 micro m or small spherical microparticles within the range 1-10 micro m. The incorporation of SA into the formulations also improved particle characteristics in terms of re-dispersibility, reduced sticking, and particle size uniformity. Both circular plasmid DNA (5 kbp) and linear salmon DNA (0.5 kbp) were efficiently adsorbed to the cationic SA microparticle surfaces. Preliminary tests on the release of DNA from spray-dried SA microparticles showed an immediate burst release, which was followed by a delayed second release phase for more than 4 weeks. The cationic SA microparticles might provide a potential drug-delivery system to improve the efficacy for protein and DNA-type therapeutics.     

Genetic Immunization Using Nanoparticles Engineered from Microemulsion Precursors

Purpose. Genetic immunization using naked plasmid DNA (pDNA) has been shown to elicit broad humoral and cellular immune responses. However, more versatile and perhaps cell-targeted delivery systems are needed. To this end, a novel process to engineer cationic nanoparticles coated with pDNA for genetic immunization was explored. Methods. Cationic nanoparticles were engineered from warm oil-in-water microemulsion precursors composed of emulsifying wax as the oil phase and cetyltrimethylammonium bromide (CTAB) as the cationic surfactant. Plasmid DNA was coated on the surface of the cationic nanoparticles to produce pDNA-coated nanoparticles. An endosomolytic lipid and/or a dendritic cell-targeting ligand (mannan) were incorporated in or deposited on the nanoparticles to enhance the in vitro cell transfection efficiency and the in vivo immune responses after subcutaneous injection to Balb/C mice. The IgG titer to expressed -galactosidase and the cytokine release from isolated splenocytes after stimulation were determined on 28 days. Results. Cationic nanoparticles (around 100 nm) were engineered within minutes. The pDNA-coated nanoparticles were stable at 37°C over 30 min in selected biologic fluids. Transmission electron microscopy showed the nanoparticles were spherical. Plasmid DNA-coated nanoparticles, especially those with both an endosomolytic lipid and dendritic cell-targeting ligand, resulted in significant enhancement in both IgG titer (over 16-fold) and T-helper type-1 (Th1-type) cytokine release (up to 300% increase) over naked pDNA. Conclusion. A novel method to engineer pDNA-coated nanoparticles for enhanced in vitro cell transfection and enhanced in vivo immune responses was reported.     

Sustained release of isoniazid from a single injectable dose of poly (DL-lactide-co-glycolide) microparticles as a therapeutic approach towards tuberculosis

Drug delivery strategies to achieve a sustained drug release and increased bioavailability involve the use of biodegradable polymeric drug carriers. Poly (DL-lactide-co-glycolide) (PLG) microparticles were investigated as carriers for isoniazid (INH). In vitro and in vivo release of INH from different formulations of PLG microparticles was examined. In vitro experiments showed a sustained release of INH up to 6 days from non-porous microparticles while porous microparticles released INH over 3 days. Both porous and non-porous microparticles released INH in plasma for up to 2 days. Hardened PLG microparticles sustained release of INH for up to 7 weeks both in vitro and in vivo. The concentrations of INH obtained at all times were much higher than the minimum inhibitory concentration (MIC) of INH. Controls injected with free INH showed release of INH in plasma for up to 12 h and in organs for up to 24 h. There was no hepatotoxicity induced as compared with control animals. Taken together these results suggest that PLG-based antitubercular drugs may serve as ideal therapeutic agents for the treatment of tuberculous infections.     

Microparticles for intranasal immunization

Of the several routes available for mucosal immunization, the nasal route is particularly attractive because of ease of administration and the induction of potent immune responses, particularly in the respiratory and genitourinary tracts. However, adjuvants and delivery systems are required to enhance immune responses following nasal immunization. This review focuses on the use of microparticles as adjuvants and delivery systems for protein and DNA vaccines for nasal immunization. In particular we discuss our own work on poly(lactide co-glycolide) (PLG) microparticles with entrapped protein or adsorbed DNA as a vaccine delivery system. The possible mechanisms involved in the enhancement of immune responses through the use of DNA adsorbed onto PLG microparticles are also discussed.     

Characterization of antigens adsorbed to anionic PLG microparticles by XPS and TOF-SIMS

The chemical composition of the surface of anionic PLG microparticles before and after adsorption of vaccine antigens was measured using X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS). The interfacial distributions of components will reflect underlying interactions that govern properties such as adsorption, release, and stability of proteins in microparticle vaccine delivery systems. Poly(lactide-co-glycolide) microparticles were prepared by a w/o/w emulsification method in the presence of the anionic surfactant dioctyl sodium sulfosuccinate (DSS). Ovalbumin, lysozyme, a recombinant HIV envelope glyocoprotein and a Neisseria meningitidis B protein were adsorbed to the PLG microparticles, with XPS and time-of-flight secondary mass used to analyze elemental and molecular distributions of components of the surface of lyophilized products. Protein (antigen) binding to PLG microparticles was measured directly by distinct elemental and molecular spectroscopic signatures consistent with amino acids and excipient species. The surface sensitive composition of proteins also included counter ions that support the importance of electrostatic interactions being crucial in the mechanism of adsorptions. The protein binding capacity was consistent with the available surface area and the interpretation of previous electron and atomic force microscope images strengthened by the quantification possible by XPS and the qualitative identification possible with TOF-SIMS. Protein antigens were detected and quantified on the surface of anionic PLG microparticles with varying degrees of efficiency under different adsorption conditions such as surfactant level, pH, and ionic strength. Observable changes in elemental and molecular composition suggest an efficient electrostatic interaction creating a composite surface layer that mediates antigen binding and release.     

Controlled Release of a Contraceptive Steroid from Biodegradable and Injectable Gel Formulations: In Vitro Evaluation

Purpose. The purpose of this study was to investigate the effects of formulation factors including varying wax concentration, drug loading and drug particle size, on drug release characteristics from both pure oil and gel formulations prepared with a combination of derivatized vegetable oil (Labrafil 1944 CS) and glyceryl palmitostearate (Precirol ATO 5), using levonorgestrel as a model drug. Methods. The effects of varying drug loadings, different drug particle sizes, and wax (Precirol) concentrations on in-vitro drug release rates were evaluated, and the mechanisms of drug release from the gels were determined. Results. Zero-order drug release rates from the 10% Precirol gel formulations containing 0.25, 0.50 and 2.00% w/v drug loadings were lower than those observed for oil formulations containing identical drug loadings. Higher zero-order release rates were observed from formulations containing smaller drug particles suspended in both oil and gel formulations. The mechanism of drug release from gels containing less than 0.25% w/w drug was diffusion-controlled. Increasing the wax concentrations in the gels from 5% w/w to 20% w/w significantly decreased the diffusivity of the drug through the gel formulations and markedly increased the force required to inject the gels from two different sizes of needles. Conclusions. This study shows how modification of the physicochemical properties of the gel formulations by changing the drug particle size, wax concentration and drug loading, affects drug release characteristics from the system.     

Alginate/Poly-L-Lysine Microparticles for the Intestinal Delivery of Antisense Oligonucleotides

Purpose. A microparticle carrier based on alginate and poly-L-lysine was developed and evaluated for the delivery of antisense oligonucleotides at the intestinal site. Formulations of oligonucleotide-loaded microparticles having differences in the carrier molecular weight and composition were characterized in vitro and in vivo. Methods. Polymeric microparticles were prepared by ionotropic gelation and crosslinking of alginate with calcium ions and poly-L-lysine. The loading of the antisense oligonucleotide into the microparticles was achieved by absorption in aqueous medium. The association capacity, loading and particle size of the microparticles were characterized. The in vivo performances of various formulations after intrajejunal administration were studied in rat and in dog models. Results. Microparticles had a sponge-like structure and an oligonucleotide loading of 27-35%. The composition of the medium affected the particle size and the in vitro release profiles. The oligonucleotide bioavailability after intrajejunal administration to rats in the presence of permeation enhancers was good for most of the tested systems. The application of microparticles in powder form compared to an equivalent suspension improved the intrajejunal bioavailability of the oligonucleotide (25% and 10% respectively) in rats. On the contrary, the intrajejunal administration to dogs resulted in poor oligonucleotide bioavailability (0.42%). Conclusions. The formulation of antisense oligonucleotides within alginate and poly-L-lysine microparticles is a promising strategy for the oral application.