Ionotropic Gelation Of Chitosan Research Articles

Impact of tannic acid on nisin encapsulation in chitosan particles

This study investigates the effect of addition of tannic acid on nisin encapsulated in chitosan matrices. Composite materials were prepared using a mild, environmentally friendly procedure, ionotropic gelation of chitosan by sodium tripolyphosphate in the presence of nisin (N) at different concentrations. In two parallel sets of preparations, tannic acid (TA) was added at 10:1 and 5:1 N:TA, respectively. The obtained particles were characterized by FTIR, SEM, size, zeta potential, encapsulation efficiency, loading capacity, and ratio of residual free amino groups. The kinetics of nisin release from the particles was studied to assess the role of TA as a potential modulator thereof. Its addition resulted in enhanced release, higher at lower N:TA ratio. An additional benefit was that TA, a strong antioxidant, imparted antioxidant activity to the composites. Antimicrobial turbidimetric tests were performed against one gram-positive bacterium (Staphylococcus aureus) and two gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), all relevant for the food, pharmaceutical, and cosmetic industries. All the composites showed synergistic effects against all the bacteria tested. The positive coaction was stronger against the gram-negative species. This is remarkable since nisin by itself has not known activity against them.

Rat Hepatocellular Primary Cells: A Cellular and Genetic Assessment of the Chitosan Nanoparticles-Induced Damage and Cytotoxicity.

Chitosan (CH) is a non-toxic vital polymer that is derived naturally from chitin. Due to its anti-bacterial and anti-fungal properties, it has attracted researchers' attention. The anti-bacterial activity of 1-3 CH is ideal in an acidic medium due to its weak solubility at pH levels higher than 6.5. The type of CH and the degree of its polymerization affect its anti-microbial activity, as well as some of its other chemical and physical properties. The present study was conducted to investigate the damage induced by chitosan nanoparticles (CHNPs) at various concentrations on the cultured rat hepatic cells. The CHNPs were synthesized by the ionotropic gelation of CH with sodium tripolyphosphate anions. Hepatic cells were cultured from tissues freshly isolated from the liver of normal laboratory rats. Cells were allowed to reach a confluence level before the treatment with CHNPs. In total, five different concentrations of CHNPs were used, and cell cytotoxicity was evaluated using the MTT assay. The genetic expression of P2Y1, P2Y2, and P2Y4 purinergic receptors was evaluated on the cellular level using Qualitative Reverse Transcription Polymerase Chain Reaction technique. The primary culture of rat hepatic cells was thoroughly exposed to a range of CHNPs. Under normal conditions, the cells showed normal cellular morphology with clearly defined borders and normal nuclear structure. Apoptotic cellular damage was observed in the cultured hepatic cells when exposed to CHNPs. Moreover, irregular cellular morphology and heavy pigmentation were noticed in the hepatic cells when exposed to a high concentration of CHNPs. Purinergic receptor gene expression indicated an inflammatory response by an increased gene fold change post-exposure to CHNPs. This study concludes that CHNPs have a strong cytotoxic effect on the cultured rat hepatic cells. Overall, CHNPs showed an inhibitory response to hepatic cells evoking a purine receptor-mediated inflammatory response.

Salicylic acid-loaded chitosan nanoparticles (SA/CTS NPs) for breast cancer targeting: Synthesis, characterization and controlled release kinetics

The present study aimed to develop an easy controlled release system for salicylic acid (SA) through ionotropic gelation of chitosan (CTS) using tripolyphosphate (TPP) as cross-linker. The synthesized nanoparticles (SA/CTS NPs) were characterized for their physicochemical properties by various techniques including Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), scanning electron microscopy (SEM) with an energy-dispersive X-ray (EDX) spectroscopy, X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD). A maximum encapsulation efficiency (EE) of 84% was obtained. The obtained material not only was tested as drug delivery system in neutral (pH 7.4) and acidic medium (pH 5.5) but also the kinetic release behavior of SA from the CTS NPs was investigated based on two models namely Korsmeyer-Peppas and Higuchi. These data demonstrated that the drug release mechanism governed by Korsmeyer-Peppas model. Finally, the cytotoxic effects of free SA and SA/CTS NPs were evaluated in-vitro against breast cancer (MDA-MB-231) cell lines by MTT assay. Results showed that SA/CTS NPs were more cytotoxic than free SA. This work suggested the potential of SA/CTS NPs system as an effective anticancer system.

Ionotropic Gelation of Chitosan Flat Structures and Potential Applications.

The capability of some polymers, such as chitosan, to form low cost gels under mild conditions is of great application interest. Ionotropic gelation of chitosan has been used predominantly for the preparation of gel beads for biomedical application. Only in the last few years has the use of this method been extended to the fabrication of chitosan-based flat structures. Herein, after an initial analysis of the major applications of chitosan flat membranes and films and their usual methods of synthesis, the process of ionotropic gelation of chitosan and some recently proposed novel procedures for the synthesis of flat structures are presented.

Optimized removal of phosphate and nitrate from aqueous media using zirconium functionalized nanochitosan-graphene oxide composite

The decontamination of excess nutrients by polysaccharide-graphene oxide composites has gained much research attention. However, the usage of synthetic polymers and toxic crosslinking agents affects the environment. This investigation is, therefore, aimed at achieving a simple, effective and nontoxic technique of fabricating nanocomposites by ionotropic gelation of chitosan and tripolyphosphate crosslinking agent. The nanochitosan-graphene oxide composite (NCS@GO) was synthesized and investigated for its potential to remove phosphate (P) and nitrate (N) from aqueous solutions. High and low amounts of zirconium (Zr) were loaded in NCT@GO composite to make it selective for the adsorbate anions. The developed nanocomposites were comparatively explored by N2 isotherms, FTIR, XRD, TGA, DTA, FESEM, EDS with mapping analysis and water regain property. Experimental design was conducted by the five-factorial central composite design-as a branch of response surface methodology (RSM). According to the design of RSM, NCS@GO/H-Zr demonstrated an excellent P and N uptake of 172.41 mgP/g and 138.88 mgN/g, reasonable pH-compatibility from 3 to 11, suitable selectivity for both adsorbates among competitor anions, desired recyclability and desorption efficiency for P and N, and retained 76% and 85% for P and N adsorption ability after ten recycles. The removal capacity of P and N anions were also assessed in bi-component systems. Thermodynamic data were considered, in which it was found that the adsorptive removal of the both anions was endothermic and spontaneous in nature. The adsorption isotherm of P and N on the surface of the NCS@GO/H-Zr was suitable for the Freundlich isotherm model, suggesting the multilayer adsorption. On the basis of kinetic studies, specific rate constants involved in the processes were calculated and the obtained result indicates that the pseudo second order kinetics was found to be a better fit. Real samples analysis indicated that the NCS@GO/H-Zr works well for removal of P and N from contaminated waters.

Functional iron chitosan microspheres synthesized by ionotropic gelation for the removal of arsenic (V) from water

Arsenic (As) is considered one of the most serious inorganic contaminants in groundwater. The maximum limit of As in drinking water is 0.010mgAs L−1 according to the World Health Organization. Functional Iron Chitosan Microspheres (F-ICM) were synthesized by ionotropic gelation of chitosan and ferric nitrate for removal of As (V) from water. The effects of iron concentration (0.7, 5.5 and 11.2gFe+3/100g chitosan), pH range (4–9), sorbent dosage, possibility of reuse and presence of interfering anions were analyzed. F-ICM were effective for As (V) adsorption in a wide range of pH with removals higher than 88.9%, reaching concentrations < 0.010mgAs L−1. A higher proportion of iron in the matrix favored the removal of As(V) in a pH range 6 to 9. Kinetic data of As(V) adsorption onto 0.7 F-ICM under all tested initial concentrations fitted well with the pseudo second-order kinetic model. Sorption isotherm experimental data were satisfactorily fitted with the Langmuir equation; the maximum sorption capacity for As(V) was 120.77mgAs(V) (gF-ICM1) and the Langmuir constant was 0.331 L mg1. A good performance of the adsorption capacity was obtained in four successive adsorption cycles of reuse and in the presence of interfering anions, indicating the high adsorption capacity of the synthesized microspheres.

The different ways to chitosan/hyaluronic acid nanoparticles: templated vs direct complexation. Influence of particle preparation on morphology, cell uptake and silencing efficiency.

This study is about linking preparative processes of nanoparticles with the morphology of the nanoparticles and with their efficiency in delivering payloads intracellularly. The nanoparticles are composed of hyaluronic acid (HA) and chitosan; the former can address a nanoparticle to cell surface receptors such as CD44, the second allows both for entrapment of nucleic acids and for an endosomolytic activity that facilitates their liberation in the cytoplasm. Here, we have systematically compared nanoparticles prepared either A) through a two-step process based on intermediate (template) particles produced via ionotropic gelation of chitosan with triphosphate (TPP), which are then incubated with HA, or B) through direct polyelectrolyte complexation of chitosan and HA. Here we demonstrate that HA is capable to quantitatively replace TPP in the template process and significant aggregation takes place during the TPP–HA exchange. The templated chitosan/HA nanoparticles therefore have a mildly larger size (measured by dynamic light scattering alone or by field flow fractionation coupled to static or dynamic light scattering), and above all a higher aspect ratio (Rg/RH) and a lower fractal dimension. We then compared the kinetics of uptake and the (antiluciferase) siRNA delivery performance in murine RAW 264.7 macrophages and in human HCT-116 colorectal tumor cells. The preparative method (and therefore the internal particle morphology) had little effect on the uptake kinetics and no statistically relevant influence on silencing (templated particles often showing a lower silencing). Cell-specific factors, on the contrary, overwhelmingly determined the efficacy of the carriers, with, e.g., those containing low-MW chitosan performing better in macrophages and those with high-MW chitosan in HCT-116.

Encapsulation of cinnamon leaf oil within chitosan: formulation and characterization

Chitosan microcapsules containing cinnamon leaf oil were formed by ionotropic gelation of chitosan by crosslinking with sodium tripolyphosphate. The microencapsulation method for oil loading was optimized by varying the amounts of polymer, oil and cross linker. The effect of varying process parameters on the encapsulation efficience (EE), oil loading capacity (LC) and release rate was monitored. The formulated microcapsules were characterized using Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR). SEM imaged clear spherical shaped oil loaded microcapsules with a crimpy surface while empty capsules had a smooth texture.FTIR spectra evidence for the successful encapsulation of oil within wall material. EE increased with increasing oil load while it decreased with increasing polymer and cross linker concentration. LC increased with increasing amounts of oil and crosslinker and showed a decrease with increasing polymer amounts. Release rate of oil increased with increasing oil loading whereas it decreased with increasing polymer and crosslinker amounts. Varying the process parameters has a direct impact on the EE, LC and release rate. The optimum formulation for cinnamon oil microcapsules is polymer, 1 g : oil, 3g: cross linker, 0.5 g with an EE of 91%, and a LC of 38%.

Development and characterization of alginate / chitosan nanoparticulate system for hydrophobic drug encapsulation

The present study was emphasized to develop Alginate/Chitosan nanoparticles capable of working as carriers of the hydrophobic drug quercetin, a polyphenolic nutraceutical belonging to flavonoid category of natural compounds, with multifaceted therapeutic applications. Accordingly, quercetin loaded nanoparticles was prepared by an ionotropic gelation of chitosan with sodium tripolyphosphate, followed by alginate polyelectrolyte complexation. Synthesised unloaded and quercetin loaded Alginate/Chitosan nanoparticles were extensively characterised for their intermolecular interaction, morphology, percentage of encapsulation and loading capacity of the drug by various physiochemical characterization techniques such as ATR-FTIR, SEM, TEM, and XRD. The nanoparticles showed excellent architecture with an average particle size of 118–255 nm, showing 82.4% of quercetin encapsulation and 46.5% of quercetin loading capacity. Our results imply that the formulated Alginate/Chitosan nanoparticles could be a promising carrier for the encapsulation of the hydrophobic bioactive compound combining safety profile with no acute systemic toxicity in animal models and enhanced protective activity of quercetin.

Layer-by-layer deposition of chitosan nanoparticles as drug-release coatings for PCL nanofibers.

Nanogels were prepared by ionotropic gelation of chitosan (CS) with tripolyphosphate (TPP). The use of such nanogels to prepare coatings by layer-by-layer deposition (LbL) was studied. The nanogels were characterized in terms of particle size, zeta-potential and stability. Nanogel suspensions were used to build polyelectrolyte multilayers on silicon wafers and on PCL fiber mats by LbL-deposition. Three different polysaccharides were used as polyanions, namely chondroitin sulfate, alginate and hyaluronic acid. The ellipsometric thickness was demonstrated to depend significantly on the type of polyanion. XPS analysis with depth profiling further substantiated the differences in the chemical composition of the films with the different polyanions. Furthermore, XPS data clearly indicated a strong penetration of the polyanions into the CS-TPP layer, resulting in a complete exchange and release of the TPP ions. The LbL-deposition also was studied with PCL fiber mats, which were modified with a chitosan-PCL-graft polymer and alginate. The possibility to create graded coatings on the fiber mats was shown employing fluorescently labelled CS-TPP nanoparticles. The potential of the coatings as drug delivery system for therapeutic proteins was exemplified with the release of Transforming Growth Factor β3 (TGF-β3). The CS-TPP nanogels were shown to encapsulate and release therapeutic proteins. In combination with the layer-by-layer deposition they will allow the creation of PCL fiber mat implants having with drug gradients for applications at tissue transitions.

Sublingual spray drug delivery of ketorolac-loaded chitosan nanoparticles

Introduction: The aim of this study was to investigate ketorolac (KT) systemic absolute bioavailability after sublingual (SL) administration in vivo to conscious rabbits. Furthermore, the study investigated the potential use of chitosan nanoparticles as a delivery system to enhance the systemic bioavailability of KT following SL administration.Methods: Ketorolac-loaded chitosan nanoparticles were prepared through ionotropic gelation of chitosan with tripolyphosphate anions. The KT-nanoparticles were administered SL as a spray to rabbits and KT plasma concentration at predetermined time points was compared to SL spray administration of KT in solution. The concentrations of KT in plasma were analyzed by ultra-performance liquid chromatography mass spectroscopy (UPLC/MS).Results: KT-loaded chitosan nanoparticles significantly (p < .05) enhanced systemic absorption with 97% absolute bioavailability as compared to 70% after SL administration of KT solution.Conclusions: The results of the present study suggest that SL absorption of KT illustrated flip-flop kinetics with prolonged persistence in the body compared to intravenous administration. Formulation of KT as chitosan nanoparticles has increased its systemic bioavailability after SL spray administration. The new delivery system could be an attractive approach for the delivery of KT.

PH-responsive chitosan nanoparticles from a novel twin-chain anionic amphiphile for controlled and targeted delivery of vancomycin

The design and synthesis of novel pH-responsive nanoantibiotics is an emerging research area to address the antibiotic resistance crisis. The purpose of this study was therefore to synthesize a new anionic gemini surfactant (AGS) that could result in the formulation of pH-responsive chitosan nanoparticles (CSNPs) to treat methicillin-resistant Staphylococcus aureus (MRSA) infections. The coupling of oleic acid with 2,2-dimethyl-5,5-bis(hydroxymethyl)-1,3-dioxane and subsequent deprotection followed by a reaction with succinic anhydride and sodium bicarbonate yielded AGS. Critical micelle concentration (CMC) was determined using conductometry and in vitro cytotoxicity was performed using a MTT assay. Vancomycin loaded CSNPs containing AGS (DL_CSSNPs) were prepared by ionotropic gelation of chitosan with pentasodium tripolyphosphate. CSNPs were characterized for size, polydispersity index (PDI), zeta potential (ZP), entrapment efficiency, surface morphology, in vitro drug release and in vitro antibacterial activity (at pH 6.5 and 7.4). Results from the in vitro antibacterial activity were further supported by an in vivo study using a mice skin infection model. The CMC of AGS was found to be 1.3mM/L and it was non-toxic. The DL_CSSNPs were spherical with size, PDI and ZP of 220.57±5.9nm, 0.299±0.004 and 21.9±0.9mV respectively. An increase in the vancomycin release from the DL_CSSNPs was observed at pH 6.5 compared to pH 7.4. The minimum inhibitory concentration values at pH 6.5 and 7.4 against MRSA were 7.81 and 62.5μg/ml respectively. In vivo antibacterial activity showed that the MRSA burden in mice treated with DL_CSSNPs was reduced by almost 8-fold compared to those treated with pure vancomycin.

Preparation of chitosan/tripolyphosphate nanoparticles with highly tunable size and low polydispersity

Nanoparticles prepared through the ionotropic gelation of chitosan with tripolyphosphate (TPP) have been extensively studied as vehicles for drug and gene delivery. Though a number of these works have focused on preparing particles with narrow size distributions, the monodisperse particles produced by these methods have been limited to narrow size ranges (where the average particle size was not varied by more than twofold). Here we show how, by tuning the NaCl concentration in the parent chitosan and TPP solutions, low-polydispersity particles with z-average diameters ranging between roughly 100 and 900nm can be prepared. Further, we explore how the size of these particles depends on the method by which the TPP is mixed into the chitosan solution, specifically comparing: (1) single-shot mixing; (2) dropwise addition; and (3) a dilution technique, where chitosan and TPP are codissolved at a high (gelation-inhibiting) ionic strength and then diluted to lower ionic strengths to trigger gelation. Though the particle size increases sigmoidally with the NaCl concentration for all three mixing methods, the dilution method delivers the most uniform/gradual size increase − i.e., it provides the most precise control. Also investigated are the effects of mixture composition and mixing procedure on the particle yield. These reveal the particle yield to increase with the chitosan/TPP concentration, decrease with the NaCl concentration, and vary only weakly with the mixing protocol; thus, at elevated NaCl concentrations, it may be beneficial to increase chitosan and TPP concentrations to ensure high particle yields. Finally, possible pitfalls of the salt-assisted size control strategy (and their solutions) are discussed. Taken together, these findings provide a simple and reliable method for extensively tuning chitosan/TPP particle size while maintaining narrow size distributions.

Insight into the ionotropic gelation of chitosan using tripolyphosphate and pyrophosphate as cross-linkers

Ionotropic gelation of chitosan by means of opposite charged ions represents an efficient alternative to covalent reticulation because of milder condition of use and, in general, higher biocompatibility of the resulting systems. In this work 90° light scattering (turbidimetry), circular dichroism (CD) and 1H NMR measurements have been performed to study the interactions between the biopolymer and ionic cross-linkers tripolyphosphate (TPP) and pyrophosphate (PPi) in dilute solutions. Thereafter, a dialysis-based technique was exploited to fabricate tridimensional chitosan hydrogels based on both polyanions. Resulting matrices showed a different mechanical behavior because of their peculiar mesh-texture at micro/nano-scale: in the present contribution we demonstrate that TPP and PPi favor the formation of homogeneous and inhomogeneous systems, respectively. The different texture of networks could be exploited in future for the preparation of systems for the controlled release of molecules.

Encapsulation and release of cladribine from chitosan nanoparticles

AbstractThis study shows the potential of chitosan (CH) nanoparticles as both an oral and IV drug delivery system using the anticancer drug cladribine as a model drug. Smooth, spherical nanoparticles were prepared by the ionotropic gelation of CH with sodium tripolyphosphate. Nanoparticle size depended on degree of hydration, drug loading, and crosslinking conditions, with the smallest nanoparticles in the size range of 212 ± 51 nm. Cladribine was entrapped in the CH matrix with an entrapment efficiency of up to 62%, depending on the initial loading. The release of cladribine followed a near‐Fickian diffusion rate over the first several hours and then reached a plateau. A second release phase began after 30–40 h of incubation in the release medium, and proceeded until ∼100 h. Loaded CH nanoparticles that were crosslinked with genipin showed a delayed release profile, with only 40% of loaded drug being released after 100 h. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013

Chitosan-based intragastric delivery of cefuroxime axetil: Development and in-vitro evaluation of mucoadhesive approach

To have advantages of reduced dosing frequency, improved bioavailability and effective delivery system of Cefuroxime Axetil, a Chitosan based intragastric sustained release microbead formulation of Cefuroxime Axetil was developed. The drug delivery system was prepared by ionotropic gelation of Chitosan in presence of sodium tripolyphosphate as polyanion and optimized by box-behnken experimental design. Response surface methodology was applied to evaluate various vitro characteristics of prepared mucoadhesive microbeads. Multiple independent variables were optimized to achieve responses of interest, thereby to get the desired sustained release profile of Cefuroxime Axetil in gastric environment.

Effects of ionic strength on the size and compactness of chitosan nanoparticles

In this work, chitosan nanoparticles were prepared by ionotropic gelation of chitosan with tripolyphosphate (TPP). The effects of the ionic strength of the solvent employed in the particle preparation on the average size and compactness of the particles were investigated. In addition, the effects of the chitosan concentration and the crosslinker to polymer ratio on the particle characteristics were studied. The chitosan–TPP nanoparticles were characterized by dynamic light scattering, zeta potential, and turbidity measurements. The compactness of the nanoparticles was estimated with a method based on the size of the nanoparticles and the turbidity of the nanoparticle suspension. All the investigated preparation parameters, i.e., the ionic strength of the solvent, the chitosan concentration, and the TPP to chitosan ratio, affected the particle characteristics. For instance, smaller and more compact particles were formed in saline solvents, compared to particles formed in pure water. Further, the addition of monovalent salt rendered it possible to prepare particles in the nanometer size range at a higher polymer concentration. Solvent salinity is thus an important parameter to address in the preparation of chitosan nanoparticles crosslinked with TPP.

Erythropoietin Encapsulation in Chitosan Nanoparticles and Kinetics of Drug Release

Recombinant human erythropoietin (rHu-EPO) is a glycoprotein, which is produced commercially from Chinese hamster ovary (CHO) cells. It is used for the therapy of renal anemia and chemotherapy-induced anemia in cancer patients. Recent evidence suggests that rHu-EPO exerts tissue protective effects via multiple mechanisms which include inhibition of apoptosis, promotion of angiogenesis and decreased inflammation. After intravenous (i.v.) injection, the blood concentration of rHu-EPO rapidly decreases due to proteolysis resulting in a relatively short half-life of 8.5 h, which necessitates regular dosing with intervals that do not exceed 7 days. It would be desirable to develop an encapsulated formulation providing controlled release of rHu-EPO to maintain therapeutic concentrations in plasma, and for potential tissue protective applications to maintain high local therapeutic concentrations in tissue while minimizing potential unwanted systemic effects such as polycythemia and platelet activation, both of which can predispose to intravascular thrombosis. Nanoparticle encapsulation of rHu-EPO can also allow for direct injection at sites of injury in specific tissues/organs, again minimizing systemic exposure of the drug. In this paper, we report the production of biopolymer nanoparticles by ionotropic gelation of chitosan with tripolyphosphate (TPP). The nanoparticle size distribution in aqueous solution was determined and rates of rHu-EPO release from chitosan-TPP nanoparticles were measured in PBS at 37°C. It was observed that almost 30% of the encapsulated rHu-EPO was released within the first 48 hours and thereafter a linear release profile was observed for up to 2 weeks. Total drug release over 15 days was 63% of the initial amount.

Intranasal drug delivery of didanosine-loaded chitosan nanoparticles for brain targeting; an attractive route against infections caused by aids viruses

The primary aim of this study was to investigate intranasal (i.n.) administration as a potential route to enhance systemic and brain delivery of didanosine (ddI). A further aim was to investigate the potential use of chitosan nanoparticles as a delivery system to enhance the systemic and brain targeting efficiency of ddI following i.n. administration. Didanosine-loaded chitosan nanoparticles, were prepared through ionotropic gelation of chitosan with tripolyphosphonate anions, and characterized in terms of their size, drug loading, and in vitro release. The nanoparticles were administered i.n. to rats, compared to i.n. and intravenous (i.v.) administration of ddI in solution. The concentrations of ddI in blood, CSF, and brain tissues were analyzed by ultra performance liquid chromatography mass spectroscopy (UPLC/MS). The brain/plasma, olfactory bulb/plasma and CSF/plasma concentration ratios were significantly higher (P < 0.05) after i.n. administration of ddI nanoparticles or solution than those after i.v. administration of didanosine aqueous solution. The ratio of ddI concentration values of the nanoparticles to the solution at 180 min post-i.n. dosing was 2.1 and 1.9 in CSF and brain, respectively. Thus, both the i.n. route of administration and formulation of ddI in chitosan nanoparticles increased delivery of ddI to CSF and brain.

Intracellular Delivery of Cysteine Protease Inhibitor Cystatin by Polymeric Nanoparticles

Two polymers chitosan and poly(lactide-co-glycolide) copolymer (PLGA) were investigated to develop nanoparticles (NPs) for delivery of protein drug substance into tumour cells. Cystatin was selected as a model protein drug due to its high potential to inhibit cysteine proteases, known to trigger the invasive process. Ionotropic gelation of chitosan with tripolyposphate and precipitation of PLGA polymer from a double emulsion system by a solvent diffusion process were used to produce 250-300 nm in diameter NPs. The cellular uptake of NPs was tested on a transformed human breast epithelial cell line, MCF-10A neoT, characterized by an increased expression of cysteine proteases and highly invasive cell phenotype. The influence of NPs on cell viability was evaluated by MTT test showing IC50 400 microg/ml for PLGA and 5 mg/ml for chitosan NPs. As determined by fluorescence microscopy chitosan NPs did not enter the cells during 1-hour incubation whereas the same amount of PLGA NPs were in the cells already after 5 min of incubation. Cystatin delivered into MCF-10A neoT cells by PLGA NPs effectively inhibited intracellular proteolytic activity of cathepsin B, as detected by specific fluorogenic substrate Z-Arg2 cresyl violet. On the contrary, free cystatin in solution did not internalise into the cells and inhibit cathepsin B. To conclude, PLGA NPs with cystatin but not chitosan NPs were targeted through endocytosis to the lysosomal compartments that are rich of proteases enzymes. Our results suggest new strategy to inactivate intracellular tumour-associated proteases, and consequently the invasion behaviour of tumour cells, which could contribute to cancer therapy.