Morten Jonas Maltesen

Spray drying of siRNA-containing PLGA nanoparticles intended for inhalation

Abstract Local delivery of small interfering RNA (siRNA) to the lungs constitutes a promising new area in drug delivery. The present study evaluated parameters of importance for spray drying of siRNA-loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) into nanocomposite microparticles intended for inhalation. The spray drying process was optimised using a statistical design of experiment and by evaluating powder characteristics upon systematic variation of the formulation parameters. Concentration, carbohydrate excipient (trehalose, lactose and mannitol) and the ratio of NP to excipient were varied to monitor the effects on moisture content, particle morphology, particle size and powder yield. The identified optimum conditions were applied for spray drying of siRNA-loaded nanocomposite microparticles, resulting in a product with a low water content (0.78% w/w) and an aerodynamic particle diameter considered suitable for inhalation. The use of mannitol in the formulation allowed a significantly lower moisture content than trehalose and lactose. The inclusion of 50% (w/w) or higher amounts of NPs resulted in a marked change in the surface morphology of the spray-dried particles. Importantly, the integrity and biological activity of the siRNA were preserved during the spray drying process. In conclusion, the present results show that spray drying is a suitable technique for producing nanocomposite microparticles comprising siRNA-containing PLGA NPs for potential use in inhalation therapy.

High loading efficiency and sustained release of siRNA encapsulated in PLGA nanoparticles: quality by design optimization and characterization.

Poly(DL-lactide-co-glycolide acid) (PLGA) is an attractive polymer for delivery of biopharmaceuticals owing to its biocompatibility, biodegradability and outstanding controlled release characteristics. The purpose of this study was to understand and define optimal parameters for preparation of small interfering RNA (siRNA)-loaded PLGA nanoparticles by the double emulsion solvent evaporation method and characterize their properties. The experiments were performed according to a 2(5-1) fractional factorial design based on five independent variables: The volume ratio between the inner water phase and the oil phase, the PLGA concentration, the sonication time, the siRNA load and the amount of acetylated bovine serum albumin (Ac-BSA) in the inner water phase added to stabilize the primary emulsion. The effects on the siRNA encapsulation efficiency and the particle size were investigated. The most important factors for obtaining an encapsulation efficiency as high as 70% were the PLGA concentration and the volume ratio whereas the size was mainly affected by the PLGA concentration. The viscosity of the oil phase was increased at high PLGA concentration, which explains the improved encapsulation by stabilization of the primary emulsion and reduction of siRNA leakage to the outer water phase. Addition of Ac-BSA increased the encapsulation efficiency at low PLGA concentrations. The PLGA matrix protected siRNA against nuclease degradation, provided a burst release of surface-localized siRNA followed by a triphasic sustained release for two months. These results enable careful understanding and definition of optimal process parameters for preparation of PLGA nanoparticles encapsulating high amounts of siRNA with immediate and long-term sustained release properties.

Quality by design – Spray drying of insulin intended for inhalation

Quality by design (QBD) refers to a holistic approach towards drug development. Important parts of QBD include definition of final product performance and understanding of formulation and process parameters. Inhalation of proteins for systemic distribution requires specific product characteristics and a manufacturing process which produces the desired product. The objective of this study was to understand the spray drying process of insulin intended for pulmonary administration. In particular, the effects of process and formulation parameters on particle characteristics and insulin integrity were investigated. Design of experiments (DOE) and multivariate data analysis were used to identify important process parameters and correlations between particle characteristics. The independent parameters included the process parameters nozzle, feed, and drying air flow rate and drying air temperature along with the insulin concentration as a formulation parameter. The dependent variables included droplet size, geometric particle size, aerodynamic particle size, yield, density, tap density, moisture content, outlet temperature, morphology, and physical and chemical integrity. Principal component analysis was performed to find correlations between dependent and independent variables. Prediction equations were obtained for all dependent variables including both interaction and quadratic terms. Overall, the insulin concentration was found to be the most important parameter, followed by inlet drying air temperature and the nozzle gas flow rate. The insulin concentration mainly affected the particle size, yield and tap density, while the inlet drying air temperature mainly affected the moisture content. No change was observed in physical and chemical integrity of the insulin molecule.

Drying methods for protein pharmaceuticals

Removal of water is a common method to prolong the storage stability of protein formulations. Traditionally, freeze-drying has been the method of choice, but spray drying and supercritical drying have gained increased interest in the past decade. The proper choice of drying technology has a significant impact on the final pharmaceutical product and on the overall economy of the process.:

Complex coacervates of hyaluronic acid and lysozyme: effect on protein structure and physical stability.

Complex coacervates of hyaluronic acid and lysozyme, a model protein, were formed by ionic interaction using bulk mixing and were characterized in terms of binding stoichiometry and protein structure and stability. The complexes were formed at pH 7.2 at low ionic strength (6mM) and the binding stoichiometry was determined using solution depletion and isothermal titration calorimetry. The binding stoichiometry of lysozyme to hyaluronic acid (870 kDa) determined by solution depletion was found to be 225.9 ± 6.6 mol, or 0.1 bound lysozyme molecules per hyaluronic acid monomer. This corresponded well with that obtained by isothermal titration calorimetry of 0.09 bound lysozyme molecules per hyaluronic acid monomer. The complexation did not alter the secondary structure of lysozyme measured by Fourier-transform infrared spectroscopy overlap analysis and had no significant impact on the Tm of lysozyme determined by differential scanning calorimetry. Furthermore, the protein stability of lysozyme was found to be improved upon complexation during a 12-weeks storage study at room temperature, as shown by a significant increase in recovered protein when complexed (94 ± 2% and 102 ± 5% depending on the polymer-protein weight to weight ratio) compared to 89 ± 2% recovery for uncomplexed protein. This study shows the potential of hyaluronic acid to be used in combination with complex coacervation to increase the physical stability of pharmaceutical protein formulations.

Analysis of insulin allostery in solution and solid state with FTIR

The insulin hexamer acts as an allosteric unit mediated by homotropic and heterotropic effects shifting the equilibrium between three distinct conformational states (T(6), R(3)T(3) and R(6)). The homotropic ligand phenol stabilises the R(6) state by binding to hydrophobic pockets only present in the R(6) state and shifts the equilibrium towards the R(6) state. The structural difference between the T(6) and R(6) state is primarily a change in the B1-B8 residues from extended conformation (T(6)) to alpha-helix (R(6)). The aim of this study was to investigate FTIR as an alternative method to monitor the T-R transition in the insulin hexamer upon phenol binding, and in addition to explore the advantage of infrared spectroscopy to measure solid state samples, and support the ability to maintain an allosteric state upon drying. The FTIR spectra of insulin in solution showed an increase in alpha-helix upon phenol binding and correlated well with the transition measured by CD yielding similar dissociation constants. Furthermore it was possible to maintain the increase in alpha-helix upon phenol binding after lyophilisation. The overall structure of the FTIR spectra changed upon lyophilisation but an increase in alpha-helix content was retained. Reconstitution of lyophilised insulin resulted in a change in structure resembling the structure of insulin prior to lyophilisation. Principal component analysis of all spectra was computed resulting in distinct clusters, and most variation in the data set could be explained by PC1 corresponding to a change in alpha-helix.

Particle engineering technologies for improving the delivery of peptide and protein drugs

Proteins and peptides are excellent therapeutic compounds which have highly specific and potent biological functions. However, the efficacious and safe delivery of peptide and protein drugs remains a major challenge due to a number of factors such as i) their intrinsic physicochemical instability, ii) their poor permeability through biological membranes, iii) their short plasma half-lives, and iv) problems with induction of immunogenicity upon repeated administration. Enabling formulation technologies are highly in demand for the successful development of various dosage forms for the effective delivery of peptide and protein drugs. Particulate delivery systems have been recognized as one of the most effective ways to improve the delivery of peptide and protein drugs. In particular, dry particle forms are very attractive because peptide and protein drugs are more stable in the solid state than in the liquid state. This review summarizes the current state of the art in particle engineering technologies enabling the formulation of peptide and protein drugs into dry particulate forms. Emphasis on the applications of spraying based technologies due to the possibility for controlling the physicochemical characteristics of the engineered particles and the potential for scaling-up for commercial scale manufacturing.