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 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.     

Chitosan Reduced Gold Nanoparticles as Novel Carriers for Transmucosal Delivery of Insulin

Purpose Colloidal metallic systems have been recently investigated in the area of nanomedicine. Gold nanoparticles have found themselves useful for diagnostic and drug delivery applications. Herein we have reported a novel method for synthesis of gold nanoparticles using a natural, biocompatible and biodegradable polymer; chitosan. Use of chitosan serves dual purpose by acting as a reducing agent in the synthesis of gold nanoparticles and also promotes the penetration and uptake of peptide hormone insulin across the mucosa. To demonstrate the use of chitosan reduced gold nanoparticles as carriers for drug delivery, we report herein the transmucosal delivery of insulin loaded gold nanoparticles. Materials and Methods Gold nanoparticles were prepared using different concentrations of chitosan (from 0.01% w/v up to 1% w/v). The gold nanoparticles were characterized for surface plasmon band, zeta potential, surface morphology, in vitro diffusion studies and fluorescence spectroscopy. The in vivo studies in diabetic male Wistar rats were carried out using insulin loaded chitosan reduced gold nanoparticles. Results Varying concentrations of chitosan used for the synthesis of gold nanoparticles demonstrated that the nanoparticles obtained at higher chitosan concentrations (>0.1% w/v) were stable showing no signs of aggregation. The nanoparticles also showed long term stability in terms of aggregation for about 6 months. Insulin loading of 53% was obtained and found to be stable after loading. Blood glucose lowering at the end of 2 h following administration of insulin loaded gold nanoparticles to diabetic rats was found to be 30.41 and 20.27% for oral (50 IU/kg) and nasal (10 IU/kg), respectively. Serum gold level studies have demonstrated significant improvement in the uptake of chitosan reduced gold nanoparticles. Conclusions The synthesis of gold nanoparticles using a biocompatible polymer, chitosan would improve its surface properties for binding of biomolecules. Our studies indicate that oral and nasal administration of insulin loaded chitosan reduced gold nanoparticles has led to improved pharmacodynamic activity. Thus, chitosan reduced gold nanoparticles loaded with insulin prove to be promising in controlling the postprandial hyperglycemia.     

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.     

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.     

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.     

胰岛素肺部给药系统研究进展

目的介绍胰岛素肺部给药系统的研究进展。方法综述了肺部给药机制、胰岛素吸入制剂/装置、药动学和药效学特点、临床应用等研究内容。结果与结论胰岛素肺部给药系统的发展用以补充和/或替代皮下注射,可能是一种有效方法,使患者获得更好地糖尿病治疗结果。     

Enhancement of Nasal Delivery of a Renin Inhibitor in the Rat Using Emulsion Formulations

Nasal absorption of O-(N-morpholino-carbonyl-3-L-phenylaspartyl-L-leucinamide of (2S,3R,4S)-2-amino-1-cyclohexyl-3,4-dihydroxy-6-methylheptane (I), a renin inhibitor, was evaluated in two rat nasal models, one involving surgery and the other requiring no surgical intervention. Oleic acid/monoolein emulsion formulations were tested along with a control PEG 400 solution. The percent absolute bioavailability of the compound was enhanced from 3–6% (PEG 400 solution) to 15–27% when the emulsion formulations were used. The different nasal model techniques (with and without surgery) did not produce any statistical difference in the absolute bioavailability values for I. Emulsion formulations did not produce appreciable damage as assessed morphologically. It is suggested that emulsion formulations containing membrane adjuvants such as oleic acid and monoolein can be used to enhance the nasal delivery of low-bioavailable, lipid-soluble drugs.____________________     

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.     

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.     

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。     

Nasal absorption and local tissue reaction of insulin nanocomplexes of trimethyl chitosan derivatives in rats

Objectives The objective of this work was to explore the potential and safety of trimethyl chitosan (TMC) and PEGylated TMC for improved absorption of insulin after nasal administration. Methods The nasal absorption of insulin nanocomplexes of TMC or PEGylated TMC was evaluated in anaesthetized rats. Concomitantly, the histopathological effects of these nanocomplexes on rat nasal mucosa were studied using a perfusion fixation technique. Key findings All insulin nanocomplexes containing TMC or PEGylated TMC showed a 34–47% reduction in the blood glucose concentration, when the insulin absorption through the rat nasal mucosa was measured indirectly. In addition, the relative pharmacodynamic bioavailability (F_dyn) of the formulations was found to be dependent upon the charge ratio of insulin and polymer, regardless of polymer structure. The F_dyn apparently decreased with increasing charge ratio of insulin : polymer. Although acute alterations in nasal morphology by the formulations were affected by the charge ratio of insulin and polymer, the formulation of insulin/PEGylated TMC nanocomplexes was shown to be less toxic to the nasal epithelial membrane than insulin/TMC nanocomplexes. Conclusions PEGylated TMC nanocomplexes were a suitable absorption enhancer for nasal delivery of insulin.     

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.     

Glibenclamide Nanocrystals in a Biodegradable Chitosan Patch for Transdermal Delivery: Engineering,Formulation, and Evaluation

Glibenclamide (GBD) nanocrystals (D₅₀ = 429 nm) were engineered by applying combined precipitation and homogenization procedures. GBD crystallinity was maintained during the nanonization process as revealed by differential scanning calorimetry and X-ray analyses. Nanonized and micronized GBD were incorporated into chitosan solutions to fabricate transdermal delivery systems (TDDSs), nano- and micro-GBD, respectively. The fabricated TDDSs displayed satisfactory physicochemical characteristics without substantial aggregation of GBD nanocrystals during the casting and drying procedures. Within 24 hours, about 85 ± 3.1% of the GBD content was released from nano-GBD, compared to 61 ± 3.9% from micro-GBD. Cumulative permeation of GBD from nano-GBD after 24 hours was 498 ± 33.35 compared to 362 ± 25.25 μg/cm² from micro-GBD. The calculated flux across rat skin for nano-GBD was 23.14 compared to 13.64 μg/cm²/h for micro-GBD, with an enhancement factor of 1.7. In vivo assessment clearly revealed the enhanced efficacy of nano-GBD to reduce blood glucose levels and counteract the induced hyperglycemia in tested animals compared to micro-GBD (p < 0.5). Simultaneously, the nano-GBD was able to maintain higher drug concentration for longer time (24 hours, p < 0.5) and minimize intense action and hypoglycemia associated with GBD oral therapy (p < 0.5).     

Chitosan-DNA Nanoparticles: The Effect of Cell Type and Hydrolysis of Chitosan on In Vitro DNA Transfection

Commercial chitosan (Ch) with low (LMWCh) and medium molecular weight (MMWCh) were hydrolyzed in diluted hydrochloric acid by heating at different temperatures. The viscosity average molecular weight of Chs was gradually decreased from 450 to 14 kDa as a function of temperature. Ch fractions were used for formation of Ch-DNA nanoparticles and tested for the ability to introduce DNA into HEK293, Swiss3T3, HeLa, and MDCK cells in vitro. The average diameter of nanoparticles was 200–220 nm. The surface charge of nanoparticles varied depending on the Ch/DNA ratio. The cell lines different response to DNA transfection with Ch fractions depended on molecular weight. HEK293 cells were efficiently transfected by nanoparticles prepared with Chs having a wide range of molecular weight (～14–195 kDa). Swiss3T3 cells were efficiently transfected by Ch polymers with about <17 kDa. In contrast, HeLa and MDCK cells were highly resistant to DNA transfection with Ch polymers. These results strongly suggest that Ch polymers may be widely used for DNA trasnfection of the mammalian cells under optimized conditions.     

壳聚糖与葡聚糖在基因递送应用中的研究进展

在基因疗法中,天然高分子多聚糖因其高生物相容性和可修饰性而成为良好的基因递送材料。壳聚糖和葡聚糖是2种重要的天然高分子多聚糖,它们本身及其衍生物都可以用做基因药物载体材料。研究者采用多种方法对壳聚糖和葡聚糖进行改良,拓宽它们在基因递送中的应用范围。本综述介绍了两者的结构和性质特点以及近年来两者及其衍生物在基因递送应用中的研究进展,最后对它们的发展前景进行了展望。     

Raman Chemical Imaging for Ingredient-specific Particle Size Characterization of Aqueous Suspension Nasal Spray Formulations: A Progress Report

Purpose This study was conducted to evaluate the feasibility of using Raman chemical imaging (i.e., Raman imaging microspectroscopy) to establish chemical identity, particle size and particle size distribution (PSD) for a representative corticosteroid in aqueous nasal spray suspension formulations. Materials and Methods The Raman imaging PSD protocol was validated using polystyrene (PS) microsphere size standards (NIST-traceable). A Raman spectral library was developed for the active and inactive compounds in the formulation. Four nasal sprays formulated with beclomethasone dipropionate (BDP) ranging in size from 1.4 to 8.3 μm were imaged by both Raman and brightfield techniques. The Raman images were then processed to calculate the PSD for each formulation. Results Within each region examined, active pharmaceutical ingredient (API) particles are unambiguously identified and the total number of those particles, particle size and PSD of API free of excipients and PSD of API particles adhered to other excipients are reported. Conclusions Good statistical agreement is obtained between the reported and measured sizes of the PS microspheres. BDP particles were clearly distinguishable from those of excipients. Raman chemical imaging (RCI) is able to differentiate between and identify the chemical makeup of multiple components in complex BDP sample and placebo mixtures. The Raman chemical imaging method (coupled Raman and optical imaging) shows promise as a method for characterizing particle size and shape of corticosteroid in aqueous nasal spray suspension formulations. However, rigorous validation of RCI for PSD analysis is incomplete and requires additional research effort. Some specific areas of concern are discussed. This article represents the personal opinions of the authors and does not necessarily represent the views or policies of the US Food and Drug Administration.     

Chitosan Nanoparticles as New Ocular Drug Delivery Systems: in Vitro Stability,in Vivo Fate,and Cellular Toxicity

PURPOSE: To assess the potential of chitosan (CS) nanoparticles for ocular drug delivery by investigating their interaction with the ocular mucosa in vivo and also their toxicity in conjunctival cell cultures. METHODS: Fluorescent (CS-fl) nanoparticles were prepared by ionotropic gelation. The stability of the particles in the presence of lysozyme was investigated by determining the size and their interaction with mucin, by measuring the viscosity of the mucin dispersion. The in vivo interaction of CS-fl nanoparticles with the rabbit cornea and conjunctiva was analyzed by spectrofluorimetry and confocal microscopy. Their potential toxicity was assessed in a human conjunctival cell line by determining cell survival and viability. RESULTS: CS-fl nanoparticles were stable upon incubation with lysozyme and did not affect the viscosity of a mucin dispersion. In vivo studies showed that the amounts of CS-fl in cornea and conjunctiva were significantly higher for CS-fl nanoparticles than for a control CS-fl solution, these amounts being fairly constant for up to 24 h. Confocal studies suggest that nanoparticles penetrate into the corneal and conjunctival epithelia. Cell survival at 24 h after incubation with CS nanoparticles was high and the viability of the recovered cells was near 100%. CONCLUSIONS: CS nanoparticles are promising vehicles for ocular drug delivery.     

Nasal Absorption in the Rat. III. Effect of Lysophospholipids on Insulin Absorption and Nasal Histology

The intranasal absorption enhancing and histological effects of a range of lysophospholipids has been investigated in the rat. Blood glucose levels fell rapidly following the administration of insulin (8 IU/kg) in combination with lysophosphatidylcholines (LPC; 0.625% w/v) which had ten or more carbon groups in their fatty acid chain. The effect of the LPC-caproyl (C6) was comparable to that of an unenhanced insulin formulation; the enhancing effect of LPC-decanoyl (C10) was similar to that of an LPC-palmitoyl/stearoyl (C16/C18) for similar concentrations. The effect of LPC-decanoyl was reduced with concentration but was still significant at 0.2% w/v (5mM). Lysophosphatidylglycerol (LPG) had a marked insulin absorption enhancing effect even at 0.0625% w/v. The histological effects of LPC-caproyl were similar to those of an unenhanced insulin formulation, while co-administration of LPC-decanoyl resulted in evidence of epithelial interaction. LPG (0.5% w/v) resulted in similar histological changes as LPC (0.625% w/v) (1), but at 0.0625% w/v no significant changes in epithelial integrity were observed. The length of the fatty acid residue of lysophospholipids was identified as an important factor for intranasal absorption enhancing activity. The nature of the polar head group may also have an influence. Increased insulin absorption was not necessarily accompanied by severe disruption of the nasal epithelium. Careful selection of lysophospholipid type and concentration may enable therapeutic drug levels to be achieved via the nasal route without prohibitive toxic effects.     

Recent Challenges in Insulin Delivery Systems: A Review

Relatively, a large percentage of world population is affected by diabetes mellitus, out of which approximately 5-10% with type 1 diabetes while the remaining 90% with type 2. Insulin administration is essential for type 1 patients while it is required at later stage by the patients of type 2. Current insulin delivery systems are available as transdermal injections which may be considered as invasive. Several non-invasive approaches for insulin delivery are being pursued by pharmaceutical companies to reduce the pain, and hypoglycemic incidences associated with injections in order to improve patient compliance. While any new insulin delivery system requires health authorities'' approval, to provide long term safety profile and insuring patients'' acceptance. The inhalation delivery system Exubera^® has already become clinically available in the United States and Europe for patients with diabetes as non-invasive delivery system.