Pullulan Acetate Research Articles

Paclitaxel-incorporated nanoparticles of hydrophobized polysaccharide and their antitumor activity

The aim of this study was to characterize paclitaxel-incorporated polysaccharide nanoparticles and evaluate their antitumor activity in vitro and in vivo. Pullulan was hydrophobically modified using acetic anhydride to make the paclitaxel-incorporated nanoparticles. Pullulan acetate (PA) was used to encapsulate paclitaxel using the nanoprecipitation method. The particles had spherical shapes under electron microscopy with sizes <100nm. The sizes of paclitaxel-incorporated nanoparticles increased to >100nm, and higher drug feeding induced higher particle size and drug content. Initial drug burst release was observed until 2 days and then the drug was continuously released over 1 week. Intrinsic cytotoxicity of empty PA nanoparticles was tested with RAW264.7 macrophage cells for biocompatibilty. The viability of RAW264.7 cells was >93% at all concentrations of empty PA nanoparticles, indicating that the PA nanoparticles are not acutely cytotoxic to normal human cells. The nanoparticles showed lower antitumor activity in vitro against HCT116 human colon carcinoma cells than that of paclitaxel itself, indicating the sustained release properties of nanoparticles. An in vivo study using HCT116 human colon carcinoma-bearing mice showed that paclitaxel-incorporated PA nanoparticles reduced tumor growth more than that of paclitaxel itself. These results indicate that PA paclitaxel-incorporated nanoparticles are a promising candidate for antitumor drug delivery.

Succinylated Pullulan Acetate Microspheres for Protein Delivery

【In order to develop new protein carrier replacing poly(DL-lactic acid-co-glycolic acid) (PLGA) microspheres, succinylated pullulan acetate (SPA) was investigated to fabricate a long term protein delivery carrier. SPA microspheres loaded with lysozyme (Lys) as a model protein drug were prepared by a water/oil/water (W/O/W) double emulsion method. An acidity test of SPA copolymers after hydrolysis was performed to estimate the change of protein stability during releasing proteins from the microspheres. There was no pH change of SPA copolymers, but pH of PLGA polymers after hydrolysis was significantly decreased to around pH 2, indicating that the long-term stability of proteins released from SPA microspheres can be guaranteed. Loading efficiency of proteins into SPA microspheres was three times higher than those into conventional PLGA microspheres, indication of inducing stronger charge interaction between proteins and succinyl groups in SPA microspheres. Although initial burst behaviors were monitored in Lys-loaded SPA microspheres due to relatively strong hydrophilic succinyl segments in SPA microspheres, initial burst issues would be circumvented if the ratio of charge density of succinyl moieties and hydrophobic acetate groups is harmonically controlled. Therefore, in this study, a new attempt of protein delivery system was made and functional SPA was successfully confirmed as a new protein carrier.】

효율적인 약물 방출 스텐트 제조를 위한 고분자 코팅물질 개발

효율적 비혈관용 약물방출 스텐트 제조를 위해 풀루란 아세테이트(pullulan acetate, PA)가 테프론(polytetrafluorethylene; PTFE)으로 피막된 스텐트(PTFE-stent)의 코팅재료로 연구되었다. 파크리탁셀 함유 PA가 코팅된 PTFE-stent의 표면, 약물 방출 거동, 세포독성이 측정되었으며, 동물실험을 통해 이의 가능성이 검토되었다. 전자현미경으로 표면을 관찰한 결과 표면이 PTFE 피막에 비해 훨씬 매끄러웠고, 약물은 80일 동안 서방적 방출 거동을 보였다. PA와 함께 코팅된 파크리탁셀의 안정성을 annexin V 결합 염색법을 통하여 측정한 결과 apoptosis의 비율이 천연 파크리탁셀과 유사한 것으로 보아 봉입된 파크리탁셀의 변성이 없음을 알 수 있었다. 소동물 실험에서는 파크리탁셀이 봉입된 PA-PTFE가 고형암의 성장을 억제하였다. 위의 결과로 보아 PA는 효율적 비혈관계 약물방출 스텐트 개발에 매우 유용한 물질이라고 기대된다.

Use of growth factor-loaded acetylated polysaccharide as a coating material to improve endothelialization of vascular stents

Endothelial cell-coated stents have attracted interest as an ideal treatment method for preventing restenosis following stent deployment to the peripheral locations. In an attempt to evaluate the proliferation and differentiation activities of human umbilical vein endothelial cells (HUVECs), the acetylated polysaccharide, pullulan acetate (PA), loaded with vascular endothelial growth factor (VEGF) (PA/V), a major potential factor influencing vascularization, was examined as a coating for peripherally placed stents. The surface of the PA/V-coated stents observed by scanning electron microscopy was smoother than that of the bare nitinol stents. In an in vitro study, the HUVECs attached to PA/V-coated stents survived and proliferated well compared to the bare stents or stents with only a PA coating (without VEGF). These results were supported by in vivo tests using nude mice. In addition, histology and immunohistochemistry analyses showed that mature endothelial cell-specific markers were expressed at high levels by HUVECs attached to PA-coated stents when VEGF was released. These results suggest that coating stents with PA/V provides a new effective approach for increasing endothelization and prevents stent restenosis.

In‐vitro Zero‐order Release of Anti‐inflammatory Drugs From Pullulan Acetate Phthalate Microspheres

Pullulan acetate phthalate (PAP) was synthesized, and applied as a wall material for microencapsulation of anti-inflammatory drugs, alclofenac, ketoprofen and ibuprofen using an oil-in-oil (%) emulsion solvent evaporation process. In relation to all drugs, the dissolution pattern from microspheres prepared with PAP which contains a phthalyl content between 13 and 16% and an acetyl content between 30 and 33%, showed an apparent zero-order release until almost 80% drug was released into the pH 6.8 medium described in the disintegration test in JP XIII. The dissolution mechanism of such an apparent zero-order dissolution profile could be explained by the Case-II transport mechanism in the category of swelling controlled system in the classification related to controlled polymeric systems.

Combination of paclitaxel- and retinoic acid-incorporated nanoparticles for the treatment of CT-26 colon carcinoma

The aim of this study was to evaluate the antitumor effect of combinatorial targeted therapy with paclitaxel and all-trans retinoic acid (ATRA) nanoparticles in vitro. Paclitaxel-incorporated pullulan acetate (PA) nanoparticles were prepared by the nanoprecipitation-solvent evaporation method. ATRA-incorporated nanoparticles were prepared by dialysis using a methoxy poly(ethylene glycol)-grafted chitosan (ChitoPEG) copolymer. Particle sizes of paclitaxel-incorporated nanoparticles and ATRA-incorporated nanoparticles were about 160 nm and 60 nm, respectively. Nanoparticles were reconstituted in various aqueous media such as deionized water, phosphate-buffered saline, and fetal bovine serum-supplemented cell culture media. The combination of paclitaxel + ATRA (10 + 10 μg/mL) delivered by nanoparticles showed a synergistic antiproliferative effect against CT26 cells that was not observed with other combinations. Furthermore, the activity of MMP-2, a key enzyme in tumor cell invasion, was significantly decreased in cells treated with the combination of paclitaxel and ATRA while other combinations and single agents did not significantly affect its activity. A matrigel assay supported these results, indicating that paclitaxel/ATRA combination nanoparticles are effective for the inhibition of the invasion of tumor cells. The results of the present study suggest that combination treatment with paclitaxel and ATRA could be an effective treatment for the inhibition of tumor cell proliferation and invasion, and that nanoparticles are promising candidates for antitumor drug delivery.

An acetylated polysaccharide-PTFE membrane-covered stent for the delivery of gemcitabine for treatment of gastrointestinal cancer and related stenosis

Gemcitabine (Gem) eluting metal stents were prepared for potential application as drug delivery systems for localized treatment of malignant tumors. Pullulan, a natural polysaccharide, was chemically acetylated (pullulan acetate; PA) by different degrees (1.18, 1.71, and 2.10 acetyl groups per glucose unit of pullulan), layered on polytetrafluoroethylene (PTFE), and applied as part of a Gem-loaded controlled-release membrane for drug-eluting non-vascular stents. PA with a higher degree of acetylation had greater drug-loading capacity with more extended release of Gem over 30 days. The released Gem accumulated in CT-26 colon cancer without systemic exposure inducing total regression of tumors. The long-term biological activity of the released Gem and apoptosis of tumor tissues following localized delivery were confirmed by annexin V binding assays and histology. The controlled release of Gem from PA-PTFE covered drug-eluting stents (DES) may increase the patency of these stents for the treatment of malignant gastrointestinal cancer as well as cancer-related stenosis.

Stability and in vivo evaluation of pullulan acetate as a drug nanocarrier

To develop pullulan acetate nanoparticles (PANs) as a drug nanocarrier, pullulan acetate (PA) was synthesized and characterized. Its acetylation degree determined by the proton nuclear magnetic resonance (1H NMR) was 2.6. PANs were prepared by the solvent diffusion method and characterized by transmission electron microscope (TEM), size distribution, and ζ potential techniques. PANs had nearly spherical shape with a size range of 200–450 nm and low ζ potentials both in distilled water and in 10% FBS. The storage stability of PANs was observed in distilled water. PANs were stored for at least 2 months with no significant size and ζ potential changes. The safety of PANs was studied through single dose toxicity test in mice, and the result showed that PANs were well tolerated at the dose of 200 mg/kg in mice. Epirubicin-loaded PANs (PA/EPI) were also prepared and characterized in this study. Moreover, the in vivo pharmacokinetics of PA/EPI was investigated. Compared with the free EPI group, the PA/EPI group exhibited higher plasma drug concentration, longer half-life time (t1/2) and the larger area under the curve (AUC). All results suggested that PANs were stable, safe, and showed a promising potential on improving the bioavailability of the loaded drug of the encapsulated drug.

Pullulan acetate coated magnetite nanoparticles for hyper-thermia: Preparation, characterization and in vitro experiments

Amphipathic polymer pullulan acetate (PA)-coated magnetic nanoparticles were prepared and characterized by various physicochemical means. The cytotoxicity and cellular uptake of the magnetic nanoparticles were examined. The hyperthermic effect of the magnetic nanoparticles on tumor cells was evaluated. Transmission electron microscopy (TEM) showed that the PA coated magnetic nanoparticles (PAMNs) had spherical morphology. Dynamic light scattering (DLS) showed that the size distribution of PAMNs was unimodal,with an average diameter of 25.8 nm ± 6.1 nm. The presence of the adsorbed layer of PA on the magnetite surface was confirmed by Fourier transform infrared (FTIR) spectroscopy. Magnetic measurements revealed that the saturation magnetization of the PAMNs reached 51.9 emu/g and the nanoparticles were superparamagnetic. Thermogravimetric analysis (TGA) showed that the Fe3O4 particles constituted 75 wt% of the PAMNs. The PAMNs had good heating properties in an alternating magnetic field. Cytotoxicity assay showed that PAMNs exhibited no significant cytotoxicity against L929 cells. TEM results showed that a large number of PAMNs were internalized into KB cells. PAMNs have good hyperthermia effect on KB cells in vitro by magnetic field induced hyperthermia. These novel magnetic nanoparticles have great potential as magnetic hyperthermia mediators. Open image in new window

Preparation of folate-modified pullulan acetate nanoparticles for tumor-targeted drug delivery

The purpose of this work was to develop a novel nano-carrier with targeting property to tumor. In this study, pullulan acetate (PA) was synthesized by the acetylation of pullulan to simplify the preparation technique of nanoparticles. Folic acid (FA) was conjugated to PA in order to improve the cancer-targeting activity. The products were characterized by proton nuclear magnetic resonance (1H NMR) spectroscopy. Epirubicin-loaded nanoparticles were prepared by a solvent diffusion method. The loading efficiencies and EPI content increased with the amount of triethylamine (TEA) increasing in some degree. FPA nanoparticles could incorporate more epirubicin than PA nanoparticles. The folate-modified PA nanoparticles (FPA/EPI NPs) exhibited faster drug release than PA nanoparticles (PA/EPI NPs) in vitro. Confocal image analysis and flow cytometry test revealed that FPA/EPI NPs exhibited a greater extent of cellular uptake than PA/EPI NPs against KB cells over-expressing folate receptors on the surface. FPA/EPI NPs also showed higher cytotoxicity than PA/EPI NPs. The cytotoxic effect of FPA/EPI NPs to KB cells was inhibited by an excess amount of folic acid, suggesting that the binding and/or uptake were mediated by the folate receptor.

Pullulan acetate nanoparticles prepared by solvent diffusion method for epirubicin chemotherapy

Pullulan acetate (PA) was synthesized by the reaction of pullulan with acetic anhydride in the presence of pyridine. PA was characterized by Fourier transform infrared (FT-IR) and proton nuclear magnetic resonance ( 1H NMR). A solvent diffusion method was employed in the current work to prepare PA nanoparticles. This technique had some advantages compared with other methods. The particle size increased from 185.7 nm to 423.0 nm with the degree of acetylation increasing from 2.71 to 3.0. Drug-loaded PA nanopaticles were prepared for controlled release of epirubicin (EPI). The drug entrapment and drug content increased with the degree substitution of PA increasing. EPI was released from the nanoparticles in a biphasic profile with a fast release rate in the first 10 h followed by a slow release in vitro. A higher cytotoxicity against KB cells was found for EPI-loaded PA nanoparticles in comparison with free EPI. Confocal laser scanning microscopy (CLSM) observations indicate that EPI-loaded nanoparticles were internalized and released in the cytoplasmic compartment.

Ionic strength-sensitive pullulan acetate nanoparticles (PAN) for intratumoral administration of radioisotope: Ionic strength-dependent aggregation behavior and 99mTechnetium retention property

In order to design an effective intratumoral radioisotope carrier, a self-assembled nanoparticle evidencing ionic strength (IS)-sensitivity from a polysaccharide derivative (pullulan acetate nanoparticle (PAN)) was prepared via dialysis. The PAN had a spherical shape in a range of size of 50–130 nm and a low critical aggregation concentration (CAC) (<8 μg/mL). With increases in the IS of the dialysis media (IS dia), the CAC of PAN was reduced gradually and the rigidity of the hydrophobic core in PAN was increased. This suggests that the property of PAN was altered more hydrophobically at high IS values. The stabilities of PANs prepared from various IS dia were also monitored with changes in the turbidity and particle size in different IS solutions. In the case of PAN prepared at an IS dia = 0.0, the turbidity was dramatically reduced with increasing IS due to the facilitation of aggregation between the particles, whereas in the other cases, these changes were negligible. This finding indicates that PAN prepared in distilled water (IS = 0.0) can be readily injected as the consequence of its nano-size, and accumulates quickly, then remains in the tumor site for a considerable period (IS = 0.15). In order to closely estimate the potential of PAN as a radioisotope carrier, the radioisotope labeling efficiency of PAN with no chelating agents was evaluated. PAN evidenced a high degree of 99mTechnetium ( 99mTc) labeling efficiency (approximately 98%). The percentage retention rate (%RR) of the 99mTc-labeled PAN was significantly longer than that of the free 99mTc ( p < 0.05), due largely to PAN's IS-sensitivity. In conclusion, PAN may constitute a new approach to the achievement of maximal radioisotope efficiency with regard to intratumoral administration.

Radioevaluation of PAMs, CMs, and PS‐Lip as an oral carrier for vaccine delivery into intestinal Peyer's patches

AbstractThe aim of the present study was to evaluate the utility of pullulan acetate microparticles (PAMs), chitosan micropaticles (CMs), and dipalmitoylphosphatidyl‐serine‐liposomes (PS‐Lip) as oral carriers for delivery to the intestinal Peyer's patches (PPs). To monitor PP delivery after oral administration, PAMs, CMs, and PS‐Lip were radiolabeled with 99mTc. Radiolabeling efficiencies of the particles were 95±2.5% (PAMs), 87±4.3% (CMs), and 77.2±5.8% (PS‐Lip). In delivery studies to the PPs, the percentage of PS‐Lip taken up to the PPs was 3.8 × 10−3±0.3% of the administered dose with PS‐Lip group showed significantly high uptake compared to the PAM and CM groups. These results suggest that PS‐Lip may be used as a potential system for developing an oral delivery carrier. Drug Dev. Res. 67:884–889, 2006. © 2007 Wiley‐Liss, Inc.

5-Fluorouracil-loaded Self-assembled pH-sensitive Nanoparticles as Novel Drug Carrier for Treatment of Malignant Tumors

In order to improve the cancer-targeting and selective activity of antineoplastic agent [5-fluorouracil (5-FU)], a novel pH-responsive drug delivery system [pullulan acetate/sulfonamide (PA/SDM) conjugate] was synthesized by a diafiltration method. Sulfonamide was grafted to the hydrophobically modified pullulan acetate to enhance the pH sensitivity for better cancer-targeting delivery. 5-FU was loaded into the self-assembled nanoparti-cles by the same method. The drug-loaded self-assembled nanoparticles were successfully obtained and characterized in terms of particle size, morphology and drug loading and release profile at various pHs. The results showed that the mean diameter of the self-assembled particles was approximately 100nm, with uniform size and good spherical morphology. The nanoparticles showed good stability at pH 7.4, which is equal to that of the normal body fluid, but shrank and aggregated below pH 6.8, which is close to the pH with tumors. The loading efficiency and concentration of released 5-FU was monitored at 269 nm on the UV/Vis spectrophotometer. The release profile was heavily pH-dependent around physiological pH, and the release rate was significantly enhanced under pH of 6.8.

플루란 아세테이트 미립구를 이용한 단백질 전달 시스템 개발

The aim of this study was to develop new protein/peptide depot system instead of poly(DL-lactic acid-coglycolic acid) (PLGA) microspheres. Pullulan was chemically modified by the addition of acetic anhydride (pullulan acetate; PA) and then investigated as new depot system for protein/peptide delivery. PA microspheres (PAM) with lysozyme as a model protein were prepared by w/o/w double emulsion method. The microspheres had a mean size of 10-50 mm with a spherical shape. The size distributions reduced with increasing the degree of acetylation. The loading efficiency of lysozyme was also increased. Lysozyme aggregation behavior in the microsphere was monitored to estimate the change of protein stability during preparation step. The ratios of protein aggregation in PAMs are lower than that of PLGA microsphere, in particular, PA 5 showed lowest as about 16%. The result indicated that the increase of acetylation suppressed the aggregation of protein. The release profiles of lysozyme from PAMs were significantly different. High acetylation effectively improved lysozyme release kinetics by reducing initial burst release and extending continuous release over a period of time. To check the effect of preservation for structural stability of lysozyme, the activity of lysozyme released from PA 5 was also observed. The activity of lysozyme was maintained almost 100% for 25 day. Therefore, PAM may become to a useful carrier for delivery of protein/peptide drugs, if it will be supported by biocompatibility and biodegradability results.

Synthesis and properties of pullulan acetate. Thermal properties, biodegradability, and a semi-clear gel formation in organic solvents

Pullulan acetate (AcPL) with various degree of substitution (DS: 1.0–3.0) was synthesized by the reaction of pullulan with acetyl chloride in the presence of pyridine. The product was characterized by gel permeation chromatography (GPC), infra-red (IR) and 1H NMR spectroscopy. The weight average molecular weights of the products did not decrease less than 190,000 (GPC) in the acetylation reaction. Thermogravimetric analysis (TGA) revealed that AcPL has a higher decomposition temperature (306–363 °C) than unmodified pullulan (295 °C). Differential scanning calorimetry analysis (DSC) revealed that all the AcPLs exhibit a clear T g, which decreased with increasing DS value in the range of DS 1.0–2.4. The AcPL with DS 2.4 showed the lowest T g (153 °C), and the AcPL with DS 3.0 had a slightly higher T g (163 °C). Tensile modulus of AcPL films was comparable to that of a popular cellulose acetate film. The biodegration rate of AcPL decreased with increasing degree of acetylation. The AcPL with DS 3.0 was found to form a semi-clear gel in organic solvents such as dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), and 1,4-dioxane.

Self-assembled polymeric nanoparticles of poly(ethylene glycol) grafted pullulan acetate as a novel drug carrier.

Self-assembling nanospheres of hydrophobized pullulan have been developed. Pullulan acetate (PA), as hydrophobized pullulan, was synthesized by acetylation. Carboxymethylated poly(ethylene-glycol) (CMPEG) was introduced into pullulan acetate (PA) through a coupling reaction using N,N'-dicyclohexyl carbodiimide (DCC). A synthesized PA-PEG-PA (abbreviated as PEP) conjugate was confirmed by Fourier transform-infrared (FT-IR) spectroscopy. Since PEP conjugates have amphiphilic characteristics in aqueous solution, polymeric nanoparticles of PEP conjugates were prepared using a simple dialysis method in water. From the analysis of fluorescence excitation spectra primarily, the critical association concentration (CAC) of this conjugate was found to be 0.0063 g/L. Observations by scanning electron microscopy (SEM) showed the spherical morphologies of the PEP nanoparticles. The particle size distribution of the PEP conjugates was determined using photon correlation spectroscopy (PCS) and the intensity-average particle size was 193.3 +/- 13.53 nm with a unimodal distribution. Clonazepam (CNZ), as a model drug, was easy to entrap into polymeric nanoparticles of the PEP conjugates. The drug release behavior was mainly diffusion controlled from the core portion.

Biocompatibility and interference eliminating property of pullulan acetate/polyethylene glycol/heparin membrane for the outer layer of an amperometric glucose sensor

This study reports the use of hydrophobically modified heparin, which is both biocompatible and permselective, as a surface coating material for glucose sensors. The biocompatibility of pullulan acetate (PA)/polyethylene glycol (PEG)/heparin membrane was investigated in terms of platelet adhesion. Scanning electron micrographs (SEM) of the membrane-coated glucose sensor after exposure to a platelet-rich plasma (PRP) solution showed that the PA/PEG/heparin was much more resistant to platelet adhesion than other membrane material such as Pellethane ® and Nafion ®. The membrane-coated glucose sensor showed an extended linearity up to 30 mM glucose with a sensitivity of 202 nA mM −1 cm −2. The anti-interference capability of the membrane was investigated, and no interference signal was observed with 2.75 mg dl −1 uric acid. The interference signals with 1.1 mg dl −1 ascorbic acid and 0.1 mM p-acetaminophen were 5 and 3 nA, respectively, and sensitivity to glucose was maintained as 87 nA/5.5 mM glucose.

Characterization of hydrophobized pullulan with various hydrophobicities

In this study, we prepared self-assembling nanospheres of hydrophobized pullulan. Pullulan acetate (PA), as hydrophobized pullulan, was synthesized by the acetylation of pullulan. PA derivatives were synthesized by changing the degree of acetylation. PA was characterized by Fourier transform infrared (FT-IR), X-ray diffractometry (XRD), and differential scanning calorimetry (DSC). The particle size distribution of the PA was determined using photon correlation spectroscopy (PCS) and the number-average particle size was found to depend upon the degree of acetylation of PA. Morphology by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) showed the PA nanospheres were spherical in shape. The fluorescence probe technique was used to study the self-association behavior of hydrophobized pullulans in water using pyrene as a hydrophobic probe. The critical association concentration (CAC) values were determined from the fluorescence excitation spectra, CAC values were dependent upon the degree of acetylation. Drug release studies using clonazepam (CNZ) as a hydrophobic model drug showed that the increased drug contents and increased degree of acetylation resulted in a slower release rate of drug from the nanospheres.

Self-assembled nanoparticles of hydrophobically-modified polysaccharide bearing vitamin H as a targeted anti-cancer drug delivery system

Vitamin H (biotin) was incorporated into a hydrophobically modified polysaccharide, pullulan acetate (PA), in order to improve the cancer-targeting activity and internalization of self-assembled nanoparticles. The biotinylated pullulan acetate (BPA) nanoparticles were prepared by a diafiltration method and the mean diameter was approximately 100 nm. Three samples of biotinylated pullulan acetate (BPA), comprising 7 (BPA 1), 20 (BPA 2), and 39 (BPA 3) vitamin H groups per 100 anhydroglucose units of PA, were synthesized. The critical aggregation concentrations (CAC) of the BPA nanoparticles in distilled water were 3.1×10 −3, 4.3×10 −3 and 6.8×10 −3 mg/ml for BPA 1, BPA 2, and BPA 3, respectively. Adriamycin (ADR) was loaded into the BPA nanoparticles as a model drug. The loading efficiencies and ADR content in the BPA nanoparticles decreased with increasing vitamin H content due to a lower hydrophobicity. The RITC-labeled BPA nanoparticles exhibited very strong adsorption to the HepG2 cells, while the RITC-labeled PA nanoparticles did not show any significant interaction. The degree of the interaction increased with increasing vitamin H content. Confocal laser microscopy also revealed that internalization of the BPA nanoparticles into the cancer cells depended on the vitamin H content.