Chitosan-Modified Dry Powder Formulations for Pulmonary Gene Delivery
Spray-drying is an effective process for preparing micron-dimensioned particles for pulmonary delivery. Previously, we have demonstrated enhanced dispersibility and fine particle fraction of spray-dried nonviral gene delivery formulations using amino acids or absorption enhancers as dispersibility-enhancing excipients. In this study, we investigate the use of the cationic polymer chitosan as a readily available and biocompatible dispersibility enhancer.
Lactose-lipid:polycation:pDNA (LPD) powders were prepared by spray-drying and post-mixed with chitosan or spray-dried chitosan. In addition, the water-soluble chitosan derivative, trimethyl chitosan, was added to the lactose-LPD formulation before spray-drying.
Spray-dried chitosan particles, displaying an irregular surface morphology and diameter of less than 2 μm, readily adsorbed to lactose-LPD particles following mixing. In contrast with the smooth spherical surface of lactose-LPD particles, spray-dried trimethyl chitosan-lactose-LPD particles demonstrated increased surface roughness and a unimodal particle size distribution (mean diameter 3.4 μm), compared with the multimodal distribution for unmodified lactose-LPD powders (mean diameter 23.7 μm). The emitted dose and in vitro deposition of chitosan-modified powders was significantly greater than that of unmodified powders. Moreover, the inclusion of chitosan mediated an enhanced level of reporter gene expression.
In summary, chitosan enhances the dispersibility and in vitro pulmonary deposition performance of spray-dried powders.
Key Wordschitosan dry powder inhaler nonviral vector spray-drying
Dry powder inhalers (DPI) have shown promise for the delivery of nucleic acids to the lung (1, 2, 3, 4). Previous studies have verified that a combination of nonviral gene therapy vector and thermoprotectant sugar carrier can maintain the structural integrity and biological activity of plasmid DNA (pDNA) following dry powder processing via lyophilization or spray-drying (5,6). Although spray-dried particles containing the well-characterized lipid:polycation:pDNA (LPD) nonviral gene vector (7) have been prepared with particle diameter within the respirable range (<5 μm), aggregation between particles provides reduced performance during the process of dose release, aerosolization, and subsequent lung deposition (5). In previous reports, amino acids, such as leucine and phenylalanine (2,3), and pulmonary absorption enhancers, particularly dimethyl-β-cyclodextrin (DMC) (4), have been shown to significantly enhance the dispersibility and in vitro pulmonary deposition of spray-dried sugar-LPD powders when incorporated as co-excipients. Unfortunately, many of these additives caused concomitant damage to the physical stability and biological activity of the LPD complex and consequently decreased the gene transfection and expression efficiency in cell cultures (2,3). As an exception, DMC was shown to increase the emitted dose, particle dispersibility, in vitro pulmonary deposition, and cell transfection efficiency of spray-dried LPD powders. Clearly, certain compounds may have the ability to enhance the dispersibility of spray-dried powders and improve the pDNA uptake and gene expression in targeted cells (4).
Chitosan (1,4-2-amino-2-deoxy-b-d-glucan) is a linear biodegradable polymer derived from the natural polymer of chitin, one of the most abundant polysaccharides in nature. Chitosan has been applied extensively in the agriculture, food, cosmetic, and pharmaceutical industries due to its exceptional biocompatibility, ready availability, and ease of chemical modification (8,9). The use of chitosan and its derivatives as cellular absorption enhancers has been comprehensively reported, with the results demonstrating that chitosan and its synthetic derivatives, particularly N-trimethyl chitosan (TMC) (10), can significantly augment the absorption of peptides, proteins, vaccines, and antibiotics in vitro (e.g., Caco-2, TR146) (11,12) or through various in vivo barriers including nasal (13), intestinal (14), urinary bladder wall (15), respiratory tract (16), ocular (17), and buccal mucosa (18).
Chitosan and its derivatives have also been widely investigated as nonviral gene carrier systems (19). For this purpose, chitosan or its derivatives are complexed with pDNA to form nanosized particulates for gene delivery to the target cells. It has been shown that chitosan materials can effectively condense DNA to form complexes with stable physiochemical properties (20,21). Furthermore, chitosan and its derivatives used in this way are reported to have excellent biocompatibility (22), as also observed in studies using chitosan as an absorption enhancer (23).
Although chitosan and its derivatives have been extensively studied both as absorption enhancers and as nonviral gene vectors, their exploitation in dry powder formulations specifically for the enhancement of powder dispersibility, airflow, and deposition has not been reported. In this paper, chitosan, spray-dried chitosan, and the water-soluble synthetic derivative TMC are employed as “dispersibility enhancers” in dry powder formulations, with the aim of increasing the functional lung deposition of the LPD vector. We demonstrate that post-mixing commercially available chitosan with spray-dried lactose-LPD powders, post-mixing spray-dried chitosan with lactose-LPD spray-dried powders, or, most simplistically, adding TMC into the spray-drying solution to prepare the spray-dried powders improves the dry powder capsule emission, dispersibility, and resulting in vitro pulmonary deposition. In addition, augmented cellular gene expression was observed in chitosan-modified preparations.
Materials and Methods
1,2-Dioleoyl-3-trimethylammoniumpropane (DOTAP) was purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Protamine sulfate (Grade X from salmon sperm) was obtained from Sigma-Aldrich Chemicals (Poole, UK). The 4.7-kbp pEGFP-N1 plasmid construct containing the green fluorescent protein (GFP) reporter gene was propagated and purified as detailed previously (24), using kanamycin to ensure selective growth of transformed bacteria.
The following chemicals were used as received: agarose LE (Promega, Southampton, UK); ethidium bromide (EtBr) solution, sodium dodecyl sulphate (SDS) (Pharmacia Biotech, St. Albans, UK); Tris base, Luria–Bertani (LB) agar, LB broth (Sigma-Aldrich); Chitosan (Chitoclear™, ∼100 kDa, Primex, Iceland). The soluble chitosan derivative trimethyl chitosan (TMC) (30% degree of substitution, ∼100 kDa) was kindly donated by Dr. Maya Thanou (formerly Welsh School of Pharmacy). Cell culture plastics were purchased from Corning-Costar (High Wycombe, UK). Dulbecco's modified Eagle's medium [DMEM; 25 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES)], fetal bovine serum, penicillin–streptomycin solution, and trypsin-ethylenediaminetetraacetic acid (EDTA) solution 1× were obtained from Life Technologies (Paisley, UK). All other chemicals were obtained from Fisher Scientific UK Ltd. (Leicestershire, UK) and were of analytical grade.
Preparation of LPD Complexes
The LPD complexes were prepared as detailed previously (5). Briefly, protamine sulphate solution (1 mg/mL) andextruded DOTAP liposomes (1 mg/mL) were added sequentially, with 10-min incubation at each step, to plasmid DNA(pEGFP-N1; 1 mg/mL stock solution) to achieve an LPD complex with a 3:2:1 mass ratio. Complexes were freshly prepared before use.
Preparation of Spray-Dried Powders
Lactose-LPD powders: LPD complex preparations comprising pDNA 400 μg, protamine sulphate 800 μg, and DOTAP 1200 μg were added to 3% w/v lactose solution (50 mL total volume) and spray-dried (Büchi B-191 mini spray-dryer, Büchi Labortechnik AG, Flawil, Switzerland) to produce a dry powder with a typical yield of approximately 30%. Standard operating conditions employed for spray-drying were inlet temperature 150°C (outlet temperature between 83 and 85°C), spray flow rate 600 L/h, aspirator setting 35 m3/h, and pump setting 450 mL/h.
Chitosan: Spray-dried chitosan powders were prepared by spray-drying 0.2% chitosan solution, pH 5.0 adjustment with acetic acid, under the abovementioned standard operating conditions.
TMC-lactose-LPD powders: The chitosan derivative TMC was added at concentrations of 0.003, 0.03, 0.1, and 0.3% (w/v) to a 3% w/v lactose solution containing LPD complexes (pDNA 400 μg, protamine sulphate 800 μg, and DOTAP 1200 μg). Dry powders were prepared by spray-drying under the standard operating conditions.
Lactose-LPD spray-dried powders were post-mixed with chitosan or spray-dried chitosan in a pestle and mortar for 60 s. Initial assessments used chitosan to lactose mass ratios ranging from 0.1:1 to 1:1.
Scanning Electron Microscopy
Selected spray-dried powders were sputter-layered with gold under partial vacuum (EMScope®; EMScope Laboratories, Ashford, UK). Representative scanning electron micrographs of the powders were taken using a Philips XL20 (Philips, Eindhoven, the Netherlands) scanning electron microscope.
Particle Size Analysis
Laser diffraction (Mastersizer 2000; Malvern Instruments, Malvern, UK) was used to determine the particle size of dry powders using a dry dispersion technique in air. Approximately 200 mg powder per sample was used to achieve the required obscuration, i.e., amount of laser light lost due to the introduction of the sample within the analyzer beam, of 0.5–5%.
Agarose Gel Electrophoresis
Gel electrophoresis was used to assess the degree of physical protection afforded to pDNA during the spray-drying process through complexation with the polycationic peptide protamine and the cationic lipid DOTAP, and the influence of chitosan and its derivative on physical stability of the LPD complex. A 1% agarose gel was prepared in 0.5× Tris-borate EDTA (TBE) buffer and immersed in 0.5× TBE buffer containing 0.5 μg/mL EtBr. Dry powders were reconstituted and mixed with gel loading buffer (10× strength, blue-orange dye containing 0.25% bromophenol blue, 40% glycerol in TBE buffer) and added to the wells in the gel at a volume equivalent to 1 μg pDNA per well. Equivalent samples were incubated with a digestion buffer (100 mM NaCl, 10 mM Tris–HCl pH 8, 25 mM EDTA pH 8, 0.5% SDS, 0.1 mg/mL proteinase K) for 35 min at 50°C to liberate pDNA from the complexing moieties. The Wizard® DNA clean-up system (Promega, Madison, WI, USA) was used to separate the released pDNA, and samples equivalent to 1 μg pDNA were loaded onto the gel. The gel was run at 100 V for 1 h and then visualized under UV light. Quantification of pDNA was carried out using Molecular Analyst® software (Bio-Rad Gel Doc 1000; Bio-Rad Laboratories, Hercules, CA, USA).
In Vitro Cell Transfection Efficiency of Dry Powders
A549 human lung epithelial carcinoma cells (European Collection of Animal Cell Cultures, Salisbury, UK) were cultured in DMEM with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin 5000 IU/mL at 37°C in a humidified atmosphere at 95% air/5% CO2. Cells were seeded onto 24-well plates (40,000 cells/cm2) and grown to 85% confluency. For transfection, cells were washed twice with prewarmed phosphate buffered saline (PBS) and exposed to selected spray-dried powders (equivalent to 5 μg pDNA per well) reconstituted in serum-depleted DMEM. The plates were incubated at 37°C for 6 h, then the surfaces were rinsed thoroughly with PBS and the cells fed with 1 mL culture medium, and returned to the incubator for a further 42 h to allow intracellular expression of the reporter plasmid (pEGFP-N1) to proceed.
In Vitro Powder Aerosolization and Deposition
The dose emission of selected spray-dried powders was determined using a one-stage impinger consisting of a United States Pharmacopeia (USP) throat attached to a pump via a filter. Powders were loaded into size 2 gelatin capsules and placed into a Spinhaler® (Fisons, Bedford, MA, USA) dry powder inhaler, located in the mouthpiece of the USP throat. The powder was aerosolized using a flow rate of 60 L/min for 5 s, corresponding to a single deep inhalation, under a controlled environment (35% relative humidity, 20°C). The percentage of powder emitted from the capsule was determined gravimetrically as the percentage of the total loaded dose released during aerosolization.
The in vitro deposition performance of the powders was characterized using a Multistage Liquid Impinger (MSLI; Westech Instrument Services Ltd., Beds, UK), as outlined previously (2, 3, 4). Briefly, powders were loaded into size 2 gelatin capsules and placed into a Spinhaler® dry powder inhaler that was attached to the MSLI via a stainless steel USP throat. Each stage of the MSLI was filled with 20 mL DMEM. The powder was aerosolized at a flow rate of 60 L/min for 5 s under a controlled environment (35% relative humidity, 20°C). Ten capsules (equivalent to 133 μg pDNA) were aerosolized into the MSLI, and the DMEM at each stage was removed for analysis. The capsule and the MSLI throat and filter were washed with DMEM (20 mL respectively). A 4-mL aliquot of the collected DMEM was placed onto A549 cells at 85% confluency in six-well cell culture plates to determine gene expression. The plates were incubated at 37°C for 6 h, then the surfaces were rinsed thoroughly with PBS and the cells fed with 4 mL culture medium, and returned to the incubator for a further 42 h as above.
Flow Cytometric Quantification of Gene Expression
Following surface rinsing with PBS, transfected cells were trypsinized with 0.3 mL trypsin-EDTA solution and resuspended in 0.6 mL culture media (DMEM/10% FBS/antibiotics). The percentage of cells displaying GFP-associated fluorescence (FL1-H) was quantified by flow cytometry (FACScan; Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) with analysis by WinMDI Software (Joseph Trotter, The Scripps Institute, La Jolla, CA, USA), as described previously (2,5).
Yield of Spray-Dried Lactose-LPD, Chitosan, and TMC-Lactose-LPD Powders
Effect of Concentration of TMC on Dry Powder Yield and Emitted Dose Following Spray-Drying
Mass TMC in solution (% w/v)
Spray-drying yield (%)
Emitted dose (%)
Significant difference (P < 0.05)
Dose Emission of Dry Powder Formulations
The emitted dose is defined as the percentage of powder loaded into the capsule that is emitted during aerosolization. The influence of addition of the chitosan derivative, TMC, on the emitted dose of the powders was investigated. Lactose-LPD capsule emission was inefficient following aerosolization, with an emitted dose of only 57.0%. In contrast, all powders containing TMC achieved an improved level of powder emission, with formulations containing 0.1% (w/v) TMC producing an optimal emitted dose of 90.8%. Consequently, this ratio of TMC to lactose (mass ratio 1:30) was employed in subsequent studies.
Spray-dried chitosan powders displayed a small particle diameter and irregular surface morphology (Fig. 2D). On mixing these particles with spray-dried lactose particles, relatively small discrete aggregates are formed (Fig. 2E). In common with the chitosan/lactose aggregates (Fig. 2C), these aggregates are segregated and are suggestive of improved powder flow properties.
In contrast with the spherical lactose-LPD particles, spray-dried TMC-lactose-LPD particles demonstrated altered surface morphology. The particles contained surface depressions and increased surface roughness (Fig. 2F), consistent with powders prepared using certain amino acids (3) and absorption enhancers (4) as co-excipients.
Particle Size Distribution of Dry Powders
Particle Size Distribution Data of Dry Powders Analyzed by Laser Diffraction
Spray-dried lactose-LPD + chitosan
Spray-dried lactose-LPD + spray-dried chitosan
To further investigate the effects of chitosan on pDNA structural integrity, equivalent LPD powder samples were incubated with digestion buffer to liberate pDNA from the condensing moieties, and the purified extracts were also analyzed by gel electrophoresis. The fluorescent bands corresponding to released pDNA fell in the linear response range of the gel documentation software (Molecular Analyst®, Bio-Rad Laboratories) allowing semiquantification. The fluorescent signal in lane 2, attributed to pDNA released from the unmodified lactose-LPD spray-dried powder following digestion and purification (Wizard® DNA clean-up system), was approximately 80% (densitometric analysis) of the fluorescent signal associated with pDNA liberated from non-spray-dried LPD complexes (not shown). This reduction in pDNA signal, also observed in our previous experiments (2, 3, 4, 5), implies that the elevated processing temperature (spray-dryer inlet temperature, 150°C; outlet temperature, 83–85°C) causes the damage to the pDNA that thereafter partially loses its biological activities. Lanes 4 and 6 show pDNA signal, following digestion and purification, of post-mixed formulations of chitosan with lactose-LPD spray-dried powders and spray-dried chitosan with lactose-LPD spray-dried powders, respectively. As with the spray-dried lactose-LPD powders, approximately 80% of the fluorescent signal is preserved, suggesting that further mixing with chitosan does not diminish the stability of the LPD complex. Addition of the soluble chitosan derivative TMC into the spray-drying solution also did not adversely affect stability of the LPD complex, with a retained fluorescent signal of 82% (lane 8).
In Vitro Transfection Efficiency
In Vitro Pulmonary Deposition
Following aerosolization of spray-dried lactose-LPD powders, maximum gene expression was observed in cells treated with capsule washings, confirming significant retention of lactose-LPD powder in the capsule following inhalation (Fig. 1). Subsequent deposition of the aerosolized lactose-LPD powders was mainly restricted to the throat and upper stages of the MSLI (corresponding to deposition sites in the upper airway), with insignificant powder deposition in stages 3 and 4 and in the device filter (related to the secondary bronchi, terminal bronchi, and alveoli regions). Dry powder formulations of spray-dried lactose-LPD containing chitosan, spray-dried chitosan, or TMC were not significantly (one-way analysis of variance, P < 0.05) retained in the capsule following aerosolization, confirming fundamentally enhanced dose emission. The reporter gene expression from the formulation containing spray-dried lactose-LPD powders post-mixed with chitosan was statistically consistent through MSLI stages 1 to 4, with approximately 10% of cells showing reporter gene expression at stage 4, equating to a particle diameter cut-off of 3.1 μm and predicted deposition in the respiratory region of secondary bronchi and below. When spray-dried lactose-LPD powders are post-mixed with spray-dried chitosan, cellular gene expression efficiency in stage 4 is increased to 20%. Spray-dried TMC-lactose-LPD powders also demonstrate improved deposition over unmodified lactose-LPD powders, with the greatest gene expression observed in stage 3, equating to the tracheal and bronchial region of the lung. All chitosan-modified formulations demonstrated enhanced deposition and subsequent gene expression in the lower stages of the MSLI, i.e., up to an 8-fold increase at stage 4.
The total gene expression collected from the capsule to the device filter for each formulation was also calculated (Fig. 7B). The cumulative gene expression data follow the sequence of spray-dried lactose-LPD < spray-dried lactose-LPD and chitosan < spray-dried TMC-lactose-LPD ≪ spray-dried lactose-LPD and spray-dried chitosan and are in close agreement with the in vitro transfection data from Fig. 5, indicating that the inhalation process did not adversely effect the biological activity of the LPD dried powders.
Chitosan is a biodegradable polymer with excellent biocompatibility and has been widely used in drug delivery systems due to its polycationic nature and the ability to further modify the reactive amino group to produce a range of chitosan derivatives. Chitosan and its derivatives have shown to be effective agents for improving the absorption of hydrophilic compounds and large biomacromolecules (i.e., antibiotics, insulin, peptides, and proteins). In this study, chitosan and its derivative TMC were shown to increase the gene expression of lipid/polycation condensed pDNA to human lung carcinoma cells. The mixture of spray-dried chitosan and spray-dried lactose-LPD demonstrated a significantly greater gene expression capability than that of spray-dried lactose-LPD post-mixed with unprocessed chitosan. As the spray-dried chitosan was polarized by acetic acid, this effect may reflect enhanced solubility of spray-dried chitosan in the culture media over commercially available chitosan. It is also possible that the reduced particle size of spray-dried chitosan (<2 μm) permits more disperse interaction with the A549 cells. It is acknowledged that chitosan relieves cellular tight junctions and enhances the transfection of biomacromolecules (26). It is not surprising that the transfection efficiency of spray-dried TMC-lactose-LPD (TMC:lactose = 1:30 w/w) is lower than that of post-mixed spray-dried chitosan with lactose-LPD (chitosan:lactose = 0.8:1 w/w) given the reduced content of chitosan in the formulation.
It should be noted that the degree of substitution in the chitosan polymer chain has an important impact on the biocompatibility of TMC, with cell (e.g., COS-7 and MCF-7) viability decreasing with increasing degree of trimethylation (27). We have tested the cytotoxicity of TMC on A549 cells using the well-characterized MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay (data not shown). Three TMC derivatives were tested, having degrees of trimethylation of 30 (TMC30), 60 (TMC60), and 90% (TMC90), respectively. Following 24 h of incubation, 50 μg/mL of TMC90 causes 70% cell death, whilst the same concentration of TMC60 mediates 34% cell death. A549 cells incubated with TMC30, however, generally remained viable (88.4% cell viability) up to a concentration of 1000 μg/mL. Chitosan derivatives with a limited degree of substitution are therefore more suitable to be used as dispersibility enhancers in spray-dried formulations for inhalation. Thanou et al. (1999) reported that TMC is nontoxic to ciliated embryo trachea when the degree of trimethylation is under 60% (28). Therefore, although further in vivo assessment would be required, including inflammatory response, the available data suggest that TMC30 could be biocompatible when used as a dispersibility enhancer in spray-dried formulations for inhalation.
A well-characterized mechanism for enhancing the dispersibility and deposition of dry powders for pulmonary administration involves the addition of relatively large carrier particles that are subsequently separated from the therapeutic “fines” during inhalation. This mechanism is employed in this paper by post-mixing spray-dried lactose-LPD particles with commercial-grade chitosan, leading to an increased emitted dose and enhanced pulmonary deposition. A second mechanism reported in this paper involves the addition of “co-fines,” e.g., spray-dried chitosan, that adsorb onto the active particles, e.g., spray-dried lactose-LPD, to form weak conglomerates with improved capsule emission and airway dispersion characteristics. The third mechanism reported in this paper involves the addition of a co-excipient before spray-drying to impart modified surface properties to the primary excipient particles. Various compounds, such as leucine, phenylalanine, and cyclodextrin (2, 3, 4), have shown to increase the dispersibility of the dry powders when employed in this fashion. These compounds enhance the flowability of the spray-dried powders through alteration of surface properties such as morphology, surface charge, surface tension, surface cohesion and adhesion, surface energy, or hydrophilicity.
In this study, we show that chitosan and its derivatives affect both powder dispersibility and cell transfection. Whilst the increase in cellular transfection represents an important advantage of these systems, the predominant effect is through dispersibility enhancement, as evidenced by non-spray-dried chitosan improving gene expression at different levels of the simulated lung (Fig. 6) without enhancing gene expression when compared with unmodified lactose-LPD in a basic cell culture environment (Fig. 5). We have also repeated this work using salbutamol as a model low-molecular-weight drug (data not shown) and observed an enhancement of powder dispersibility similar to that shown in this paper. In addition, previous publications using amino acids (3) and absorption enhancers (4) have shown that dispersibility-enhancing excipients that modify the dry powder surface characteristics and aggregation behavior are able to increase nonviral gene transfer to the lower regions of the lung without any effect on cell transfection.
As absorption enhancers, chitosan and its derivatives have been widely studied for therapeutic drug delivery via various administration routes (e.g., oral, nasal); however, their function as dispersibility enhancers in dry powder formulations for pulmonary deposition has not been reported previously. Our results demonstrate that both chitosan and its soluble derivative TMC can significantly increase the emitted dose (>90%) and the in vitro pulmonary deposition (FPF >35%) of dry powder dispersions. Given their additional value in enhancing cellular uptake of materials and their impressive biocompatibility, these materials demonstrate potential for inclusion in dry powder formulations for the pulmonary deposition of pharmaceutically active agents.
The authors are grateful for financial support from The Cardiff Young Researcher Initiative. Many thanks are also given to Dr. Maya Thanou for her kind donation of chitosan derivatives.
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