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

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

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.     

Enhanced oral bioavailability of insulin using PLGA nanoparticles co-modified with cell-penetrating peptides and Engrailed secretion peptide (Sec)

Abstract

Biodegradable polymer nanoparticle drug carriers are an attractive strategy for oral delivery of peptide and protein drugs. However, their ability to cross the intestinal epithelium membrane is largely limited. Therefore, in the present study, cell-penetrating peptides (R8, Tat, penetratin) and a secretion peptide (Sec) with N-terminal stearylation were introduced to modify nanoparticles (NPs) on the surface to improve oral bioavailability of peptide and protein drugs. In vitro studies conducted in Caco-2 cells showed the value of the apparent permeability coefficient (P_app) of the nanoparticles co-modified with Sec and penetratin (Sec-Pen-NPs) was about two-times greater than that of the nanoparticles modified with only penetratin (Pen-NPs), while the increase of transcellular transport of nanoparticles modified together with Sec and R8 (Sec-R8-NPs), or Sec and Tat (Sec-Tat-NPs), was not significant compared with nanoparticles modified with only R8 (R8-NPs) or Tat (Tat-NPs). Using insulin as the model drug, in vivo studies performed on rats indicated that compared to Pen-NPs, the relative bioavailability of insulin for Sec-Pen-NPs was 1.71-times increased after ileal segments administration, and stronger hypoglycemic effects was also observed. Therefore, the nanoparticles co-modified with penetratin and Sec could act as attractive carriers for oral delivery of insulin.     

Titanium dioxide nanoparticles exhibit genotoxicity and impair DNA repair activity in A549 cells

Titanium dioxide nanoparticles (TiO(2)-NPs) are produced in large quantities, raising concerns about their impact for human health. The aim of this study was to deeply characterize TiO(2)-NPs genotoxic potential to lung cells, and to link genotoxicity to physicochemical characteristics, e.g., size, specific surface area, crystalline phase. A549 cells were exposed to a panel of TiO(2)-NPs with diameters ranging from 12 to 140 nm, either anatase or rutile. A set of complementary techniques (comet and micronucleus assays, gamma-H2AX immunostaining, 8-oxoGuanine analysis, H2-DCFDA, glutathione content, antioxidant enzymes activities) allowed us to demonstrate that small and spherical TiO(2)-NPs, both anatase and rutile, induce single-strand breaks and oxidative lesions to DNA, together with a general oxidative stress. Additionally we show that these NPs impair cell ability to repair DNA, by inactivation of both NER and BER pathways. This study thus confirms the genotoxic potential of TiO(2)-NPs, which may preclude their mutagenicity and carcinogenicity.     

Meloxicam β-cyclodextrin transdermal gel: physicochemical characterization and in vitro dissolution and diffusion studies

The aim of the study was to develop a Meloxicam (ME) transdermal gel formulations based on complexation with β-cyclodextrin. ME β-Cyclodextrin gel formulations were prepared using four different gel bases with different concentrations and different permeation enhancers. The developed formulations were examined for their in vitro characteristics and their diffusion through a mouse skin. The gel formulations were prepared successfully. Physicochemical characterization of ME β-CD complex in solution state by phase solubility revealed 1:1 M complexation of ME with β-Cyclodextrin. ME release profiles from the inclusion complex were superior over ME alone. Hydroxypropyl methyl cellulose 15% w/w gel base was proven to be a suitable base for ME inclusion complex formulation as it provides a high drug release than other studied bases. ME β-CD complex gel formulations containing oleic acid (1% w/w) or (5% w/w) cineol used as permeation enhancers in (15% w/w) HPMC gel base were proven to provide a higher diffusion rate of the drug through the mouse skin. This is very promising in providing analgesic activity of meloxicam via topical route of administration.     

Bioadhesive transdermal film containing caffeine

The aim of this work was to investigate in vitro the transdermal permeation of caffeine from a new bioadhesive film, using rabbit ear skin as a barrier. The effects of film composition and of the presence of penetration enhancers in the formulation were studied. The obtained fluxes were compared with those shown by commercial formulations. The results obtained indicate that the bioadhesive film gave rise to a higher transdermal permeation compared to a commercial gel and to a saturated solution of caffeine in water. Additionally, the film did not present the typical time lag of solution and gel. Another peculiar feature of the film is that the percentage of permeated active substance is much higher than that obtained from commercial formulations. Finally, it was possible to modulate caffeine permeation from the film by adding different enhancers/solvents.     

Intranasal bioavailability of insulin powder formulations: effect of permeation enhancer-to-protein ratio.

The intranasal administration of powder formulations containing insulin and the permeation enhancer sodium tauro-24,25-dihydrofusidate (STDHF) were investigated in the sheep model. Both the hypoglycemic response and the serum insulin levels increased as the mole ratio of STDHF to insulin was increased from 0 to 16.8. In vitro dissolution rates of the powders and the rapid tmax (approximately 5 min) observed after intranasal administration suggest that the absorption of insulin is not dissolution limited. The bioavailabilities (F) of the powder formulations ranged from 2.9 to 37.8%. In comparison, the F values for a solution formulation with a STDHF:insulin ratio of 8.4 administered as either drops or spray were 15.7 and 37.4%, respectively. The permeation enhancer STDHF increases mucosal permeability and reduces the average molecular weight of the insulin species.     

羧甲基壳聚糖-聚乙二醇作为胰岛素载体的研究

目的：制备胰岛素-羧甲基壳聚糖-聚乙二醇纳米粒。方法：利用红外光谱（FTIR）和核磁共振氢谱（¹H-NMR）对羧甲基壳聚糖-聚乙二醇的结构进行表征,用粒度分析仪测定纳米粒的粒径分布及电位,采用动态透析法考察纳米粒的释药性能,用CCK-8试剂盒检测纳米粒细胞毒性,以糖尿病小鼠为模型,研究纳米粒的降血糖作用。结果：聚乙二醇成功接枝到羧甲基壳聚糖上,包埋胰岛素的纳米粒的平均粒径为（257.5±12.1）nm,Zeta电位为（-15.2±0.3）mV,负载胰岛素的羧甲基壳聚糖-聚乙二醇纳米粒在中性释放介质中,5 h内胰岛素的释放速度较快,之后8 h趋于平稳,胰岛素的累计释放量可达到80%,CCK-8试剂盒显示纳米粒对L929细胞基本无细胞毒性,50 U·kg^-1的纳米粒溶液经灌胃给药后,血糖浓度明显降低。结论：胰岛素-羧甲基壳聚糖-聚乙二醇纳米粒基本无毒性,具有良好的生物相容性,对糖尿病小鼠有效发挥降血糖作用。     

Preparation and evaluation of procaine gels for the enhanced local anesthetic action

To develop the new procaine gel formulations with a suitable bioadhesive property, the gel was formulated using hydroxypropyl methylcellulose (HPMC) and poloxamer containing an enhancer and the local anesthetic action were evaluated. As the drug concentration in the gels and the temperature of surrounding solutions increased, the drug release increased. The activation energy of drug permeation was 4.35 kcal/mol for procaine. The effects of permeation enhancers on the permeation rate of drug through skin were studied using various enhancers, such as the glycols, the non-ionic surfactants, and the bile salts. Among the enhancers used, polyoxyethylene 2-oleyl ether showed the most enhancing effects on drug permeation through skin. The analgesic activity was examined using a tail-flick analgesimeter. From the area under the efficacy curve of the rat-tail flick tests, procaine gel containing polyoxyethylene 2-oleyl ether showed about 1.77-fold increase in analgesic activity compared with the control. These results support that the enhanced local anesthetic gels containing an enhancer could be developed using the bioadhesive polymer gels based on HPMC and poloxamer.     

Formulation,in vitro evaluation and kinetic analysis of chitosan–gelatin bilayer muco-adhesive buccal patches of insulin nanoparticles

The present study was performed to optimise the formulation of a muco-adhesive buccal patch for insulin nanoparticles (NPs) delivery. Insulin NPs were synthesised by an ionic gelation technique using N-di methyl ethyl chitosan cysteine (DMEC-Cys) as permeation enhancer biopolymer, tripolyphosphate (TPP) and insulin. Buccal patches were developed by solvent-casting technique using chitosan and gelatine as muco-adhesive polymers. Optimised patches were embedded with 3?mg of insulin-loaded NPs with a homogeneous distribution of NPs in the muco-adhesive matrix, which displayed adequate physico-mechanical properties. The drug release characteristics, release mechanism and kinetics were investigated. Data fitting to Peppas equation with a correlation coefficient indicated that the mechanism of drug release followed an anomalous transport that means drug release was afforded through drug diffusion along with polymer erosion. In vitro drug release, release kinetics, physical and mechanical studies for all patch formulations reflected the ideal characteristics of this buccal patch for the delivery of insulin NPs.     

Insulin-loaded alginic acid nanoparticles for sublingual delivery

Abstract

Alginic acid nanoparticles (NPs) containing insulin, with nicotinamide as permeation enhancer were developed for sublingual delivery. The lower concentration of proteolytic enzymes, lower thickness and enhanced retention due to bioadhesive property, were relied on for enhanced insulin absorption. Insulin-loaded NPs were prepared by mild and aqueous based nanoprecipitation process. NPs were negatively charged and had a mean size of ～200?nm with low dispersity index. Insulin loading capacities of >95% suggested a high association of insulin with alginic acid. Fourier Transform Infra-Red Spectroscopy (FTIR) spectra and DSC (Differential Scanning Calorimetry) thermogram of insulin-loaded NPs revealed the association of insulin with alginic acid. Circular dichroism (CD) spectra confirmed conformational stability, while HPLC analysis confirmed chemical stability of insulin in the NPs. Sublingually delivered NPs with nicotinamide exhibited high pharmacological availability (>100%) and bioavailability (>80%) at a dose of 5?IU/kg. The high absolute pharmacological availability of 20.2% and bioavailability of 24.1% in comparison with subcutaneous injection at 1?IU/kg, in the streptozotocin-induced diabetic rat model, suggest the insulin-loaded alginic acid NPs as a promising sublingual delivery system of insulin.     

Effect of Size on the Biodistribution and Blood Clearance of Etoposide-Loaded PLGA Nanoparticles

Approaches used to avoid uptake of the injected particles by the reticuloendothelial system include modification of the particle properties such as surface charge and particle size. In the present study the effect of mean particle size of etoposide-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) of sizes 105 nm ((99m)Tc-Eto-PLGA NP(105)) and 160 nm ((99m)Tc-Eto-PLGA NP(160)) on biodistribution and blood clearance were studied after intravenous administration of the radiolabeled formulations and compared to that of free drug ((99m)Tc-Eto). It was found that etoposide-loaded PLGA NPs of size 105 nm were present in the blood at higher concentrations up to 24 h and were able to reduce their uptake by the reticuloendothelial system as compared to that of etoposide-loaded PLGA NPs of size 160 nm and pure drug. Moreover, the pure drug ((99m)Tc-Eto) did not cross the blood-brain barrier, whereas (99m)Tc-Eto-PLGA NP(105) showed relatively high concentrations of 0.58% of injected dose in brain in 1 h (8-fold higher), 0.6% in 4 h (20-fold higher) and 0.22% in 24 h (10-fold higher) than the concentration of (99m)Tc-Eto-PLGA NP(160). In bone, concentration of (99m)Tc-Eto-PLGA NP(105) was about 7.2 times higher than the concentration of (99m)Tc-Eto in 24 h. The study concludes that NPs of size ～100 nm can be used for long-term circulation without the need for surface modification. Such NPs could be exploited for use in leukemia therapy for providing sustained release of etoposide by long-term circulation. LAY ABSTRACT: Approaches used to avoid uptake of the injected particles by the reticuloendothelial system include modification of the particle properties such as surface charge and particle size. In the present study the effect of mean particle size of etoposide-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) of sizes 105 nm and 160 nm on biodistribution studies after intravenous administration in mice and blood clearance studies after intravenous administration in rats was studied. It was found that etoposide-loaded PLGA-NPs of size 105 nm were present in the blood at higher concentrations up to 24 h and were able to reduce their uptake by the reticuloendothelial system as compared to that of etoposide-loaded PLGA-NP of size 160 nm and pure drug. Moreover, the NPs of size 105 nm had greater uptake in bone and brain, in which concentration of free drug and NPs of size 160 nm was negligible. The study concludes that NPs of size ～100 nm can be used for long-term circulation without the need for surface modification.     

胰岛素经口腔给药对正常大鼠的降血糖作用

目的　研究胰岛素溶液 (insulinsolution ,INS SOL)经正常大鼠口腔给药后的降血糖作用。方法　以血糖水平为指标 ,考察各种吸收促进剂经正常大鼠口腔给药后对INS SOL降血糖作用的影响 ,以皮下注射为对照 ,计算不同条件下INS SOL的药理生物利用度 (pharmacologicalbioavailability ,PA)。 结果　不加吸收促进剂的条件下 ,10U·kg-1的INS SOL经口腔给药后的生物利用较低 (PA =6 9% )。十二烷基硫酸钠 (5 % ,PA =14 5 % ) ,苄泽 78(5 % ,PA =2 0 6 % ) ,脱氧胆酸钠 (5 % ,PA =16 5 % )和卵磷脂(10 % ,PA =13 8% )均增加INS SOL的降血糖作用。苄泽78(5 % )可使INS SOL(5U·kg-1)的PA最高达到 33%。结论　在适当的吸收促进剂的作用下INS SOL经口腔给药后具有明显的降血糖效果。     

Nanovesicles System for Rapid-Onset Sublingual Delivery Containing Sodium Tanshinone IIA Sulfonate: In vitro and In Vivo Evaluation

A novel formulation based on nanovesicles system for rapid-onset sublingual delivery of hydrophilic drug (sodium tanshinone IIA sulfonate, STS) was investigated. The nanovesicles system was composed of 1.5% soybean lecithin, 6% propylene glycol, and penetration enhancers (1% sodium dodecyl sulfate and 0.03% hyaluronan acid). The STS-loaded nanovesicles with an average diameter of 133 ± 9.04 nm and high entrapment efficiency of 85.65 ± 3.89% were characterized. The effects of permeation enhancers on the penetration of STS formulations were investigated using Franz diffusion cells in vitro, showing 86.1–235.8 times higher permeation rate than that of normal STS solution. The rapid symptom relief effect of the nanovesicles system on acute myocardial infarction rabbits was evaluated by in vivo study, ST-segment deviation(S and T wave abnormality in electrocardiogram) was attenuated markedly and rapidly within 5 min, infarct size of heart was significantly reduced and the biochemical indicators were substantially decreased, compared with the control groups (p < 0.05). This study provided a promising tool for the future sublingual delivery of hydrophilic compounds with the noninvasive and rapid onset clinical effect. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2332–2340, 2013     

Relative hypoglycemic effect of insulin suppositories in diabetic beagle dogs: optimization of various concentrations of sodium salicylate and polyoxyethylene-9-lauryl ether.

The effect of insulin suppositories containing different amounts and concentrations of sodium salicylate (50, 100 mg) and polyoxyethylene-9-lauryl ether (POELE 1, 3, 4%), respectively, on the plasma glucose concentration of diabetic beagle dogs was investigated after rectal administration. Comparison of the effects of these formulations was made with that produced after subcutaneous insulin injections. Insulin suppositories containing sodium salicylate (50 mg) produced a maximum reduction of plasma glucose concentration (Cmax) of 55 +/- 11%, an area under the curve (AUC) of 252 +/- 59% reduction h; and a relative hypoglycemia (RH) of 49 +/- 12% relative to subcutaneous injection of insulin (4 U/kg). Increasing sodium salicylate to 100 mg/suppository did not improve the hypoglycemic effect of insulin suppositories further. Investigation of the influence of insulin suppositories containing different concentrations of the nonionic surfactant POELE (1, 3, 4%) showed that; the suppositories containing the lowest concentration (1%) produced the highest hypoglycemic effect with a Cmax of 68%, AUC of 332 +/- 67% reduction h, and RH of 55 +/- 11%. Incorporation of sodium salicylate 50mg in insulin suppositories containing 1% POELE did not improve further the effects found with these suppositories. In conclusion, a relative hypoglycemic effect of about 50-55% can be achieved using insulin suppositories containing Witepsol W35 as a base, insulin (5 U/kg), and sodium salicylate (50 mg) or POELE (1%) as rectal absorption enhancers.     

Rat epidermal keratinocyte organotypic culture (ROC) compared to human cadaver skin: the effect of skin permeation enhancers.

The objective of this study was to evaluate the response of the rat epidermal keratinocyte organotypic culture (ROC) to permeation enhancers, and to compare these responses to those in human cadaver skin. Different concentrations of two mixtures for enhancing permeation were investigated, sodium dodecyl sulfate:phenyl piperazine and methyl pyrrolidone:dodecyl pyridinium chloride, using skin impedance spectroscopy and two experimental compounds, the lipophilic corticosterone and the hydrophilic sucrose. The chemical irritation effects of the formulations were evaluated based on leakage of lactate dehydrogenase enzyme (LDH) and cellular morphological perturbation. This study provides evidence for direct correlations of permeation/permeation, impedance/impedance and permation/impedance between the culture model and human skin. The only exception was the enhancer induced permeation of sucrose which was 1-40-fold higher in ROC compared to human skin, reflecting the more disordered lipid organization in stratum corneum and consequently the greater number of polar pathways. LDH leakage and cellular morphology indicated that it was possible to differentiate between safe permeation enhancers from irritating agents. This is not only the first study to have compared the enhancer effects on a cultured skin model with human skin, but also it has demonstrated enhancer induced irritation using an artificial skin model.     

格列本脲乳胶剂的制备及初步药效学

目的制备格列本脲乳胶剂,并观察其对正常大鼠的降血糖作用。方法采用改进的Franz扩散池,考察不同浓度二乙胺、不同有机胺和促进剂对格列本脲透皮吸收的影响,并评价优选处方对正常大鼠的降血糖作用。结果当二乙胺和氮酮的质量分数分别为0.70%和3%时,格列本脲乳胶剂的稳态渗透速率最高,并且能显著降低正常大鼠空腹血糖(P<0.01)。结论格列本脲乳胶剂的降血糖作用显著,有望成为一种治疗糖尿病的新型制剂。     

Nanoparticle surface charges alter blood-brain barrier integrity and permeability

PURPOSE: The blood-brain barrier (BBB) presents both a physical and electrostatic barrier to limit brain permeation of therapeutics. Previous work has demonstrated that nanoparticles (NPs) overcome the physical barrier, but there is little known regarding the effect of NP surface charge on BBB function. Therefore, this work evaluated: (1) effect of neutral, anionic and cationic charged NPs on BBB integrity and (2) NP brain permeability. Methods: Emulsifying wax NPs were prepared from warm oil-in-water microemulsion precursors using neutral, anionic or cationic surfactants to provide the corresponding NP surface charge. NPs were characterized by particle size and zeta potential. BBB integrity and NP brain permeability were evaluated by in situ rat brain perfusion. RESULTS: Neutral NPs and low concentrations of anionic NPs were found to have no effect on BBB integrity, whereas, high concentrations of anionic NPs and cationic NPs disrupted the BBB. The brain uptake rates of anionic NPs at lower concentrations were superior to neutral or cationic formulations at the same concentrations. CONCLUSIONS: (1) Neutral NPs and low concentration anionic NPs can be utilized as colloidal drug carriers to brain, (2) cationic NPs have an immediate toxic effect at the BBB and (3) NP surface charges must be considered for toxicity and brain distribution profiles.     

Absorption of insulin from pluronic F-127 gels following subcutaneous administration in rats.

The main objective of this work was to evaluate the use of Pluronic (PF127) gels, polylactic-co-glycolic acid (PLGA) nanoparticles and their combination for parenteral delivery of peptides and proteins having short half-lives using insulin as a model drug. The in vitro insulin release profiles of various PF127 formulations were evaluated at 37 degrees C using a membraneless in vitro model. In vivo evaluation of the serum glucose and insulin levels was performed following subcutaneous administration of various insulin formulations in normal rats. The in vitro results demonstrated that the higher the concentration of PF127 in the gel, the slower the release of insulin from the matrices, independent of the vehicle used. The acute hypoglycemic peak resulting from administration of an insulin solution between 0.5 and 2.0 h after administration (peak at 1 h) is replaced after administration of insulin-PLGA nanoparticles by an almost constant hypoglycemic effect with a slower recovery of the serum glucose levels at about 2 h after administration. By loading insulin into PF127 gels, a slower and more prolonged hypoglycemic effect of insulin was obtained in inverse proportion to the polymer concentration. PF127 gel formulations containing insulin-PLGA nanoparticles had the most long-lasting hypoglycemic effects of all formulations. From the current in vitro and in vivo study, we concluded that PF127 gel formulations containing either drug or drug-nanoparticles could be useful for the preparation of a controlled delivery system for peptides and proteins having short half-lives. Copyright