In-vitro Cellular Uptake and Transport Study of 9-Nitrocamptothecin PLGA Nanoparticles Across Caco-2 Cell Monolayer Model (original) (raw)
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International Journal of Nanomedicine, 2010
The purpose in this study was to investigate poly(ethylene glycol)-modified poly (d,l-lactide-co-glycolide) nanoparticles (PLGA-PEG-NPs) loading 9-nitrocamptothecin (9-NC) as a potent anticancer drug. 9-NC is an analog of the natural plant alkaloid camptothecin that has shown high antitumor activity and is currently in the end stage of clinical trial. Unfortunately, at physiological pH, these potent agents undergo a rapid and reversible hydrolysis with the loss of antitumor activity. Previous researchers have shown that the encapsulation of this drug in PLGA nanoparticles could increase its stability and release profile. In this research we investigated PLGA-PEG nanoparticles and their effect on in vitro characteristics of this labile drug. 9-NC-PLGA-PEG nanoparticles with particle size within the range of 148.5 ± 30 nm were prepared by a nanoprecipitation method. The influence of four different independent variables (amount of polymer, percent of emulsifier, internal phase volume, and external phase volume) on nanoparticle drug-loading was studied. Differential scanning calorimetry and X-ray diffractometry were also evaluated for physical characterizing. The results of optimized formulation showed a narrow size distribution, suitable zeta potential (+1.84), and a drug loading of more than 45%. The in vitro drug release from PLGA-PEG NPs showed a sustained release pattern of up to 120 hours and comparing with PLGA-NPs had a significant decrease in initial burst effect. These experimental results indicate that PLGA-PEG-NPs (versus PLGA-NPs) have a better physicochemical characterization and can be developed as a drug carrier in order to treat different malignancies. Keywords: long circulating nanoparticle, 9-nitrocamptothecin, in vitro characterization Camptothecin (CPT) and its derivatives play an important role in the treatment of different malignancies. 1,2 They all have a unique mechanism of action, targeting the nuclear enzyme of DNA topoisomerase I (topo I). CPT inhibits this enzyme by blocking the rejoining step of the cleavage/relegation reaction of topo I, resulting in accumulation of a covalent reaction intermediate, the cleavage complex. The primary mechanism of cell killing by CPT is S-specific killing through potentially lethal collision between advancing replication forks and topo-I cleavable complexes. 3,4,5 In the structure of these analogues, common features include an aromatic 5-ring system and a lactone moiety which is crucial for their antitumor activity. 4 9-Nitrocamptothecin (9-NC) is a novel, lipophilic derivative of CPT which has a high antitumor activity which has been used in several clinical trial studies in order to treat advanced pancreatic carcinoma, ovarian epithelial cancer and leukemia. 2 Unfortunately, the lactone form depends on the pH of the medium and easily inverts to carboxylate form in alkaline pHs, especially when exposed to serum albumin proteins, which remove its antitumor properties (see Figure 1). 4,6
Anti-Cancer Drugs, 2008
The objective of this study was to evaluate the cytotoxicity and pharmacokinetics of total and lactone forms of 9-nitrocamptothecin (9-NC), an effective antineoplastic drug, after intravenous injection of drug incorporated into poly (DL-lactic-glycolic acid) nanoparticles (NPs). Drug-loaded NPs (9-NC.NP) were prepared by the nanoprecipitation method and examined for particle characteristics and in-vitro release in phosphate buffered saline. The best formulation showed a narrow size with an average diameter of 207 ± 26 nm and a drug loading of more than 33.5%. The drug release profile showed a sustained 9-NC release up to 160 h. For a pharmacokinetic study, the concentration of 9-NC as the lactone form (9-NC.lac) and as the total of the lactone and carboxylate forms (9-NC.tot) in plasma was determined by using reverse-phase high performance liquid chromatography after intravenous administration of 9-NC.NP and a control solution to cannulated Wistar rats. In-vitro cytotoxic activity of 9-NC.NP and control solution was evaluated on the human ovarian cancer cell line (A2780sn) by MTT cell cytotoxicity assay. Results of in-vivo studies showed that NP encapsulation markedly increased the plasma concentration of both lactone and total forms of 9-NC compared with free drug. In comparison with free drug, NPs resulted in 3.63-fold and 5.40-fold increases in area under the plasma concentration-versus-time curve (AUC 0-N) for lactone and total forms of 9-NC, respectively. The values of mean residence time and elimination half-life (T 1/2) were also significantly higher for NPs than for free drug. The in-vitro cytotoxicity study revealed that the IC 50 value of NPs decreased 10-fold compared with the drug solution. Prepared NPs described here were considered potentially useful in both stabilizing and delivering 9-NC and enhancing the efficacy of this drug for cancer treatment for which high drug retention in the body, protection from the drug-active lactone form, and gradual drug release appeared to be related.
Poly (D,L-lactide-co-glycolide) nanoparticles:Uptake by epithelial cells and cytotoxicity
Nanoparticles as drug delivery systems offer benefits such as protection of the encapsulated drug against degradation, site-specific targeting and prolonged blood circulation times. The aim of this study was to investigate nanoparticle uptake into Caco-2 cell monolayers, their co-localization within the lysosomal compartment and their cytotoxicity in different cell lines. Rhodamine-6G labelled poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles were prepared by a double emulsion solvent evaporation freeze-drying method. Uptake and co-localisation of PLGA nanoparticles in lysosomes were visualized by confocal laser scanning microscopy. The cytotoxicity of the nanoparticles was evaluated on different mammalian cells lines by means of Trypan blue exclusion and the MTS assay. The PLGA nanoparticles accumulated in the intercellular spaces of Caco-2 cell monolayers, but were also taken up transcellularly into the Caco-2 cells and partially co-localized within the lysosomal compartment indicating involvement of endocytosis during uptake. PLGA nanoparticles did not show cytotoxic effects in all three cell lines. Intact PLGA nanoparticles are therefore capable of moving across epithelial cell membranes partly by means of endocytosis without causing cytotoxic effects. Keywords: biocompatible polymers, Caco-2 cells, cellular uptake, cytotoxicity, PLGA nanoparticles
Biomaterials, 2005
This study evaluated cellular uptake of polymeric nanoparticles by using Caco-2 cells, a human colon adenocarcinoma cell line, as an in vitro model with the aim to apply nanoparticles of biodegradable polymers for oral chemotherapy. The feasibility was demonstrated by showing the localization and quantification of the cell uptake of fluorescent polystyrene nanoparticles of standard size and poly(lactic-co-glycolic acid) (PLGA) nanoparticles coated with polyvinyl alcohol (PVA) or vitamin E TPGS. Coumarin-6 loaded PLGA nanoparticles were prepared by a modified solvent extraction/evaporation method and characterized by laser light scattering for size and size distribution, scanning electron microscopy (SEM) for surface morphology, zeta-potential for surface charge, and spectrofluorometry for fluorescent molecule release from the nanoparticles. The effects of particle size and particle surface coating on the cellular uptake of the nanoparticles were quantified by spectrofluorometric measurement. Cellular uptake of vitamin E TPGS-coated PLGA nanoparticles showed 1.4 folds higher than that of PVA-coated PLGA nanoparticles and 4-6 folds higher than that of nude polystyrene nanoparticles. Images of confocal laser scanning microscopy, cryo-SEM and transmission electron microscopy clearly evidenced the internalization of nanoparticles by the Caco-2 cells, showing that surface modification of PLGA nanoparticles with vitamin E TPGS notably improved the cellular uptake. It is highly feasible for nanoparticles of biodegradable polymers to be applied to promote oral chemotherapy.
Lupeol exhibits anti-inflammatory effects; unfortunately it shows low water solubility. An alternative to overcome this is the development of nanomaterials. Several methods for nanomaterial production are available. One of them is emulsification/solvent-evaporation. The objective of the present work was to evaluate physical properties, transport and in vitro modulator effects on NF-kB of poly (lactide-co-glycolide) (PLGA) nanoparticles loaded with lupeol. Nanonutraceuticals were prepared with 16% (w/v) of lupeol. Size distribution and morphology were measured by particle size analyzer and TEM. In vitro release of lupeol was studied by three different models: Higuchi, Siepmann & Peppas, and Power law. Transport of nanonutraceutical was studied in a Caco-2 cell model and by GCeMS. Modulator effect on NK-kB was studied by western blot analysis. Nanonutraceuticals were 10% larger than the nanoparticles without lupeol (372 vs 337 nm) and presented a broader size distribution (0.28 vs 0.22). TEM results displayed spherical structures with a broader size distribution. Entrapment efficiency of lupeol was 64.54% and it in vitro release data fitted well to the Power law and Higuchi equation (R > 0.84e0.84). Strong regulation of NF-kB of nanonutraceutical was observed. It was not observed any transport across the Caco-2 cell model at the different experimental conditions.
PLGA nanoparticles containing various anticancer agents and tumour delivery by EPR effect
Advanced Drug Delivery Reviews, 2011
As mortality due to cancer continues to rise, advances in nanotechnology have significantly become an effective approach for achieving efficient drug targeting to tumour tissues by circumventing all the shortcomings of conventional chemotherapy. During the past decade, the importance of polymeric drugdelivery systems in oncology has grown exponentially. In this context, poly(lactic-co-glycolic acid) (PLGA) is a widely used polymer for fabricating 'nanoparticles' because of biocompatibility, long-standing track record in biomedical applications and well-documented utility for sustained drug release, and hence has been the centre of focus for developing drug-loaded nanoparticles for cancer therapy. Such PLGA nanoparticles have also been used to develop proteins and peptides for nanomedicine, and nanovaccines, as well as a nanoparticle-based drug-and gene-delivery system for cancer therapy, and nanoantigens and growth factors. These drug-loaded nanoparticles extravasate through the tumour vasculature, delivering their payload into the cells by the enhanced permeability and retention (EPR) effect, thereby increasing their therapeutic effect. Ongoing research about drug-loaded nanoparticles and their delivery by the EPR effect to the tumour tissues has been elucidated in this review with clarity.
Effect of Physico-Chemical Properties of Nanoparticles on Their Intracellular Uptake
International Journal of Molecular Sciences, 2020
Cellular internalization of inorganic, lipidic and polymeric nanoparticles is of great significance in the quest to develop effective formulations for the treatment of high morbidity rate diseases. Understanding nanoparticle–cell interactions plays a key role in therapeutic interventions, and it continues to be a topic of great interest to both chemists and biologists. The mechanistic evaluation of cellular uptake is quite complex and is continuously being aided by the design of nanocarriers with desired physico-chemical properties. The progress in biomedicine, including enhancing the rate of uptake by the cells, is being made through the development of structure–property relationships in nanoparticles. We summarize here investigations related to transport pathways through active and passive mechanisms, and the role played by physico-chemical properties of nanoparticles, including size, geometry or shape, core-corona structure, surface chemistry, ligand binding and mechanical effect...
Study on uptake of PLA-Pluronic P85-PLA nanoparticles with Caco-2 cells
Proceedings of the 2015 International Conference on Advanced Engineering Materials and Technology, 2015
We have shown that insulin-loaded Poly(lactic acid)-b-Pluronic-b-poly(lactic acid) nanoparticles (PLA-P85-PLA-NPs) had the hypoglycemic effect after oral administration on diabetic mice. In the present work, free insulin (INS) as controls, the influence of some factors such as time, temperature, pH level, and INS concentration on the uptake efficacy of PLA-P85-PLA-NPs as drug carriers were investigated through a Caco-2 cells. The micrographs of Caco-2 cell model were observed by microscopy. The cytotoxicity of PLA-P85-PLA-NPs in vitro was evaluated by MTT assay. The insulin concentrations in Caco-2 cells were determined by high-performance liquid chromatography (HPLC). Compared with INS groups, PLA-P85-PLA-NPs uptake was not controlled by pH levels. PLA-P85-PLA-NPs can be uptake by Caco-2 cells, and the absorption of PLA-P85-PLA-NPs is dependent on the concentration and incubation time. INS-loaded PLA-P85-PLA-NPs significantly enhanced insulin absorption. These results suggest that PLA-P85-PLA-NPs were more efficient than individual drugs into Caco-2 cells in a short period of time, and PLA-P85-PLA-NPs might be a useful drug carrier for proteins and peptides.
Antitumoral activity of camptothecin-loaded nanoparticles in 9L rat glioma model
International Journal of Pharmaceutics, 2011
Camptothecin (CPT), a plant alkaloid, is a potent anticancer drug in cell culture studies but it is clinically inactive due to rapid hydrolysis under physiological conditions. The drug exists in two forms depending on the pH value, an active lactone form at pH below 5 and an inactive carboxylate form at basic pH and this is a reversible reaction. In this study, nanoparticulate delivery systems were developed with either amphiphilic cyclodextrins, poly(lactide-co-glycolide) or poly-caprolactone in order to maintain the active lactone form and prevent the drug from hydrolysis. All nanoparticles were prepared with nanoprecipitation technique. Mean particle sizes were 130-280 nm and surface charges were negative. The encapsulation efficiency was significantly higher for amphiphilic cyclodextrin nanoparticles when compared to polymeric nanoparticles. Nanoparticle formulations based on cyclodextrins showed a controlled release profile extended up to 12 days. 6-O-Capro--cyclodextrin (1.44 g/60 L CPT) and concentrated 6-O-Capro--cyclodextrin (2.88 g/60 L CPT) nanoparticles significantly modified the growth or lethality of the 9L gliomas, since the median survival time was 26 days for the untreated group and between 27 and 33 days for amphiphilic cyclodextrin nanoparticle groups. These results indicate that, CPT-loaded amphiphilic cyclodextrin nanoparticles may provide a promising carrier system for the effective delivery of CPT in comparison to polymeric analogues.