Alginate and chitosan particles as drug delivery system for cell therapy
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- Ciofani, G., Raffa, V., Menciassi, A. et al. Biomed Microdevices (2008) 10: 131. doi:10.1007/s10544-007-9118-7
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Drug-carrying microstructures which have a size similar to biological structures are very attractive to encapsulate drugs and protect them during the transit in the human body. This paper describes polymeric (alginate and chitosan) particles (average radius 500 nm) produced by homogenization techniques. In vitro studies performed on cell lines demonstrate the effectiveness of such particles for intracellular drug delivery. Our experiments suggest that cellular up - take increases linearly with particle concentration in the growth medium, and the internalization process has a first order kinetics (characteristic time around 0.5 h−1). In addition, the particles degrade within 24 h from the up-take without side effects for cell viability.
KeywordsDrug delivery systemAlginate particlesChitosan particlesCellular up-take
Micro- and nano-scale drug delivery systems are ideal candidates to provide breakthrough therapeutic solutions (Ferrari 2005). Being of the same size as biological entities, they have the ability to enter cells, as cells typically internalize materials with sizes in the range of tens to hundreds of nanometers (Gao et al. 2005).
In the past few decades, particulate drug delivery systems have attracted a considerable research interest as carriers for small and large molecules. Particulate systems have been used as a physical approach to alter and improve the pharmacokinetic and pharmacodynamic properties of various types of drug molecules. They have been used in vivo to protect the drug entity in the systemic circulation, to restrict the drug access to the target site and to deliver it at a controlled and sustained rate. Various polymers have been used in the formulation of particles for drug delivery research to increase therapeutic benefit, while minimizing side effects (Zhang et al. 2001; Jahanshahi et al. 2004).
Particles are defined as particulate dispersions or solid particles with a size in the range between 10 and 1,000 nm. The drug is dissolved, entrapped, encapsulated or attached to the nanoparticle matrix (Mohanraj and Chen 2006).
Particles formulated from biocompatible and biodegradable polymers are being extensively investigated for different therapeutic applications, such as for sustained drug, vaccine and gene delivery (Moghimi et al. 2001). For a number of these applications, it is important to study the kinetics of cellular and tissue up-take, intracellular distribution and retention, and in vivo biodistribution (Panyam and Labhasetwar 2003). The need for particles as biodegradable and non toxic drug delivery system was firstly formulated in 1978 by Marty (Marty et al. 1978). Since then, numerous synthetic and natural polymers were adopted for the production of biodegradable micro- and nanoparticles. Poly-ɛ-caprolactone (PCL), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and their co-polymers poly(lactide-co-glycolide) (PLGA) and natural polymers such as proteins (albumin and gelatin) and polysaccharides (dextran, alginate, and chitosan) are the most widely used starting materials (Hans and Lowman 2006). Alginate and chitosan microparticles (tens or hundreds of microns) have been employed, e.g. for colon specific delivery (Wittaya-Areekul et al. 2006). Requirements for an ideal targeting system are the following: (1) biocompatibility, biodegradability, and low antigenicity, (2) protection of the drug, (3) maintenance of the integrity till the target is reached, (4) avoidance of side effects, (5) membrane passage, (6) target recognition and association, (7) controlled drug release, and (8) elimination upon drug release. While many of these points are widely explored in literature, vector interactions at cell level is not yet deeply investigated.
In this paper up-take properties of alginate and chitosan sub-micron particles (∼500 nm in radius) are investigated and compared.
Among polymers, alginate has several unique properties that have allowed to use it as a matrix for the entrapment and/or delivery of a variety of biological agents (Chretien and Chaumeil 2005). Alginate is a co-polymer extracted from some types of brown algae and it is made up of two uronic acids: d-mannuronic acid and l-guluronic acid. Polyvalent cations are responsible for interchain and intrachain reticulations because they are tied to the polymer when two guluronic acid residuals are close. The reticulation process consists of the simple substitution of sodium ions with calcium ions (Gombotz and Wee 1998). The relatively mild gelation process has enabled not only proteins, but also cells (Murtas et al. 2005) and DNA (Douglas and Trabizian 2005) to be incorporated into alginate matrices with retention of full biological activity.
Chitosan is a cationic natural biopolymer produced by alkaline N-deacetylation of chitin, the most abundant natural polymer after cellulose. There are at least four methods described in literature for chitosan-based system production: ionotropic gelation, microemulsion, emulsification solvent diffusion and polyelectrolyte complex (Tiyaboonchai 2003). Thanks to its good biocompatibility, biodegradability, low toxicity and low cost, it has been extensively used over a wide range of applications, such as drug carriers, wound-healing agents, chelating agents, membrane filter for water treatment, and biodegradable coating or film for food packaging (Tokura et al. 1996; Kwunchit and Bernd 1997; Wang et al. 2005; Yang et al. 2007).
In this paper alginate and chitosan particles are realized and characterized via Focused Ion Beam (FIB) microscopy. After the optimization of particles realization procedure, in vitro assays of cellular up-take are performed on Crandell feline fibroblast kidney cell line and immortalized mouse hippocampal cells HN9.10e: internalization kinetics is analysed, considering the particles concentration - dependent up-taking behaviour. In vitro tracking as well as intracellular drug delivery monitoring are performed by using fluorescein loaded particles. Finally, particles internalization is monitored via a lysosomal marker that binds acidic cellular organelles involved in the up-take and endocytic pathway of external substaces inside cells (Campbell and Reece 2002).
2 Materials and methods
2.1 Particles realization
Alginate particles were realized by a homogenization process and reticulation with calcium ions (Ciofani et al. 2007). 5 ml of a 1% alginate (71240 from Fluka) solution was added drop by drop to 25 ml of an oil phase (iso-octane and Tween 85, a surfactant) and homogenized with a Ultra Turrax T18 basic (purchased from Ika) at 15,000 rpm for 2 min; cross-linking was then performed by the addition drop by drop of 5 ml of a 100% CaCl2 aqueous solution and stirring for 2 min. The system was allowed to settle and the obtained particles were cleaned by centrifuging three times in distilled water at 900 g. Final product was resuspended in 1 ml of distilled water. Procedure yield (calculated as ratio between weight of employed polymer and weight of obtained particles) was about 70%.
A similar procedure was carried out in order to obtain chitosan particles: 5 ml of a 1% chitosan solution (417963 from Sigma) in acetic acid (5%) was homogenised for 2 min in the oil phase; particles precipitation was performed with addition of 5 ml of a 100% KOH solution. Procedure yield was about 35%.
Fluorescent particles were obtained with the same procedures but with the addition of 0.1% fluorescein sodium salt to the aqueous phases.
To characterize the particles, a drop of the final suspension was posed on a silicon substrate and dried before imaging with a FIB microscope, a system that enables imaging, localized milling and deposition of conductors and insulators with high precision (Raffa et al. 2005). The FIB system used for the imaging in the present work is a FEI 200 (Focused Ion Beam Localized milling and deposition) delivering a 30 keV beam of gallium ions (Ga+), with beam currents varying between 1 pA and 11 nA.
Size and size distribution analysis of particles were carried out by specific imaging and CAD softwares (Adobe PhotoShop, AutoCAD).
2.2 Cell cultures
Both Crandell feline fibroblast kidney cell line, CrFK (ATCC CCL-94), and immortalized mouse hippocampal cells HN9.10e (Lee et al. 1990; donated by Dr. Mercedes Garcia, University of Pisa, Italy) were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, 100 IU/ml penicillin, 100 μg/ml streptomycin and 2 mM l-glutamine. Cells were maintained at 37°C in a satured humidity atmosphere containing 95% air/5% CO2.
For in vitro assays cells were seeded in 24-well plates (30000 cells per plate) and grown with particle - modified culture medium (particles concentrations of 10, 25, 50 and 100 μg/ml).
Cells were incubated in the particle - modified growth medium for 4 h, after replaced by fresh medium for the following analysis. Before optical imaging cells were rinsed three times in phosphate buffer saline solution (PBS) in order to remove not up-taken particles.
Kinetics and concentration-dependent analysis was easily performed counting particles inside cells (n = 100) by using a Nikon TE2000U fluorescent microscope equipped with Nikon DS-5MC USB2 cooled CCD camera. Image analysis was performed with NIS Elements imaging software.
Lysosomes tracking assays were performed on HN9.10e cells which are sensitive to LysoTracker dye (L12491) from Invitrogen). The LysoTracker probe is a fluorescent acidotropic probe for labelling and tracking acidic organelles in live cells. When it accumulates in cellular compartments with low internal pH, the probe fluoresces. Cells were incubated two hours in a growth medium modified with LysoTracker probe at ratio 1:2,500.
HN9.10e cells were chosen because they show a pH change that can be detected via Lysotracker dye. The possibility to differentiate these cells with retinoic acid (Lee et al. 1990), moreover, offers the opportunity to test retinoic acid loaded nanovectors as drug delivery systems for neuronal active factors.
CrFK cells were chosen because their very large surface allows an optimal optical inspection of up-taken particles; moreover the ability of virus-based vectors to transduce CrFK cell line is well known (Ikeda et al. 2002); this cell line is a good candidate in order to test our particles as enhanced vectors for gene therapy.
3 Experiments and results
3.1 Particle analysis and internalization phenomena
Cells were cultured in a growth medium added with particles at a concentration of 50 μg/ml. After 4 h of incubation cells were analysed via optical microscopy; the modified growth medium was thus substituted with particle-free fresh medium and cells were monitored for the following 20 h. Results are briefly described for different combination of polymer type and cell line employed.
HN9.10e incubated with alginate particles
HN9.10e incubated with chitosan particles
A similar behaviour occurs for chitosan particles but the up-take increases [up to eight particles per cell in Fig. 2(d)]. This result should be explained with the electrostatic interaction between positive-charged amino-groups of chitosan and negative-charged cell membrane which enhances particle internalization (Harush-Frenkel et al. 2007).
CrFK incubated with alginate particles
CrFK incubated with chitosan particles
Chitosan particles up-take is again very large [up to 10 particles per cell, Fig. 3(d)]. Figure 3(f) shows that after further 20 h of incubation in the particle-free growth medium, most of internalized particles are degraded, although a few of them are already inside cells.
Experimental results allow to conclude that our particles are readily up-taken by cells, especially chitosan ones; particles degradation starts immediately after cell internalization, is time dependent as shown in the previous Figs. 2 and 3 and it finishes after approximately 20 h from the free-particles medium change.
3.2 Concentration- and time-dependent up-take
Once proved particles internalization, the following step was to investigate up-take kinetics.
Data at different concentrations confirm that the up-take is higher for chitosan particles, especially in CrFK which are larger and spreader cells and they offer a bigger surface area to particle interactions.
Time-dependent up-take of chitosan particles was studied both on HN9.10e and CrFK cells.
Cells were incubated with particle-modified medium (concentration of 100 μg/ml). Every hour, for 4 h, medium was removed, cells washed three times with PBS and observed. After microscope observation, cells were re-incubated with particle-modified medium. Results are showed in Fig. 4(b).
Both C and k′ can be calculated from the fitting showed by Fig. 4(b). k′ constant represents the characteristic time of the particles up-take. Similar values for HN9.10e and CrFK cells are extrapolated from data of Fig. 4(b) (respectively k′ = 0.49 h−1 and k′ = 0.53 h−1). C is related to the maximum number of internalized particles and it is again in agreement with previous results (C = 13.89 for HN9.10e cells and C = 17.80 for CrFK cells).
3.3 Polymeric particles as a drug delivery system
In order to validate the particles described in this paper as drug delivery systems, in vitro tests were carried out by entrapping a fluorescent molecule (fluorescein sodium salt, C20H10Na2O5) in particles. Fluorescein is a fluorophore, which has absorption maxima at 493.5 and 460 nm and is widely employed in assessing tear turnover and corneal permeability (Nelson 1995). It is a cell impermeable molecule, therefore spontaneous internalization of fluorescein by cells must be excluded.
These results suggest that alginate and chitosan particles described in this paper are efficient carriers for small molecules. Internalization dynamic allowed passage inside cells of otherwise impermeable drugs, such as fluorescein. Positive charged amino-groups of chitosan allow again higher internalization rate and final amount compared to alginate particles.
3.4 Lysosomes tracking assays on HN9.10e cells
Lysosomes are organelles that contain digestive enzymes (acid hydrolases, at pH 4.8). They digest excess or worn out organelles, food particles, and engulfed viruses or bacteria. The membrane surrounding a lysosome prevents the digestive enzymes inside from destroying the cell. Lysosomes fuse with vacuoles and dispense their enzymes into the vacuoles, digesting their contents (Campbell and Reece 2002). The lysosomes are used for the digestion of macromolecules from phagocytosis (ingestion of other dying cells or larger extracellular material), endocytosis (where receptor proteins are recycled from the cell surface), and autophagy (where old or unneeded organelles or proteins, or microbes which have invaded the cytoplasm are delivered to the lysosome).
Increased number of lysosomes and other acidic compartment denotes a degradation activity inside cells. Since complete digestion of particles seems to occur following the substitution of particle-modified medium by fresh medium (Section 3.1), further assays that evidence such activity were carried out and described below.
The higher fluorescence intensity in the chitosan particles treated cells demonstrated again the higher internalization of chitosan particles compared to alginate ones.
In the past 30 years, the explosive growth of nanotechnology has fostered challenging innovations in pharmacology, which is in the process of revolutionizing the delivery of biologically active compounds. The main input of today’s nanotechnology in pharmacology is that it allows real progresses to achieve temporal and spatial site-specific delivery. This advance was made possible by the development of various types of micro- and nanosystems resulting from researches based on multidisciplinary approaches. Starting from the first liposomes proposed in 1974 by Gregoriadis (Gregoriadis et al. 1974) there was an explosion in the number of microsystems suitable for drug delivery, which are either made of lipids or composed of polymers (Couvreur and Vauthier 2006).
This paper describes new polymeric particles: alginate and chitosan based particles. Alginate and chitosan particles were realized by homogenization techniques and, respectively, by ionotropic gelation and precipitation. Particles with mean radius of 600 nm were obtained.
Experimental data demonstrate that these polymeric particles are an effective mean to release intracellularly drugs with high throughput but without side effects.
A comparative study of internalization of polymeric nanoparticles was carried out demonstrating that chitosan nanoparticles are easier to be internalised because of their surface cationic features; internalization studied performed on CrFK and HN9.10e cells demonstrated that cellular up - take increases linearly with nanoparticles concentration in the growth medium, and internalization process shows a first order kinetics with a characteristic time around 0.5 h−1. In addition lysosomal tracking assays show that nanoparticles degradation occurs within 24 h from the up-taking. Finally, in order to show the effectiveness of the particles as drug delivery systems, particles filled with a fluorescent drug (fluorescein sodium salt) were successfully tested. Drug release inside the cell could be tuned by changing particles preparation parameters. For example, it was demonstrated (Ciofani et al. unpublished) that by increasing polymer concentration in the water phase of the emulsion, a slower release can be achieved. Moreover drug release is influenced by particles dimensions: the bigger are the particles, the slower is the release; obviously particles dimensions have to be in a range suitable for cellular up-taking.
Next studies will be focused on applying our particles as non-viral gene carriers for cell transfection of CrFK cells and as intracellular delivery systems of retinoic acid for HN9.10e cell differentiation.
The activity presented in this work has been partially supported by the IIT (Italian Institute of Technology) Network and the NINIVE (Non Invasive Nanotransducer for In Vivo gene thErapy, STRP 033378) project, cofinanced by the 6FP of the European Commission.