Effect of ginsenoside Rg_3 nanostructured lipid carrier modified by pullulan on promoting absorption and its anti-tumor evaluation in vitro / 中国中药杂志

Ginsenoside Rg_3 is widely used in clinical practice as an anti-tumor adjuvant drug, but its application is limited due to its poor oral absorption. In this study, we intended to construct a ginsenoside Rg_3 nanostructured lipid carrier modified by the pullulan(PUL-Rg_3-NLC) to improve the adhesion properties of ginsenoside Rg_3, promote the drug uptake and improve the anti-tumor efficacy. PUL-Rg_3-NLC was characterized by morphology, particle size and Zeta potential. In vivo adhesion characteristics were evaluated by oral gavage tests, and the results were verified from multiple perspectives in combination with in vitro uptake behavior and in vitro pharmacodynamics. The results showed that PUL-Rg_3-NLC, with a particle size of(102±1.89) nm, was characterized by gastric adhesion and could be retained in gastric tissues for a long time, and its uptake by BGC-823 cells was promoted mainly through the pathway mediated by the caveolin-mediated endocytosis. In vitro MTT, cell apoptosis, wound-healing assay and invasion assay all showed some anti-tumor effects. Therefore, PUL-Rg_3-NLC can significantly promote the adhesion of Rg_3 in the stomach, promote the uptake of drugs by gastric cancer cells, and improve the anti-tumor effect. This study can provide some reference for the adjuvant treatment of gastric cancer.

Desenvolvimento e avaliação de nova preparação de anfotericina B e/ou cetoconazol em dispersão de bixina, pullulan e trealose / Development and evaluation of novel preparation of amphotericin B and/or ketoconazole in dispersion of bixin, pullulan and trehalose

Muitos pacientes acometidos por infecções fúngicas sucumbem devido a não eficácia dos antibióticos ou por toxicidade dos mesmos. Anfotericina B é um dos antifúngicos mais eficientes do mercado apesar de sua alta toxicidade, tem estrutura poliênica e é um composto insolúvel em água, sendo necessário o uso de adjuvantes e novas tecnologias para preparo de formulações eficazes. Cetoconazol é um composto imidazólico, também com ação antifúngica de grande espectro de ação e difícil solubilização em meio aquouso, porém solúvel somente em baixos valores de pH. Estudos têm demonstrado a utilização de bixina na preparação de dispersões aquosas de compostos insolúveis ou pouco solúveis em água. Bixina é o principal composto das cascas de semente de Bixa orellana (urucum), sendo um carotenoide insolúvel em água, porém, permite preparações na forma de nanodispersões aquosas com incorporação de fármacos apolares ou lipofílicos. O objetivo deste trabalho foi preparar anfotericina B e cetoconazol na forma de nanodispersões a partir de bixina, utilizando pullulan e trealose como adjuvantes e avaliar estabilidade e eficácia antimicrobiana por ensaios físico-químicos e microbiológicos. Pullulan é um polissacarídeo constituído por unidades de maltotriose, com propriedades adesivas e capacidade de formar biofilmes, enquanto trealose é um composto com duas unidades de glicose, com boa estabilidade em faixas de pH de 3 a 10 e capaz de suportar altas temperaturas, como processos de esterilização por calor úmido. Ensaios físico-químicos demonstraram boa manutenção das características das preparações propostas neste projeto, como, por exemplo, diâmetro hidrodinâmico e potencial zeta das estruturas das nanodispersões de bixina e antifúngicos e também eficácia antimicrobiana frente a Candida albicans ATCC 10231. Os resultados apresentam perspectivas para aprimoramentos de formulações com fármacos pouco solúveis ou insolúveis em água, pesquisa de novos biomateriais e outras aplicações nas áreas farmacêutica e cosmética

Many patients with fungal infections succumb due to ineffectiveness or toxicity of antibiotics. Amphotericin B is one of the most efficient antifungals on the market despite its high toxicity. It presents polyenic structure and is a water-insoluble compound. In this case, it is necessary to use adjuvants and new technologies to prepare effective formulations. Ketoconazole is an imidazolic compound, also with broad spectrum antifungal action and difficult solubilization in aqueous medium but it is soluble at low pH values. Studies have demonstrated the use of bixin in the preparation of aqueous dispersions of insoluble or poorly soluble compounds in water. Bixin is the main compound of Bixa orellana (annatto) seed husks, being a water-insoluble carotenoid, but it allows preparations in the form of aqueous nanodispersions with incorporation of apolar or lipophilic drugs. The objective of this work was to prepare amphotericin B and ketoconazole as nanodispersions from bixin, using pullulan and trehalose as adjuvants and to evaluate them under aspects of stability and efficacy by physicochemical and microbiological assays. Pullulan is a polysaccharide consisting of maltotriose units with adhesive properties and ability to form biofilms, while trehalose is a compound with two glucose units with good stability at pH ranges from 3 to 10 and capable of withstanding high temperatures such as processes of sterilization by moist heat. Physicochemical tests demonstrated good maintenance of the characteristics of the preparations proposed in this project, such as hydrodynamic diameter and zeta potential of bixin and antifungal nanodispersions and also antimicrobial efficacy against Candida albicans ATCC 10231. The results present prospects for improvement. of poorly soluble or water-insoluble drug formulations, research on new biomaterials and other applications in the pharmaceutical and cosmetic fields

Formulation Optimization of Hydrophobically-Pullulan Nanoparticles by Box-Behnken Response Surface Methodology / 中国药学杂志

OBJECTIVE: To optimize the preparation of self-assembled doxorubicin-loaded cholesterol-modified pullulan nanoparticles(DOX-CHSP-SAN) by response surface methodology. METHODS: DOX-CHSP-SAN was prepared by dialysis method with the amphiphilic carrier of cholesterol-modified pullulan (CHSP) and the model drug of doxorubicin(DOX). Box-Behnken Design (BBD) response surface method was used to optimize the preparation process of DOX-CHSP-SAN. The morphology, average particle size, Zeta potentia, the encapsulation efficiency, drug loading of doxorubicin and the drug release characteristics in vitro were determined. RESULTS: The optimized DOX-CHSP-SAN has a round spherical shape with an average particle size of (117.1±3.4) nm, PDI of (0.205±0.023), Zeta potential of (-26.1±0.135) mV, the encapsulation efficiency was (68.17±0.93)%, and the drug loading was (6.84±0.47)%. The in vitro release behavior in PBS of pH 7.4 was consistent with the Weibull equation. CONCLUSIO:N The DOX-CHSP-SAN prepared by the dialysis method and optimized by the response surface method is round and smooth, the particle size is small and uniform, the encapsulation efficiency and the drug loading amount are high, and the in vitro release has the sustained release characteristics.

Preparation of biotin-grafted pullulan acetate nanoparticles conjugated with CD3 antibodies and their uptake of T cells / 医学研究生学报

Objective Cytotoxic T lymphocytes are the main effector cells of anti-tumor immunity. Active targeting of nanoparticles to T cells and activation of T cells can be achieved by conjugation with specific antibodies. We prepared the biotin-grafted pullulan acetate nanoparticles conjugated with CD3 (Bio-PA-CD3 NPs), and explored their effects on the proliferation, cytokine secretion and uptake of CD8+T cells.Methods We prepared Bio-PA NPs by the dialysis method, conjugated CD3 antibodies to the surface of NPs to make Bio-PA-CD3 NPs, and measured the diameter and Zeta potential of the NPs. We evaluated the effects of the NPs on the proliferation of CD8+T cells and the secretion of cytokines by CCK-8 assay and ELISA, respectively, and quantitatively analyzed the cellular uptakes of the Bio-PA-CD3 NPs by the flow cytometry.Results The Bio-PA-CD3 NPs exhibited regular spherical shapes of even size and with no adhesion. The content of CD3 antibodies on the surface of the NPs decreased with the increased degree of biotin substitution. The CD3 contents of the Bio-PA-CD3 NPs with biotin substitution degrees of 1.6%, 5.4% and 6.3% were (36.1±4.4), (21.4±4.3) and (10.3±4.7) μg/mg, respectively. Compared with Bio-PA NPs, Bio-PA-CD3 NPs at a certain concentration significantly enhanced the proliferation of CD8+T cells in vitro and promoted the secretion of IFN-γ, TNF-β and IL-2 cytokines. The Bio-PA-CD3 NPs manifested a higher cellular uptake with the increased content of CD3 antibodies.Conclusion The Bio-PA-CD3 NPs we prepared could be a promising agent to enhance the immune effect of T cells.

Efficient production of pullulan by Aureobasidium pullulans grown on mixtures of potato starch hydrolysate and sucrose

Abstract Pullulan is a natural exopolysaccharide with many useful characteristics. However, pullulan is more costly than other exopolysaccharides, which limits its effective application. The purpose of this study was to adopt a novel mixed-sugar strategy for maximizing pullulan production, mainly using potato starch hydrolysate as a low-cost substrate for liquid-state fermentation by Aureobasidium pullulans. Based on fermentation kinetics evaluation of pullulan production by A. pullulans 201253, the pullulan production rate of A. pullulans with mixtures of potato starch hydrolysate and sucrose (potato starch hydrolysate:sucrose = 80:20) was 0.212 h−1, which was significantly higher than those of potato starch hydrolysate alone (0.146 h−1) and mixtures of potato starch hydrolysate, glucose, and fructose (potato starch hydrolysate:glucose:fructose = 80:10:10, 0.166 h−1) with 100 g L−1 total carbon source. The results suggest that mixtures of potato starch hydrolysate and sucrose could promote pullulan synthesis and possibly that a small amount of sucrose stimulated the enzyme responsible for pullulan synthesis and promoted effective potato starch hydrolysate conversion effectively. Thus, mixed sugars in potato starch hydrolysate and sucrose fermentation might be a promising alternative for the economical production of pullulan.

Pullulan as a stabilizer agent of polymeric nanocapsules for drug delivery

ABSTRACT Polymeric stabilizers have received attention in the preparation of nanostructured systems due to their ability to enhance formulation stability. Considering this, the objective of this work was to prepare poly(ε-caprolactone) nanocapsules using the pullulan as a polymeric stabilizer. The nanocapsules were prepared using the interfacial deposition method of preformed polymers and they were characterized in terms of pH, average diameter, polydispersity index, zeta potential, beclomethasone dipropionate content, encapsulation efficiency, photostability and drug release profiles. The formulations showed physicochemical characteristics consistent with nanocarriers for drug delivery such as: average diameter lower than 270 nm, polydispersity indexes lower than 0.2, negative zeta potential (-22.7 to -26.3 mV) and encapsulation efficiencies close to 100%. In addition, the nanocapsules were able to delay the beclomethasone dipropionate photodegradation under UVC radiation and by the dialysis bag diffusion technique, the nanocapsules were able to prolong the drug release. Thus, pullulan could be considered an interesting excipient to formulate polymeric nanocapsules.

Diseño de un sistema bioadhesivo de clorhexidina empleando pullulan como matriz para uso en mucosa oral / Design of a bioadhesive system with chlorhexidine using pullulan as matrix for use in oral mucous

Se desarrollaron sistemas poliméricos bioadhesivos del tipo película polimérica y comprimido empleando el biopolímero pullulan, para el transporte de digluconato de clorhexidina, el cual es un principio activo utilizado como alternativa terapéutica en el tratamiento de la gingivitis y de la enfermedad periodontal. Inicialmente, se evaluó la capacidad del polímero para formar películas y tabletas, luego, fueron propuestas formulaciones de cada uno de los sistemas. A las películas y comprimidos obtenidos se les determinaron propiedades mecánicas y de transporte, actividad antiséptica, caracterización de las microestructuras obtenidas, además, se comprobó la liberación del fármaco desde los sistemas estudiados. Con las formulaciones seleccionadas se determinó su capacidad mucoadhesiva in vitro, empleando como sustrato mucosa oral porcina.

As a therapeutic alternative in the transport of active substances in treatment of gingivitis and periodontal disease, bioadhesive polymeric systems type polymeric film and tablet were developed using the biopolymer pullulan. First, the ability of pullulan polymer to form films and tablets was evaluated and formulations were proposed for each system. Mechanical and transport properties, as well as antiseptic activity and microstructures characteristics were determined for these polymeric systems. Drug release behavior in the studied systems was also verified. The in vitro mucoadhesive capacity was determined with the formulations selected, using porcine oral mucous membrane like substrate.

Interaction between bovine serum albumin and pullulan acetate nanoparticles / 国际生物医学工程杂志

Objective To investigate the interaction of pullulan acetate nanoparticles (PANs) and bovine serum albumin (BSA),and to provide the basis for in vivo pharmacokinetic study of PANs.Methods Mixed solutions of different concentration of PANs and BSA solution was prepared,the interaction was studied by fluorescence quenching method and circular dichroism (CD) measurement.Apparent quenching constant (Kq) between the PANs and BSA was calculated,and the anti-stress effect induced by urea and heating of PANs-BSA solution was observed.Results The fluorescence spectrum of BSA was affected by PANs on density-related manner,and Kq calculated by the modified Stern-Volmer plot increased from 2.64×104 to 3.55 ×l05 with the concentration of PANs increased from 0.015 mg/ml to 0.25 mg/ml.With the denaturation of urea or heating,the CD spectrum of PANs-BSA and free BSA had the similar variation tendency.Conclusions The fluorescence spectrum display results show that interaction between PANs and BSA exists,but the interaction does not protect the BSA from degeneration by urea and heating.

Cellular uptake of Tumor-targeted nanoparticles derived from pullulan acetate conjugate / 医学研究生学报

Objective Folate receptors ( FRs) , overexpressed on the surface of a variety of tumor cells , are potential targets for tumor targeting therapy .This study aimed to prepare an FR-mediated drug nanocarrier with folate conjugated pullulan acetate ( FPA) to target chemotherapeutic agents to FR-overexpressed tumor cells and investigate its tumor-suppressing effect in vitro. Methods The cytotoxicity of epirubicin-loaded FPA nanoparticles ( FPA/EPI NP) against HepG2 and Hela cells was evaluated by MTS assay.The HepG2 and Hela cells were divided into five groups to be treated with NPs (NP control), chlorpromazine, chloro-quine, amiloride, and folate, respectively, followed by detection of the fluorescence intensity by flow cytometry . Results FPA/EPI NP was successfully formulated into NPs , with the mean particle size of (268.5 ±12.0) nm, by dialysis with an almost spherical shape . The drug-loading rate and entrapment rate of FPA/EPI NP were (6.45 ±1.04) and (72.45 ±11.50) %, respectively.The survival rates of the HepG2 and Hela cells were both >95%after 24 hours of incubation with FPA NP at 5, 40, 200, 400 and 1000μg/mL and 90.0%after 72 hours.The survival rates of the HepG2 cells treated with 5, 40, 200, 400 and 1000μg/mL FPA/EPI NP for 24 hours were (92.3 ±5.2), (70.4 ±4.6), (50.0 ±4.0), (41.1 ±4.1) and (27.0 ±3.6) %, respectively.Compared with the NP control group, the Hela cells of the chlorpromazine , amiloride and folate groups showed a significantly lower rate of NP uptake (P<0.05), and so did the HepG2 cells pretreated with chlorpromazine or amilo-ride (P<0.05).At 72 hours, the half maximal inhibitory concentrations (IC50) of FPA/EPI NP against HepG2 and Hela cells were 168 and 105μg/mL, respectively . Conclusion Clathrin-mediated endocytosis and macropinocytosis are involved in the internaliza-tion of FPA/EPI NP in HepG2 cells, while clathrin-and FR-mediated endocytosis in that of Hela cells .FPA NP may serve as a new drug carrier for tumor-targeted therapy .

Influence of molecular weight and spacer length on the properties of modified pullulan carriers / 国际生物医学工程杂志

Objective To investigate the influence of pullulan molecular weight and spacer length on the properties of modified pullulan carriers including morphologies,sizes and in vitro release behaviours of drug-loading carriers.Methods Using cholesterol as hydrophobic ligand,succinic anhydride and 1,6-hexyldiisocyanate as spacers,hydrophobic modified pullulans with different molecular weights were prepared.Self-assembled nanoparticles were then formed in the aqueous solution,and drug-loaded nanoparticles were prepared by dialysis method.The influence of pullulan molecular weight and spacer length on the loading-content,morphologies and in vitro release behaviours of drug-loading nanoparticles were then investigated in detail.Results Self-assembled nanoparticles could be formed by the cholesterol-modified pullulan,and doxorubicin and mitoxantrone could be loaded into cholesterol-modified pullulan to form nanoparticles.Pullulan molecular weight and spacer length show influences on sizes,morphologies and stabilities of pullulan nanoparticles and drug-loaded nanocarriers.Conclusions Before drug loading,nanoparticles with larger moleculare weight and shorter spacer length are more stable in solution,while after drug loading,the influences of these two factors on the nanoparticles are drug-type depended.

Rsearch progress on breeding of pullulan high-yield strain without melanin / 中国生化药物杂志

Pullulan is a linear glucosic polysaccharide produced by the polymorphic fungus Aureobasidium Pullulans, which has long been applied for various applications in medical and food industry due to its security, stability and low adhesive ability.At present, the two problems in restricting pullulan industrial production are the low polysaccharide production and melanin secreted which is hard to erase completely, giving the following process some problem.As a starting point, this review article collects and analyzes the progress on the breeding of pullulan high-yield strain without melanin in recent years, in order to find more efficient strains breeding methods, laying a foundation for further breeding of pullulan high-yield strain without melanin.

Advances of the graft copolymers of pullulan as pharmaceutical carriers / 中国药科大学学报

@#Graft copolymerization is one of the most effective methods to improve the performance of pullulan and extend its applications. This paper attempts to review the recent progresses in the preparation and application of graft copolymers of pullulan as pharmaceutical carriers from different synthesized polymers and the properties of graft copolymers of pullulan. The problems of pullulan based graft copolymers in drug release system are expounded, and the future development in this field direction is presented.

Pullulan: an advantageous natural polysaccharide excipient to formulate tablets of alendronate-loaded microparticles

This work reports the preparation of tablets by direct compression of sodium alendronate-loaded microparticles, using pullulan as filler. The tableting properties of pullulan were compared with those of microcrystalline cellulose and lactose. Pullulan tablets showed low variations in average weight, thickness and drug content. Moreover, these tablets exhibited a higher hardness compared to the other excipients. In vitro release studies showed that only pullulan was capable to maintain gastroresistance and release properties of microparticles, due to its ability to protect particles against damage caused by compression force. Thus, pullulan was considered an advantageous excipient to prepare tableted microparticles.

Neste trabalho relata-se a preparação de comprimidos pela compressão direta de micropartículas contendo alendronato de sódio, utilizando o pullulan como diluente. As propriedades dos comprimidos de pullulan foram comparadas com as de comprimidos de celulose microcristalina e de lactose. Os comprimidos de pullulan mostraram baixa variação no peso médio, espessura e teor. Por outro lado, estes apresentaram altos valores de dureza comparados aos preparados com os outros excipientes. Através dos estudos de liberação in vitro pode-se observar que apenas o pullulan foi capaz de manter a gastrorresistência e as propriedades de liberação das micropartículas, o que se deve à sua capacidade de proteger as partículas do dano causado pela força de compressão. Dessa forma, o pullulan foi considerado um excipiente vantajoso para a preparação de comprimidos microparticulados.

Production of pullulan by a thermotolerant Aureobasidium pullulans strain in non-stirred fed batch fermentation process

Total 95 isolates of Aureobasidium pullulans were isolated from different flowers and leaves samples, out of which 11 thermotolerant strains produced pullulan. One thermotolerant non-melanin pullulan producing strain, designated as RG-5, produced highest pullulan (37.1±1.0 g/l) at 42ºC, pH 5.5 in 48h of incubation with 3% sucrose and 0.5% ammonium sulphate in a non-stirred fed batch fermentor of 6 liters capacity. The two liters of initial volume of fermentation medium was further fed with the 2 liters in two successive batches at 5 h interval into the fermentor. The sterile air was supplied only for 10h at the rate of 0.5 vvm.

The role of microbial pullulan, a biopolymer in pharmaceutical approaches: A review.

The article presents an overview of the latest advances in investigations and application of the pullulan from microbes (Auriobasidium sps.) and its derivatives in the biomedical and pharmaceutical field and how this polysaccharide can be of use as a promising biomaterial in the coming future. So many papers were published during the last four decades indicated that pullulan the unique biomaterial which has so many applications in food, cosmetics, biomedical and pharmaceutical industries . The unique linkage α (1→4) and α (1→6) pattern of pullulan endows the polymer with distinctive physical traits, including adhesive properties and the capacity to form fibers. Due to its non-toxic, non-immunogenic, non-mutagenic and non-carcinogenic nature recently there is an attempt to explore this polysaccharide for various medical applications including targeted drug and gene delivery, tissue engineering, wound healing and also even in diagnostic applications like perfusion, receptor, and lymph node target specific imaging and vascular compartment imaging using it as quantum dots.

Retention Characteristics of Tc-99m-Pullulan-Derivatives in CT26 Tumor of Mice / 대한핵의학회잡지

OBJECTIVE: Pullulan derivatives (PD) can be used to make self-assembled hydrogel nanoparticles which are responsive to ionic strength. The aim of this study is to evaluate the potential of PD as a retaining carrier of radioisotope inside tumors. MATERIALS AND METHODS: Four types of PD were evaluated which included pullulan acetate (PA), succinylated PA (SPA), PA-DTPA and SPA-DTPA conjugates. They were radiolabeled with Tc-99m. Labelling efficiencies were determined at 30 min, 1, 2, 4 and 12 hours after radiolabeling. CT-26 colon cancer cells were subcutaneously injected into Balb/c mice. After 2 weeks of subcutaneous injection, Tc-99m-labelled PD (Tc-99m-PD) were injected into the tumors. Whole body images of mice were obtained at 30 min, 1, 2, and 12 hr after intratumoral injection. All twenty mice were grouped into four groups by largest diameter; control A (largest diameter = 5 mm, n = 5), control B (largest diameter = 10 mm, n = 5), pullulan A (largest diameter = 5 mm, n = 5), pllulan B (largest diameter = 10 mm, n = 5). Dynamic images were obtained for 1 hour after intratumoral injection. Static images were obtained at 1 hr, 2 hr, 3 hr and 4 hr after intratumoral injection with Tc-99m pertechnetate and Tc-99m-PA. Target-to-background ratios and retention rates were calculated. RESULTS: Labeling efficiencies of PA, SPA, PA-DTPA and SPA-DTPA were 94.5 +/- 5.9%, 97.8 +/- 3.5% 94.2 +/- 3.8%, and 92.5 +/- 6.2%, respectively (p> 0.05). Percent retention rates (%RR) of PA and PA-DTPA were significantly higher than those of control, however, those of SP-DTPA and SPA became similar to control at 4 and 12 hr, respectively. %RR of pullulan A and pullulan B at 1, 4 and 8 hr is significantly higher than that of control (p < 0.05). However, %RR between pullulan A and pullulan B were similar. CONCLUSION: The ionic strength dependent PD-nanoparticles are retained in the tumor. No difference of %RR according to tumor size was noted. Therapeutic application of PD labelled with beta- or alpha- emitting radionuclides can be expected.

FERMENTATION PRODUCTION OF PULLULAN FROM ISO-STARCH-HYDROLYZATE / 微生物学通报

Utilized Sucrose as carbon source for fermentation production of pullulan, the yield were 60%. But when Starch-hydrolyzate as an alternative of sucrose as carbon source, the yield were less than 45%. utilize some carbon sources containing fructose, the yield tend upwards. We utilized isostarch-hydrolyzate as only carbon source and got an yield as similar as that sucrose did.