Development and characterization of chitosan succinate microspheres for the improved oral bioavailability of insulin

The present study describes the fabrication of insulin loaded chitosan succinate microspheres to improve the efficacy of orally administered insulin. Chitosan succinate polymer was synthesized and its microspheres were prepared by emulsion phase separation technique. The microspheres were characterized by FT-IR spectroscopy, scanning electron microscopy, particle size, X-ray diffraction, and swelling index. Insulin was loaded into the microspheres by passive absorption technique. The ability of microspheres to protect insulin from gastric enzymatic degradation was investigated. Stability of insulin in the microspheres was determined by gel electrophoresis and circular dichroism (CD). In vitro release studies were performed under simulated gastric and intestinal pH conditions (pH 2.0 and pH 7.4). The pharmacokinetic parameters were monitored after oral administration of insulin loaded chitosan succinate microspheres, chitosan succinate-insulin solution, as well as after subcutaneous injection of insulin to diabetic rats. The degree of succinate substitution in the synthesized polymer was 16%. The prepared microspheres were spherical with an average diameter of 49 +/- 2 microm. The insulin-loading capacity was 62%. Chitosan succinate microspheres were found to protect the degradation of insulin from gastric enzymes. The encapsulated insulin was quickly released in simulated intestinal fluid (SIF, pH 7.4), whereas a small fraction of insulin was released in simulated gastric fluid (pH 2.0). The relative pharmacological efficacy for chitosan succinate microspheres (16 +/- 4%) was almost fourfold higher than the efficacy of the chitosan succinate-insulin solution administration (4 +/- 1.5%). The results suggest that chitosan succinate microspheres could be used as a potential carrier for oral insulin delivery.     

盐酸尼卡地平核壳型纳米囊的制备及其药剂学性质研究

目的:制备盐酸尼卡地平核壳型纳米囊并考察其药剂学性质。方法:采用逐层静电自组装法制备盐酸尼卡地平核壳型纳米囊,考察纳米囊形态、粒径、载药量等,计算其在人工胃液、肠液中的累积释放度,并与原料药比较。结果:所制纳米囊呈球形,平均粒径为200nm,载药量最高值为2.512%;纳米囊在人工胃液、肠液中12h累积释放度分别为18.64%、70%,原料药在人工肠液中3h时已达到87%。结论:所制盐酸尼卡地平核壳型纳米囊具有良好的药剂学性质。     

Application of surface‐coated liposomes for oral delivery of peptide: Effects of coating the liposome's surface on the GI transit of insulin

We prepared two kinds of surface‐coated liposomes and investigated their potencies as oral dosage forms for peptide drugs by focusing on their effects on the gastrointestinal (GI) transit of drugs. The surface of the liposomes was coated with poly(ethylene glycol) 2000 (PEG‐Lip) or the sugar chain of mucin (Mucin‐Lip). As a model peptide drug, insulin was encapsulated in these liposomes. Coating the surface with poly(ethylene glycol) was found to reduce the transit rate of liposomes in the small intestine after oral administration to rats in vivo. Mucin‐Lip was retained in the stomach longer than PEG‐Lip or uncoated liposomes. The effect of surface coating on the intestinal transit of liposomes was determined by means of in situ single pass perfusion in the rat small intestine. Statistical moment analysis was applied to the outflow pattern of both liposomes and encapsulated insulin. The mean transit time (MTT) and deviation of transit time (DTT) in the intestinal tract were calculated. The MTT of PEG‐Lip was much longer than those of uncoated liposomes and Mucin‐Lip and was significantly shortened after removal of the intestinal mucous layer. These results indicated that PEG‐Lip interacts strongly with the intestinal mucous layer, leading to its slow transit in the intestine. In contrast, coating the liposome's surface with mucin did not affect either the MTT or DTT of liposomes in the intestine. This result is in accordance with the in vivo observation that Mucin‐Lip was highly retained in the stomach, but not in any region of the small intestine in vivo. Both the MTT and DTT values of insulin encapsulated in PEG‐Lip and Mucin‐Lip were almost the same as those of liposomes themselves, suggesting that surface‐coated liposomes retained insulin in the intestinal tract. However, MTT and DTT of insulin were significantly shorter than those of uncoated liposomes because these liposomes degraded and released significant amounts of insulin during single pass perfusion. The ability of surface‐coated liposomes, especially of PEG‐Lip, to interact with the mucus layer and slow the transit rate in the GI tract is considered desirable for oral delivery of peptide drugs. Modification of the liposomal surface with appropriate materials, therefore, should be an effective method by which to achieve the oral delivery of peptide drugs.     

Visualization of Insulin-Loaded Nanocapsules: In Vitro and in Vivo Studies After Oral Administration to Rats

Purpose. Biodegradable poly(isobutylcyanoacrylate) nanocapsules have been recognized as a promising carrier for oral administration of peptides and proteins. In the present study, we investigate the fate of insulin-loaded nanocapsules by fluorescence and transmission electron microscopy (TEM) after intragastric force-feeding to rats. Methods. Insulin-, Texas-red^®-labeled insulin, or gold-labeled insulin-loaded nanocapsules were first characterized. Rats received a single dose of nanocapsules (diameter 60-300 nm, 57 IU insulin/kg) by intragastric force-feeding. After 90 min, ileum was isolated and prepared for fluorescence and transmission electron microscopy. Results. Nanocapsules were observed on both sides of the gut epithelium and in blood capillaries. In M-cell-free epithelium, apparently intact nanocapsules could be seen in the underlying tissue, suggesting they could cross the epithelium and carry the encapsulated peptide. In M-cell-containing epithelium, nanocapsules appeared degraded in the vicinity of macrophages. It is noteworthy that intestinal absorption of nanocapsules was observed without artifacts forcing the nanocapsules to stay in the gut. Conclusions. Based on TEM observations, this study shows the intestinal absorption of biodegradable nanocapsules leading to the transport of insulin across the epithelium mucosa. The fate of the nanocapsules appeared different depending on the presence or the absence of M cells in the intestinal epithelium.     

Effective in-vivo utilization of lipid-based nanoparticles as drug carrier for carvedilol phosphate

Objectives Lipid nanoparticles as carrier for oral drug administration improve gastrointestinal solubility of poorly soluble drugs and thus enhance bioavailability. However, basic drugs may undergo rapid dissolution from such solid dispersions in the stomach and precipitate in the intestine due to their higher solubility in acidic medium. Therefore, the objective of this work was to study the enhancement in bioavailability of carvedilol phosphate (basic drug) by providing an alkaline gastric environment to drug‐loaded solid lipid nanoparticles. Methods An alkaline gastric environment in rats was created and maintained with oral administration of an antacid suspension 5 min before and 30 min post dosing. Key findings The formulation administered orally exhibited enhanced bioavailability (～27%) when compared with drug suspension and sustained release behaviour when compared with formulation under ideal gastric conditions. The enhanced bioavailability is due to the presence of lipid nanoparticles as drug carrier while the sustained‐release characteristic may be attributed to the presence of antacid, which resulted in elevation of gastric pH and reduced the drug's solubility. Conclusions It may be concluded that although lipid nanoparticles can be instrumental in improving bioavailability, additional sustained release may be achieved by targeting intestinal release of basic drugs from lipid vehicles, which is possible by incorporating them into suitable enteric‐coated formulations.     

In-vitro release and oral bioactivity of insulin in diabetic rats using nanocapsules dispersed in biocompatible microemulsion

This study evaluated the potential of poly(iso-butyl cyanoacrylate) (PBCA) nanocapsules dispersed in a biocompatible microemulsion to facilitate the absorption of insulin following intragastric administration to diabetic rats. Insulin-loaded PBCA nanocapsules were prepared in-situ in a biocompatible water-in-oil microemulsion by interfacial polymerisation. The microemulsion consisted of a mixture of medium-chain mono-, di- and tri-glycerides as the oil component, polysorbate 80 and sorbitan mono-oleate as surfactants and an aqueous solution of insulin. Resulting nanocapsules were approximately 200 nm in diameter and demonstrated a high efficiency of insulin entrapment (> 80%). In-vitro release studies showed that PBCA nanocapsules could suppress insulin release in acidic media and that release at near neutral conditions could be manipulated by varying the amount of monomer used for polymerisation. Subcutaneous administration of insulin-loaded nanocapsules to diabetic rats demonstrated that the bioactivity of insulin was largely retained following this method of preparing peptide-loaded nanocapsules and that the pharmacodynamic response was dependent on the amount of monomer used for polymerisation. The intragastric administration of insulin-loaded nanocapsules dispersed in the biocompatible microemulsion resulted in a significantly greater reduction in blood glucose levels of diabetic rats than an aqueous insulin solution or insulin formulated in the same microemulsion. This study demonstrates that the formulation of peptides within PBCA nanocapsules that are administered dispersed in a microemulsion can facilitate the oral absorption of encapsulated peptide. Such a system can be prepared in-situ by the interfacial polymerisation of a water-in-oil biocompatible microemulsion.     

Calcitonin and Insulin in Isobutylcyanoacrylate Nanocapsules: Protection Against Proteases and Effect on Intestinal Absorption in Rats

Abstract— One of the major limiting steps for the absorption of peptide drugs from the intestine is proteolytic degradation. To slow this degradation, human calcitonin was trapped in polyacrylamide nanoparticles, and human calcitonin and insulin were encapsulated with polyisobutylcyanoacrylate. Human calcitonin trapped in polyacrylamide nanoparticles showed no delayed release characteristics and thus would not provide protection from proteases. Proteolytic degradation of human calcitonin and insulin in polyisobutylcyanoacrylate nanocapsules was slower than the free peptides in solution. The plasma pharmacokinetic profiles were consistent with increased survival time of the peptides in the intestine, with higher plasma concentrations of the peptides in the later time samples compared with the controls. However, the nanocapsules gave no significant overall enhancement of peptide absorption. This led to the conclusion that the nanocapsules released the peptides into the intestinal lumen, with small amounts then being absorbed but the rest largely degraded.     

Chitosan microparticles containing plasmid DNA as potential oral gene delivery system.

The potential of chitosan as a polycationic gene carrier for oral administration has been explored since 1990s. Chitosan has been shown to effectively bind DNA in saline or acetic acid solution and protect DNA from nuclease degradation. In this study, pDNA (plasmid DNA) was encapsulated in chitosan microparticles. Chitosan-DNA microparticles were prepared using a complex coacervation process and stability of plasmid DNA was investigated in this complex. The chitosan-DNA microparticles could protect the encapsulated plasmid DNA from nuclease degradation. Release of pDNA from microparticles was studied in simulated gastric, simulated intestinal medium and acidic PBS (phosphate buffer saline) (pH 4.5) buffer at 37 degrees C, and released pDNA was assayed spectrophotometrically. In vitro release of pDNA from chitosan microparticles was dependent on pH, as the pH of the release medium increased release profile decreased. In in vivo-animal studies blue color was observed with X-gal (4-chloro-5-bromo-3-indolyl-beta-galactosidase) staining of histological stomach and small intestine sections after oral administration of pDNA-chitosan microparticles as an indicator of exogeneous gene expression.     

Novel technique of insulin loading into porous carriers for oral delivery

The increasing demand for oral macromolecule delivery encouraged the development of microencapsulation technologies to protect such drugs against gastric and enzymatic degradation. However, microencapsulation often requires harsh conditions that may jeopardize their biological activity. Accordingly, many trials attempted to load macromolecules into porous drug carriers to bypass any formulation induced instability. In this study, we prepared chitosan coated porous poly (d, l-lactide-co-glycolide) (PLGA) microparticles (MPs) loaded with insulin using a novel loading technique; double freeze-drying. The results showed a significant increase in drug loading using only 5 mg/ml initial insulin concentration and conveyed a sustained drug release over uncoated MPs. Furthermore, SEM and confocal microscopy confirmed pore blocking and insulin accumulation within the MPs respectively. The oral pharmacodynamic data on rats also proved the preservation of insulin bioactivity after formulation. Finally, the new coating technique proved to be efficient in producing robust layer of chitosan with higher insulin loading while maintaining insulin activity.     

A Delivery System for Oral Administration of Proteins/Peptides Through Bile Acid Transport Channels

Proteins and peptides are poorly absorbed via oral administration because of the gastrointestinal tract environment and lysosomal digestion after apical endocytosis. A delivery system, consisting of a deoxycholic acid–conjugated nanometer-sized carrier, may enhance the absorption of proteins in the intestine via the bile acid pathway. Deoxycholic acid is first conjugated to chitosan. Liposomes are then prepared and loaded with the model drug insulin. Finally, the conjugates are bound to the liposome surface to form deoxycholic acid and chitosan conjugate–modified liposomes (DC-LIPs). This study demonstrates that DC-LIPs can promote the intestinal absorption of insulin via the apical sodium-dependent bile acid transporter, based on observing fluorescently stained tissue slices of the rat small intestine and a Caco-2 cell uptake experiment. Images of intestinal slices revealed that excellent absorption of DC-LIPs is achieved via apical sodium-dependent bile acid transporter, and a flow cytometry experiment proved that DC-LIPs are a highly efficient delivery carrier. Caco-2 cells were also used to study the lysosome escape ability of DC-LIPs. We learned from confocal microscopy photographs that DC-LIPs can protect their contents from being destroyed by the lysosome. Finally, according to pharmacokinetic analyses, insulin-loaded DC-LIPs show a significant hypoglycemic effect with an oral bioavailability of 16.1% in rats with type I diabetes.     

Oral delivery of insulin using pH-responsive complexation gels.

The goal of oral insulin delivery devices is to protect the sensitive drug from proteolytic degradation in the stomach and upper portion of the small intestine. In this work, we investigate the use of pH-responsive, poly(methacrylic-g-ethylene glycol) hydrogels as oral delivery vehicles for insulin. Insulin was loaded into polymeric microspheres and administered orally to healthy and diabetic Wistar rats. In the acidic environment of the stomach, the gels were unswollen due to the formation of intermolecular polymer complexes. The insulin remained in the gel and was protected from proteolytic degradation. In the basic and neutral environments of the intestine, the complexes dissociated which resulted in rapid gel swelling and insulin release. Within 2 h of administration of the insulin-containing polymers, strong dose-dependent hypoglycemic effects were observed in both healthy and diabetic rats. These effects lasted for up to 8 h following administration.     

Enteric coated insulin pellets: development, drug release and in vivo evaluation

Pellets with human insulin as a model drug were prepared by an extrusion and spheronization process to investigate the oral application of peptides. The described process proved suitable for preparing small batches of about 50 g in laboratory scale. The developed formulation was completed by the addition of aprotinin as protease inhibitor and sodium cholate as an intestinal absorption promoter to enhance oral bioavailability of insulin. In order to protect the peptide against the gastric juice the pellets were coated with shellac in a fluid-bed ball coater. Pharmaceutical properties of the produced batches were examined by analysis of contents and dissolution tests. Dissolution of insulin in simulated gastric juice of pH 1.2 was prevented by shellac. On the other hand, a rapid and complete release of the molecular-dispersed insulin from the pellets was found in simulated intestinal fluid (pH 7.5) with simultaneous efficiency of the protease inhibitor against added enzyme activity. Despite promising in vitro results no significant absorption of insulin was detected in vivo after oral application of the pellets to streptozotocin diabetic rats. High sensitivity to enzymatic degradation and low ability to cross the intestinal wall are discussed as limiting factors for the insufficient absorption of insulin in vivo.     

Chitosan phthalate microspheres for oral delivery of insulin: preparation, characterization, and in vitro evaluation

Chitosan phthalate polymer was synthesized and its microspheres were prepared by emulsion phase separation technique. The characterization of microspheres was determined by means of FTIR spectroscopy, electron microscopy, particle size, and zeta potential. The insulin was loaded to the microspheres by passive absorption technique. The peptic and tryptic enzymes degradation of insulin in microspheres was investigated. The in vitro release behavior of the microspheres was investigated under different pH conditions (pH 2.0 and pH 7.4). The degree of phthalate substitution in the synthesized polymer was 20%. The prepared microspheres were spherical with an average diameter 46.34 μ m. The insulin-loading capacity was 62%. Chitosan phthalate microspheres protect the insulin from gastric enzymes degradation that may enhance the oral stability of insulin. The encapsulated insulin was quickly released in a phosphate buffer saline (pH 7.4), whereas a small amount of insulin was released under acidic condition (0.1N HCl; pH 2.0) because under acidic conditions, carboxylic groups present in the system exist in nonionized form and are poorly hydrophilic. However, in alkaline conditions, it exists in ionized form and is considerably hydrophilic. The results suggest that chitosan phthalate microspheres may be used as a potential carrier for oral insulin delivery.     

Effect on cerebral blood flow of orally administered indomethacin-loaded poly(isobutylcyanoacrylate) and poly(DL-lactide) nanocapsules

Nanocapsules, containing indomethacin, were prepared either by interfacial polymerization of isobutylcyanoacrylate monomers or by interfacial deposition of a performed (DL-lactide) polymer. In-vitro release of indomethacin from nanocapsules was dependent on the pH of the sink solution and was enhanced by addition of albumin. A decrease in cerebral blood flow was noted 15 min after oral administration to rats of indomethacin nanocapsules (5 mg kg-1) and lasted over 3 h. Empty nanocapsules had no effect. Since release of indomethacin from nanocapsules is unlikely to occur in the lumen of the stomach, due to unsuitable pH conditions, and nanocapsules have been previously shown to be able to cross the intestinal barrier, to reach the villi vessels intact and to protect against the ulcerating effect of the free drug, it is suggested that the rapid onset of the pharmacological effect was sufficiently induced by free indomethacin released in the plasma following absorption of the intact nanocapsules.     

Investigation of pluronic and PEG-PE micelles as carriers of meso-tetraphenyl porphine for oral administration

Meso-tetraphenyl porphine (mTPP) is a highly lipophilic, fluorescent porphyrin derivate and it is used as photosensitizer on the treatment of malign neoplasms. The aim of this study was to prepare mTPP loaded pluronic F127 and polyethylene glycol-distearoyl phosphatidylethanolamine (PEG(2000)-DSPE) micelles to evaluate polymeric micelles potential for the transport of drugs through intestinal mucosa. Transport and bioadhesion behaviors of polymeric micelles was investigated using Caco-2 cell monolayer and everted rat intestine models. In order to show that Caco-2 cells can be used as a transport model cytotoxicity of formulations was tested. Cell viability was more than 80%, showing that Caco-2 cells will keep their viability during the transport studies demonstrating that prepared formulations can be securely used as oral drug carrier systems. Plain micelles were labeled with a fluorescent agent rhodamine-phosphatidylethanolamine (Rh-PE) and their transport through Caco-2 cells was investigated beside mTPP loaded micelles. At the end of 4h transport study through Caco-2 cells, cumulative transport (%) of fluorescent agents were around 14% and 1% in Rh-PE labeled and mTPP loaded micelles This difference was attributed to the different placement of mTPP and Rh-PE in the micellar core. Drug transport was not estimated in everted rat intestine model but the bioadhesion was 79% and 70% for mTPP loaded pluronic F127 and PEG(2000)-DSPE micelles. These good bioadhesion rates are promising for oral drug delivery.     

Development and in-vivo evaluation of insulin-loaded chitosan phthalate microspheres for oral delivery

Novel chitosan phthalate microspheres containing insulin were prepared by emulsion cross-linking technique. The feasibility of these microspheres as oral insulin delivery carriers was evaluated. The pH-responsive release behaviour of insulin from microspheres was analysed. The ability of chitosan phthalate-insulin microspheres to enhance intestinal absorption and improve the relative pharmacological availability of insulin was investigated by monitoring the plasma glucose and insulin level of streptozotocin-induced diabetic rats after oral administration of microspheres at insulin dose of 20 IU kg(-1). In simulated gastric fluid (pH 2.0), insulin release from the microspheres was very slow. However, as the pH of the medium was changed to simulated intestinal fluid (pH 7.4), a rapid release of insulin occurred. The relative pharmacological efficacy for chitosan phthalate microspheres (18.66 +/- 3.84%) was almost four-fold higher than the efficacy of the chitosan phthalate-insulin solution administration (4.08 +/- 1.52%). Chitosan phthalate microspheres sustained the plasma glucose at pre-diabetic level for at least 16 h. These findings suggest that the microsphere is a promising carrier as oral insulin delivery system.     

Efficacy and Mechanism of Action of Chitosan Nanocapsules for Oral Peptide Delivery

Purpose We have previously shown that high molecular weight (MW > 100 kDa) chitosan nanocapsules are efficient vehicles for improving the oral absorption of salmon calcitonin (sCT). In the present work, our objectives were, first, to investigate the influence of some formulation parameters on the efficacy of chitosan nanocapsules as carriers for the oral administration of sCT and, second, to elucidate the mechanism of interaction of chitosan nanocapsules with intestinal model cell lines. Methods sCT-loaded chitosan nanocapsules were prepared by the solvent displacement technique. They were characterized for their size, zeta potential, and sCT loading. The ability of chitosan nanocapsules to enhance the oral absorption of sCT was investigated in rats by monitoring the serum calcium levels. Finally, the mechanism of interaction of chitosan nanocapsules with the Caco-2 cell model or in the coculture of Caco-2 with HT29-M6 cells was investigated by confocal fluorescence microscopy. Results Chitosan nanocapsules presented a particle size in nanometer range, a positive surface charge, and an efficient encapsulation of sCT. Following oral administration to rats, all formulations of nanocapsules exhibited the ability to reduce calcemia levels; however, the intensity of the response varied depending on the formulation conditions. With regard to the mechanism of interaction of chitosan nanocapsules with cell culture, the xz images evidenced that chitosan nanocapsules interact and remain associated to the apical side of both model cell cultures. In addition, chitosan nanocapsules showed a preferable association to the mucus-secreting cells (HT29-M6). Conclusions Chitosan nanocapsules are able to enhance and prolong the intestinal absorption of sCT and this effect could be mainly ascribed to their mucoadhesive character and intimate interaction with the intestinal barrier.     

Effect of matrix composition,sphere size and hormone concentration on diffusion coefficient of insulin for controlled gastrointestinal delivery for diabetes treatment

Oral insulin administration is limited due to its degradation by proteases. The hormone was encapsulated in spheres made of either pure calcium alginate (ALG) or its association with whey protein isolate (WPI-ALG) in order to minimise loss in the stomach region while allowing liberation in the maximum absorption area, located in the intestine. Diffusion coefficients for both matrix compositions were determined in vitro for gastric pH (5.88 and 10.26?×?10^?12?m²?s^?1) and intestinal pH (21.11 and 79.29?×?10^?12?m²?s^?1). Higher initial insulin concentrations and lower diameters accelerated its release, confirming Fickian behaviour. The analytic model exhibited a good fit in most cases. Computer simulations revealed that ALG spheres are more convenient for oral administration because they release more insulin in the intestine than the WPI-ALG ones, thus supporting its therapeutic viability for the purpose of reducing stress in those who depend on insulin.     

HA-GMS-INS胰岛素口服纳米给药系统研究

目的:建立透明质酸-单硬脂酸甘油酿-胰岛素(HA-GMS-INS)口服纳米给药系统,并进行成药性研究.方法:利用酿化反应合成载体HA-GMS,单因素法筛选制备工艺,通过红外光谱和核磁共振氢谱对结构进行表征;采用低能乳化法制备HA-GMS-INS,用多分散激光粒度仪和透射电镜测定纳米乳的粒径及形态,并对胰岛素的体外释放性...     

Macropored microparticles with a core–shell architecture for oral delivery of biopharmaceuticals

Microparticles (MPs) have been extensively researched as a potential drug delivery vehicle. Here, we investigated the fabrication of MPs with pH-responsive macropores and evaluated their potential applicability in developing solid oral drug formulations. Our previous study showed that macropored MPs, made of Eudragit^® L100-55, could encapsulate 100 nm, 1 µm, and 4 µm sized fluorescent beads—model drugs that are mimicking vaccines, bacteria, and cells. In the present study, closed-pored MPs after freeze-drying were coated with a gastric soluble Eudragit^® EPO layer to protect MPs in the simulated pregastric environment. Subsequently, drug encapsulated MPs maintained their intact closed-pored structure in the simulated gastric environment and exhibited a rapid release in the simulated intestine environment. Our MP system was found to provide a significantly higher level of protection to the encapsulated lactase enzyme compared to the control sample (i.e. without using MPs). Real-time fluorescence microscopy analysis showed that macropored MPs released encapsulated drugs in a burst-release pattern and in a size-independent manner. This work shows that our proposed EPO-coated MPs with pH-responsive macropores can meet the challenges posed by the multiple physiological environments of the digestive tract and be used in developing highly effective solid oral drug/vaccine formulations.