Formulation of pH responsive peptides as inhalable dry powders for pulmonary delivery of nucleic acids

Nucleic acids have the potential to be used as therapies or vaccines for many different types of disease, but delivery remains the most significant challenge to their clinical adoption. pH responsive peptides containing either histidine or derivatives of 2,3-diaminopropionic acid (Dap) can mediate effective DNA transfection in lung epithelial cells with the latter remaining effective even in the presence of lung surfactant containing bronchoalveolar lavage fluid (BALF), making this class of peptides attractive candidates for delivering nucleic acids to lung tissues. To further assess the suitability of pH responsive peptides for pulmonary delivery by inhalation, dry powder formulations of pH responsive peptides and plasmid DNA, with mannitol as carrier, were produced by either spray drying (SD) or spray freeze drying (SFD). The properties of the two types of powders were characterised and compared using scanning electron microscopy (SEM), next generation impactor (NGI), gel retardation and in vitro transfection via a twin stage impinger (TSI) following aerosolisation by a dry powder inhaler (Osmohaler™). Although the aerodynamic performance and transfection efficacy of both powders were good, the overall performance revealed SD powders to have a number of advantages over SFD powders and are the more effective formulation with potential for efficient nucleic acid delivery through inhalation.     

The impact of drying method and formulation on the physical properties and stability of methionyl human growth hormone in the amorphous solid state

The objective of this work was to investigate the impact of drying method and formulation on the physical stability (aggregation) and selected important physical properties of dried methionyl human growth hormone (Met-hGH) formulations. Solutions of Met-hGH with different stabilizers were dried by different methods (freeze drying, spray drying, and film drying), with and without surfactant. Properties of the dried powders included powder morphology, specific surface area (SSA), protein surface coverage, thermal analysis, and protein secondary structure. Storage stability of Met-hGH in different formulations was also studied at 50 degrees C and at 60 degrees C for 3 months. The dried powders displayed different morphologies, depending mainly on the method of drying and on the presence or absence of surfactant. Film dried powders had the lowest SSA (approximately 0.03 m(2)/g) and the lowest total protein surface accumulation (approximately 0.003%). Surfactant caused a reduction in the SSA of both spray dried and freeze dried powders. Spray dried powders showed greater protein surface coverage and SSA relative to the same formulations dried by other means. Greater in-process perturbations of protein secondary structure were observed with polymer excipients. Formulation impacted physical stability. In general, low molecular weight stabilizers provided better stability. For example, the aggregation rate at 50 degrees C of Met-hGH in a freeze dried trehalose-based formulation was approximately four times smaller than the corresponding Ficoll-70-based formulation. Drying method also influenced physical stability. In general, the film dried preparations studied showed superior stability to preparations dried by other methods, especially those formulations employing low molecular weight stabilizers.     

Electrohydrodynamic Comminution: A Novel Technique for the Aerosolisation of Plasmid DNA

Purpose Naked plasmid DNA (pDNA) is a potential gene transfer agent for lung gene therapies but cannot be aerosolised without degradation using conventional nebulisation devices. This study investigated the viability of an alternative nebulisation technique, electrohydrodynamic (EHD) comminution for the aerosol delivery of naked DNA in vivo.Methods Naked pDNA was aerosolised using jet and ultrasonic nebulisers, and by EHD comminution. Degradation associated with the aerosolisation process was investigated using gel electrophoresis and by transfection studies in cell culture. Optimised formulations for EHD aerosolisation of pDNA were developed and in vivo deposition and reporter gene expression were investigated in mice.Results Unlike conventional nebulisation devices, EHD comminution of plasmids up to 15 kb in size resulted in no detectable pDNA degradation. EHD formulations containing up to 1 mg/ml pDNA were developed and shown to produce monodisperse aerosols suitable for targeted lung delivery in humans. Aerosolisation studies in vivo demonstrated detectable levels of pDNA deposition and measurable luciferase reporter gene expression in the lungs of exposed mice.Conclusions This study demonstrates for the first time that respirable aerosols of naked pDNA can be generated without plasmid degradation and that EHD comminution is an appropriate technique for the aerosolisation of delicate gene transfer agents.     

Aerosolisation of beclomethasone dipropionate using spray dried lactose/polyethylene glycol carriers.

The aim of this study was to characterize the physical properties of spray dried lactose in the presence of different polyethylene glycols (PEG 400, PEG 3000 and PEG 6000) and to evaluate their performance as carriers for dry powder inhaler (DPI) formulations. The efficiency of spray dried lactose/PEG carriers in aerosolisation of beclomethasone dipropionate (BD), a model hydrophobic drug, was compared to Pharmatose 325 M (L325), spray dried lactose alone (SDL), and also a sieved (< 38 microm) fraction of alpha-lactose monohydrate (SL). In vitro deposition analysis was performed using a twin stage liquid impinger at a flow rate of 60 l/min through a Spinhaler. The deposition profiles of the drug from binary formulations composed of BD and spray dried lactose/PEG carriers were also compared to ternary formulations containing large and fine lactose carriers. Differential scanning calorimetry and X-ray diffraction data showed the presence of alpha-anhydrous lactose in spray dried lactose/PEG crystalline powders. Spray drying of lactose in the presence of PEG 400 resulted in the production of a powder (SDL-PEG400) with lower alpha-lactose monohydrate content, and also smaller particle size distribution than those obtained in the presence of PEG 3000 (SDL-PEG3000) or PEG 6000 (SDL-PEG6000). All formulations showed different deposition profiles, except those containing SDL-PEG3000 or SDL-PEG6000 which exhibited similar data. The fine particle fraction of aerosolised BD varied from 6.26 +/- 1.07 (for L325) to 25.87 +/- 5.33 (for SDL-PEG3000). All deposition profiles of BD aerosolised from SDL-PEG3000 were significantly higher (P < 0.01) than those produced by binary and ternary formulations containing L325, a coarse lactose commercially available for DPI formulations. The differences observed in deposition data for various carriers were interpreted according to their physical properties. It was concluded that particle size distribution, morphology and specific surface texture of SDL-PEG3000 and SDL-PEG6000 were important factors influencing their efficiency as small carriers for DPI formulations.     

Effects of lipid formulations on clove extract spray dried powders: comparison of physicochemical properties,storage stability and in vitro intestinal permeation

Abstract

Clove is an aromatic plant spice with potent antioxidant and anti-inflammatory activity. Eugenol is the main compound which contributes to such medicinal and nutritional benefits. To date, the formulation of unstable, volatile and poorly water-soluble compounds remains a challenging task. Lipid formulations can be used to improve physicochemical and biopharmaceutical properties of poorly soluble compounds. The aim of this study is to investigate the effects of lipids, such as Gelucire and Compritol on physicochemical properties; stability and in vitro intestinal permeation of spray dried powdered formulations loaded with clove’s bioactive compounds. Results showed that eugenol retention in spray-dried powders could be correlated with antioxidant activity and with mass recovery after spray drying. Adding Gelucire but not Compritol to clove extract formulations, improved solubility of spray dried powders. Stability test in high humidity environment (63.5% RH) suggested that formulations including both Gelucire and Compritol were significantly more stable compared to the formulation without any lipid at the two tested temperatures (25?°C and 40?°C). This suggests that lipid additions to clove (Syzygium aromaticum) extract formulations provide protective effects for the spray dried powders in high-humidity environments. In addition, results from in vitro intestinal permeation studies suggested that eugenol uptake, was not being hindered by transporters nor was the absorption being affected by lipid formulations.     

Drying of a plasmid containing formulation: chitosan as a protecting agent

Background

Along with research on development of more efficient gene delivery systems, it is necessary to search on stabilization processes to extend their active life span. Chitosan is a nontoxic, biocompatible and available gene delivery carrier. The aim of this study was to assess the ability of this polymer to preserve transfection efficiency during spray-drying and a modified freeze-drying process in the presence of commonly used excipients.

Methods

Molecular weight of chitosan was reduced by a chemical reaction and achieved low molecular weight chitosan (LMWC) was complexed with pDNA. Obtained nanocomplex suspensions were diluted by solutions of lactose and leucine, and these formulations were spray dried or freeze dried using a modified technique. Size, polydispersity index, zeta potential, intensity of supercoiled DNA band on gel electrophoresis, and transfection efficiency of reconstituted nanocomplexes were compared with freshly prepared ones.

Results and conclusion

Size distribution profiles of both freeze dried, and 13 out of 16 spray-dried nanocomplexes remained identical to freshly prepared ones. LMWC protected up to 100% of supercoiled structure of pDNA in both processes, although DNA degradation was higher in spray-drying of the nanocomplexes prepared with low N/P ratios. Both techniques preserved transfection efficiency similarly even in lower N/P ratios, where supercoiled DNA content of spray dried formulations was lower than freeze-dried ones. Leucine did not show a significant effect on properties of the processed nanocomplexes. It can be concluded that LMWC can protect DNA structure and transfection efficiency in both processes even in the presence of leucine.     

Drying-Induced Variations in Physico-Chemical Properties of Amorphous Pharmaceuticals and Their Impact on Stability II: Stability of a Vaccine

Objectives To investigate the impact of drying method on the storage stability of dried vaccine formulations. Materials and Methods A sucrose-based formulation of a live attenuated virus vaccine of a parainfluenza strain, with and without surfactant, was dried from by different methods; freeze drying, spray drying and foam drying. Dried powders were characterized by differential scanning calorimetry, specific surface area (SSA) analysis and by electron spectroscopy for chemical analysis (ESCA) to evaluate vaccine surface coverage in the dried formulations. Dried formulations were subjected to storage stability studies at 4, 25 and 37°C. The vaccine was assayed initially and at different time points to measure virus-cell infectivity, and the degradation rate constant of the vaccine in different dried preparations was determined. Results SSA was highest with the spray dried preparation without surfactant (∼ 2.8 m²/g) and lowest in the foam dried preparations (with or without surfactant) (∼ 0.1 m²/g). Vaccine surface coverage was estimated based on ESCA measurements of nitrogen content. It was predicted to be highest in the spray dried preparation without surfactant and lowest in the foam with surfactant. Stability studies conducted at 25°C and 37°C showed that the vaccine was most stable in the foam dried preparation with surfactant and least stable in spray dried preparations without surfactant and in all freeze dried preparations regardless of the presence of surfactant. Addition of surfactant did lower the SSA and vaccine surface coverage in freeze dried preparations but still did not improve storage stability. Conclusions In drying methods that did not involve a freezing step, good storage stability of Medi 534 vaccine in the dried form was found with low SSA and low vaccine surface accumulation, both of which integrate into low fraction of vaccine at the surface. Ice appears to be a major destabilizing influence.     

Tris(2-aminoethyl)amine-based α-branched fatty acid amides – Synthesis of lipids and comparative study of transfection efficiency of their lipid formulations

The synthesis of a new class of cationic lipids, tris(2-aminoethyl)amine-based α-branched fatty acid amides, is described resulting in a series of lipids with specific variations in the lipophilic as well as the hydrophilic part of the lipids. In-vitro structure/transfection relationships were established by application of complexes of these lipids with plasmid DNA (pDNA) to different cell lines. The α-branched fatty acid amide bearing two tetradecyl chains and two lysine molecules (T14diLys) in mixture with the co-lipid 1,2-di-[(9Z)-octadec-9-enoyl]-sn-glycero-3-phosphoethanolamine (DOPE) (1/2, n/n) exhibits effective pDNA transfer in three different cell lines, namely Hep-G2, A549, and COS-7. The presence of 10% serum during lipoplex incubation of the cells did not affect the transfection efficiency. Based on that, detailed investigations of the complexation of pDNA with the lipid formulation T14diLys/DOPE 1/2 (n/n) were carried out with respect to particle size and charge using dynamic light scattering (DLS), ζ-potential measurements, and transmission electron microscopy (TEM). Additionally, the lipoplex uptake was investigated by confocal laser scanning microscopy (CLSM). Overall, lipoplexes prepared from T14diLys/DOPE 1/2 (n/n) offer large potential as lipid-based polynucleotide carriers and further justify advanced examinations.     

Preparation and Evaluation of Controlled Release Microparticles for Respiratory Protein Therapy

A series of microparticle formulations, designed for controlled release pulmonary therapy, were evaluated in terms of their physical properties, aerosol performance, lung epithelial cell toxicity, and controlled release profile. A protein, bovine serum albumin (BSA) was chosen as a model macromolecule active ingredient which was coprocessed, using spray drying, with varying concentrations of the release modifier, polyvinyl alcohol (PVA). The spray dried microparticles were tested for their physico-chemical characteristics (e.g., size distribution, morphology and density), in vitro aerosolisation performance using a 5-stage Marple Miller Impactor (MMI) and in vitro release profiles by a custom-built diffusion cell (in 100 mL phosphate buffer pH 7.4). The toxicity of PVA on lung epithelial cells was investigated using a human alveolar basal epithelium A549 cell line. Analysis of the particle size data indicated that all the spray dried BSA/PVA samples had similar size distributions with a median particle diameter (d_0.5) across all samples of 2.79 ± 0.11 µm. All formulations had relatively good aerosolisation performance when compared to conventional dry powder inhalation (DPI) formulations although increasing PVA percentage had a negative effect on the aerosol performance in vitro. Analysis of the difference and similarity factors for the release profiles indicated significant differences with respect to PVA concentration. Furthermore, cell toxicity analysis indicated PVA to have limited effect on cell viability after 24 h exposure. A series of protein-based inhalation formulations have been developed and tested, and shown to be suitable for controlled release in the respiratory tract. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:2709–2717, 2009     

Characterization and aerodynamic evaluation of spray dried recombinant human growth hormone using protein stabilizing agents

The effect of the protein stabilizers on the stability and aerosol performance of spray dried recombinant human growth hormone (SD rhGH) was investigated. rhGH solution was spray dried alone, with polysorbate 20 (at three concentrations of 0.05%, 0.01%, and 0.005%), Zn(2+) (by Zn(2+):rhGH molar ratio of 2:1 and 4:1), and/or lactose (by lactose:rhGH weight ratio of 2:1). Size exclusion chromatography (SEC) analysis of spray dried powders demonstrated that of all the potential protein stabilizers, the combination of polysorbate 20 (0.05%), Zn(2+) (Zn(2+):rhGH molar ratio of 2:1) and lactose (lactose:rhGH weight ratio of 2:1) was the most effective at protecting rhGH against aggregation during spray drying. The results of circular dichroism (CD) analysis revealed that using of polysorbate 20 (in all concentrations) and Zn(2+) (by Zn(2+):rhGH molar ratio of 2:1) together in the formulations would preserve rhGH conformational stability during the process. The particle size distribution data obtained by laser diffraction method showed all SD rhGH formulations had volume median diameter and mean diameter below 5mum. The characterization of the aerosol performance of the spray dried powders by Andersen cascade impactor (ACI) showed that by increasing the concentration of polysorbate 20 in the formulations the aerodynamic efficiency of the resultant particles was reduced. In conclusion, the optimum amounts of polysorbate 20, Zn(2+) and lactose satisfied both physical stability during spray drying process (2.37% aggregation) and good aerosol performance (fine particle fraction; FPF=38.52%).     

Optimization of Lipid Composition in Cationic Emulsion as In Vitro and In Vivo Transfection Agents

Purpose. To enhance in vitro and in vivo transfection activity by optimizing lipid composition of cationic lipid emulsions.Methods. Various emulsion formulations having different cationic lipids as emulsifiers, and additional helper lipids as co-emulsifiers, were prepared. The stability of the emulsion and its complex with DNA was investigated by measuring the particle size change in phosphate buffer saline (PBS) over a period of 20 days. The activity of the emulsions in transfecting pCMV-beta into COS-1 cells in the presence or absence of 80% serum was evaluated. We also evaluated in vivo transfection activity using intravenously administered pCMV-Luc+ as a reporter gene.Results. Among the cationic emulsifiers, 1,2-dioleoyl-sn-glycero-3-trimethylammonium-propane (DOTAP) formed the most stable and efficient emulsion gene carrier. Addition of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increased in vitro transfection activity, but slightly compromised the stability of the emulsion. The loss was compensated for by including small amounts of Tween 80 in the emulsion. The in vitro and in vivo transfection activities were also increased by adding Tween 80. Even though in vitro transfection activity of liposomes was high in the absence of serum, the transfection activity of emulsions was far greater than that of liposomes in the presence of serum and for in vivo applications.Conclusions. By including DOPE as an endosomolytic agent and Tween 80 as a stabilization agent, the cationic emulsion becomes a more potent gene carrier for in vitro and in vivo applications, especially in the presence of serum.     

Development of nanosomes using high‐pressure homogenization for gene therapy

Objectives The aim of this project was to develop a novel lipid‐based formulation suitable for gene therapy. Methods Novel nanosize liposome (nanosome) formulations containing pDNA (plasmid DNA) were developed using high‐pressure homogenization (HPH). The effect of lipid concentration was studied at two levels: 3 mm and 20 mm . The preformed nanosomes were incubated for 18–20 h with pDNA or pDNA/protamine sulfate (PS) complex. The physical properties of the pDNA nanosomes were compared by particle size distribution and zeta‐potential measurements. Their biological properties were also compared by pDNA efficiency of encapsulation/complexation, integrity, nuclease digestion, transfection efficiency and cell cytotoxicity. Key findings pDNA nanosomes prepared with 20 mm lipid (nanosomes : pDNA : PS at a ratio of 8.6 : 1 : 2) had particle sizes of 170–422 nm (90% confidence). The zeta‐potential of the formulation was 49.2 ± 1.5 mV, and the pDNA encapsulation/complexation efficiency was ～98%. pDNA nanosomes prepared with 3 mm lipid (nanosomes : pDNA : PS at a ratio of 2.09 : 1 : 2) had particle sizes of 140–263 nm (90% confidence). The zeta‐potential of this formulation was 36.4 ± 1.2 mV, and the pDNA encapsulation/complexation efficiency was ～100%. However, a comparison of the efficiency of transfection indicated that pDNA nanosomes prepared with low‐concentration lipids (3 mm ) showed significantly higher transfection efficiency compared with the pDNA nanosomes prepared with high‐concentration lipids (20 mm ), as well as those prepared with Fugene‐6 (a commercially available transfection reagent). This particular formulation (pDNA nanosomes, 3 mm lipids) also showed significantly less cytotoxicity compared with the other pDNA nanosome formulations. Conclusions To conclude, these results indicate that condensing pDNA with PS followed by subsequent complexation with low‐concentration nanosomes generated from HPH can produce a pDNA nanosome formulation that will boost transfection efficiency, while minimizing cytotoxicity. This new technology appears to be an efficient tool for future commercial or large‐scale manufacture of DNA delivery systems for gene therapy.     

Excipient-free nanoporous microparticles of budesonide for pulmonary delivery

The aim of this study was to investigate the application of a spray-drying process for the production of nanoporous microparticles (NPMPs) to budesonide, and to characterise the particles produced in terms of their suitability for pulmonary delivery.Budesonide was spray dried with and without ammonium carbonate from ethanol/water or methanol/water solutions. The solid-state characteristics and micromeritic (particle size, density, surface area) properties of spray dried powders were assessed. In vitro deposition studies were performed to assess aerosol performance.The densities of the NPMPs were significantly lower and the surface areas significantly higher than for non-porous spray dried or micronised material. NPMPs of budesonide demonstrated improved aerosolisation properties compared to spray dried non-porous, micronised material and two budesonide commercial products. All spray dried materials were amorphous in nature. The glass transition temperature (90 °C) was sufficiently high to suggest good physical stability at room temperature. When stored at 25 °C/60% RH NPMPs showed a reduced tendency to recrystallise compared to the equivalent non-porous spray dried powder. The physical stability and amorphous nature of NPMPs was retained, under these storage conditions for at least one year and the in vitro aerosolisation properties were not affected by the storage conditions.Excipient-free porous microparticles, prepared by the novel process described, show good potential for drug delivery by oral inhalation with improved in vitro deposition properties compared to non-porous particles.     

Development of directly compressible powders via co-spray drying.

Continuous production of directly compressible powders was achieved by coprocessing acetaminophen and carbohydrates via spray drying. Binary and ternary powder mixtures containing drug substance and carbohydrates were prepared by co-spray drying and evaluated on spray drying processibility, powder hygroscopicity, flowability, and compactability. The influence of process parameters during spray drying on the compaction behaviour of drug/excipient mixtures was investigated via Heckel analysis. Erythritol, lactose, maltodextrin, and mannitol were efficient in co-spray drying with acetaminophen. However, lactose mixtures showed poor flowability. Spray dried mixtures containing mannitol and erythritol were characterised as non-hygroscopic, highly dense, and good flowing powders. Mannitol increased tablet tensile strength in contrast with the poor compactability of erythritol. Maltodextrin was selected for further experiments because it provided excellent tablet tensile strength. The use of erythritol, maltodextrin and mannitol in binary drug/excipient mixtures resulted in high process yields. Compacts of erythritol, mannitol, and maltodextrin were characterised by higher tablet tensile strength at higher spray drying temperatures due to the increased particle fragmentation of erythritol and mannitol mixtures and to the increased plastic deformation of maltodextrin formulations. A combination of erythritol, maltodextrin, and mannitol was selected for further formulation and process optimisation of co-spray dried powders for direct compression.     

Colistin Powders with High Aerosolisation Efficiency for Respiratory Infection: Preparation and In Vitro Evaluation

In many respiratory infections caused by multi-drug-resistant Gram-negative bacteria, colistin is often the last-line drug for treatment despite its nephrotoxicity when administered parenterally. Inhalation therapy of colistin has great potential to improve the efficacy while reducing adverse effects. In this study, inhalable powder formulations of colistin (sulphate) were produced via spray drying. The colistin powders were found to have intact antimicrobial activity against Acinetobacter baumannii measured by broth micro-dilution. Both the raw material and spray-dried formulations were amorphous and absorbed significant amount of water up to 30% (w/w) at relative humidity (RH) of at least 70%. The spray-dried formulations were physically stable in the amorphous form at 60% RH and 25°C, having a high aerosol efficiency (emitted dose >86% and fine particle fraction total >83%) which remained unchanged after a 3-month storage. Storage at an elevated RH of 75% resulted in the aerosolisation performance significantly decreased, and at RH 90%, the formulation particles fused together (but without re-crystallisation). Although spray drying has been extensively used for generating inhalable drug particles, this is the first report that colistin powder can be physically stable in the amorphous form at ambient conditions, indicating that spray-drying approach is suitable for producing inhalable colistin powder formulation.     

Caffeine microparticles for nasal administration obtained by spray drying

This study investigated the possibility to use spray drying technique to prepare powders formulations containing caffeine intended for nasal delivery. Spray dried powders containing caffeine and excipients, as filler and shaper agents, were prepared. Powders were investigated for particle size, morphology and delivery properties from Monopowder P nasal insufflator, assessing the influence of each excipient on microparticles characteristics. The results showed that the excipients strongly affected microparticle properties. Size, shape and agglomeration tendency are relevant characteristics of spray dried nasal powder.     

Radio Frequency - Assisted Ultrasonic Spray Freeze Drying for Pharmaceutical Protein Solids

This study examined physical stability of spray freeze dried (SFD) bovine serum albumin (BSA) solids produced using the radio frequency (RF)-assisted drying technique. BSA formulations were prepared with varying concentrations of trehalose and mannitol, using an excipient-free formulation as control. These formulations were produced using either traditional ultrasonic spray freeze drying (SFD) or RF-assisted ultrasonic spray freeze drying (RFSFD). The dried formulations were then characterized using Karl Fischer moisture content measurement, powder X-ray diffraction (PXRD), size exclusion chromatography (SEC), and solid-state hydrogen/deuterium exchange with mass spectrometry (ssHDX-MS). Moisture content did not have a good correlation with the physical stability of the formulations measured by SEC. ssHDX-MS metrics such as deconvoluted peak areas of the deuterated samples showed a satisfactory correlation (R² = 0.914) with the SEC stability data. RFSFD improved the stability of formulations with 20 mg/ml of trehalose and no mannitol, and had similar stability with all other formulations as compared to SFD. This study demonstrated that RFSFD technique can significantly reduce the duration of primary drying cycle from 48.0 h to 27.5 h while maintaining or improving protein physical stability as compared to traditional lyophilization.     

Spray-drying preparation of microparticles containing cationic PLGA nanospheres as gene carriers for avoiding aggregation of nanospheres

Preparation of nano-sized particles using lyophilization, which is a standard drying technique for high-molecular-weight compounds such as bioactive peptides, proteins, plasmid DNA and siRNA, often results in particle aggregation. In this study, spray-drying was applied for preparation of cationic PLGA nanospheres as gene delivery vectors in order to minimize aggregation and loss of gene transfection efficiency. PLGA nanoparticle emulsions were prepared by dropping an acetone/methanol mixture (2/1) containing PLGA and a cationic material, such as PEI, DOTMA, DC-Chol or CTAB, into distilled water with constant stirring. The PLGA nanosphere emulsion was dried with mannitol by spray-drying, and mannitol microparticles containing PLGA nanospheres were obtained. Mean particle diameter of spray dried PLGA particles was 100-250 nm, which was similar to that of the nano-emulsion before drying, whereas the lyophilized PLGA particles showed increased particle diameter due to particle aggregation. PEI, DOTMA and DC-Chol were useful for maintaining nanoparticle size and conferring positive charge to nanospheres. Transfection of pDNA (pCMV-Luc) using these spray-dried cationic PLGA nanospheres yielded high luciferase activity in COS-7 cells, particularly with PLGA/PEI nanospheres. The present spray-drying technique is able to provide cationic PLGA nanospheres, and may improve redispersal and handling properties.     

Microdialysis sampling and the clinical determination of topical dermal bioequivalence

The influence of the deposition pattern and spray characteristics of nasal powder formulations on the insulin bioavailability was investigated in rabbits. The formulations were prepared by freeze drying a dispersion containing a physical mixture of drum dried waxy maize starch (DDWM)/Carbopol^® 974P (90/10, w/w) or a spray-dried mixture of Amioca^® starch/Carbopol^® 974P (25/75, w/w). The deposition in the nasal cavity of rabbits and in a silicone human nose model after actuation of three nasal delivery devices (Monopowder, Pfeiffer and experimental system) was compared and related to the insulin bioavailability. Posterior deposition of the powder formulation in the nasal cavity lowered the insulin bioavailability.

To study the spray pattern, the shape and cross-section of the emitted powder cloud were analysed. It was concluded that the powder bulk density of the formulation influenced the spray pattern. Consequently, powders of different bulk density were prepared by changing the solid fraction of the freeze dried dispersion and by changing the freezing rate during freeze drying. After nasal delivery of these powder formulations no influence of the powder bulk density and of the spray pattern on the insulin bioavailability was observed.     

Freeze-Dried Mannitol for Superior Pulmonary Drug Delivery via Dry Powder Inhaler

Purpose

To show for the first time the superior dry powder inhaler (DPI) performance of freeze dried mannitol in comparison to spray dried mannitol and commercial mannitol.

Methods

Different mannitol powders were sieved to collect 63–90 μm particles and then analyzed in terms of size, shape, surface morphology, solid state, density, flowability. Salbutamol sulphate-mannitol aerosol formulations were evaluated in terms of homogeneity, SS-mannitol adhesion, and in vitro aerosolization performance.

Results

Freeze dried mannitol demonstrated superior DPI performance with a fine particle fraction believed to be highest so far reported in literature for salbutamol sulphate under similar protocols (FPF?=?46.9%). To lesser extent, spray dried mannitol produced better aerosolization performance than commercial mannitol. Freeze dried mannitol demonstrated elongated morphology, α-+β-+δ- polymorphic forms, and poor flowability whereas spray dried mannitol demonstrated spherical morphology, α-+β- polymorphic forms, and excellent flowability. Commercial mannitol demonstrated angular morphology, β- polymorphic form, and good flowability. Freeze dried mannitol demonstrated smoother surface than spray dried mannitol which in turn demonstrated smoother surface than commercial mannitol. FPF of SS increased as mannitol powder porosity increase.

Conclusions

Freeze drying under controlled conditions can be used as a potential technique to generate aerodynamically light mannitol particles for superior DPI performance.