Comparison of In vitro Nanoparticles Uptake in Various Cell Lines and In vivo Pulmonary Cellular Transport in Intratracheally Dosed Rat Model
© to the authors 2008
Received: 7 July 2008
Accepted: 18 August 2008
Published: 9 September 2008
In present study, the potential drug delivery of nanoformulations was validated via the comparison of cellular uptake of nanoparticles in various cell lines and in vivo pulmonary cellular uptake in intratracheally (IT) dosed rat model. Nanoparticles were prepared by a bench scale wet milling device and incubated with a series of cell lines, including Caco-2, RAW, MDCK and MDCK transfected MDR1 cells. IT dosed rats were examined for the pulmonary cellular uptake of nanoparticles. The processes of nanoparticle preparation did not alter the crystalline state of the material. The uptake of nanoparticles was observed most extensively in RAW cells and the least in Caco-2 cells. Efflux transporter P-gp did not prevent cell from nanoparticles uptake. The cellular uptake of nanoparticles was also confirmed in bronchoalveolar lavage (BAL) fluid cells and in bronchiolar epithelial cells, type II alveolar epithelial cells in the intratracheally administrated rats. The nanoparticles uptake in MDCK, RAW cells and in vivo lung epithelial cells indicated the potential applications of nanoformulation for poorly soluble compounds. The observed limited direct uptake of nanoparticles in Caco-2 cells suggests that the improvement in oral bioavailability by particle size reduction is via increased dissolution rate rather than direct uptake.
KeywordsCellular uptake Nanoparticles Intratracheally dosed rat model P-glycoprotein
In association with slow dissolution characteristics, poor permeability and/or the involvement of efflux transporters, poorly aqueous soluble/permeable drugs present a challenging problem for drug formulation development due to the limitation of drug absorption in the gastrointestinal (GI) tract. In an environment of ever increasing drug entities with these characteristics where conventional formulation techniques are not efficient to develop poorly water-soluble compounds into drug products , novel approaches to overcome these factors are of great importance. Among the various solubility/dissolution rate enhancement methodologies available, particle size reduction is most commonly employed by formulators to improve bioavailability. Particle size reduction leads to increased dissolution and solubility characteristics and offers improvement in bioavailability as outlined by the Ostwald-Feundlich equations . In addition, size reduction to the nanometer range of 10–1000 nm, termed nanoparticles , has been shown to greatly improve exposure . An outstanding feature of nanoparticles is the greater surface area consequently resulting in the increase in saturation solubility and the increase in dissolution rate of the compounds. Recently, nanoparticles have been reported to cross the intestinal epithelial barrier or rapidly diffuse from the lungs into the systemic circulation [5, 6]; however, the route, mechanism and extents to which this occurs are not yet entirely clear.
The pharmaceutical industry has invested heavily in the area of non-invasive delivery systems for GI poorly absorbed or unstable molecules. One of the most important aspects of systemic or local drug delivery routes has been targeting drug delivery into the lungs. Accordingly, techniques and new drug delivery devices intended to deliver drugs into the lungs have been widely developed in the last few years. Cellular uptake studies have demonstrated that besides macrophages, other cell lines like cancer cells and epithelial cells are also able to take up nanoparticles [7–9]. A hypothesis, which has not been widely investigated so far, is that the variations of nanoparticles uptake in vivo are observed in different tissue or cell barriers. To elucidate the hypothesis, in this study, we investigated the uptakes and transport of water-insoluble nanoparticles in various cell lines and in a nanoparticle IT injected rat model.
Materials and Methods
For particle size reduction, a bench scale wet milling (micronization) device was used . To make the stock nanosuspension formulation (20 mg/mL) pyrene (GC grade from Fluka Chemical, Switzerland) or charcoal (acid washed with hydrochloric acid, cell culture tested, Sigma-Aldrich), an appropriate amount of glass beads, and 0.1% (w/w) Tween 80 in phosphate buffered saline (pH 7.4) were added in a scintillation vial. The mixture was then stirred at 1200 rpm for a period of 48 h with occasional shaking. The stock formulation was harvested and the potency of suspension and supernatant were examined by HPLC/UV.
Evaluation of Solid State Properties
Powder X-ray diffraction (PXRD) was used to confirm the solid state properties pre- and post-milling of pyrene, and conducted with a Bruker D-8 Advance diffractometer. The system used a copper X-ray source maintained at 40 kV and 40 mA to provide radiation with intensity weighted average of 1.54184 Å (Kαave). A scintillation counter was used for detection. Data were collected using a step scan of 0.02° per point with a 1 s/point counting time over a range of 3°–35° two-theta. In-house fabricated aluminum inserts or inserts with a Hasteloy sintered filter (0.45 μm) pressed in the center and held in Bruker plastic sample cup holders were utilized for all analyses. Dry pyrene was run as received without hand grinding. Suspensions were filtered onto sintered filters under vacuum. Particle size distribution was determined on a Beckman Coulter LS 230 particle size analyzer using a small volume accessory. Distributions from 2000 μm to 0.04 μm were generated using Mie scattering theory and a polarization intensity differential scattering obscuration optical model (PIDS) with sample obscurations held between 45% and 55%. There was no absorption by pyrene at the scattering wavelengths so the average index of refraction was determined by optical microscopy using index matching fluids (Cargille catalog #18005).
Caco-2 cells (Pfizer Global batch) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, 1% non-essential amino acids, 1% Glutamax, 1 mM sodium pyruvate, and 0.06 mg/mL Gentamicin. MDCK and MDCK-MDR1 cells were cultured in minimum essential medium (MEM) with Earle’s salts andl-glutamine containing 10% fetal bovine serum (FBS), 100 units penicillin, and 100 μg/mL streptomycin. The murine macrophage-like cells (RAW cells, ATCC TIB 71) were cultured in DMEM supplemented with 4 mMl-glutamine and 10% (v/v) FBS. All media and reagents were obtained from the Gibco BRL (Carlsbad, CA).
Cells were seeded at a density of 1 × 106cells/mL in a glass chamber slide (Nalge Nunc International, NY) with regular changes in media. The uptake experiments were conducted after cell reaching confluence in a chamber slide. For nanoparticle uptake, the cells were washed with fresh medium and medium was replaced with nanoparticle suspension (0.05 mg/mL). The cells then were incubated at 37 °C in a humidified 5% CO2/95% air atmosphere. At 2 and 4 h post-incubation, the glass slide chambers were completely washed with HBSS buffer to remove the non-specific binding particles. The cells were fixed with either 10% paraformaldehyde (for Pyrene nanoparticles) for 30 min or the fixative from the Diff-Quik staining kits (for charcoal nanoparticles). The fixed cells were stained with the Diff-Quik following the manufacturer’s instruction (Dade Behring Inc, DE). Microscopic analysis was conducted on a Nikon E600 polarizing microscope equipped with a Nikon DXM 1200 digital camera and filters for light polarization.
Intratracheally Instilled Nanoparticles
Male Sprague Dawley (SD) rats (~350–400 g) from Charles Rivers Labs were anesthetized with 4–5% Isoflurane anesthesia for oro-tracheal administration of 0.5% Tween 80 vehicle and nano-suspension (4 mg/rat). The rats were positioned to allow visualization of their vocal cords and trachea using an otoscope. A Hamilton syringe was used to inject 100 μL of pyrene nanosuspension directly into the trachea. At 30 min and 120 min after oro-tracheal dosing, rats were euthanized with 30 mg/kg pentobarbitol (Sleepaway) injection intraperitoneally. The throat was incised exposing the trachea and a cannula inserted for bronchoalveolar lavage (BAL). BAL fluid collection was performed through four instillations of 2.5 mL each, 10 mL in total. After each BAL was recovered, the fluid was placed in a 15 mL conical tube on ice. The BAL fluid was centrifuged at 900g for 15 min at 4 °C to precipitate the cells. After being placed on glass slides, the cells were fixed with 10% paraformaldehyde for 30 min and then stained with the Diff-Quik kit. A similar protocol was conducted for charcoal nanoparticles and was followed by the histopathological examination. The Pfizer Institutional Animal Care and Use Committee (IACUC) reviewed and approved the animal use in these studies. The Association for Assessment and Accreditation of Laboratory Animal Care, International fully accredits the Pfizer animal care and use program.
At necropsy, the entire lung pluck with trachea was removed. Lung lobes were instilled with approximately 10 mL of 10% neutral buffered formalin (NBF). The trachea was clamped with a bull dog style clip to retain instilled formalin throughout fixation. Lungs were fixed for 24 h in 10% NBF. Lung lobes were cassetted individually to maintain identity and processed whole on a Sakura VIP 5 series by dehydrating through a series of graded ethanol solutions, cleared with xylene and impregnated with paraffin. Lung lobes were embedded ventral aspect down in block. Sections of 4 μm thickness were cut to expose intrapulmonary airways for each lung lobe. Sectioned tissues were heated in a 60° oven for a minimum of 1 h, stained via automated linear stainer with hematoxylin–eosin and coverslipped. The processed glass slides were evaluated under Olympus light microscope and the images were captured by Spot Insight Firewire Camera and analyzed by Spotsoftware Advanced (Diagnostic Instruments, Inc., Sterling Heights, MI).
Results and Discussion
Wet Milling Preparation and the Solid State Properties of Nanoparticles
Nanoparticles Uptake in Epithelial Cell Lines
The oral bioavailability of a poorly absorbed molecule can be improved by size reduction to the nanoparticle range. Leroux et al.  have demonstrated significant improvement in bioavailability for HIV-1 protease inhibitors using pH sensitive nanoparticles, as the smaller particle size can increase surface area resulting in an increased dissolution rate and bioavailability. Transmucosal passage of microparticles from the intestinal lumen to the systemic circulation has been also observed . In addition, by reducing the size of particles to the sub-micron level, enhanced uptake of intact polymeric particles was observed in pre-clinical experiments [14, 15]. However, the relevant importance and mechanism of directly cellular nanoparticles uptake in overall improvement of drug absorption or targeting delivery remains unclear.
We know that ATP binding cassette (ABC) transporters are present in virtually every cell and they play a central role in physiology. They may be pivotal in the protection of against xenobiotics entering the organs or cells . Moreover, multiple efflux transporters are identified in Caco-2 cell and intestinal epithelial cells, and are responsible for restricting intestine absorptions for their substrates . However, the effects of efflux transporter(s) on nanoparticle uptake remain unknown. Recently, MDCK cells genetically modified to overexpress human P-glycoprotein (P-gp) have been shown to effectively discriminate compounds that cross the BBB but are not P-gp substrates from those that cross the BBB but are P-gp substrates. This, along with the ability to assess P-gp transport, makes them a very useful in vitro tool to assess the BBB permeation of compounds and the extent of outwardly directed active efflux . In addition, the delivery of pulmonary drugs to the site of action may also depend on the presence and activity of many ABC transporters . Even though there is no direct evidence showing that efflux transporters might prevent nanoparticles uptake from the cells, it would be of further interest to investigate if the efflux mechanism on the cells barriers (e.g. BBB or chemotherapy) could be bypassed by using nanoparticles as a carrier system to enhance uptake of the agents which are otherwise poorly deposited because of the transporter mediated efflux . To test the hypothesis, a similar experiment protocol was applied to a MDCK-MDR1 cell that was genetically engineered to overexpress P-gp. Not surprisingly, a similar pattern of nanoparticle uptake was found in both the MDCK and MDCK-MDR1 cells (Fig. 3). Significant MDCK and MDCK-MDR1 cell engulfment of nanoparticles suggested that nanoformulation might be a useful tool to overcome the BBB and/or efflux transporters in chemotherapy via transcytosis mechanism.
Nanoparticles Uptake in Mouse Macrophage Cells
Pulmonary Cellular Uptake of Nanoparticles in Intratracheally Dosed Rat Model
In conclusion, while the uptake of small molecule, crystalline nanoparticles in caco-2 cells, which represents a GI absorption model, was limited, a greater uptake was observed in MDCK and MDCK cells overexpressing P-gp. In addition, extensive uptake was observed in mouse macrophage-like RAW cell, suggesting that nanoparticle uptake is highly dependent on the cell type. The improvement of oral bioavailability by particle size reduction is via increased dissolution rate rather than direct uptake; however, the approach of nanoparticle delivery might further improve efficacy and practicability of inhaled delivery and/or to overcome efflux transporter barriers in chemotherapy or CNS delivery.
Madin-Darby canine kidney epithelial cell
- RAW cell:
The murine macrophage-like cell lines
Human colon adenocarcinoma cell
We would like to thank Drs. Timothy G Heath and Joeseph Fleishaker for their helpful comments and suggestions on our study.
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