Nasal Delivery of Insulin Using Novel Chitosan Based Formulations: A Comparative Study in Two Animal Models Between Simple Chitosan Formulations and Chitosan Nanoparticles

Purpose. To investigate whether the widely accepted advantages associated with the use of chitosan as a nasal drug delivery system, might be further improved by application of chitosan formulated as nanoparticles. Methods. Insulin-chitosan nanoparticles were prepared by the ionotropic gelation of chitosan glutamate and tripolyphosphate pentasodium and by simple complexation of insulin and chitosan. The nasal absorption of insulin after administration in chitosan nanoparticle formulations and in chitosan solution and powder formulations was evaluated in anaesthetised rats and/or in conscious sheep. Results. Insulin-chitosan nanoparticle formulations produced a pharmacological response in the two animal models, although in both cases the response in terms of lowering the blood glucose levels was less (to 52.9 or 59.7% of basal level in the rat, 72.6% in the sheep) than that of the nasal insulin chitosan solution formulation (40.1% in the rat, 53.0% in the sheep). The insulin-chitosan solution formulation was found to be significantly more effective than the complex and nanoparticle formulations. The hypoglycaemic response of the rat to the administration of post-loaded insulin-chitosan nanoparticles and insulin-loaded chitosan nanoparticles was comparable. As shown in the sheep model, the most effective chitosan formulation for nasal insulin absorption was a chitosan powder delivery system with a bioavailability of 17.0% as compared to 1.3% and 3.6% for the chitosan nanoparticles and chitosan solution formulations, respectively. Conclusion. It was shown conclusively that chitosan nanoparticles did not improve the absorption enhancing effect of chitosan in solution or powder form and that chitosan powder was the most effective formulation for nasal delivery of insulin in the sheep model.     

胰岛素壳聚糖胶态纳米粒的制备及体外释药性能

采用离子趋向凝胶化法制备了胰岛素壳聚糖胶态微粒,并考察了外观、粒径和体外释药性能.所得产品呈球形,表面光滑圆整,平均粒径为276nm,多分散系数为0.08.体外释药呈pH依赖性,释药速度受载药量及泊洛沙姆188含量的影响.     

Enhancement of Nasal Absorption of Insulin Using Chitosan Nanoparticles

Purpose. To investigate the potential of chitosan nanoparticles as a system for improving the systemic absorption of insulin following nasal instillation. Methods. Insulin-loaded chitosan nanoparticles were prepared by ionotropic gelation of chitosan with tripolyphosphate anions. They were characterized for their size and zeta potential by photon correlation spectroscopy and laser Doppler anemometry, respectively. Insulin loading and release was determined by the microBCA protein assay. The ability of chitosan nanoparticles to enhance the nasal absorption of insulin was investigated in a conscious rabbit model by monitoring the plasma glucose levels. Results. Chitosan nanoparticles had a size in the range of 300–400 nm, a positive surface charge and their insulin loading can be modulated reaching values up to 55% [insulin/nanoparticles (w/w): 55/100]. Insulin association was found to be highly mediated by an ionic interaction mechanism and its release in vitro occurred rapidly in sink conditions. Chitosan nanoparticles enhanced the nasal absorption of insulin to a greater extent than an aqueous solution of chitosan. The amount and molecular weight of chitosan did not have a significant effect on insulin response. Conclusions. Chitosan nanoparticles are efficient vehicles for the transport of insulin through the nasal mucosa.     

Chitosan and Chitosan/Ethylene Oxide-Propylene Oxide Block Copolymer Nanoparticles as Novel Carriers for Proteins and Vaccines

Purpose. The aim of this study was to investigate the interaction between the components of novel chitosan (CS) and CS/ethylene oxide-propylene oxide block copolymer (PEO-PPO) nanoparticles and to evaluate their potential for the association and controlled release of proteins and vaccines. Methods. The presence of PEO-PPO on the surface of the nanoparticles and its interaction with the CS was identified by X-ray photoelectron spectroscopy (XPS). The mechanism of protein association was elucidated using several proteins, bovine serum albumin (BSA), and tetanus and diphtheria toxoids, and varying the formulation conditions (different pH values and concentrations of PEO-PPO), and the stage of protein incorporation into the nanoparticles formation medium. Results. BSA and tetanus and diphtheria toxoids were highly associated with CS nanoparticles partly due to electrostatic interactions between the carboxyl groups of the protein and the amine groups of CS. PEO-PPO also interacted electrostatically with CS, thus competing with the proteins for association with CS nanoparticles. A visible amount of PEO-PPO was projected towards the outer phase of the nanoparticles. Proteins were released from the nanoparticles at an almost constant rate, the intensity of which was closely related to the protein loading. Furthermore, the tetanus vaccine was released in the active form for at least 15 days. Conclusions. CS and CS/PEO-PPO nanoparticles prepared by a very mild ionic crosslinking technique are novel and suitable systems for the entrapment and controlled release of proteins and vaccines.     

Chitosan as a Novel Nasal Delivery System for Peptide Drugs

A nasal solution formulation of the cationic material chitosan was shown to greatly enhance the absorption of insulin across the nasal mucosa of rat and sheep. The absorption promoting effect was concentration dependent with the optimal efficacy obtained for concentrations higher than 0.2% and 0.5% in rats and sheep, respectively. The absorption promoting effect was reversible with time in a pulse-chase study. Histological examination of the nasal mucosa of rats exposed to a chitosan solution for 60 minutes showed little change.     

Optimization of Chitosan Succinate and Chitosan Phthalate Microspheres for Oral Delivery of Insulin using Response Surface Methodology

In the present study, a Box-Behnken experimental design was employed to statistically optimize the formulation parameters of chitosan phthalate and chitosan succinate microspheres preparation. These microspheres can be useful for oral insulin delivery system. The effects of three parameters namely polymer concentration, stirring speed and cross linking agent were studied. The fitted mathematical model allowed us to plot response surfaces curves and to determine optimal preparation conditions. Results clearly indicated that the crosslinking agent was the main factor influencing the insulin loading and releasing. The in vitro results indicated that chitosan succinate microspheres need high amount of crosslinking agent to control initial burst release compared to chitosan phthalate microspheres. The reason may be attributed that chitosan succinate is more hydrophilic than chitosan phthalate. The relative pharmacological efficacy for chitosan phthalate and chitosan succinate microspheres (18.66 ± 3.84%, 16.24 ± 4%) was almost three-fold higher than the efficacy of the oral insulin administration (4.68 ± 1.52%). These findings suggest that these microspheres are promising carrier for oral insulin delivery system.     

Alginate/Chitosan Nanoparticles are Effective for Oral Insulin Delivery

Purpose To evaluate the pharmacological activity of insulin-loaded alginate/chitosan nanoparticles following oral dosage in diabetic rats. Methods Nanoparticles were prepared by ionotropic pre-gelation of an alginate core followed by chitosan polyelectrolyte complexation. In vivo activity was evaluated by measuring the decrease in blood glucose concentrations in streptozotocin induced, diabetic rats after oral administration and flourescein (FITC)-labelled insulin tracked by confocal microscopy. Results Nanoparticles were negatively charged and had a mean size of 750 nm, suitable for uptake within the gastrointestinal tract due to their nanosize range and mucoadhesive properties. The insulin association efficiency was over 70% and insulin was released in a pH-dependent manner under simulated gastrointestinal conditions. Orally delivered nanoparticles lowered basal serum glucose levels by more than 40% with 50 and 100 IU/kg doses sustaining hypoglycemia for over 18 h. Pharmacological availability was 6.8 and 3.4% for the 50 and 100 IU/kg doses respectively, a significant increase over 1.6%, determined for oral insulin alone in solution and over other related studies at the same dose levels. Confocal microscopic examinations of FITC-labelled insulin nanoparticles showed clear adhesion to rat intestinal epithelium, and internalization of insulin within the intestinal mucosa. Conclusion The results indicate that the encapsulation of insulin into mucoadhesive nanoparticles was a key factor in the improvement of its oral absorption and oral bioactivity.     

壳寡糖-水杨酸接枝物纳米粒用于释放阿霉素的研究

目的 考察壳寡糖/水杨酸纳米粒负载碱化阿霉素的可能性,评价制备而得的微粒给药系统理化性质及其体外释放行为。方法 以碳二亚胺为交联偶合剂合成壳寡糖/水杨酸接枝共聚物,三硝基苯磺酸法测定水杨酸接枝率。运用超声分散法制备壳寡糖/水杨酸空白纳米粒,芘荧光法测定纳米粒临界聚集浓度,动态光散射法测定微粒粒径和表面电位,MTT法考察空白纳米粒的细胞毒性。以碱化阿霉素为模型药物,透析法制备壳寡糖/水杨酸载药纳米粒,经透射电镜考察载药纳米粒的形态,对其体外释放行为进行了研究。结果 合成得到的壳寡糖分子量=9000/水杨酸理论投料量=50%的实际接枝率为16.92%,空白纳米粒的临界聚集浓度为867.0 μg/mL,空白纳米粒的粒径和表面Zeta电位分别为434.0 nm和48.6 mV,对人肝癌细胞Hep-G2的半数抑制浓度为1745μg/mL。在碱化阿霉素理论投药量为10%时壳寡糖/水杨酸载药纳米粒的实际载药量为8.52%,包封率为93.15%。;载药纳米粒的粒径和表面电位分别为214.2 nm和33.6 mV。体外释放结果表明药物的释放呈现pH敏感性;并主要以溶蚀的方式从载体内部释放出来。结论 壳寡糖/水杨酸接枝物可以有效包裹碱化阿霉素并成为粒径均一的纳米粒给药系统。载药纳米粒具有pH敏感和缓释作用。壳寡糖/水杨酸接枝物有望成为潜在的难溶性药物的载体材料。     

壳聚糖载药纳米粒研究进展

目的介绍壳聚糖载药纳米粒近年来的研究进展。方法总结壳聚糖纳米粒的制备方法、释药特性、生物摄取及其应用。结果不同的制备方法可得到不同粒径和表面特性的壳聚糖纳米粒。壳聚糖纳米粒改变了壳聚糖的摄取机制，广泛应用于药物的器官靶向、DNA转染效率提高、药物的非注射途释给药等方面。结论壳聚糖纳米粒作为一种新型的药物载体，具有重要的研究开发价值。     

Uptake of Chitosan and Associated Insulin in Caco-2 Cell Monolayers: A Comparison Between Chitosan Molecules and Chitosan Nanoparticles

PURPOSE: To evaluate the uptake of chitosan molecules (fCS) and nanoparticles (fNP), and their ability to mediate insulin transport in Caco-2 cell monolayers. METHODS: Cell-associated fCS and fNP were evaluated by fluorometry, trypan blue quenching, and confocal microscopy using FITC-labeled chitosan. Chitosan-mediated transport of FITC-labeled insulin was studied in Caco-2 cell monolayers cultured on permeable inserts. RESULTS: Caco-2 cells showed twofold higher association with fNP than fCS after 2-h incubation with 1 mg/ml samples. fNP uptake was a saturable (Km 1.04 mg/ml; Vmax 74.15 microg/mg/h), concentration- and temperature-dependent process that was inhibited by coadministered chlorpromazine. fCS uptake was temperature dependent, but was less sensitive to concentration and was inhibited by filipin. Postuptake quenching with 100 microg/ml of trypan blue suggests a significant amount of intracellular fNP, although the bulk of fCS was extracellular. Internalized fNP were located by confocal microscopy at 15 microm from the apical membrane, but there was no apparent breaching of the basal membrane. This might explain the failure of the nanoparticles to mediate significant insulin transport across the Caco-2 cell monolayer. CONCLUSIONS: Formulation of chitosan into nanoparticles transforms its extracellular interactions with the Caco-2 cells to one of cellular internalization via clathrin-mediated endocytosis.     

基于纳米金的肿瘤靶向给药系统的研究进展

化疗是目前临床癌症治疗的主要手段之一,主要通过静脉给药细胞毒药物,在杀伤肿瘤细胞的同时,往往会导致健康器官和组织的全身细胞毒性.最新研究表明,纳米金是一种高效的抗肿瘤药物载体,能够携带药物穿透血管和组织屏障进入肿瘤病灶,并特异性积蓄于肿瘤组织,有效降低化疗药物的机体不良反应.此外,金纳米粒子具有易修饰性,相比于其他纳米...     

Insulin Nanoparticles: A Novel Formulation Approach for Poorly Water Soluble Zn-Insulin

PURPOSE: To determine the feasibility of using wet milling technology to formulate poorly water soluble zinc-insulin as a stable, biologically active, nanoparticulate dispersion. METHODS: The feasibility of formulating zinc-insulin as a nanoparticulate dispersion using wet milling technology was studied. An insulin nanoparticulate formulation was reproducibly obtained after milling zinc-insulin in the presence of F68, sodium deoxycholate and water at neutral pH. The physical and chemical properties of these peptide particles were studied using electron microscopy, laser light scattering, HPLC and SDS-PAGE. To verify efficacy, hyperglycemic rats were dosed subcutaneously and intraduodenally with nanoparticles or solubilized insulin. Glucose and insulin levels were monitored on blood samples collected throughout the study. RESULTS: Zn-insulin (mean size = 16.162 microm) was processed using milling technology to form an aqueous-based nanoparticle dispersion with a mean particle size of less than 0.150 microm. The formulation was homogeneous and exhibited a unimodal particle size distribution profile using laser light diffraction techniques. Insulin, processed as a peptide-particle dispersion, was shown to be comparable to unprocessed powder using HPLC and SDS-PAGE. In addition, HPLC analyses performed on samples, heat-treated at 70 degrees C for 100 minutes, demonstrate that under conditions which effect the solubilized peptide, formulated as a peptide-particle dispersion, insulin was chemically stable. Also, when stored refrigerated, the insulin dispersion was chemically and physically stable. Finally, peptide particles of insulin, dosed subcutaneously and intraduodenally, were effective at lowering blood glucose levels of hyperglycemic rats. CONCLUSION: Water insoluble Zn-insulin can be formulated as a stable, biologically active nanometer-sized peptide particle dispersion using wet media milling technology.     

金纳米粒子的研究进展

目的:迅速发展的纳米技术近年来的在纳米研究领域的蓬勃发展中起到了推波助澜的作用。特别是,它的应用在医药领域中。金纳米粒子纳米粒子不断的扩大基础知识更好地了解金纳米粒子的属性,金纳米粒子实验意味着纳米技术前沿不断的改变。目前似乎要提高金纳米粒子在治疗癌症的应用程序,包括诊断,监控和治疗疾病。     

胰岛素纳米粒制剂研究进展

目的介绍近年来胰岛素纳米粒制剂研究进展。方法查阅国内外胰岛素纳米粒的相关文献资料，从吸收机制、制备、检测方法及其发展等方面进行整理和归纳。结果与结论目前胰岛素纳米粒的研究已广泛开展，取得了令人瞩目的成就，发展空间广阔。     

Development of Novel Chitosan Derivatives as Micellar Carriers of Taxol

Purpose. To develop an intravenous injectable carrier composed of chitosan derivatives for taxol. Methods. A chitosan with lauryl groups attached to amino groups to provide the hydrophobic moieties and, carboxymethyl groups attached to hydroxy groups to provide the hydrophilic moieties (N-lauryl-carboxymethyl-chitosan = LCC), was newly synthesized. The solubility of taxol in LCC micelles in aqueous solution was examined. The hemolysis test of LCC and the growth inhibition experiment of taxol-loading micelle using KB cells were also performed as in vitro assay. Results. It was found that LCC solubilized taxol by forming micelles with particle sizes less than l00nm. This particle size was considered effective for passive targeting for tumors. The concentration of taxol in the micellar solution was very high, with a maximum of 2.37mg/ mL. This maximum was 1000 times above that in a saturated solution of taxol at pH 7.4. Hemolysis testing as an in vitro assay indicated that LCC was safer than Polysorbate 80 (TO-10M) as intravenous surfactant in terms of induction of membrane damage. As judged by cytostatic activity against KB cells, taxol retained activity even when included in LCC micelles. LCC-entrapped taxol was more effective in cytostatic activity than free taxol in low concentrations. Conclusions. The results of solubilization capacity examination, hemolysis testing, and cytostatic activity suggest that LCC may be useful as a carrier of taxol.     

A Novel Approach to the Oral Delivery of Micro- or Nanoparticles

A novel oral multiple-unit dosage form which overcame many of the problems commonly observed during the compression of microparticles into tablets was developed in this study. Micro- or nano-particles were entrapped in beads formed by ionotropic gelation of the charged polysaccharide, chitosan or sodium alginate, in solutions of the counterion, tripolyphosphate (TPP) or calcium chloride (CaCl₂), respectively. The described technique did not change the physical properties of the microparticles, and it allowed a high microparticle loading (up to 98%). The ionic character of the polymers allowed pH-dependent release of the microparticles. Chitosan beads disintegrated and released the microparticles in 0.1 N HC1, while calcium alginate beads stayed intact in 0.1 N HC1 but rapidly disintegrated in simulated intestinal fluids. Coating the calcium alginate beads with cellulose acetate phthalate resulted in an enteric drug delivery system. Scanning electron microscopy and dissolution and disintegration tests were used to characterize the microparticle-containing beads. The disintegration time of the beads was studied as a function of the solution viscosity of the polysaccharide, gelation time, counterion concentration, and method of drying.     

壳聚糖介导的靶向药物传递系统

目的:综述了以壳聚糖或其衍生物为基础的多种特殊靶向载体系统的研究现状。方法:以国内外近年有代表性的文献为依据,进行分析、整理和归纳。结果与结论:由于壳聚糖具有一些独特的物理和生物特性,并且通过修饰后增加了壳聚糖的一些性质,使其成为优良的药物载体,从而实现药物的靶向性。这一载体材料近年来逐渐受到重视,具有良好的开发和应用前景。     

Polyalkylcyanoacrylate Nanoparticles as Polymeric Carriers for Antisense Oligonucleotides

Adsorption of oligothymidylates on polyisobutyl- or polyisohexylcyanoacrylate nanoparticles was achieved in the presence of hydrophobic cations such as tetraphenylphosphonium chloride or quaternary ammonium salts. Results suggested that oligonucleotide adsorption on the nanoparticles was mediated by the formation of ion pairs between the negatively charged phosphate groups of the nucleic acid chain and the hydrophobic cations. The adsorption efficiency of oligonucleotide–cation complexes on nanoparticles was found to be highly dependent upon several parameters: oligonucleotide chain length, nature of the cyanoacrylic monomer, hydrophobicity of cations used as ion-pairing agents, and ionic concentration of the medium. Carrier capacity of polyisohexylcyanoacrylate nanoparticles for oligothymidylates (16 nucleotides) complexed with cetyltrimetylammonium bromide in the presence of 0.15 M NaCl was determined to be 5 µmol/g polymer. The in vitro protection of oligothymidylates adsorbed to nanoparticles against degradation by a 3-exonuclease (snake venom phosphodiesterase) was also demonstrated. These results showed that nanoparticles can be considered as convenient carriers for the protection and delivery of Oligonucleotides to cells in culture and for future applications in vivo.     

离子凝胶法制备壳聚糖纳米粒的影响因素研究

目的 探讨离子凝胶法制备壳聚糖纳米粒(CS-NPs)的影响因素.方法 用碱降解法制备高脱乙酰度的壳聚糖(CS),并以之为材料,采用离子凝胶法制备CS-NPs,以微粒的平均粒径、分散度和Zeta电位为指标,考察CS及三聚磷酸钠(TPP)的质量浓度、CS/TPP质量比、CS溶液pH值和CS溶液温度对制备CS-NPs的影响....     

Gelatin‐coated Gold Nanoparticles as Carriers of FLT3 Inhibitors for Acute Myeloid Leukemia Treatment

This study presents the design of a gold nanoparticle (AuNPs)—drug system with improved efficiency for the treatment of acute myeloid leukemia. The system is based on four different FLT3 inhibitors, namely midostaurin, sorafenib, lestaurtinib, and quizartinib, which were independently loaded onto gelatin‐coated gold nanoparticles. Detailed investigation of the physicochemical properties of the formed complexes lead to the selection of quizartinib—loaded AuNPs for the in vitro evaluation of the biological effects of the formed complex against OCI‐AML3 acute myeloid leukemia cells. Viability tests by MTT demonstrated that the proposed drug complex has improved efficacy when compared with the drug alone. The obtained results constitute a premise for further in vivo investigation of such drug vehicles based on AuNPs. To the best of our knowledge, this is the first study that investigates the delivery of the above‐mentioned FLT3 inhibitors via gelatin‐coated gold nanoparticles.