N-palmitoyl Chitosan Research Articles

An explorative study of biotin functionalized magnetic hybrid particles

In the field of biomedicine, magnetic particles have a significant role, owing to their low production cost and the possibility to control their characteristics, like size, shape and ability to tailor the cellular interactions, in order to promote their involvement in the development of novel therapies. Biofunctionalisation is by far, the most studied aspect, since it provides multifunctionality (e.g. increased biocompatibility and biodistribution, or cytotoxicity and toxic selectivity mechanisms). In this framework, this explorative study is devoted to development and characterization of the biotin functionalized magnetic hybrid particles, comprised of a hydrophilic magnetite, synthesized by co-precipitation, in the presence of Pluronic F-127, and a biotinylated N-palmitoyl chitosan matrix. Microscopy images, corroborated with FT-IR spectroscopy results and thermal analysis data confirmed the hybrid structure - multiple magnetite crystallites entrapped in the biofunctionalized polymer matrix, whilst the magnetic assays indicated adequate magnetic properties for biomedical applications. In vitro investigations highlighted that biotin functionalized magnetic hybrid particles are biodegradable and biocompatible, whilst hemocompatibility assessment revealed no significant modifications of blood coagulation times. To conclude, biotin functionalized magnetic hybrid particles exhibited promising assets for future biomedical applications as drug delivery systems or theranostic agents.

Synthesis and evaluation of novel Docetaxel-loaded magnetic composites based on chitosan biotinylated derivative

This study describes a simple approach to obtain novel Docetaxel (DCX)-loaded magnetic composites, based on biotinylated N-palmitoyl chitosan and double-layered hydrophobic magnetite, using a combination of self-assembly and ionic gelation methods. Their physicochemical features (size, surface charge and composition) were determined by specific means. Magnetic composites exhibited a high saturation magnetization of 40 emu/g and superparamagnetic behaviour. In vitro investigations indicated their capacity to deliver DCX up to 72 h, to be biodegraded and cytotoxic on breast cancer cell lines.

Paclitaxel-Loaded Magnetic Nanoparticles Based on Biotinylated N-Palmitoyl Chitosan: Synthesis, Characterization and Preliminary In Vitro Studies.

A considerable interest in cancer research is represented by the development of magnetic nanoparticles based on biofunctionalized polymers for controlled-release systems of hydrophobic chemotherapeutic drugs targeted only to the tumor sites, without affecting normal cells. The objective of the paper is to present the synthesis and in vitro evaluation of the nanocomposites that include a magnetic core able to direct the systems to the target, a polymeric surface shell that provides stabilization and multi-functionality, a chemotherapeutic agent, Paclitaxel (PTX), and a biotin tumor recognition layer. To our best knowledge, there are no studies concerning development of magnetic nanoparticles obtained by partial oxidation, based on biotinylated N-palmitoyl chitosan loaded with PTX. The structure, external morphology, size distribution, colloidal and magnetic properties analyses confirmed the formation of well-defined crystalline magnetite conjugates, with broad distribution, relatively high saturation magnetization and irregular shape. Even if the ability of the nanoparticles to release the drug in 72 h was demonstrated, further complex in vitro and in vivo studies will be performed in order to validate the magnetic nanoparticles as PTX delivery system.

Enzymatically mediated, physiologically triggered N-palmitoyl chitosan hydrogels with temporally modulated high injectability

Injectable hydrogels ideally are low-viscosity liquids when being pushed through a syringe needle and quickly form gels after injection. Physiologically triggered injectable hydrogels based on stimuli-responsive polymers induce sol to gel transitions via pH or temperature alteration, however, such mild environmental stimuli are often prone to be inefficient for triggering the rapid and drastic transition from low-viscosity liquids to form-stable hydrogels. Here, an enzyme mediation strategy to regulate, transmit, and amplify the environmental stimuli is presented for efficient formation of hydrogels with high injectability and stability. The urease-regulated biocatalytic reaction initially inhibited at slightly acidic pH and room temperature is integrated with a pH-responsive system of aqueous N-palmitoyl chitosan (NPCS), acquiring temporally modulated high injectability. After injection to physiological environments, the enzyme activity is significantly enhanced with the elevated pH and temperature, resulting in fast enzymatic generation of a base, which in turn triggers the efficient hydrogelation of aqueous NPCS.

Evaluation of Chitosan Derivative Microparticles Encapsulating Superparamagnetic Iron Oxide and Doxorubicin as a pH-Sensitive Delivery Carrier in Hepatic Carcinoma Treatment: An in vitro Comparison Study.

We developed a novel, pH-sensitive drug delivery microparticle based on N-palmitoyl chitosan (NPCS) to transport the superparamagnetic iron oxide (SPIO) and anticancer drug doxorubicin (DOX). The characteristics of NPCS were characterized through nuclear magnetic resonance. Our results based on testing of volume swelling in multiple pH aqueous solutions revealed that the modified chitosan had a pH-sensitive property. The morphology and size of the DOX-SPIO/NPCS microparticles were investigated using transmission electron microscopy and scanning electron microscopy. The statistical result of microparticles had diameter of 185 ± 87 nm. Surface chemical moieties of DOX-SPIO/NPCS microparticles were confirmed using attenuated total reflection Fourier transform infrared spectroscopy and indicated the existence of mostly hydrophilic groups such as -OH, -C=O, and -C-O-C-. Transmission electron microscopy revealed the dark contrast of SPIO dots encapsulated in the NPCS matrix. Nuclear magnetic resonance T2-weighted magnetic resonance imaging confirmed that the produced DOX-SPIO/NPCS microparticles still exhibited T2 relaxation durations as short as 37.68 ± 8.69 ms (under administration of 2.5 μg/mL), which is comparable to the clinically required dosage. In the drug release profile, the DOX-SPIO/NPCS drug delivery microparticle was accelerated in an acidic environment (pH 6.5) compared with that in a basic environment. Microparticles in a cytotoxicity assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay) revealed that DOX-SPIO/NPCS microparticles had better antitumor ability than did free-form of DOX. Additionally, microparticles loaded with 0.5–5 μg/mL DOX in an acidic environment (pH 6.5) demonstrated higher efficacy against Hep G2 cell growth, possibly because of the swelling effect of NPCS, resulting in volume expansion and easy drug release. Accordingly, these large DOX-SPIO/NPCS microparticles showed potential for application as a pH-sensitive drug delivery system and as chemoembolization particles for hepatic carcinoma therapy.

Folate-conjugated nanobubbles selectively target and kill cancer cells via ultrasound-triggered intracellular explosion

With the rapid development of cancer-targeted nanotechnology, a variety of nanoparticle-based drug delivery systems have clinically been employed in cancer therapy. However, multidrug resistance significantly impacts the therapeutic efficacy. Physical non-drug therapy has emerged as a new and promising strategy. This study aimed to determine whether novel folate-nanobubbles (F-NBs), combined with therapeutic ultrasound (US), could act as a safe and effective physical targeted cancer therapy. Using folate-conjugated N-palmitoyl chitosan (F-PLCS), we developed novel F-NBs and characterised their physicochemical properties, internalization mechanism, targeting ability, therapeutic effects, and killing mechanism. The results showed that the novel F-NBs selectively accumulated in FR-positive endothelial cells and tumour cells via FR coupled with clathrin- and caveolin-mediated endocytosis in vitro and in vivo. In addition, the F-NBs killed target cells by an intracellular explosion under US irradiation. Hoechst/PI staining demonstrated that apoptosis and necrosis accounted for a large proportion of cell death in vivo. F-NBs combined with US therapy significantly inhibited tumour growth and improved the overall survival of tumour-bearing mice. Under US irradiation, the novel F-NBs selectively killed FR-positive tumour cells in vitro and in vivo via intracellular explosion and therefore is a promising alternative for targeted cancer treatment.

Biotinylated N-palmitoyl chitosan for design of drug loaded self-assembled nanocarriers

Owing to its remarkable properties, biotin has been proposed as a very active targeting molecule in anti-cancer formulations. In this study, biotinylated N-palmitoyl chitosan has been prepared and investigated as suitable polymeric derivative for preparation of docetaxel self-assembled nanocarriers with applications in breast cancer treatment. Biotinylated N-palmitoyl chitosan has been obtained through the reaction of N-palmitoyl chitosan with biotin, via carbodiimide chemistry; its structure has been confirmed by FT-IR, 1H NMR spectroscopy and thermal analyses. The self-aggregation behaviour of the polymer in aqueous was determined using fluorescence spectroscopy. Docetaxel loaded self-assembled nanoparticles based on biotinylated N-palmitoyl chitosan, prepared by combining the dialysis method with ultrasonication, were nearly spherical in shape, with narrow size distribution, in the range of 300–400nm and positive charged. Nanoparticles exhibited a good drug loading ability and a pH-dependent release profile of drug, susceptibility to biodegradation and hemocompatibility, properties which can be exploited in drug delivery applications.

Design and evaluation of biofunctionalized magnetic nanoparticles for biomedical applications

Event Abstract Back to Event Design and evaluation of biofunctionalized magnetic nanoparticles for biomedical applications Vera Balan1, Maria Butnaru1, Ovidiu Bredetean1 and Liliana Verestiuc1 1 Gr.T.Popa University of Medicine and Pharmacy, Faculty of Medical Bioengineering, Romania INTRODUCTION Recently, biofunctionalized magnetic nanoparticles are investigated for cancer theragnosis applications[1]. These “intelligent” particles are comprised by a magnetic core, a biocompatible surface coating, a therapeutic agent, and a recognition layer represented by a suitable receptor attached[2]. Amphiphilic derivative chitosan was used to design nanoparticles as a cancer drug delivery system with promising results regarding tumor growth inhibition[3]. Recent studies have revealed that biotin receptors are overexpressed on the surface of tumoral cells, especially breast cancer cells and imply that biotin can be used as a tumor targeting approach for various anti-cancer drugs[4]. Doxorubicin (DOX) is known as one of the most important chemotherapeutic agent against breast, ovarian or lung cancer cells. In this context, the main objective of this study was to design biofunctionalized magnetic nanostructures based on magnetite and N-palmitoyl chitosan, with dual entrapment (therapeutic agent and magnetic material) and evaluate their physico-chemical and biological characteristics. EXPERIMENTAL METHODS Biofunctionalized magnetic nanoparticles (BMNs) have been prepared in two steps: first, nanostructures based on N-palmitoyl chitosan, loaded with doxorubicin and hydrophobic magnetite have been obtained by a double emulsion method; in the next step, magnetic nanoparticles have been functionalized with biotin via carbodiimide chemistry. BMNs structure have been investigated and confirmed by Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction analysis. Particles size and zeta potential were measured with a Malvern Zetasizer NanoS instrument; Magnetic properties have been evaluated using a vibrating sample magnetometer (Lakeshore VSM 7400 System) and the surface morphology was studied by transmission electron microscopy (TEM; PHILIPS CM 20). Drug loading efficiency and in vitro kinetic of drug release has been assessed by UV-VIS Spectroscopy. Internalization of biofunctionalized magnetic nanoparticles was investigated by fluorescence microscopy on MCF-7 cells (Fig 1). RESULTS AND DISCUSSION A new type of biofunctionalized magnetic nanoparticles with submicron size, positive zeta potential, dual entrapment ability, cytotoxic effects on MCF7 cell line human breast adenocarcinoma, redispersion ability and suitable magnetic properties (good magnetic saturation and superparamagnetic behaviour) have been prepared. FT-IR confirmed the structure of biofunctionalized magnetic nanocarriers: biotin, N-palmitoyl chitosan, magnetite and doxorubicin. TEM image has confirmed that hydrophobic magnetite was effectively incorporated into the magnetic nanoparticles. Fig. 1. The fluorescent microscopy image of BMNs in contact with MCF cells CONCLUSIONS These particles exhibit a multitude of good assets together with suitable biological behavior that sustain future experiments aiming to confirm the suitability of proposed magnetic nanoparticles as drug delivery system for breast cancer chemotherapy. This work was financially supported by the Romanian Ministry of Education; grant PN II- PT-PCCA-2011-3.2-0428 - INTERBIORES.

Doxorubicin-loaded magnetic nanocapsules based on N-palmitoyl chitosan and magnetite: Synthesis and characterization

Doxorubicin-loaded magnetic nanocapsules, based on N-palmitoyl chitosan and magnetite, have been prepared using a double emulsion method. The morphology and size of the nanocapsules have been investigated by transmission and scanning electron microscopy. Magnetic nanocapsules were nearly spherical in shape, with a mean diameter in the nanometer range, and positive zeta potential. Fourier Transform Infrared Spectroscopy (FTIR), X-ray diffraction (XRD) and Energy Dispersive X-ray analysis (EDX) confirmed the structure of composite magnetic nanocapsules. Magnetic nanocapsules exhibited suitable magnetic saturation, superparamagnetic behavior and good ability to incorporate a chemotherapeutic agent, properties which can be exploited in many different areas of biomedicine.

High molecular weight chitosan derivative polymeric micelles encapsulating superparamagnetic iron oxide for tumor-targeted magnetic resonance imaging.

Magnetic resonance imaging (MRI) contrast agents based on chitosan derivatives have great potential for diagnosing diseases. However, stable tumor-targeted MRI contrast agents using micelles prepared from high molecular weight chitosan derivatives are seldom reported. In this study, we developed a novel tumor-targeted MRI vehicle via superparamagnetic iron oxide nanoparticles (SPIONs) encapsulated in self-aggregating polymeric folate-conjugated N-palmitoyl chitosan (FAPLCS) micelles. The tumor-targeting ability of FAPLCS/SPIONs was demonstrated in vitro and in vivo. The results of dynamic light scattering experiments showed that the micelles had a relatively narrow size distribution (136.60±3.90 nm) and excellent stability. FAPLCS/SPIONs showed low cytotoxicity and excellent biocompatibility in cellular toxicity tests. Both in vitro and in vivo studies demonstrated that FAPLCS/SPIONs bound specifically to folate receptor-positive HeLa cells, and that FAPLCS/SPIONs accumulated predominantly in established HeLa-derived tumors in mice. The signal intensities of T2-weighted images in established HeLa-derived tumors were reduced dramatically after intravenous micelle administration. Our study indicates that FAPLCS/SPION micelles can potentially serve as safe and effective MRI contrast agents for detecting tumors that overexpress folate receptors.

One-step biofunctionalization of quantum dots with chitosan and N-palmitoyl chitosan for potential biomedical applications.

Carbohydrates and derivatives (such as glycolipids, glycoproteins) are of critical importance for cell structure, metabolism and functions. The effects of carbohydrate and lipid metabolic imbalances most often cause health disorders and diseases. In this study, new carbohydrate-based nanobioconjugates were designed and synthesized at room temperature using a single-step aqueous route combining chitosan and acyl-modified chitosan with fluorescent inorganic nanoparticles. N-palmitoyl chitosan (C-Pal) was prepared aiming at altering the lipophilic behavior of chitosan (CHI), but also retaining its reasonable water solubility for potential biomedical applications. CHI and C-Pal were used for producing biofunctionalized CdS quantum dots (QDs) as colloidal water dispersions. Fourier transform infrared spectroscopy (FTIR), thermal analysis (TG/DSC), surface contact angle (SCA), and degree of swelling (DS) in phosphate buffer were used to characterize the carbohydrates. Additionally, UV-Visible spectroscopy (UV-Vis), photoluminescence spectroscopy (PL), dynamic light scattering (DLS), scanning and transmission electron microscopy (SEM/TEM) were used to evaluate the precursors and nanobioconjugates produced. The FTIR spectra associated with the thermal analysis results have undoubtedly indicated the presence of N-palmitoyl groups “grafted” to the chitosan chain (C-Pal) which significantly altered its behavior towards water swelling and surface contact angle as compared to the unmodified chitosan. Furthermore, the results have evidenced that both CHI and C-Pal performed as capping ligands on nucleating and stabilizing colloidal CdS QDs with estimated average size below 3.5 nm and fluorescent activity in the visible range of the spectra. Therefore, an innovative “one-step” process was developed via room temperature aqueous colloidal chemistry for producing biofunctionalized quantum dots using water soluble carbohydrates tailored with amphiphilic behavior offering potential applications as fluorescent biomarkers in the investigation of glycoconjugates for the nutrition, biology, pharmaceutical, and medicine fields.

Intracellularly monitoring/imaging the release of doxorubicin from pH-responsive nanoparticles using Förster resonance energy transfer

Stimuli-responsive nanoparticles (NPs) have been receiving much attention as a drug-delivery vehicle for therapeutic applications; once internalized into cells, the intracellular fate of NPs and their drug release behavior in response to local stimuli must be understood for efficient delivery of therapeutics. In this study, we prepared pH-responsive doxorubicin (DOX)-loaded NPs, made of N-palmitoyl chitosan bearing a Cy5 moiety (Cy5–NPCS), as an anticancer delivery device. The results of our molecular dynamic simulations showed that the ability of Cy5–NPCS to self-associate offered the close proximity between the donor (DOX) and the acceptor (Cy5) required for Förster resonance energy transfer (FRET), while the pH-driven structure transition prescribed the on-to-off switch of the energy transfer. The caveolae-mediated pathway played a major role in the internalization of NPCS NPs. Using the concept of FRET, we found that the DOX fluorescence in the cytosol was first seen when NPCS NPs were present in the slightly acidic early endosomes. Following NPCS NPs trafficking into a more acidic organelle (late endosomes/lysosomes), a more evident release of DOX into the cytosol was observed; the released DOX was then gradually accumulated in the cell nuclei, leading to a significant cytotoxicity. Understanding the fate of NPs with respect to their intracellular localization and drug release behavior is crucial for the rational design of drug carriers.

A Dual-Emission Förster Resonance Energy Transfer Nanoprobe for Sensing/Imaging pH Changes in the Biological Environment

A dual-emission nanoprobe that can sense changes in the environmental pH is designed based on the concept of pH-responsive Förster resonance energy transfer induced by the conformational transition of an associating polyelectrolyte, N-palmitoyl chitosan, bearing a donor (Cy3) or an acceptor (Cy5) moiety. We demonstrate that the developed pH-responsive nanoprobe can be used to ratiometrically image and thus discriminate the pH changes in the biological environment at different length scales.

Preparation and evaluation of nanometer-scale bubbles with surfaces of N-palmitoyl chitosan

Objective To develop nanometer-scale bubbles with surfaces of N-palmitoyl chitosan(PLCS) as ultrasound contrast agent and evaluate its characteristics and acoustic effects in vivo. Methods The PLCS nanobubbles were prepared using a cutting technique at differential high-frequency of shear speed. Both optical and transmission electron micrography were performed to determine the nanobubble size and morphology. Concentration, size-distribution and zeta potential of the PLCS nanobubbles were measured by cell counting chamber, Malvern lazer particle analyzer and zeta-sizer at 1-day, 45-day and 90-day. The acoustic effects of the PLCS nanobubbles on myocardium and renal tissue in 6 normal rats were observed using bolus infusion of the nanobubbles intravenously. The maximum video intensity(VI) was measured.Results The PLCS nanobubbles with nice round-shape and uniform site-distribution were demonstrated.The mean diameter,concentration and zeta potential of the PLCS nanobubbles were (617 ± 12) nm, (7.2 ±0.6) × 109/ml and (52.9 ± 1.3)mV at the 1-day,and all of parameters did not change significantly in 45-day and 90-day ( P ＞ 0. 05). A significant contrast-enhancement was noted on myocardium and renal tissue during infusion of the nanobubbles. VI on both tissues was (15.6 ± 1.1)GU and (27.3 ± 2.5)GU,respectively. The visual contrast-enhancement last up to (10 ± 2)min. Conclusions The PLCS nanometerscale bubbles have excellent physical-features and contrast-enhanced ultrasound effects in vivo. It may develop as a novel contrast ultrasound agent which could cross endothelial cell membrances. Key words: Ultrasonography; Microbubbles; Nanostructures; N-palmitoyl chitosan

The characteristics, cellular uptake and intracellular trafficking of nanoparticles made of hydrophobically-modified chitosan

It has been reported that nanoparticles (NPs) prepared by hydrophobically-modified polymers could accumulate passively in the tumor tissue; however, their cellular uptake mechanism and intercellular trafficking pathway have never been understood. This study was designed to address these concerns, using NPs prepared by a hydrophobically-modified chitosan ( N-palmitoyl chitosan, NPCS). Molecular dynamic simulations found that a degree of substitution (DS) of 5% of palmitoyl groups on its backbone was sufficient to allow NPCS to form NPs, due to a significant increase in the intra- and intermolecular hydrophobic interactions. With an increase of DS, there were more palmitoyl groups present on the surface of NPs which were then able to interact with the cell membranes. A greater extent of cellular uptake of NPCS NPs was observed with increasing the DS on NPCS. The internalization of NPCS NPs was clearly related with the lipid raft-mediated routes; with increasing the DS on NPCS, the caveolae-mediated endocytosis became more important. The results obtained in the intracellular trafficking study showed that NPCS NPs entered cells via caveolae and transiently localized to caveosomes before trafficking to the endosomal pathway. These results suggest that the prepared NCPS NPs may serve as a carrier for intracellular delivery of therapeutic agents.

Preparation, characterization and pharmacokinetics of N-palmitoyl chitosan anchored docetaxel liposomes

The objective of this work was to investigate the preparation, characterization and pharmacokinetics of N-palmitoyl chitosan anchored docetaxel liposomes. To decrease toxic effects and improve antitumour efficacy of the drug, docetaxel has been incorporated in liposomes; the formulation, stability and pharmacokinetics of plain docetaxel liposomes (PDLs), PEGylated docetaxel liposomes (PEGDLs) and N-palmitoyl chitosan anchored docetaxel liposomes (NDLs) were compared. NDL was more stable than PDL and PEGDL in-vitro, especially in the presence of serum at 37 degrees C. The concentration of docetaxel in the plasma of rats after intravenous administration of docetaxel injection, PDL, PEGDL and NDL was studied by RP-HPLC. The pharmacokinetic behaviour of docetaxel injection, PDL, PEGDL and NDL were significantly different. These findings suggest that anchored liposomes could increase the stability of docetaxel in-vivo, as compared with plain liposomes, but the improvement was not more significant than PEGylated liposomes. N-Palmitoyl chitosan as a new polymeric membrane to anchor liposome was useful to stabilize liposomes containing anti-tumour drug.

PH-triggered injectable hydrogels prepared from aqueous N-palmitoyl chitosan: In vitro characteristics and in vivo biocompatibility

In-situ forming hydrogels triggered by environmental stimuli have emerged as a promising injectable strategy targeted for various biomedical applications. However, several drawbacks associated with temperature-stimulated hydrogels have been reported. Employing a hydrophobically-modified chitosan ( N-palmitoyl chitosan, NPCS), we developed a pH-triggered hydrogel system which showed a rapid nanostructure transformation within a narrow pH range (pH 6.5–7.0). NPCS in an aqueous environment was found to be a shear-thinning fluid and exhibited an instant recovery of its elastic properties after shear thinning, thereby making it an injectable material. Additionally, aqueous NPCS, an associating polyelectrolyte, can be rapidly transformed into hydrogel triggered simply by its environmental pH through a proper balance between charge repulsion and hydrophobic interaction. This in-situ hydrogel system was shown to be nontoxic. Subcutaneous injection of aqueous NPCS (pH 6.5) into a rat model resulted in rapid formation of a massive hydrogel at the location of injection. The implanted hydrogel was found to be degradable and was associated with an initial macrophage response which decreased with time as the degradation proceeded. These results suggested that the developed NPCS hydrogel may be used as an injectable drug/cell delivery system.

Novel Polymer Micelles Prepared from Chitosan Grafted Hydrophobic Palmitoyl Groups for Drug Delivery

Chitosan-based polymer micelles have a splendid outlook for drug delivery owing to the interesting properties, abundance, and low cost of chitosan. A new method of preparation of water-soluble N-palmitoyl chitosan (PLCS) which can form micelles in water is developed in this paper. The preparation of PLCS was carried out by swollen chitosan coupling with palmitic anhydride in dimethyl sulfoxide (DMSO). The degree of substitution (DS) of PLCS was in the range of 1.2-14.2%, and the critical aggregation concentration (CAC) of PLCS micelles was in the range of 2.0 x 10(-3) to 37.2 x 10(-3) mg/mL. The properties of PLCS micelles such as encapsulation capacity and controlled release ability of hydrophobic model drug ibuprofen (IBU) were evaluated. Experimental results indicated that the loading capacity (LC) of PLCS was approximately 10%. The drug release strongly depended on pH and temperature: low pH and high temperature accelerated drug release markedly. Moreover, the IR, 1H NMR, and TEM of PLCS, IBU-loaded PLCS, and a PLCS-IBU physical mixture have been measured to show that IBU is loaded by PLCS micelles.