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.     

Formulation optimization and characterization of spray dried microparticles for inhalation delivery of doxycycline hyclate

The local delivery of antibiotics in the treatment of infectious respiratory diseases is an attractive alternative to deliver high concentration of antimicrobials directly to the lungs and minimize systemic side effects. In this study, inhalable microparticles containing doxycycline hyclate, sodium carboxymethylcellulose, leucine and lactose were prepared by spray drying of aqueous ethanol formulations. Box-Behnken design was used to study the influence of various independent variables such as polymer concentration, leucine concentration, ethanol concentration and inlet temperature of the spray dryer on microparticle characteristics. The microparticles were characterized in terms of particle morphology, drug excipient interaction, yield, entrapment efficiency, Carr's index, moisture content, thermal properties, X-ray powder diffraction, aerosolization performance and in vitro drug release. The effect of independent variables on spray dryer outlet temperature was also studied. The overall shape of the particles was found to be spherical like doughnuts in the size range of 1.16-5.2 μm. The optimized formulation (sodium carboxymethylcellulose concentration 14% w/v, leucine concentration 33% w/v, ethanol concentration 36% v/v, inlet temperature of 140°C) exhibited the following properties: yield 56.69%, moisture content 3.86%, encapsulation efficiency 61.74%, theoretical aerodynamic diameter 3.11 μm and Carr's index 23.5% at an outlet temperature 77°C. The powders generated were of a suitable mass median aerodynamic diameter (4.89 μm) with 49.3% fine particle fraction and exhibited a sustained drug release profile in vitro.     

Non-viral dried powders for respiratory gene delivery prepared by cationic and chitosan loaded liposomes

The aim of this work was to investigate lipid-based dried powders as transfection competent carriers capable of promoting the expression of therapeutic genes. The lipid-based vectors were prepared by combining different cationic lipids 1,2-dioleoyl-3-trimethylammoniumpropane chloride (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 3beta(N(N',N-dimethylaminoethane) carbamoyl) cholesterol hydrochloride (DC-Chol) or by mixing of anionic lipids (1,2-dimyristoyl-sn-glycero-3-phospocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol sodium salt (DMPG) and chitosan salts. Spray drying of the formulations was performed using carbohydrates as thermoprotectant excipients and some amino acids as aerosolisation enhancers. Both the lipidic vectors and the dried powders were characterized for morphology, size, zeta potential (Z-potential) and a yield of the process. Agarose gel electrophoresis was used to examine the structural integrity of dehydrated plasmid DNA (pDNA). The biological functionality of the powders was quantified using the in vitro cell transfection. Among the several lipids and lipid-polymer mixtures tested, the best-selected formulations had spherical shape, narrow size distribution (mean diameter<220 nm, P.I.<0.250), a positive zeta-potential (>25 mV) with a good yield of the process (>65%). The set-up spray drying parameters allowed to obtain good yield of the process (>50%) and spherically shaped particles with the volume-weighted mean diameter (d[4,3])<6 microm in the respirable range. The set-up conditions for the preparation of the lipid dried powders did not adversely affect the structural integrity of the encapsulated pDNA. The powders kept a good transfection efficiency as compared to the fresh colloidal formulations. Lipid-based spray dried powders allowed the development of stable and viable formulations for respiratory gene delivery. In vitro dispersibility and deposition studies are in progress to determine the aerosolisation properties of the powders.     

Spray drying of budesonide, formoterol fumarate and their composites-II. Statistical factorial design and in vitro deposition properties

The aim of this study was to investigate the effect of changing spray drying parameters on the production of a budesonide/formoterol fumarate 100:6 (w/w) composite. The systems were spray dried as solutions from 95% ethanol/5% water (v/v) using a Büchi 191-Mini Spray Dryer. A 2(5-1) factorial design study was undertaken to assess the consequence of altering spray drying processing variables on particle characteristics. The processing parameters that were studied were inlet temperature, spray drier airflow rate, pump rate, aspirator setting and feed concentration. Each batch of the resulting powder was characterised in terms of thermal and micromeritic properties as well as an in vitro deposition by twin impinger analysis. Overall, the parameter that had the greatest influence on each response investigated was production yield - airflow (higher airflow giving greater yields), median particle size - airflow (higher airflow giving smaller particle sizes) and Carr's compressibility index - feed concentration (lower feed concentration giving smaller Carr's indices). A six- to seven-fold difference in respirable fraction can be observed by changing the spray drying process parameters. The co-spray dried composite system which displayed best in vitro deposition characteristics, showed a 2.6-fold increase in respirable fraction in the twin impinger experiments and better dose uniformity compared with the physical mix of micronised powders.     

Development of spray-dried acetaminophen microparticles using experimental designs

Experimental factorial designs were built to investigate the effects of five parameters on production yields and moisture contents of spray-dried products. These factors concerned both the solution feed (drug concentration, colloidal silica concentration and polymer/drug ratio) and the spray dryer (inlet temperature and feed rate). Three formulations containing cellulose derivatives and acetaminophen were tested. The aim of the study was to optimize the operating conditions to maximize production yields while minimizing moisture contents. First screening experiments consisting of fractional factorial designs revealed the most significant factors to be inlet temperature, feed rate and their interaction for both formulations containing sodium carboxymethylcellulose and feed rate and colloidal silica concentration for the formulation containing microcrystalline cellulose. Then, the optimal operating conditions were estimated by response surface methodology. Central rotational composite designs showed quadratic models were adequate. New assays were carried out using these last conditions to evaluate both the repeatability and reproducibility of the spray-drying technique. Yields above 80% and moisture content of approximately 1% were reached. The characterization of microparticles revealed the poor flowability of the spray-dried products due to significant cohesiveness and very small size (less than 55 microm).     

New insights into respirable protein powder preparation using a nano spray dryer

In this study the Nano Spray Dryer B-90 (BüCHI Labortechnik AG, Flawil, Switzerland) was evaluated with regard to the drying of proteins and the preparation of respirable powders in the size range of 1-5 μm. β-galactosidase was chosen as a model protein and trehalose was added as a stabilizer. The influence of inlet temperature, hole size of the spray cap membrane and ethanol concentration in the spray solution was studied using a 33 full factorial design. The investigated responses were enzyme activity, particle size, span, yield and shelf life. Furthermore, the particle morphology was examined. The inlet temperature as well as the interaction of inlet temperature and spray cap size significantly influenced the enzyme activity. Full activity was retained with the optimized process. The particle size was affected by the hole size of the spray cap membrane and the ethanol content. The smallest cap led to a monodisperse particle size distribution and the greatest yield of particles of respirable size. Higher product recovery was achieved with lower inlet temperatures, higher ethanol contents and smaller cap sizes. Particle morphology differed depending on the cap size. The protein exhibited higher storage stability when spray dried without ethanol and when a larger spray cap size was used.     

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.     

Microparticle-mediated transfection of non-phagocytic cells in vitro

DNA-loaded microparticles represent an attractive delivery system to target professional antigen presenting cells (APC) for the delivery of DNA vaccines. Microparticles exhibiting a positively charged surface were prepared by the incorporation of two selected cationic polymers into a poly(D, L-lactide-co-glycolide) polymer (PLGA) core. The toxicity of the different formulations was checked in two cell lines and was found to be comparable to plain PLGA particles. Increased toxicity of some formulations was observed in primary macrophages (Mphi) with high phagocytosis activity. Plasmid DNA was efficiently adsorbed to the microparticle surfaces, and the different formulations were checked for their transfection efficiency in phagocytic and non-phagocytic cells. Interestingly, the most pronounced gene transfer efficiency was observed in a non-phagocytic 293 cell line when compared to a macrophage cell line and primary Mphi. Possible mechanisms include the dissociation of DNA-polymer complex and subsequent transfection of the cells. Microscopic observation of fluorescent-labeled DNA in primary Mphi revealed large amounts of DNA entering the cells, but no detectable DNA inside the nuclei. We conclude that phagocytic professional APC represent a group of cells, which is especially difficult to transfect when compared to other cell types. The administration of DNA in vivo is likely to predominantly result in the transfection of non-lymphoid cells unless there is a possibility to provide efficient targeting and trafficking of the DNA to the nucleus of professional APC. Although DNA-loaded PEI and DAEM microparticles resulted in significant transfection of cells, toxicity and transfection efficiency was not superior to that of DNA complexed with soluble PEI and DAEM.     

Microparticle-mediated Transfection of Non-phagocytic Cells In Vitro

DNA-loaded microparticles represent an attractive delivery system to target professional antigen presenting cells (APC) for the delivery of DNA vaccines. Microparticles exhibiting a positively charged surface were prepared by the incorporation of two selected cationic polymers into a poly (d, l -lactide-co-glycolide) polymer (PLGA) core. The toxicity of the different formulations was checked in two cell lines and was found to be comparable to plain PLGA particles. Increased toxicity of some formulations was observed in primary macrophages (M Φ) with high phagocytosis activity. Plasmid DNA was efficiently adsorbed to the microparticle surfaces, and the different formulations were checked for their transfection efficiency in phagocytic and non-phagocytic cells. Interestingly, the most pronounced gene transfer efficiency was observed in a non-phagocytic 293 cell line when compared to a macrophage cell line and primary M Φ. Possible mechanisms include the dissociation of DNA-polymer complex and subsequent transfection of the cells. Microscopic observation of fluorescent-labeled DNA in primary M Φ revealed large amounts of DNA entering the cells, but no detectable DNA inside the nuclei. We conclude that phagocytic professional APC represent a group of cells, which is especially difficult to transfect when compared to other cell types. The administration of DNA in vivo is likely to predominantly result in the transfection of non-lymphoid cells unless there is a possibility to provide efficient targeting and trafficking of the DNA to the nucleus of professional APC. Although DNA-loaded PEI and DAEM microparticles resulted in significant transfection of cells, toxicity and transfection efficiency was not superior to that of DNA complexed with soluble PEI and DAEM.     

Modulation of Polyplex Release from Biodegradable Microparticles through Poly(ethylenimine) Modification and Varying Loading Concentration

Purpose

This work investigates the effects of hyaluronic acid (HA) conjugated onto branched poly(ethylenimine) (bPEI) and varying loading concentrations of these polymers complexed with DNA on their release from poly(DL-lactic-co-glycolic acid) (PLGA) microparticles and the transfection of target cells.

Methods

To examine the effect of alteration of the gene delivery polymer on the system, we observed the morphology, size, loading efficiency, polymer and DNA release, and the transfection efficiency for the microparticles formed with three internal phase loading concentrations during microparticle formation.

Results

Addition of HA to this vector allowed for increased loading concentration within these systems and significantly altered release kinetics without changing the morphology of the particles. The incorporation of HA onto the bPEI backbone significantly increased the transfection efficiency of the complexes released from the corresponding microparticle formulation.

Conclusions

The results show that the modification of bPEI with HA and the concentration of loaded polymer/DNA complexes can significantly alter the entrapment and release profiles from PLGA microparticles. This is significant in that it offers insight into the effects of modification of gene delivery vectors on a controlled release system designed to achieve a sustained therapeutic response.     

PEGylated quaternized copolymer/DNA complexes for gene delivery

The aim of this study was to improve the colloidal stability, decrease unspecific interactions with cells and blood components of a novel gene delivery system composed of epsilon-caprolactone and quaternized epsilon-caprolactone. For this purpose, diblock 50/50 copolymer was used to generate complexes with DNA by either the solvent evaporation technique and by dialysis. The size, surface charge and degree of interaction of the plasmid-loaded formulations were measured. Then, polyplexes were combined with a poly(CL)-b-PEG copolymer to create a hydrophilic corona on the surface of the complexes. The cytotoxicity, transfection efficiency and cellular uptake of polyplexes and their association with PEG were evaluated on HeLa cells. The dialysis method did not allow to reduce the size of complexes as compared to the solvent evaporation method. The zeta potential of polyplexes became positive from a charge ratio of 4. The degree of interaction of copolymer with plasmid DNA was very high. Cytotoxicity and transfection efficiency were found to be comparable to polyethylenimine 50 kDa. Association of polyplexes with poly(CL)-b-PEG copolymer led to a small increase in particle size and a sharp decrease of charge surface. Cytotoxicity, transfection efficiency and cellular uptake were significantly reduced relative to unshielded copolymer/DNA complexes. The PEGylated formulations may be an attractive approach for an in vivo application.     

Optimisation of spray drying process conditions for sugar nanoporous microparticles (NPMPs) intended for inhalation

The present study investigated the effect of operating parameters of a laboratory spray dryer on powder characteristics, in order to optimise the production of trehalose and raffinose powders, intended to be used as carriers of biomolecules for inhalation.The sugars were spray dried from 80:20 methanol:n-butyl acetate (v/v) solutions using a Büchi Mini Spray dryer B-290. A 2⁴ factorial design of experiment (DOE) was undertaken. Process parameters studied were inlet temperature, gas flow rate, feed solution flow rate (pump setting) and feed concentration. Resulting powders where characterised in terms of yield, particle size (PS), residual solvent content (RSC) and outlet temperature. An additional outcome evaluated was the specific surface area (SSA) (by BET gas adsorption), and a relation between SSA and the in vitro deposition of the sugar NPMPs powders was also investigated.The DOE resulted in well fitted models. The most significant factors affecting the characteristics of the NPMPs prepared, at a 95% confidence interval, were gas flow: yield, PS and SSA; pump setting: yield; inlet temperature: RSC.Raffinose NPMPs presented better characteristics than trehalose NPMPs in terms of their use for inhalation, since particles with larger surface area resulting in higher fine particle fraction can be produced.     

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.     

Development of albumin microspheres containing Sp H1-DNA complexes: A novel gene delivery system

This study investigated the stability and transfection efficiency of plasmid DNA (pDNA) and sea urchin sperm histone H1 (Sp H1) complexes embedded in albumin microsphere formulations. Sp H1 increased the stability and transfection efficiency of pDNA, while providing a favourable sustained pDNA release profile. Encapsulating Sp H1-complexed pDNA into albumin microspheres further protected the pDNA from physical stress and heparin treatment. When compared with free pDNA encapsulated in albumin microspheres, the Sp H1-pDNA microsphere formulations exhibited decreased hydrophilicity, slower pDNA release profiles, protection against heparin-induced degradation of embedded pDNA and increased stability against physical stress. These results indicate that complex formation of pDNA with Sp H1 facilitates intracellular DNA transfer and that albumin microspheres-Sp H1-pDNA gene delivery formulations are suitable for controlled-release delivery of pDNA while offering protection of the pDNA from degradation and maintaining pDNA biological activity.     

Effect of sugars on storage stability of lyophilized liposome/DNA complexes with high transfection efficiency

Cationic lipid-based gene delivery systems have shown promise in transfecting cells in vitro and in vivo. However, liposome/DNA complexes tend to form aggregates after preparation. Lyophilization of these systems, therefore, has become of increasing interest. In this study, we investigated the feasibility of preserving complexes as a dried preparation using a modified dehydration rehydration vesicle (DRV) method as a convenient and reliable procedure. We also studied storage stability of a lyophilized novel cationic gene delivery system incorporating sucrose, isomaltose and isomaltotriose. Liposomes were composed of 3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and L-dioleoylphosphatidylethanolamine (DOPE), plus sucrose, isomaltose or isomaltotriose. Lyophilized liposome/DNA complexes were stored at -20, 25, 40 and 50 degrees C and their stability was followed for 50 days. Liposome/DNA complexes with sucrose could be stored even at 50 degrees C without large loss of transfection efficiency. The transfection efficiency of formulations stored at various temperatures indicated that the stabilizing effect of sugars on plasmid DNA was higher in the following order: isomaltotriose相似文献   

Hyaluronic acid-modified redox-sensitive hybrid nanocomplex loading with siRNA for non-small-cell lung carcinoma therapy

A novel hyaluronic acid (HA)-modified hybrid nanocomplex HA-SeSe-COOH/siR-93C@PAMAM, which could efficiently deliver siRNA into tumor cells via a redox-mediated intracellular disassembly, was constructed for enhanced antitumor efficacy. Thereinto, siR-93C (siRNA) and positive PAMAM were firstly mixed into the electrostatic nano-intermediate, and then diselenide bond (-SeSe-)-modified HA was coved to shield excessive positive charges. This hybrid nanocomplex displayed uniform dynamic sizes, high stability, controlled zeta potential and narrow PDI distribution. Moreover, the -SeSe- linkage displayed GSH/ROS dual responsive properties, improving intracellular trafficking of siRNA. In vitro assays in A549 cell line presented that HA-SeSe-COOH/siR-93C@PAMAM has low cytotoxicity, rapid lysosomal escape and significant transfection efficiency; besides, an efficient proliferation inhibition ability and enhanced apoptosis. Furthermore, in animal studies, this negative-surfaced hybrid nanocomplex showed a prolonged circulation in blood and improved inhibition of tumor growth. All these results verified our hypothesis in this study that diselenide bonds-modified HA could promote not only stability and safety of nanoparticles in vivo but also intracellular behavior of siRNA via redox-dual sensitive properties; furthermore, this hybrid nanocomplex provided a visible potential approach for siRNA delivery in the antitumor field.     

Preparation and characterization of spray dried inhalable powders containing chitosan nanoparticles for pulmonary delivery of isoniazid

The aim of this study was to prepare spray dried inhalable powders containing isoniazid-loaded chitosan/tripolyphosphate (TPP) nanoparticles for sustained delivery of the drug to the lung. Nanoparticles were prepared by ionic gelation method. In-vitro drug release study indicated that the rate of drug release from nanoparticles was decreased by increasing the amount of chitosan. Entrapment of isoniazid into chitosan/TPP nanoparticles decreased minimum inhibitory concentrations (MIC) of the drug against mycobacterium avium intracellulare. Nanoparticles were spray dried using excipients such as lactose, mannitol and maltodextrin alone or with leucine. Results showed that the obtained powders had different aerosolization property. It was observed that by adding leucine, the particle size of microparticles deceased and the process yield and fine particle fraction (FPF) increased significantly. The in-vitro deposition data indicated that spray drying of isoniazid-loaded nanoparticles with lactose in the presence of leucine resulted in the production of inhalable powders with the highest FPF (45%).     

Chitosan-based formulations for delivery of DNA and siRNA

Among non-viral vectors, chitosan and chitosan derivatives have been developed in vitro and in vivo for DNA and siRNA delivery systems because of their cationic charge, biodegradability and biocompatibility, as well as their mucoadhesive and permeability-enhancing properties. However, the transfection efficiency of chitosan is too low for clinical application. Studies indicated that the transfection efficiency depends on a series of chitosan-based formulation parameters, such as the Mw of chitosan, its degree of deacetylation, the charge ratio of chitosan to DNA/siRNA (N/P ratio), the chitosan salt form used, the DNA/siRNA concentration, pH, serum, additives, preparation techniques of chitosan/nucleic acid particles and routes of administration. In this paper, chitosan-based formulations for the delivery of DNA and siRNA were reviewed to facilitate the process of chitosan vector development for clinical application. In addition to formulation optimization, chitosan structure modification or additive incorporation is an effective way to improve the stability of the polyplex in biological fluids, enhance targeted cell delivery and facilitate endo-lysosomal release of the complex. In summary, the transfection efficiency of chitosan-based delivery systems can be adjusted by changing formulation-related parameters.     

Microparticle size control and glimepiride microencapsulation using spray congealing technology

Drug-free microparticles were prepared using a spray congealing process with the intention of studying the influence of processing parameters. By varying the atomizing pressure and liquid feed rate, microparticles with median sizes (d_(0.5)) from 58 to 278 μm were produced, with total process yields ranging from 81% to 96%. An increased liquid feed rate was found to increase microparticle size, and higher atomizing pressures were found to decrease microparticle size. Greater change in microparticle size was achieved by varying atomizing pressure, which can be considered a dominant process parameter regarding microparticle size. In addition, microparticles with glimepiride, a model poorly water-soluble drug, were prepared by spray congealing using three different hydrophilic meltable carriers: Gelucire^® 50/13, poloxamer 188, and PEG 6000. Spherical microparticles with relatively smooth surfaces were obtained, with no drug crystals evident on the surfaces of drug-loaded microparticles. XRPD showed no change in crystallinity of the drug due to the technological process of microparticle production. All glimepiride-loaded microparticles showed enhanced solubility compared to pure drug; however, Gelucire^® 50/13 as a carrier represents the most promising approach to the dissolution rate enhancement of glimepiride. The influence of storage (30 °C/65% RH for 30 days) on the morphology of glimepiride/Gelucire^® 50/13 microparticles was studied, and the formation of leaf-like structures was observed (a “blooming” effect).     

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%).