Influence of dextran on the bioadhesive properties of poly(anhydride) nanoparticles

This work describes the bioadhesive properties of poly(methyl vinyl ether-co-maleic anhydride) (Gantrez AN) nanoparticles (NP) associated with various types of dextran (two hydroxyl-functionalized dextrans and one amino-derivative of dextran). The association of dextran to the polymer was performed either prior NP formation or by the attachment of dextran to the surface of the just formed NP. The amount of dextran associated to the nanoparticles was quantified by a HPLC/ELSD method and dextran presence in the nanoparticles was confirmed by IR spectroscopy, ¹H NMR and in vitro agglutination assay. The in vivo bioadhesion study has demonstrated significantly higher adhesive interactions with the gastrointestinal tract of rats for all types of dextran associated nanoparticles compared with control nanoparticles. For nanoparticles associated with the aminated-dextran, the curves of bioadhesion were characterized by a maximum of adhesion just after administration followed by a rapid and constant decline with time. On the contrary, nanoparticles associated to conventional dextrans displayed a maximum bioadhesion between 1 and 3 h post-administration. These results encourage us for further use of these systems for oral delivery of drugs.     

Surface modification of poly(lactic acid) nanoparticles by covalent attachment of thiol groups by means of three methods

The aim of the present work was to find a suitable method for the introduction of thiol functions on the surface of poly(DL-lactic acid) (PLA) nanoparticles. Three different approaches were investigated. The modification of the surface involves the activation of PLA carboxylic acid groups followed by the attack of a nucleophile such as cysteine (method #1) or cystamine (method #2 and #3) that provide thiol functions via an amide bond. For the conjugation with cystamine, a second reaction step is required to expose the sulfhydryl function of cystamine that is otherwise protected in a disulfide bond. 1,4-Dithio-DL-threitol (DTT) (method #2) or Tris(2-carboxyethyl)-phosphine hydrochloride (method #3) were evaluated for their ability to reduce this linkage. Method #1 allowed a maximum of 8.5+/-2.8 mmol of thiol functions per mol of PLA to be attached on the surface of the nanoparticles. Method #2 allowed the introduction of a greater number of thiol functions (up to 190+/-15 mmol per mol of PLA). However, this latter method has a major drawback: DTT interacts strongly with the nanoparticle matrix during the reduction step. Method #3 has the advantage over method #2 in that it allowed a significant number of thiol functions to be covalently bound to the particles (up to 107.6+/-0.6 mmol per mol of PLA) without the problem of undesired interaction between DTT and the nanoparticle matrix. The introduction of thiol groups onto the surface of PLA nanoparticles is possible with all three suggested methods. The method #3 provides a straight forward approach for the substitution of carboxylic acid groups with a high number of activated sulfhydryl at the surface of PLA nanoparticles.     

姜黄素PLGA纳米粒的制备及制剂学性质分析

目的制备姜黄素(Curcumin,Cur)聚乳酸羟基乙酸共聚物(PLGA)纳米粒(Cur-PLGA-NPs)并对其理化性质进行考察。方法采用改良的自乳化溶剂挥发法制备纳米粒,通过正交设计,以粒径、包封率和载药量为评价指标优化处方工艺。结果制备Cur-PLGA-NPs的优化条件为PLGA 100 mg,泊洛沙姆188浓度1.0%,丙酮与乙醇体积比3∶1,有机相体积15 m L。按优化条件所制备的Cur-PLGA-NPs粒径为(120.33±2.44)nm,多分散系数为0.10±0.02,包封率为84.50%±1.13%,载药量为4.75%±0.22%。结论采用改良的自乳化溶剂挥发法成功制备了Cur-PLGA-NPs,为后续"纳米粒-脂质体系统"的研究奠定了基础,有望实现药物在肝脏的浓集。     

Polyelectrolyte complex nanoparticles of amino poly(glycerol methacrylate)s and insulin

Amino poly(glycerol methacrylate)s (PGOHMAs) were synthesized from linear or star-shaped poly(glycidyl methacrylate)s (PGMA)s via ring opening reactions with 1,2-ethanediamine, 1,4-butanediamine and diethylenetriamine, respectively. The resulting cationic polymers were employed to form polyelectrolyte complexes (PECs) with insulin. Parameters influencing complex formation were investigated by dynamic light scattering (DLS). PECs in the size range of 100-200 nm were obtained under optimal conditions, i.e., the pH value of PECs was 5.58-6.27, the concentration of NaCl was 0.02 mol/L, and insulin-polymer weight ratio was 0.8. The insulin association efficiency (AE) of current system increased with zeta potentials of PECs. Circular dichroism (CD) analysis corroborated that the structure of insulin in the PEC nanoparticles was preserved after lyophilization. Fourier transform infrared (FT-IR) and X-ray diffraction (XRD) experiments demonstrated that weak physical interactions between insulin and amino PGOHMAs play an important role in the formation of PECs. The release of insulin depends on both structure and architecture of amino PGOHMAs. These PECs would be potentially useful for mucosal administration.     

Clonazepam release from core-shell type nanoparticles of poly(-caprolactone)/poly(ethylene glycol)/poly(-caprolactone) triblock copolymers

The triblock copolymer based on poly(-caprolactone) (PCL) as hydrophobic part and poly(ethylene glycol) (PEG) as hydrophilic one was synthesized and characterized. Core-shell type nanoparticles of poly(-caprolactone)/poly(ethylene glycol)/poly(-caprolactone) (CEC) block copolymer were prepared by a dialysis technique. According to the amphiphilic characters, CEC block copolymer can self-associate at certain concentration and their critical association concentration (CAC) was determined by fluorescence probe technique. CAC value of the CEC-2 block copolymer was evaluated as 0.0030 g/l. CAC values of CEC block copolymer decreased with the increase of PCL chain length, i.e. the shorter the PCL chain length, the higher the CAC values. From the observation of transmission electron microscopy (TEM), the morphologies of CEC-2 core-shell type nanoparticles were spherical shapes. Particle size of CEC-2 nanoparticles was 32.3±17.3 nm as a monomodal and narrow distribution. Particle size, drug loading, and drug release rate of CEC-2 nanoparticles were changed by the initial solvents and the molecular weight of CEC. The degradation behavior of CEC-2 nanoparticles was observed by ¹H NMR spectroscopy. It was suggested that clonazepam (CNZ) release kinetics were dominantly governed by diffusion mechanism.     

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.     

Aerosol delivery of spermine-based poly(amino ester)/Akt1 shRNA complexes for lung cancer gene therapy

Polyethylenimine (PEI) has been commonly used as a cationic polymeric gene carrier due to high transfection efficiency, however, its cytotoxicity has hindered the practical application. In this study, we report the development of poly(amino ester) (PAE) based on glycerol propoxylate triacrylate (GPT) and spermine (SPE) as an alternative gene carrier for lung cancer therapy. GPT-SPE copolymer was prepared by Michael addition reaction between GPT and SPE, and the efficacy was evaluated using shAkt1 as a model therapeutic gene. The molecular weight and composition were characterized using gel permeability chromatography (GPC) and ¹H-nuclear magnetic resonance (¹H-NMR), respectively. The GPT-SPE could effectively condense DNA with about 163 nm size and protect the DNA from nucleases. GPT-SPE/DNA complexes showed excellent transfection with low toxicity both in vitro and in vivo. Furthermore, aerosol delivery of GPT-SPE/Akt1 shRNA complexes significantly suppressed lung tumorigenesis in K-ras^LA1 lung cancer model mice. These results suggest that GPT-SPE can be used in shRNA-based lung cancer gene therapy.     

Immunotoxicity of poly (lactic-co-glycolic acid) nanoparticles: influence of surface properties on dendritic cell activation

Abstract

Modified nanoparticles (NPs) can interact with the immune system by causing its activation to fight tumors or for vaccination. During this activation, dendritic cells (DCs) are effective in generating robust immune response. However, the effect of nanomaterials on dendritic cell (DC) maturation, and the associated adjuvant effect, should be assessed as a novel biocompatibility criteria for biomaterials since immune consequences may constitute potential complications in nanomedicine. Among emerging biomaterials, poly(lactic-co-glycolic acid) NPs (PLGA NPs) are widely explored for various applications in which the degree of desired adjuvant effect may vary. As contradictory results are reported regarding their effects on DCs, we aimed at clarifying this point with particular emphasis on the relative impact of particle surface properties. To that end, NP uptake and effects on the viability, phenotype, and secretory activity of DC primary cultures. Intracellular signaling pathways were explored and evaluated. Immature human and murine DCs were exposed to cationic, neutral, or anionic PLGA NPs. Particle uptake was assessed by both confocal microscopy and flow cytometry. Cell viability was then evaluated prior to the study of maturation by examination of both surface marker expression and cytokine release. Our results demonstrate that PLGA NPs are rapidly engulfed by DCs and do not exert cytotoxic effects. However, upon exposure to PLGA NPs, DCs showed phenotypes and cytokine secretion profiles consistent with maturation which resulted, at least in part, from the transient intracellular activation of mitogen-activated protein kinases (MAPKs). Interestingly, NP-specific stimulation patterns were observed since NP surface properties had a sensible influence on the various parameters measured.     

Poly(L-lactic acid)/polyethylenimine nanoparticles as plasmid DNA carriers

Non-viral vectors such as liposomes, polycations, and nanoparticles have been used as gene delivery systems. In this study, we prepared and characterized biodegradable poly(L-lactic acid) (PLA)/polyethylenimine (PEI) nanoparticles as gene carriers. pCMV/β-gal and pEGFP-C1 were utilized as model plasmid DNAs (pDNA). Nanoparticles were prepared using a double emulsion-solvent evaporation technique, and their pDNA binding capacity was assessed by agarose gel electrophoresis. Transfection was studied in HEK 293 and HeLa cell lines, and the transfection efficiencies were determined by β-galactosidase assay or flow cytometry. Three kinds of PLA/PEI systems were studied by varying the molecular weight of PEI. The PLA/PEI 25K system had a higher transfection efficiency than the PLA/PEI 0.8K or PLA/PEI 750K systems. The transfection efficiency was found to be dependent on the ratio of PLA/PEI nanoparticles to pDNA with an optimum ratio of 60:1 (w/w). The cytotoxicity was dependent on the quantity of PLA/PEI nanoparticles used, but it was comparable to that of commercial Lipofectin™. These results demonstrate the potential of PLA/PEI nanoparticles as gene carriers.     

Design and evaluation of novel pH-sensitive chitosan nanoparticles for oral insulin delivery

Chitosan nanoparticles (CS NPs) have been commonly regarded as potential carriers for the mucosal delivery of therapeutic peptides because of their biocompatibility, bioadhesion and permeation enhancing properties. However, they have limited colloidal stability and readily dissociate and dissolve in the acidic gastric conditions. In the current study, CS NPs were formulated by ionic cross-linking with hydroxypropyl methylcellulose phthalate (HPMCP) as a pH-sensitive polymer and evaluated for the oral delivery of insulin. In vitro results revealed a superior acid stability of CS/HPMCP NPs with a significant control over insulin release and degradation in simulated acidic conditions with or without pepsin. Furthermore, fluorescently-labeled CS/HPMCP NPs showed a 2- to 4-fold improvement in the intestinal mucoadhesion and penetration compared to CS/TPP NPs as evidenced by quantitative fluorescence analysis and confocal microscopy. After s.c. injection to rats, no significant difference in the hypoglycemic effect of insulin solution or insulin-loaded CS/HPMCP NPs was observed, confirming the physico-chemical stability and biological activity of the entrapped peptide. Following peroral administration, CS/HPMCP NPs increased the hypoglycemic effect of insulin by more than 9.8 and 2.8-folds as compared to oral insulin solution and insulin-loaded CS/tripolyphosphate (TPP) NPs, respectively.     

Drug release from the enzyme-degradable and pH-sensitive hydrogel composed of glycidyl methacrylate dextran and poly(acrylic acid)

Hydrogels composed of glycidyl methacrylate dextran (GMD) and poly(acrylic acid, PAA) were prepared by UV irradiation method for colon-specific drug delivery. GMD was synthesized by coupling of glycidyl methacrylate to dextran in the presence of 4-(N,N-dimethylamino)pyridine. GMD was photo-polymerized by ammonium peroxydisulfate as initiating system in phosphate-buffered solution (0.1 M, pH 7.4). And then, acrylic acid monomer was added and subsequently heat-polymerized by 2,2'-azobisisobutyronitrile as an initiator. The hydrogels exhibited high swelling ratio (about 20) at 37 degrees C, and showed a pH-dependent swelling behavior. In addition, the swelling ratio of the hydrogel was remarkably enhanced to about 45 times in the presence of dextranase at pH 7.4. The swelling-deswelling behavior proceeded reversibly for the GMD/PAA hydrogels between pH 2 and pH 7.4. Release of 5-aminosalicylic acid from the GMD/PAA hydrogels was evaluated in simulated gastrointestinal pH fluids in the absence or presence of dextranase. We concluded that the hydrogels prepared could be used as a dual-sensitive drug carrier for sequential release in gastrointestinal tract.     

Combined hydroxypropyl-β-cyclodextrin and poly(anhydride) nanoparticles improve the oral permeability of paclitaxel

The aim of this work was to study the effect of the combination between 2-hydroxypropyl-β-cyclodextrin (HPCD) and bioadhesive nanoparticles on the encapsulation and intestinal permeability of paclitaxel (PTX). In this context, a solid inclusion complex between PTX and HPCD was prepared by an evaporation method. Then, the complex was incorporated in poly(anhydride) nanoparticles by a solvent displacement method. The resulting nanoparticles, PTX–HPCD NP, displayed a size of about 300 nm and a drug loading of about 170 μg/mg (500-fold higher than in the absence of HPCD). The effect of these nanoparticles on the permeability of intestinal epithelium was investigated using the Ussing chamber technique. The apparent permeability (P_app) of PTX was found to be 12-fold higher when formulated as PTX–HPCD NP than when formulated as Taxol^® (control). Furthermore, when interaction between nanoparticles and the mucosa was avoided, the permeability of PTX significantly decreased. In summary, the association between PTX–HPCD and poly(anhydride) nanoparticles would induce a positive effect over the intestinal permeability of paclitaxel, being the bioadhesion a mandatory condition in this phenomena.     

Acid-cleavable ketal containing poly(β-amino ester) for enhanced siRNA delivery

The safe and effective intracellular delivery of nucleic acids remains the most challenging obstacle to the broad application of gene therapy in clinic. Endosomal escape of nucleic acids is also a major barrier for efficient gene delivery. Ketal linkage is known to readily cleave at the acidic pH of endosomal compartments. Here, we report ketal containing poly(β-amino ester) (KPAE) as an acid-cleavable non-viral siRNA delivery system. KPAE efficiently condensed siRNA into nanocomplexes with a diameter of ∼150 nm, which are stable under neutral conditions but rapidly dissociate to release siRNA at acidic pH. KPAE had a buffering capacity due to the presence of secondary amines in its backbone, confirmed by acid–base titration. Moreover, the studies of confocal fluorescence imaging using calcein and LysoTracker Red revealed that KPAE disrupted endosomes by colloid osmotic mechanism and “proton sponge” effects. Cell culture studies demonstrated that KPAE can deliver tumor necrosis factor-α (TNF-α) siRNA to lipopolysaccharide (LPS)-stimulated macrophages and significantly inhibit the expression of TNF-α. The results demonstrate that acid-cleavable KPAE has great potential as gene delivery systems based on its excellent biocompatibility, pH sensitivity and high gene delivery efficiency.     

乳酸-羟基乙酸共聚物微球的研究进展

通过整理和归纳国内外文献,介绍乳酸-羟基乙酸(PLGA)载药微球的制备方法和作为药物载体的应用。     

pH-sensitive nanoparticles for improving the oral bioavailability of cyclosporine A

The purpose of this work was to improve the oral bioavailability of cyclosporine A (CyA) by preparation the CyA-pH sensitive nanoparticles. The CyA-pH sensitive nanoparticles were prepared by using poly(methacrylic acid and methacrylate) copolymer. The characterization and the dispersion state of CyA at the surface or inside the polymeric matrices of the nanoparticles were investigated. The in vitro release studies were conducted by ultracentrifuge method. The bioavailability of CyA from nanoparticles and Neoral microemulsion was assessed in Sprague–Dawley (SD) rats at a dose of 15 mg/kg. The particle size of the nanoparticles was within the range from 37.4 ± 5.6 to 106.7 ± 14.8 nm. The drug entrapped efficiency was very high (from 90.9 to 99.9%) and in all cases the drug was amorphous or molecularly dispersed within the nanoparticles polymeric matrices. In vitro release experiments revealed that the nanoparticles exhibited perfect pH-dependant release profiles. The relative bioavailability of CyA was markedly increased by 32.5% for CyA-S100 nanoparticles (P < 0.05), and by 15.2% and 13.6% for CyA-L100-55 and CyA-L100 nanoparticles respectively, while it was decreased by 5.2% from CyA-E100 nanoparticles when compared with the Neoral microemulsion. With these results, the potential of pH-sensitive nanoparticles for the oral delivery of CyA was confirmed.     

Novel approach for delivery of insulin loaded poly(lactide-co-glycolide) nanoparticles using a combination of stabilizers

Insulin stability during microencapsulation and subsequent release is essential for retaining its biological activity. The successful delivery of insulin relies on the proper selection of stabilizers in addition to other parameters. Attempts were made to address the problem with a few combination of stabilizers for maintaining the integrity of insulin during formulation and delivery. Insulin loaded nanoparticles with different stabilizers such as pluronic F68, trehalose, and sodium bicarbonate were prepared by the double emulsion evaporation method using two different copolymer ratios of poly(DL-lactide-co-glycolide) (50:50 and 85:15). The presence of stabilizers in the nanoparticles resulted in an increase in the particle size but a reduction of encapsulation efficiency. Insulin release rate was comparatively higher for the batches containing stabilizers when compared with controls for both the copolymer ratios. Also the presence of stabilizers resulted in sustained release of insulin resulting in prolonged reduction of blood glucose levels in streptozotocin induced diabetic rats. From the in vitro and in vivo studies, we concluded that a combination of stabilizers results in beneficial effects without compromising the advantages of delivery systems.     

Clonazepam release from poly(DL-lactide-co-glycolide) nanoparticles prepared by dialysis method

Aim of this work is to prepare poly(DL-lactide-co-glycolide) (PLGA) nanoparticles by dialysis method without surfactant and to investigate drug loading capacity and drug release. The size of PLGA nanoparticles was 269.9±118.7 nm in intensity average and the morphology of PLGA nanoparticles was spherical shape from the observation of SEM and TEM. In the effect of drug loading contents on the particle size distribution, PLGA nanoparticles were monomodal pattern with narrow size distribution in the empty and lower drug loading nanoparticles whereas bi- or trimodal pattern was showed in the higher drug loading ones. Release of clonazepam from PLGA nanoparticles with higher drug loading contents was slower than that with lower loading contents.     

Novel,dynamic on-line analytical separation system for dissolution of drugs from poly(lactic acid) nanoparticles

A novel method for investigating drug release in a dynamic manner from nanoparticles including, but not limited to, biodegradable poly(lactic acid) (PLA) is reported. The PLA nanoparticles were prepared by the nanoprecipitation method. Two poorly soluble drugs, beclomethasone dipropionate (BDP) and indomethacin, were encapsulated into PLA nanoparticles, and their dissolution from the nanoparticles were followed in a dynamic way. The on-line method comprised a short column (vessel) packed with the PLA nanoparticles, on-line connected to an analytical liquid chromatographic column via a multiport switching valve equipped with two loops. The system allowed monitoring of the drug release profiles in real time, and the conditions for the drug release could be precisely controlled and easily changed. The effects of solvent composition and temperature on the rate of dissolution of the drugs from the PLA nanoparticles were investigated. The system proved to be linear for the drugs tested over the concentration range 10–3000 ng (n = 6, R² = 0.999 and 0.997 for indomethacin and beclomethasone, respectively) and repeatable (RSD of peak areas <0.5%). The recoveries of the dissolution study were quantitative (120 and 103% for indomethacin and beclomethasone, respectively).     

Poly (amino acid) micelle nanocarriers in preclinical and clinical studies

Polymeric micelles are expected to increase the accumulation of drugs in tumor tissues utilizing the EPR effect and to incorporate various kinds of drugs into the inner core by chemical conjugation or physical entrapment with relatively high stability. The size of the micelles can be controlled within the diameter range of 20 to 100 nm, to ensure that the micelles do not pass through normal vessel walls; therefore, a reduced incidence of the side effects of the drugs may be expected due to the decreased volume of distribution.

These are several anticancer agent-incorporated micelle carrier systems under clinical evaluation. Phase 1 studies of a CDDP incorporated micelle, Nc-6004, and an sN-38 incorporated micelle, NK012, are now underway. A phase 2 study of a PTX incorporated micelle, NK105, against stomach cancer is also underway.     

新型复方氨基酸(19) 丙谷二肽注射液临床耐受性试验

目的评价健康人体对新型复方氨基酸(19)丙谷二肽注射液(创伤用)的耐受性。方法 48名健康受试者,男女各半,分三部分试验,均经静脉给药。滴速耐受性试验纳入24名受试者,随机分为3组,滴速设为1、1.5、2 mL·min~(-1);剂量耐受剂量试验纳入18名受试者,随机分为3组,剂量分别为500、750、1 000 mL;全静脉营养液滴注试验纳入6名受试者,滴速不超过3 mL·min~(-1)。观察受试者用药前后生命体征、心电图及实验室检查的变化,并记录不良事件。结果滴速耐受性试验、剂量耐受剂量试验和全静脉营养液滴注试验中受试者用药前后生命体征、心电图和实验室检查均未出现有临床意义的改变。共有13名受试者出现轻度不良事件,表现为血管胀痛、头胀、眼干等,未予处理,均自行缓解。结论健康受试者静脉滴注新型复方氨基酸(19)丙谷二肽注射液(创伤用),滴速1～2 mL·min~(-1)、剂量500～1 000 mL,全静脉营养液1700 mL,滴速≤3 mL·min~(-1),可安全耐受。