Insulin-egg yolk dispersions in self microemulsifying system

Formulation of insulin into a microemulsion very often presents a physicochemical instability during their preparation and storage. In order to overcome this lack of stability and facilitate the handling of these colloidal systems, stabilization of insulin in presence of hydrophobic components of a microemulsion appears as the most promising strategy. The present paper reports the use of egg yolk for stabilization of insulin in self microemulsifying dispersions. Insulin loaded egg yolk self microemulsifying dispersions were prepared by lyophilization followed by dispersion into self microemulsifying vehicle. The physicochemical characterization of selfmicroemulsifying dispersions includes such as insulin encapsulation efficiency, in vitro stability of insulin in presence of proteolytic enzymes and in vitro release. The biological activity of insulin from the dispersion was estimated by enzyme-linked immunosorbant assay and in vivo using Wistar diabetic rats. The particle size ranged 1.023±0.316 μm in diameter and insulin encapsulation efficiency was 98.2±0.9 %. Insulin hydrophobic self microemulsifying dispersions suppressed insulin release in pH 7.4 phosphate buffer and shown to protect insulin from enzymatic degradation in vitro in presence of chymotripsin. Egg yolk encapsulated insulin was bioactive, demonstrated through both in vivo and in vitro.     

Influence of microencapsulation method and peptide loading on formulation of poly(lactide-co-glycolide) insulin nanoparticles

Insulin stability during microencapsulation and subsequent release is essential for retaining its biological activity. Therefore we investigated a novel solid/oil/water anhydrous encapsulation method with a combination of stabilizers for maintaining the integrity of insulin during formulation and delivery. Two methods were used for preparation of nanoparticles, namely water/oil/water solvent evaporation and s/o/w anhydrous encapsulation to study the influence of the microencapsulation method on nanoparticle characteristics such as size and morphology, drug content, encapsulation efficiency, and in vitro and in vivo release profile. Poly (lactic-co-glycolic) acid (PLGA) with co-polymer ratio 50:50 was selected to prepare drug-loaded nanoparticles. When nanoparticles were prepared by solvent evaporation higher encapsulation efficiencies could be obtained, e.g. 74 +/- 13 with 5% target loading, whereas with 12% target loading, encapsulation efficiency was 27 +/- 8.6. The s/o/w method has a direct influence on the evaluation parameters where very poor encapsulation efficiencies 11 +/- 6.8 (max) were observed. The presence of stabilizers in the nanoparticles resulted in an increase in particle size but a reduction of encapsulation efficiency. Insulin release rate was comparatively higher for the batches prepared by the w/o/w method containing stabilizers than the s/o/w method. 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, it can be concluded that careful selection of processing conditions and combination of stabilizers also result in beneficial effects without compromising the advantages of these delivery systems.     

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.     

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.     

Formulation pH modulates the interaction of insulin with chitosan nanoparticles

Previous studies on chitosan-insulin nanoparticles have reported diverse encapsulation efficiency and insulin release profiles despite similar formulation and preparation procedures. This study examined the efficiency and mechanism of association of insulin with chitosan nanoparticles in the pH range of 2.3 to 6.3. Nanoparticles of 237 to 235 nm were prepared by ionotropic gelation of chitosan with tripolyphosphate counterions. Insulin was quantified by an RP-HPLC method. The insulin association efficiency (AE) spanned a broad range from 2 to 85%, and was highly sensitive to formulation pH. Highest AE was measured at insulin loading concentrations >/= 4.28 U/mL and pH 6.1, close to the pI of native insulin and the pK(a) of chitosan. This association, attributed to physical adsorption of insulin through hydrophobic interactions with chitosan, was labile, and the associated insulin rapidly and completely released by dilution of the nanoparticles in aqueous media of pH 2 to 7.4. AE obtained at pH 5.3 was less than half that measured at pH 6.1 at corresponding insulin concentration, but the association at pH 5.3 appeared to be based on stronger interactions, because the release of insulin was pH-dependent and recovery was less than 25% even upon disintegration of the chitosan matrix. Interaction of insulin with the chitosan nanoparticles rendered the protein more susceptible to acid and enzymatic hydrolyses, the effects being more predominant in nanoparticles prepared at pH 5.3 than at pH 6.1.     

壳聚糖-甲基丙烯酸甲酯共聚物纳米粒的制备和表征

目的制备壳聚糖-甲基丙烯酸甲酯共聚物纳米粒。方法用自由基聚合法合成壳聚糖-甲基丙烯酸甲酯共聚物,该聚合物在水中形成具有疏水核心、亲水表面的纳米粒。测定纳米粒的形态、粒径和表面电位(Zeta电位),并研究了壳聚糖含量、聚合物总浓度和引发剂浓度对纳米粒粒径的影响。以胰岛素为模型药物,研究纳米粒的包封和释药性能。结果纳米粒呈球形,粒径均匀,表面荷正电。胰岛素的包封率可达90%以上。pH 5.8的磷酸盐缓冲液中胰岛素的释放较慢,16 h释放量为66.8%。结论该纳米制剂具有较好的物理性能和体外缓释特性。     

Pelleted bioadhesive polymeric nanoparticles for buccal delivery of insulin: preparation and characterization

The study was an attempt to develop an alternative buccal delivery system for insulin. Insulin bearing nanoparticles were prepared by the emulsion internal phase evaporation method. The effect of some formulation variables viz., polymer/drug ratio and emulsifier concentration was studied on particle size and entrapment efficiency. Nanoparticles were pelleted to impart three-dimensional structural conformity and coherence thereby facilitating buccal application. Solid lateral and horizontal sedimentaton in the pellet can be avoided by nanoparticulation and ensuring uniform drug distribution throughout the pellet. The in vitro studies of the pellets included bioadhesion and drug release profile. In vivo studies were performed on diabetic rats. A significant hypoglycemic response was observed after 7 h, without any detectable fluctuation in blood glucose profile and risk of hypoglycemia.     

Design and evaluation of a novel nanoparticulate-based formulation encapsulating a HIP complex of lysozyme

Formulation development of protein therapeutics using polymeric nanoparticles has found very little success in recent years. Major formulation challenges include rapid denaturation, susceptibility to lose bioactivity in presence of organic solvents and poor encapsulation in polymeric matrix. In the present study, we have prepared hydrophobic ion pairing (HIP) complex of lysozyme, a model protein, using dextran sulfate (DS) as a complexing polymer. We have optimized the process of formation and dissociation of HIP complex between lysozyme and DS. The effect of HIP complexation on enzymatic activity of lysozyme was also studied. Nanoparticles were prepared and characterized using spontaneous emulsion solvent diffusion method. Furthermore, we have also investigated release of lysozyme from nanoparticles along with its enzymatic activity. Results of this study indicate that nanoparticles can sustain the release of lysozyme without compromising its enzymatic activity. HIP complexation using a polymer may also be employed to formulate sustained release dosage forms of other macromolecules with enhanced encapsulation efficiency.     

Alginate microspheres prepared by internal gelation: development and effect on insulin stability

Recombinant human insulin was encapsulated within alginate microspheres by the emulsification/internal gelation technique with the objective of preserving protein stability during encapsulation procedure. The influence of process and formulation parameters was evaluated on the morphology and encapsulation efficiency of insulin. The in vitro release of insulin from microspheres was studied under simulated gastrointestinal conditions and the in vivo activity of protein after processing was assessed by subcutaneous administration of extracted insulin from microspheres to streptozotocin-induced diabetic rats. Microspheres mean diameter, ranging from 21 to 287 microm, decreased with the internal phase ratio, emulsifier concentration, mixer rotational speed and increased with alginate concentration. Insulin encapsulation efficiency, near 75%, was not affected by emulsifier concentration, mixer rotational speed and zinc/insulin hexamer molar ratio but decreased either by increasing internal phase ratio and calcium/alginate mass ratio or by decreasing acid/calcium molar ratio and alginate concentration. A high insulin release, above 75%, was obtained at pH 1.2 and under simulated intestinal pH a complete dissolution of microspheres occurred. Extracted insulin from microspheres decreased hyperglycemia of diabetic rats proving to be bioactive and showing that encapsulation in alginate microspheres using the emulsification/internal gelation is an appropriate method for protein encapsulation.     

Thiolated Eudragit nanoparticles for oral insulin delivery: Preparation, characterization and in vivo evaluation

In the present study thiolated Eudragit L100 (Eul) based polymeric nanoparticles (NPs) were employed to develop an oral insulin delivery system. Sulfydryl modification was achieved by grafting cysteine to the carboxylic acid group of Eudragit L100, which displayed maximum conjugate level of 390.3±13.4μmol thiol groups per gram. Eudragit L100-cysteine (Eul-cys) and Eul nanoparticles were prepared by the precipitation method, in which reversible swelling of pH-sensitive material was used for insulin loading and release. Nanoparticles were characterized in terms of their particle size, morphology, loading efficiency (LE%) and in vitro insulin release behavior. The NPs had an average size of 324.2±39.0nm and 308.8±35.7nm, maximal LE% of 92.2±1.7% and 96.4±0.5% for Eul-cys and Eul, respectively. The release profile of NPs in vitro showed pH-dependent behavior. Circular dichroism (CD) spectroscopy analysis proved that the secondary structure of the insulin released from NPs was unchanged compared with native insulin. The mucoadhesion study in vitro showed that Eul-cys NPs produced a 3-fold and 2.8-fold increase in rat jejunum and ileum compared with unmodified polymer NPs, respectively, which was due to the immobilization of thiol groups on Eudragit L100. Oral administration of insulin-loaded Eul-cys NPs produced a higher and prolonged hypoglycemic action, and the corresponding relative bioavailability of insulin was found to be 7.33±0.33%, an increase of 2.8-fold compared with Eul NPs (2.65±0.63%). This delivery system is a promising novel tool to improve the absorption of protein and peptide drugs in the intestinal tract.     

Improved bioavailability of orally administered mifepristone from PLGA nanoparticles

The objective of this study was to prepare an oral dosage formulation of mifepristone that will improve the oral bioavailability of mifepristone and sustain the release of mifepristone for at least 3 days to effectively control reproduction, especially in coyotes. Nanoparticles containing mifepristone were prepared from dl-lactide/glycolide copolymers (PLGA). Encapsulation efficiency of the nanoparticles was determined by HPLC. In vitro release study was done in 30% isopropyl alcohol in water. In vivo bioavailability study was performed in male rats. Mifepristone and drug-loaded 50/50 PLGA, M(W) 4.4kDa, nanoparticles (equivalent to 100mg/kg mifepristone) were administered orally to rats. The concentration of mifepristone in serum at different time intervals was determined by HPLC. The average sizes of 50/50 PLGA (M(W) 4.4 and 13kDa) nanoparticles containing mifepristone were 516 and 468nm, respectively. The drug encapsulation efficiency was 75.6% at 20% drug loading in 50/50 PLGA (M(W) 4.4kDa) nanoparticles. In vitro cumulative release of mifepristone from the 50/50 PLGA (M(W) 4.4 and 13kDa) nanoparticles with 20% drug loading was 60% and 48% in 72h, respectively. In vivo studies in rats demonstrated that PLGA-1A-nanoparticles increase the bioavailability of mifepristone. We are currently using the nanoparticles containing mifepristone for efficacy studies in coyotes.     

Nanoparticles of quaternized chitosan derivatives as a carrier for colon delivery of insulin: ex vivo and in vivo studies

The aim of the present study was to develop insulin nanoparticulate systems by using chitosan (CS), triethylchitosan (TEC) and dimethyl-ethylchitosan (DMEC, a new quaternized derivative of chitosan) for colon delivery. The nanoparticles were prepared by the polyelectrolyte complexation (PEC) method. Particle size distribution, zeta potential and polydispersity index of the nanoparticles were determined using dynamic light scattering technique. Transmission electron microscopy (TEM) was also used to observe the morphology of the nanoparticles. It was found that the nanoparticles carried positive charges and showed a size distribution in the range of 170-270 nm with spherical morphology and smooth surface structure. The amount of insulin loaded into the nanoparticles was determined by measuring the association efficiency and also the content of insulin in the nanoparticles. Insulin loading was found to be more than 80% for all of the nanoparticles. In vitro release studies showed a small burst effect at the beginning and then a sustained release characteristic for 5h. Ex vivo investigations revealed better insulin transport across the colon membrane of rats for nanoparticles made with quaternized derivatives than those made of chitosan. In vivo studies in rats have showed enhanced colon absorption of insulin by using these nanoparticles compared to free insulin in diabetic rats. The insulin absorption from the rat's colon was evaluated by its hypoglycemic effect.     

Effect of poly(ethylene glycol) content and formulation parameters on particulate properties and intraperitoneal delivery of insulin from PLGA nanoparticles prepared using the double-emulsion evaporation procedure

Abstract

Context: Size, encapsulation efficiency and stability affect the sustained release from nanoparticles containing protein-type drugs.

Objectives: Insulin was used to evaluate effects of formulation parameters on minimizing diameter, maximizing encapsulation efficiency and preserving blood glucose control following intraperitoneal (IP) administration.

Methods: Homogenization or sonication was used to incorporate insulin into poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles with increasing poly(ethylene glycol) (PEG) content. Effects of polymer type, insulin/polymer loading ratio and stabilizer in the internal aqueous phase on physicochemical characteristics of NP, in vitro release and stability of encapsulated insulin were investigated. Entrapment efficiency and release were assessed by radioimmunoassay and bicinconnic acid protein assay, and stability was evaluated using SDS-PAGE. Bioactivity of insulin was assessed in streptozotocin-induced, insulin-deficient Type I diabetic mice.

Results: Increasing polymeric PEG increased encapsulation efficiency, while the absence of internal stabilizer improved encapsulation and minimized burst release kinetics. Homogenization was shown to be superior to sonication, with NP fabricated from 10% PEG–PLGA having higher insulin encapsulation, lower burst release and better stability. Insulin-loaded NP maintained normoglycaemia for 24?h in diabetic mice following a single bolus, with no evidence of hypoglycemia.

Conclusions: Insulin-loaded NP prepared from 10% PEG–PLGA possessed therapeutically useful encapsulation and release kinetics when delivered by the IP route.     

醋酸布舍瑞林纳米粒的制备及体外释放特性研究

目的:制备醋酸布舍瑞林纳米粒并考察其体外释药特性。方法:以复乳法制备纳米粒。采用高效液相色谱法测定其含量并计算制剂包封率及载药量,透析袋法考察制剂体外释药特性。结果:所制制剂外观圆整,醋酸布舍瑞林检测浓度的线性范围为0.1～8.0μg·mL-(1r=0.999 9),平均回收率为105.38%,日内RSD<1.78%,日间RSD<0.93%。制剂包封率为(63.37±0.29)%,载药量为(1.03±0.09)%,在pH 7.4的磷酸盐缓冲液中72 h的累积释药百分率为62.35%。结论:该制剂制备工艺简单可行,具有明显的缓释效果。     

Stealth nanoparticles coated with heparin as peptide or protein carriers

Nanoparticles (prepared from a mixture of polyester and a polycationic polymer) loaded with insulin were prepared by a double emulsion method followed by evaporation solvent. Low molecular weight heparin (LMWH) was bound by electrostatic interactions onto the surface of the particles to confer Stealth properties. These nanoparticles were characterized in vitro (mean diameter, zeta potential, encapsulation efficiency, and release kinetics) and compared with conventional (without LMWH) and unloaded nanoparticles. The pharmacokinetics of insulin were studied after intravenous injection into diabetic rats in the form of Stealth or conventional nanoparticles or as a solution. Stealth nanoparticles allowed an increase in the elimination half-life of insulin, showing that the hydrophilic layer of LMWH was able to limit recognition by the mononuclear phagocytosis system in vivo. However, complement activation studies (CH50) did not reveal significant difference between Stealth and conventional nanoparticles.     

载阿霉素介孔二氧化硅纳米粒的细胞毒性及细胞摄取

目的制备载阿霉素的介孔二氧化硅纳米粒(mesoporous silica nanoparticles,MSN),对其理化性质及细胞摄取行为进行初步研究。方法通过聚合法制备MSN,透射电镜表征纳米粒的形态,动态光散射粒径测定仪测定粒子的平均粒径及分布。紫外分光光度计测定阿霉素的负载行为,MTT比色分析法研究粒子的细胞毒性,激光共聚焦显微镜观察其人乳腺癌MCF-7细胞对载药粒子的摄取。结果纳米粒分布均一,平均粒径约70 nm(PDI<0.1),载药量质量分数达到20%。MCF-7细胞对载药粒子的摄取较快,空白纳米粒具有较低的细胞毒性。结论介孔二氧化硅纳米粒具有较高的药物载药量和良好的生物相容性,能较快地被对人乳腺癌MCF-7细胞摄取,有望成为一种新型的药物化疗载体。     

Preparation and in vitro evaluation of heparin-loaded polymeric nanoparticles

Nanoparticles of a highly soluble macromolecular drug, heparin, were formulated with two biodegradable polymers (poly-E-caprolactone [PCL] and poly (D, L-lactic-co-glycolic-acid) 50/50 [PLAGA]) and two nonbiodegradable positively charged polymers (Eudragit RS and RL) by the double emulsion and solvent evaporation method, using a high-pressure homogenization device. The encapsulation efficiency and heparin release profiles were studied as a function of the type of polymers employed (alone or in combination) and the concentration of heparin. Optimal encapsulation efficiency was observed when 5000 IU of heparin were incorporated in the first emulsion. High drug entrapment efficiency was observed in both Eudragit RS and RL nanoparticles (60% and 98%, respectively), compared with PLAGA and PCL nanoparticles (<14%). The use of the two types of Eudragit in combination with PCL and PLAGA increased the encapsulation efficiency compared with these two biodegradable polymers used alone; however, the in vitro drug release was not modified and remained low. On the other hand, the addition of esterase to the dissolution medium resulted in a significant increase in heparin release. The in vitro biological activity of released heparin, evaluated by measuring the anti-Xa activity by a colorimetric assay, was conserved after the encapsulation process.     

载氟尿嘧啶纳米粒的制备及SGC-7901胃癌细胞株的摄取实验

目的:制备载氟尿嘧啶的纳米粒,并考察其体外释放性能和胃癌细胞株对其的摄取能力。方法:以聚乙二醇单甲醚-聚乳酸乙醇酸-聚赖氨酸-(缬氨酸-精氨酸-甘氨酸-天冬氨酸-谷氨酸)环肽(PEAL-cRGD)为载体,用乳化-溶剂蒸发法制备氟尿嘧啶纳米粒,以粒径和包封率为评价指标,通过正交试验设计筛选最优制备工艺,并测定SGC-7901胃癌细胞株对该纳米粒的摄取能力。结果:以最优工艺制备的纳米粒为大小均匀的球形,粒径(172.9±2.3)nm,包封率(75.31±1.91)%,在pH7.4磷酸盐缓冲液中192h累积释放率为(58.63±2.47)%。SGC-7901胃癌细胞株能有效摄取该纳米粒。结论:本研究制备的氟尿嘧啶纳米粒包封率较高,粒径较小,能促进胃癌细胞对氟尿嘧啶的摄取。     

载米托蒽醌PLA-PLL-RGD纳米粒的制备

采用乳化-溶剂蒸发法制备载盐酸米托蒽醌的聚乳酸-聚赖氨酸-精氨酰-甘氨酰-门冬氨酸纳米粒,以粒径和包封率为评价指标,用正交设计优化制备工艺.结果表明,照优化方案制备的纳米粒呈大小均匀的圆球形,平均粒径(186±2.37)nm,平均包封率(94.7±1.46)%,在pH 7.4磷酸盐缓冲液中192h累积释放量为61%.     

黄芩素固体脂质纳米粒包封率测定及体外释放度考察

目的测定黄芩素固体脂质纳米粒的包封率,并考察其体外释放规律。方法溶剂扩散法制备脂质纳米粒,高速离心法分离纳米粒和游离药物,HPLC法测定包封率并考察其体外释放规律。结果测得纳米粒的平均包封率为60.73%,其体外释放规律符合H iguch i动力学方程。结论黄芩素固体脂质纳米粒有较高的包封率,在体外具有良好的缓释作用。