Self-aggregated nanoparticles of cholesterol-modified chitosan conjugate as a novel carrier of epirubicin

Cholesterol-modified chitosan conjugate with succinyl linkages (CHCS) was synthesized and characterized by fourier transform infrared (FTIR) and proton nuclear magnetic resonance (¹H NMR). The degree of substitution (DS) of cholesterol moiety determined by elemental analysis was 7.3%. The self-aggregation behavior of CHCS was evaluated by the fluorescence probe technique and the critical aggregation concentration (CAC) was 1.16 × 10⁻² mg mL⁻¹ in 0.1 M acetic acid solution. CHCS formed monodisperse self-aggregated nanoparticles with a roughly spherical shape and a mean diameter of 417.2 nm by probe sonication in aqueous media. Epirubicin (EPB), as a model anticancer drug, was physically entrapped inside CHCS self-aggregated nanoparticles by the remote loading method and the characteristics of EPB-loaded CHCS self-aggregated nanoparticles were analyzed using dynamic laser light scattering (DLLS), transmission electron microscopy (TEM) and fluorescence spectroscopy. EPB-loaded CHCS self-aggregated nanoparticles were almost spherical in shape and their size increased from 338.2 to 472.9 nm with the EPB-loading content increasing from 7.97% to 14.0%. The release behavior of EPB from CHCS self-aggregated nanoparticles was studied in vitro by dialysis method. The results showed that EPB release rate decreased with the pH increase of the release media. In phosphate buffered saline (PBS, pH 7.4), the EPB release was very slow and the total release amount was about 24.9% in 48 h.     

Self-aggregated nanoparticles from methoxy poly(ethylene glycol)-modified chitosan: synthesis; characterization; aggregation and methotrexate release in vitro

Methoxy poly(ethylene glycol)-grafted-chitosan (mPEG-g-CS) conjugates were synthesized by formaldehyde linking method and characterized by Fourier transform infrared (FT-IR) and proton nuclear magnetic resonance (¹H-NMR). The degree of substitution (DS) of methoxy poly (ethylene glycol) (mPEG) in the mPEG-g-CS molecules determined by ¹H-NMR ranged from 19% to 42%. The critical aggregation concentration (CAC) was determined by fluorescence spectroscopy using pyrene as fluorescence probe and its value was 0.07 mg/mL in water. mPEG-g-CS formed monodisperse self-aggregated nanoparticles with a roughly spherical shape and a mean diameter of 261.9 nm were prepared by the dialysis method. mPEG-g-CS self-aggregated nanoparticles were used as carriers of poorly water-soluble anticancer drug methotrexate (MTX). MTX was physically entrapped inside mPEG-g-CS self-aggregated nanoparticles by dialysis method and the characteristics of MTX-loaded mPEG-g-CS self-aggregated nanoparticles were analyzed using dynamic laser light scattering (DLLS), transmission electron microscopy (TEM). Moreover, in vitro release behavior of MTX was also investigated and the results showed that MTX was continuously released more than 50% in 48 h.     

Self-assembly of an amphiphilic derivative of chitosan and micellar solubilization of puerarin

A kind of amphiphilic derivatives of chitosan (2-hydroxyl-3-butoxyl)-propylcarboxymethyl-chitosan (HBP-CMCHS), has been synthesized, and the critical micelle concentration (cmc) of HBP-CMCHS was detected by the fluorescence method. The puerarin-loaded HBP-CMCHS micellar system was prepared by physical entrapped method. Result showed that when adding the same amount of puerarin, the solubilizing capacity was enhanced by increasing the concentration of HBP-CMCHS and temperature. Puerarin-loaded micellar system of HBP-CMCHS was characterized by TEM and DLS. TEM photograph revealed that the micelles were spherical and puerarin was solubilized in the cores of the spherical polymeric micelles. DLS showed that after solubilization the size of the micelles became bigger. In vitro tests showed that puerarin was slowly released from micellar solution and the release lasted up to 60 h by means of the dialysis method.     

Micellar Solubilization and In Vitro Release of Silymarin in the self-Aggregates of an Amphiphilic Derivative of Chitosan

Summary: The amphiphilic derivatives of chitosan, (2-hydroxyl-3-butoxyl)-propylcarboxymethyl-chitosan (HBP-CMCHS), can form micelles with the inner core of hydrophobic segments and the outer shell of hydrophilic segments. The typical poor water-soluble drug silymarin was solubilized in the HBP-CMCHS micellar by physical entrapped method. Results showed that the solubilizing capacity was enhanced by increasing the concentration of HBP-CMCHS with the same dosage of silymarin. Silymarin-loaded micellar system of HBP-CMCHS was characterized by TEM and DLS. TEM photograph revealed that the micelles were spherical and silymarin was solubilized in the cores of the spherical polymeric micelles. DLS showed that after solubilization the size of the micelles became bigger. In vitro tests showed that silymarin was slowly released from micellar solution and the release lasted up to 40 h by means of the dialysis method.     

Preparation and properties of ionically cross‐linked chitosan nanoparticles

Chitosan nanoparticles were fabricated by a method of tripolyphosphate (TPP) cross‐linking. The influence of fabrication conditions on the physical properties and drug loading and release properties was investigated by transmission electron microscopy (TEM), dynamic light scattering (DLS), and UV–vis spectroscopy. The nanoparticles could be prepared only within a zone of appropriate chitosan and TPP concentrations. The particle size and surface zeta potential can be manipulated by variation of the fabrication conditions such as chitosan/TPP ratio and concentration, solution pH and salt addition. TEM observation revealed a core–shell structure for the as‐prepared nanoparticles, but a filled structure for the ciprofloxacin (CH) loaded particles. Results show that the chitosan nanoparticles were rather stable and no cytotoxicity of the chitosan nanoparticles was found in an in vitro cell culture experiment. Loading and release of CH can be modulated by the environmental factors such as solution pH and medium quality. Copyright © 2008 John Wiley & Sons, Ltd.     

Characterization and release of triptolide-loaded poly (d,l-lactic acid) nanoparticles

Triptolide (TP), which has immunosuppressive effect, anti-neoplastic activity, anti-fertility function and severe toxicities on digestive, urogenital, blood circulatory system, was used as a model drug in this study. TP-loaded poly (d,l-lactic acid) (PLA) nanoparticles were prepared by the modified spontaneous emulsification solvent diffusion method (modified-SESD method). Dynamic light scattering system (DLS), transmission electron microscope (TEM), atomic force microscopy (AFM), differential scanning calorimetry (DSC), X-ray powder diffractometry and Fourier transform infra-red spectroscopy (FT-IR) were employed to characterize the nanoparticles fabricated for size and size distribution, surface morphology, the physical state of drug in nanoparticles, and the interaction between the drug and polymer. Encapsulation efficiency (EE) and the in vitro release of TP in nanoparticles were measured by the reverse phase high-performance liquid chromatography (RP-HPLC). The produced nanoparticles exhibited a narrow size distribution with a mean size of approximately 150 nm and polydispersity index of 0.088. The morphology of the nanoparticles exhibited a fine spherical shape with smooth surfaces without aggregation or adhesion. TP-entrapped in nanoparticles was found in the form of amorphous or semicrystalline. It was found that a weak interaction existed between the drug and polymer. In all experiments, more than 65% of EE were obtained. The in vitro release profile of TP from nanoparticles exhibited a typical biphasic release phenomenon, namely initial burst release and consequently sustained release. In this case, the particle size played an important role for the drug release. The modified-SESD method was a potential and advantage method to produce an ideal polymer nanoparticles for drug delivery system (DDS).     

Long circulating 10-hydroxycamptothecin-loaded nanoparticles fabricated from poly(ethylene glycol)/poly(L-lactic acid) multiblock copolymers

A series of poly(ethylene glycol) (PEG)/poly(L-lactic acid) (PLLA) multiblock copolymers were facilely synthesized using triphosgene as coupling agent. With the resulting multiblock copolymers, 10-hydroxycamptothecin (HCPT)-loaded nanoparticles were successfully prepared by dialysis method. The results obtained from dynamic light scattering (DLS) measurements confirmed that HCPT-loaded nanoparticles had the size of less than 200 nm and the average diameter decreased with increasing PLLA content. TEM images demonstrated that most of the drug-loaded nanoparticles had a distinct spherical shape and smooth surface without any aggregation. Atomic force microscopy (AFM) images further indicated that the nanoparticles were in spherical shape with smooth surface, no drug crystal was visualized on their surface. To investigate the drug state in HCPT-loaded nanoparticles, differential scanning calorimetry (DSC) and X-ray powder diffraction (XRD) measurements were carried out. The results from these tests suggested that HCPT was molecularly dispersed in the amorphous polymer matrix. Drug loading content and in vitro drug release behavior of HCPT-loaded nanoparticles showed dependence on polymer composition. Cytotoxicity test indicated that HCPT-loaded nanoparticles exhibited greatly superior cytotoxicity compared to free HCPT due to its molecular dispersion in the polymer matrix. Furthermore, the nanoparticles significantly increased the duration of the drug in circulation. All these results demonstrated that PEG/PLLA nanoparticles have great potential as promising delivery system for poorly soluble antitumor drugs.     

Long circulating 10-hydroxycamptothecin-loaded nanoparticles fabricated from poly(ethylene glycol)/poly(L-lactic acid) multi-block copolymers

A series of poly(ethylene glycol) (PEG)/poly(L-lactic acid) (PLLA) multi-block copolymers were facilely synthesized using triphosgene as coupling agent. With the resulting multi-block copolymers, 10-hydroxycamptothecin (HCPT)-loaded nanoparticles were successfully prepared by dialysis method. The results obtained from dynamic light scattering (DLS) measurements confirmed that HCPT-loaded nanoparticles had the size of less than 200 nm and the average diameter decreased with increasing PLLA content. TEM images demonstrated that most of the drug-loaded nanoparticles had a distinct spherical shape and smooth surface without any aggregation. Atomic force microscopy (AFM) images further indicated that the nanoparticles were in spherical shape with smooth surface, no drug crystal was visualized on their surface. To investigate the drug state in HCPT-loaded nanoparticles, differential scanning calorimetry (DSC) and X-ray powder diffraction (XRD) measurements were carried out. The results from these tests suggested that HCPT was molecularly dispersed in the amorphous polymer matrix. Drug loading content and in vitro drug release behavior of HCPT-loaded nanoparticles showed dependence on polymer composition. Cytotoxicity test indicated that HCPT-loaded nanoparticles exhibited greatly superior cytotoxicity compared to free HCPT due to its molecular dispersion in the polymer matrix. Furthermore, the nanoparticles significantly increased the duration of the drug in circulation. All these results demonstrated that PEG/PLLA nanoparticles have great potential as promising delivery system for poorly soluble antitumor drugs.     

Nanoparticles from Chitosan

Novel biodegradable nanoparticles were synthesized by chemical modification of the chitosan linear chain. A natural dicarboxylic acid (malic acid) was used as a crosslinking agent for intramolecular covalent condensation reaction to obtain hydrophilic nanoparticles based on chitosan. A variety of methods including, solubility studies, laser light scattering (DLS), transmission electron microscopy (TEM) and nuclear magnetic resonance (NMR) was used to characterize the crosslinked macromolecules. The prepared biodegradable chitosan nanoparticles, soluble in aqueous media, might be useful for various biomedical applications, like injectable drug- or gene-delivery systems.     

Structural and dynamical properties of aqueous mixtures of pectin and chitosan

In this study phase separation, structure, and dynamics of aqueous pectin-chitosan mixtures of different ratios and a pure aqueous pectin sample have been investigated under various conditions by turbidimetry, SANS and dynamic light scattering (DLS). Only the mixture with r = 0.75 gelled upon decreasing the temperature ((r ≡ m_pectin/(m_pectin + m_chitosan), where m denotes the mass of the considered component). The pure pectin sample (r = 1) did not gel and the decrease in temperature seemed to promote phase separation. The addition of chitosan reduced the tendency of pectin to phase separate in the mixtures of pectin and chitosan. The general trend when cooling the samples was that the turbidity and the growth of the turbidity became more pronounced as the amount of pectin in the mixture was increased. The wavelength dependence of the turbidity indicated a change of the conformation of pectin chains from an extended form to a more compact structure in pectin solutions without chitosan as the temperature decreased. This was not observed for the mixture of pectin and chitosan. SANS measurements revealed excess scattered intensity in the low wave vector area with the strongest upturn for the pure pectin sample (r = 1). DLS experiments showed longer slow relaxation times after a temperature quench for all samples, with the most pronounced effect for the mixture of pectin and chitosan with r = 0.75. The synergism between pectin and chitosan at high pectin contents (r = 0.75) generated large association complexes over time.     

Synthesis and characterization of PEG modified N-trimethylaminoethylmethacrylate chitosan nanoparticles

Chitosan-N-trimethylaminoethylmethacrylate chloride-PEG (CS-TM-PEG) copolymers were synthesized in order to improve the solubility of chitosan in physiological environment, and enhance the biocompatibility of quaternized chitosan. The result of ¹H NMR confirmed that PEG had been combined with amino groups of quaternized chitosan. The profile of hemolysis assay showed that Chitosan-N-trimethylaminoethylmethacrylate chloride (CS-TM) copolymer exhibited hemolytic activity from 10.31% to 13.58%, while CS-TM-PEG copolymer had hemolytic activity from 4.76% to 7.05% at copolymer concentrations from 250 to 2000 μg/ml. Through PEG modification, the hemolytic activity could be reduced to a half. CS-TM-PEG copolymer-insulin nanoparticles were prepared based on ionic gelation process of positively charged copolymers and negatively charged insulin. The nanoparticles were characterized in terms of particle size, TEM, association efficiency and in vitro release. These nanoparticles were 200-400 nm in size and insulin association efficiency of optimal formulations was found up to 90%. In vitro release showed that the nanoparticles provided an initial burst release followed by a sustained release with the sensitivity of ionic strength and pH values.     

Synthesis and characterization of thermo-responsive copolymeric nanoparticles of poly(methyl methacrylate-co-N-vinylcaprolactam)

Copolymeric nanoparticles of methyl methacrylate (MMA) and N-vinylcaprolactam (VCL) were prepared through free radical polymerization using hydrogen peroxide and l-ascorbic acid as a redox initiator in o/w microemulsion containing sodium dodecyl sulphate (SDS). The copolymers were characterized by FTIR and gel permeation chromatography (GPC) and composition of copolymer was determined by ¹H NMR spectroscopy. Reactivity ratio was determined by linear least square and non-linear least square methods. The morphology and particle size distribution of copolymer latexes was determined through transmission electron microscopy (TEM) and dynamic light scattering (DLS). Copolymers were of less than 50 nm size with spherical morphology and latexes were stable for more than 6 months. Phase transition temperature measured through UV-vis spectrometry, for the synthesized copolymer indicates their potential use in biosensors and targeted drug delivery system. Cytotoxicity of nanoparticles was determined by MTT assay on B16F10 melanoma cell lines. Cell viability data shows the IC₅₀ values of copolymeric nanoparticles to be in the range of 0.01-0.1 mg/mL.     

Synthesis of gold nanoparticle necklaces using linear-dendritic copolymers

Linear-dendritic copolymers containing hyperbranched poly(citric acid) and linear poly(ethylene glycol) blocks (PCA-PEG-PCA) were used as reducing and capping agents to synthesize and support gold nanoparticles (AuNPs). PCA-PEG-PCA copolymers with 1758, 1889 and 3446 molecular weights, called A1, A2 and A3 through this work, respectively, were synthesized using 2, 5, and 10 citric acid/PEG molar ratios. The diameter of A1, A2 and A3 in a fresh water solution was investigated using dynamic light scattering (DLS) and it was between 1.8 and 2.8 nm. AuNPs were simply synthesized and supported by addition a boiling aqueous solution of HAuCl₄ to aqueous solutions of A1, A2 and A3. Supported AuNPs were stable in water for several months and agglomeration was not occurred. The loading capacity of A1, A2 and A3 and the size of synthesized AuNPs were investigated using UV spectroscopy and transmission electron microscopy (TEM). It was found that the loading capacity of PCA-PEG-PCA copolymers depend on the concentration of copolymers and the size of their poly(citric acid) parts directly. For example average loading capacities for 400 μM concentration of A1, A2 and A3 were 32.24, 37.4 and 41.52 μM, respectively, and average loading capacities for 400, 200 and 100 μM concentration of A1 were 32.24, 20.28 and 9.1 μM, respectively. Interestingly there was a reverse relation between the size of synthesized AuNPs and size of poly(citric acid) parts of PCA-PEG-PCA copolymers.     

Preparation and characterization of nanoparticles formed by chitosan-caseinate interactions

Intermacromolecular complexation between chitosan and sodium caseinate in aqueous solutions was studied as a function of pH (3–6.5), using absorbance measurements (at 600 nm), dynamic light scattering (DLS), and transmission electron microscopy (TEM). The chitosan–caseinate complexes formed were stable and soluble in the pH range 4.8–6.0. In this pH range, the biopolymers had opposite charges. At higher concentrations of chitosan (0.15 wt%), the soluble complexes associated to form larger particles. DLS data showed that, between pH 4.8 and 6.0, the particles formed by the complexation of chitosan and caseinate had sizes between 250 and 350 nm and these nanoparticles were visualized using negative staining TEM. Above pH 6.0, the nanoparticles associated to form larger particles, causing phase separation. Addition of NaCl increased the particle size. The pH dependence of the zeta potential of the mixture solutions was appreciably different from that of the pure protein and pure chitosan solutions.     

Self-aggregates of deoxycholic acid-modified chitosan as a novel carrier of adriamycin

A novel and simple method for delivery of adriamycin (ADR) was developed using self-aggregates of deoxycholic acid-modified chitosan. Deoxycholic acid was covalently conjugated to chitosan via EDC-mediated reaction to generate self-aggregated chitosan nanoparticles. ADR was physically entrapped inside the self-aggregates and the characteristics of ADR-loaded chitosan self-aggregates were analyzed by dynamic light scattering, fluorescence spectroscopy, and atomic force microscopy (AFM). The maximum amount of entrapped ADR reached 16.5 wt% of chitosan self-aggregates, suggesting a loading efficiency of 49.6 wt%. The size of ADR-loaded self-aggregates increased with increasing the loading content of ADR. AFM images showed spherical shape of ADR-loaded self-aggregates, and ADR was slowly released from chitosan self-aggregates in PBS solution (pH 7.2). Received: 24 April 2000/Accepted: 11 July 2000     

Preparation and characterization novel polymer-coated magnetic nanoparticles as carriers for doxorubicin

The poly(lactide-co-glycolide)-coated magnetic nanoparticles (PLGA MNPs) were prepared as carriers of doxorubicin (PLGA-DOX MNPs) through water-in-oil-in-water (W/O/W) emulsification method. The characteristics of PLGA-DOX MNPs were measured by using transmission electron microscopy (TEM) and vibrating-sampling magnetometry (VSM). It was found that the synthesized nanoparticles were spherical in shape with an average size of 100 ± 20 nm, low aggregation and good magnetic responsivity. Meanwhile, the drug content and encapsulation efficiency of nanoparticles can be achieved by varying the feed weight ratios of PLGA and DOX particles. These PLGA-DOX MNPs also demonstrated sustained release of DOX at 37 °C in buffer solution. Besides, influence of drug-loaded nanoparticles on in vitro cytotoxicity was determined by MTT assay, while cellular apoptosis was detected by Annexin V-FITC apoptosis detection kit. The results showed that PLGA-DOX MNPs retained significant antitumor activities. Therefore, PLGA-DOX MNPs might be considered a promising drug delivery system for cancer chemotherapy.     

Biosynthesis and structural characteristics of selenium nanoparticles by Pseudomonas alcaliphila

In this paper, we report that selenium (Se) nanoparticles were first biosynthesized by Pseudomonas alcaliphila with a simple and eco-friendly biological method. The structural characteristics of Se nanoparticles were examined. The results showed that spherical particles appeared with diameters ranging from 50 to 500 nm during incubation and Se nanorods were present after incubating in an aqueous reaction solution for 24 h. However, the formation of Se nanorods was interrupted when 5% (w/v) poly(vinyl pyrrolidone) (PVP) was added in the aqueous reaction solution, obtaining stable spherical Se nanoparticles with a diameter of about 200 nm.     

壳聚糖纳米粒子荧光探针的制备和表征

通过低分子量的壳聚糖(LCS)聚阳离子与三聚磷酸钠(TPP)的静电作用制备纳米级壳聚糖微球,并利用壳聚糖链上丰富的氨基与荧光素异硫氰酸酯(FITC)反应从而制备纳米壳聚糖微球荧光探针(NFCS)。结果表明,当壳聚糖分子量为60000,LCS与TPP的质量比为6∶1时,可得到粒度均一的球形纳米粒子,平均粒径为40±3 nm。荧光倒置显微镜观察证实FITC结合到壳聚糖微球上。荧光光谱分析显示NFCS的最大激发波长、最大发射波长与游离态FITC无显著差异。光漂白实验证实NFCS的稳定性比游离态FITC有显著提高。     

Detection of glucose in synthetic tear fluid using dually functionalized gold nanoparticles

A simple fluorescent sensing of glucose in aqueous fluids (e.g. tear fluid) using dually functionalized gold nanoparticles is presented. As a first step gold nanoparticles (AuNPs) were synthesized using oxidised dextran which acted both as reducing and stabilizing agent. Aminophenyl boronic acid was conjugated onto AuNPs by Schiff's base formation and the formed Schiff's base was stabilized by sodium borohydride reduction. Rhodamine B isothiocyanate (RBITC) was then assembled onto the modified AuNPs. The fluorescence of RBITC was nearly quenched and found to be revived when glucose was added. It is reasoned that the glucose binding induces restructuring of the surface assembly resulting in an overall increase in the size and thereby enhancing the distance between the gold core and fluorophore. TEM image and size measurements using dynamic light scattering (DLS) in fact, reflected this possibility. The increase in fluorescence was proportional with the concentration of glucose enabling quantitative detection. A good linearity was observed between the fluorescence intensity and glucose concentration in a range of 0.025-0.125 μM with detection limit of 0.005 ± 0.002 μM. The potential of the method was demonstrated by measuring glucose in real tear fluids collected from volunteers. The method is extremely sensitive and can be employed to measure low concentration of glucose in aqueous fluids such as tear.     

pH-Responsive Self-assembled Nanoparticles of Simulated P(AA-co-SA)-g-PEG for Drug Release

Simulated graft copolymer of poly(acrylic acid-co-stearyl acylate) [P(AA-co-SA)] and poly(ethylene glycol) (PEG) was synthesized, where acrylic acid, stearyl acylate and PEG was employed as the pH-sensitive, hydrophobic and hydrophilic segment, respectively. Polymeric nanoparticles prepared by the dialysis of simulated graft copolymer solution in dimethylformamide against citrate buffer solution with different pH values were characterized by transmission electron microscopy (TEM), fluorescence technique and laser light scattering (LLS). TEM image revealed the spherical shape of the self-aggregates, which was further confirmed by LLS measurements. The critical aggregation concentration increased markedly (10 to 150 mg/L) with increasing pH (4.6 to 7.0), consistent with the de-protonation of carboxylic groups at higher pH. The hydrodynamic radius of polymeric nanoparticles decreased from 118 nm at pH 3.4 to 90 nm at pH 7.0. The controlled release of indomethacin from those nanoparticles was investigated, and the self-assembled nanoparticles exhibited improved performance in controlled drug release.