Chitosan nanoparticle-based neuronal membrane sealing and neuroprotection following acrolein-induced cell injury
© Cho et al; licensee BioMed Central Ltd. 2010
Received: 19 October 2009
Accepted: 29 January 2010
Published: 29 January 2010
The highly reactive aldehyde acrolein is a very potent endogenous toxin with a long half-life. Acrolein is produced within cells after insult, and is a central player in slow and progressive "secondary injury" cascades. Indeed, acrolein-biomolecule complexes formed by cross-linking with proteins and DNA are associated with a number of pathologies, especially central nervous system (CNS) trauma and neurodegenerative diseases. Hydralazine is capable of inhibiting or reducing acrolein-induced damage. However, since hydralazine's principle activity is to reduce blood pressure as a common anti-hypertension drug, the possible problems encountered when applied to hypotensive trauma victims have led us to explore alternative approaches. This study aims to evaluate such an alternative - a chitosan nanoparticle-based therapeutic system.
Hydralazine-loaded chitosan nanoparticles were prepared using different types of polyanions and characterized for particle size, morphology, zeta potential value, and the efficiency of hydralazine entrapment and release. Hydralazine-loaded chitosan nanoparticles ranged in size from 300 nm to 350 nm in diameter, and with a tunable, or adjustable, surface charge.
We evaluated the utility of chitosan nanoparticles with an in-vitro model of acrolein-mediated cell injury using PC -12 cells. The particles effectively, and statistically, reduced damage to membrane integrity, secondary oxidative stress, and lipid peroxidation. This study suggests that a chitosan nanoparticle-based therapy to interfere with "secondary" injury may be possible.
Substantial cellular injury caused by acute mechanical, chemical, or biological insult is initially associated with an instantaneous loss of plasma membrane integrity. This facilitates the unregulated exchange of intracellular/extracellular ion species, and subsequently leads to cell death [1, 2]. The failure of this functional barrier further induces incomplete oxygen metabolism within the cell, and accelerates the production of highly reactive oxygen species (so-called "free radicals") such as superoxide anions, hydrogen peroxide, and hydroxyl radicals. The biochemical reactivity of such reactive oxygen species (ROS) triggers the deterioration of lipids within the inner domain of the cell membrane (lipid peroxidation or LPO) producing endogenous toxins composed of mainly aldehydes - especially acrolein [3–7]. Unfortunately, the continuing production of toxic aldehydes feed back to attack mitochondria, yet untouched healthy membrane, and pass through intact cell membranes to attack even nearby healthy cells. As a consequence, the initial loss of membrane integrity in response to the "primary injury" is the crucial step initiating a "secondary injury" process in the nervous system. Unchecked, these self - reinforcing processes influence further collapse of mitochondria and oxidative metabolism, continued deterioration of the cell membrane, the further production of endogenous toxins, and ultimately, cell death. In the past decade, significant neuroprotection was achieved by the topical, intravenous, or even subcutaneous application of water-soluble polymers, such as poloxamer 188, 1100, or polyethylene glycol (PEG), as a rescue strategy to alleviate cell and tissue damage following traumatic insults [8–16]. Such versatile polymers have demonstrated their capability to induce functional recovery at the cell level by initiating spontaneous molecular rearrangement of the lipid bilayers through membrane fusion and integration .
This membrane-based recovery is initially seen as an erasure of defects in the axolemma whose integrity is the basis for action potential conduction. Measurement of a rapid (minutes) recovery of compound action potentials traversing crushed or cut guinea pig spinal cord in organ culture [14, 17] and an equally rapid recovery of somatosensory evoked potentials traversing the spinal cord lesion in "whole animal" guinea pig injury models reveals the initial consequences of acute neuroprotection/neurorepair by direct application - or intravenous injection - of these polymers . The longer term, and stable, repair of critical anatomy has been documented by intracellular tracing in severely injured guinea pig spinal cord and adult rat brain [18, 19]. This in turn is responsible for significant behavioral recovery of spinal cord and brain - mediated animal behavior [12, 20, 21]. PEG and its derivatives have preferable molecular weights and concentration ranges enabling them to carry out this neuroprotection, which significantly narrows the therapeutic window, and possibly their efficacy without side effects in clinical trials . For example, higher concentration of PEG dissolved in sterile saline produce viscosity problems for injectable solutions whereas low molecular weight PEGs (< ~1000 kD) may produce ethylene glycol poisoning [23, 24]. Additionally, it has been observed that bulky chains of poly (ethylene oxide) tend to inhibit the efficient entry of such synthetic polymers into cells where continued beneficial activity is desired. According to a previous study, PEG was accumulated only on the outside of cells for hours instead of internalizing, where it can produce other beneficial effects by acting on damaged mitochondria .
Synthesis and characterization of hydralazine-loaded chitosan nanoparticles
In vitro release of hydralazine
The effect of hydralazine-loaded chitosan nanoparticles on acrolein-mediated cell injury
The inhibition of plasma membrane peroxidation by the application of hydralazine-loaded chitosan nanoparticle
Chitosan as a membrane "fusogen"
Recently, we have learned that chitosan, as a component of an injectable solution, can serve as a novel therapeutic agent after severe compression injury of guinea pig spinal cord (unpublished observations). In such injuries, progressive destruction of cells and tissues occurs after mechanical trauma, however critical anatomy and function was restored by the injection of chitosan. Chitosan accumulation typically occurs around the defect area in cells and tissues by hydrophobic interactions. Conversely, at intact membranes, high surface densities of lipid moieties inhibit the penetration of chitosan [36–38]. The mechanism underlying chitosan-mediated membrane sealing and specific targeting of injured membranes is still being investigated; however a clear demonstration of the latter observation is significantly persuasive that this approach holds unexpected merit in dealing with cell and tissue trauma.
The evidence presented in this report suggests the potential usefulness of chitosan - as a nanoparticle base likely due to its two-pronged attack on cell damage. We emphasize that chitosan nanoparticles themselves are capable of restoring cell viability by first mediating the sealing of damaged membrane. We additionally showed the application of chitosan achieved neuroprotection by interfering with the generation of ROS and LPO. This is also due to its membrane reconstruction properties rather than an ability to directly scavenge these toxins. However upon conjugation with hydralazine, its potential therapeutic effects are dramatically enhanced. Chitosan treatment alone did not provide neuroprotection after exposure to acrolein - even in high concentrations (unpublished observations). This fact suggested the inclusion of hydralazine in the chitosan nanoparticle to directly provide this function.
Acrolein-induced progressive neuronal degeneration and rescue by hydralazine
It is widely accepted that traumatic injuries often deteriorate cell membrane integrity through multiple mechanisms involving several biological processes and biochemistries. Steady and progressive membrane rupture and concomitant loss of intracellular contents initially causes serious derangement of ionic species. Especially, the unregulated influx of Ca2+ into cytoplasm plays a key role in destabilizing and collapsing the cytoarchitecture as well as membrane integrity by activating numerous catabolic physiological and biochemical processes such as ROS mediated LPO. There is little evidence that free radicals (with half lives less than seconds - even fractions of a second) are directly toxic to cells. It is the downstream end product of ROS stimulated LPO that produces actual toxins such as a variety of aldehydes - acrolein being the most potent. Compared to free radicals, acrolein has a half-life many orders of magnitude longer . Indeed, highly reactive acrolein actively forms biomolecule conjugates with proteins, DNA, and glutathione, which further stimulates the generation of ROS and LPO. The cytotoxicity of acrolein is known to be concentration-dependent. After exposure to 75 - 100 μM acroleoin, a majority of the PC 12 cell population was dead within 4 hr . In previous reports, we have shown that the toxicity of acrolein can be significantly diminished after application of an acrolein-trapping agent such as hydralazine. Hydralazine, a well-known nucleophile drug, is able to immediately inhibit acrolein cytotoxicity by forming "hydrazones" through a scavenging reaction (Figure 1) [42–44].
Notes on the fabrication, characterization, and use of hydralazine-loaded chitosan nanoparticles in CNS trauma and neurodegenerative diseases
There are many studies to take advantage of colloidal particles as drug carriers. The use of colloidal drug carriers can facilitate not only site-specific drug delivery in a designated region but also facilitate drug bioavailability and therapeutic efficacy. On the basis of these considerations, a variety of biocompatible and biodegradable materials such as synthetic or natural polymers, lipids, or solid (metal, semiconductor, magnetic, or insulator) components have been intensively investigated [45, 46].
First, liposome-based colloidal carriers have already commercialized. Despite these advances, liposomes still have some technical weakness with regard to the limitations in reproducibility, stability, and low drug encapsulation efficacy [47, 48]. Solid particles such as silica particles have been proposed as alternative drug carriers that could potentially overcome such conventional problems. Silica particles with well-established synthesis methods are easily incorporated with other substances through surface modification, bioconjugation, and entrapment .
On the other hand, the use of biodegradable polymeric drug carriers, either synthetic or natural, shows great potential in controlled drug release by tuning their biodegradability. Of these, chitosan is the one of the most extensively studied - in particular, since it is composed of reactive hydroxyl and amine groups, it tends to adhere well to negatively charged surfaces, thus increasing cellular transport .
Because it is an antihypertensive drug, the systemic exposure of hydralazine will provoke problems related to blood pressure management in hypotensive trauma patients [51, 52]. The use of our particulated system is capable of significantly reducing systemic exposure of drug since it is delivered to the intended site of action. In spite of its potent therapeutic effects reported here in vitro, our major challenge is to first, enhance the entrapment efficacy of hydralazine in the chitosan nanoparticle, and second, move the improved system to animal models of spinal cord and brain injury [12, 19].
The first issue arises due to the existence of charge repulsion between cationic chitosan polymer (pKa = 6.5) and positively charged hydralazine (pKa = 7.1). In an attempt to enhance hydralazine encapsulation, two different kinds of polyanions were employed. Interestingly, the encapsulation of hydralazine was particularly dependent on the ionic interaction formed between chitosan and polyanions, where the amount of negatively charged groups of TPP and DS existed per mole was critical for hydralazine association. As a consequence, Chi-TPP formulation showed entrapment efficiency of 15.8% with slightly increased positively charged surface property, whereas Chi-DS exhibited an entrapment ratio of 23.5% with negative surface, characteristics as shown in (Figure 3c). In both cases, after entrapment of hydralazine, the nanoparticle formula showed some increase in diameter and zeta potential value, indicating the presence of drug through inter and intramolecular cross-linking. Both particles, Chi-TPP and Chi-DS, displayed similar trends in release behavior, where an initial burst release could likely be a result of the desorption of hydralazine (which is mostly attached on the surface of particles) and later progressive degradation of chitosan. Such a reduced release could be attributed to an increased viscosity resulting from the concentration ratio of chitosan and the polyanion, where a dense layer of chitosan might cause a greater density of crosslinking and consequently interfere with a swelling effect.
In summary, moving forward with this line of investigation requires exploring the stability of drug-loaded chitosan nanoparticles on the basis of pH or chemical crosslinking for a variety of compounds of interest to us. Various molecular weight chitosan samples or water-soluble chitosan molecules will be employed in this ongoing research. The second challenge requires a diversion towards toxicology studies to decipher any potential side effects or unforeseen problems associated with the chitosan particles or their cargo of hydralazine. Subsequently, we have much experience in the development of behavioral, physiological, and anatomical models of SCI and TBI, as well as access to veterinary clinical models of neurotrauma to move these notions forward.
Materials and methods
Preparation of chitosan nanoparticles: Chi-DS and Chi-TPP
Chitosan with 85% deacetylation degree and of medium weight (Chi, M.W. 200,000 Da), dextran sulfate (DS, M.W. 9,000 ~ 20,000 Da), and sodium tripolyphosphate (TPP, M.W. 367.8 Da) were purchased from Fluka/Sigma-Aldrich. Two kinds of chitosan particles were synthesized: Chi-DS and Chi-TPP. Briefly, Chi-DS was prepared by complexation of Chi and DS as described previously, where chitosan was dissolved at 0.10% (w/v) with a 1% aqueous acetic acid solution while DS was prepared in deionized water at the concentration of 0.5 mg/ml [26, 28]. Equivalent volumes of chitosan and the DS solution were mixed by magnetic stirring at room temperature. Once the nanoparticle suspension started to form, the mixture was stirred for another 20 min. The formation of Chi-TPP nanoparticles was initiated by ionic gelation mechanism based on the interaction of cations and anions [27, 33]. Chi-TPP nanoparticles were formed spontaneously when equal volume of Chi (1.75 mg/ml) and TPP (2 mg/ml) solution were prepared and stirred at room temperature. Hydralazine-loaded chitosan nanoparticles were then immediately prepared by incorporating equivalent volume of a Chi acidic solution (1.75 mg/ml) and an aqueous TPP solution (2 mg/ml) or aqueous DS solution (0.5 mg/ml) containing hydralazing (1 mg/ml) while stirring with a magnetic bar.
Characterization of chitosan nanoparticles
Particle size and zeta potential measurements were carried out with a zeta-potential/particle size analyzer (Zetasizer). To begin, samples were diluted in deionized water and measured in an automatic mode. All measurements were performed in three ~ five repetitions. The morphology of chitosan nanoparticles was observed by transmission electron microscopy (JEOL 2000FX).
Encapsulation and release of hydralazine from particles
To observe the release behavior of hydralazine from chitosan nanoparticles, a modified Krebs' solution (pH 7.2) that contained 124 mM NaCl, 2 mM KCl, 1.2 mM KH2PO4, 1.3 mM MgSO4, 2 mM CaCl2, 26 mM NaHCO3 was used. The release of hydralazine suspended in this Krebs' solution was observed as a function of the concentration of incorporated hydralazine. The released hydralazine was extracted at a different time-interval and centrifuged to permit measurement by UV spectroscopy. The concentration was then calculated by linear equation to determine the hydralazine release curve.
PC 12 cells with a density of 1 × 106 cells/mL were grown in Dulbecco's modified eagle's medium (DMEM; Invitrogen) supplemented with 12.5% horse serum, 2.5% fetal bovine serum, 50 U/ml penicillin, and 5 mg/ml streptomycin - at an incubator setting of 5% CO2 and 37°C. After trypsinization and centrifugation, cell pellets were resuspended in Hank's balanced salt solution (HBSS) for the exposure to acrolein and subsequent treatment with chitosan nanoparticles. Acrolein (100 μM) was prepared fresh daily in PBS solution. The Chi-TPP/Hy or Chi-DS/Hy suspensions were applied at a concentration of 20 μl/ml in medium with a delay of 15 min after the application of acrolein.
Determining the integrity of cell membranes: The lactate dehydrogenase (LDH) exclusion assay
MTT assay for cellular viability
Upon exposure to acrolein, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium (MTT, Sigma) assay was used to determine the overall viability of the sample cells as it is an indicator of oxidative metabolism. The assay assesses the activity of a mitochondrial dehydrogenase enzyme which is detectable only from viable cells by the color change of tetrazolium rings from pale yellow MTT to dark blue formazan crystals. Quantification of these shifts was evaluated by spectrophotometric measurement. PC 12 cells were seeded in 12-well plates at 1 × 106 cells/well in HBSS. MTT reconstituted in PBS was added to each well. After incubation, formazan crystals were pelleted by centrifugation, and dissolved in a MTT solubilization solution. The absorbance was read at 550 nm minus the background at 660 nm.
Cell mortality using the Live - Dead cell assay
To assess the impact of acrolein exposure on living cells, the determination of the number of live and dead cells in the total cell population was performed by the application of a two-color fluorescence assay (Biovision Inc.). This assay can selectively stain the live and dead cells using calcein AM (Cal AM) and ethidium homodimer (EthD-1). Cal AM typically crosses the cell membrane into living cells and consequently the cleaved calcein fluorophore is released which possess a strong green fluorescence with an emission wavelength of ~525 nm and an excitation wavelength of ~485 nm. In contrast, EthD-1 can only diffuse into dead or dying cells and produces a red fluorescence at ~625 nm when excited at ~525 nm. The test PC 12 cells were cultured in 12-well plate at 1 × 106 cells/well in HBSS, and subsequently incubated in Cal AM and EthD-1 for 5 minute. Using fluorescent microscopy, cell viability was calculated and expressed as a percentage.
Measurement of reactive oxygen species (ROS) and lipid peroxidation (LPO)
The production of reactive oxygen species accelerates as a result of cell membrane damage which in turn drives LPO and toxic aldehyde production. Therefore a comparison of the levels of ROS and LPO following acrolein-exposure and post-treatment with chitosan nanoparticles provides clues for examining the state of cell deterioration in response to oxidative stress. The cultured cells were immersed in 1 mL of PBS with hydroethidium (HE, Invitrogen) at a final concentration of 1 μM for 10 min in the dark. Concentrations of ROS weredetermined by the chemi-luminescence assay based on the conversion of hydroethidine (HE) to ethidium (E+) in the presence of intracellular superoxide anion (O2-). Subsequently, samples were analyzed with epifluorescence on an Olympus BX61 microscope using a standard rhodamine cube (545/590 nm excitation/emission respectively), and quantified using Image J software (NIH) to measure the amount of ethidium bromide uptake. LPO was measured using a lipid hydroperoxide assay (Cayman Chem. Co.). Once cells were exposed to acrolein, the level of hydroperoxides was directly measured utilizing their redox reactions with ferrous ions. Subsequently, the absorbance of each sample was read at 500 nm using a spectrophotometer (SLT spectra plate reader). Lipid peroxidation was calculated and expressed as a percentage of control values.
One-way ANOVA was used to determine the statistical significance between control and experimental groups using InStat software. Results are expressed as mean ± SD. P ≤ 0.05 was considered statistically significant.
We would gratefully like to thank Debra Bohnert for her expert handling of whole animal experiments. We appreciate the excellent illustrations and graphics of Michel Schweinsberg, and financial support from the General Funds of the Center for Paralysis Research, The State of Indiana, and a generous endowment from Mrs. Mari Hulman George.
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