Insulin Association Efficiency Research Articles

Peroral delivery of insulin using chitosan derivatives: A comparative study of polyelectrolyte nanocomplexes and nanoparticles

Polymeric delivery systems based on nanoparticles (NP) have emerged as a promising approach for peroral insulin delivery. Using a trimethyl chitosan (TMC) and a PEG- graft-TMC copolymer, polyelectrolyte complexes (PEC) and nanoparticles were prepared and their properties were compared. The amount of insulin was quantified by HPLC and the stability of PEC and NP upon exposure to simulated gastrointestinal (GI) fluid was monitored by dynamic laser light scattering. It was shown that polymer/insulin (+/−) charge ratio played an important role in PEC and NP formation. Stable, uniform, and spherical PEC/NP with high insulin association efficiency (AE) were formed at or close to optimized polymer/insulin (+/−) charge ratio, depending on polymer structure. All PEC were more stable in pH 6.8 simulated intestinal fluid (SIF) than NP. The PEC also appeared to play some role in protecting insulin from degradation at higher temperature and with proteolytic enzyme more efficiently than NP. On the basis of these results, polyelectrolyte complexation can be suggested as a potentially useful technique for generating insulin delivery systems for peroral administration.

Synthesis and characterization of PEG modified N-trimethylaminoethylmethacrylate chitosan nanoparticles

Chitosan- N-trimethylaminoethylmethacrylate chloride–PEG (CS-TM–PEG) copolymers were synthesized in order to improve the solubility of chitosan in physiological environment, and enhance the biocompatibility of quaternized chitosan. The result of 1H NMR confirmed that PEG had been combined with amino groups of quaternized chitosan. The profile of hemolysis assay showed that Chitosan- N-trimethylaminoethylmethacrylate chloride (CS-TM) copolymer exhibited hemolytic activity from 10.31% to 13.58%, while CS-TM–PEG copolymer had hemolytic activity from 4.76% to 7.05% at copolymer concentrations from 250 to 2000 μg/ml. Through PEG modification, the hemolytic activity could be reduced to a half. CS-TM–PEG copolymer–insulin nanoparticles were prepared based on ionic gelation process of positively charged copolymers and negatively charged insulin. The nanoparticles were characterized in terms of particle size, TEM, association efficiency and in vitro release. These nanoparticles were 200–400 nm in size and insulin association efficiency of optimal formulations was found up to 90%. In vitro release showed that the nanoparticles provided an initial burst release followed by a sustained release with the sensitivity of ionic strength and pH values.

Probing insulin’s secondary structure after entrapment into alginate/chitosan nanoparticles

The aim of the present study was to probe the structural integrity of insulin after being entrapped into chitosan/alginate nanoparticles produced by ionotropic polyelectrolyte pre-gelation. By manipulating the alginate:chitosan mass ratio and the pH during nanoparticle production, desired nanoparticles with a mean size of 850 (±88) nm and insulin association efficiency of 81 (±2)% were obtained. Insulin secondary structure was assessed by Fourier transform infrared (FTIR) and circular dichroism (CD) after entrapment into nanoparticles and after release from the particles under gastrointestinal simulated conditions. FTIR second-derivative spectra and area-overlap compared to an insulin standard confirmed that no significant conformational changes of insulin occurred in terms of α-helix and β-sheet content. Far-UV-CD spectra corroborated the preservation of insulin structure during the nanoparticle production procedure. The presented nanoparticulate system is a promising carrier for insulin oral delivery since it preserves insulin structure and therefore also, potentially, its bioactivity.

Development and Comparison of Different Nanoparticulate Polyelectrolyte Complexes as Insulin Carriers

The overall objective of our research is to produce polyanion/chitosan nanoparticulate oral delivery systems for insulin. Specific objectives of the present study were to study dextran sulfate or alginate complexation with chitosan on mean particle size, insulin association efficiency, loading capacity and release profile. Nanoparticles were formed by ionotropic complexation and coacervation between polyanions (dextran sulfate and alginate) and chitosan. Diameter was evaluated with photon correlation spectroscopy, polymer interaction was confirmed by DSC and FTIR and particle morphology was assessed by SEM and TEM. Mean nanoparticle diameter ranged from 423 to 850 nm, insulin association efficiency from 63 to 94% and loading capacity from 5 to 13%. Dextran sulfate provided highest insulin association efficiency and retention of insulin in gastric simulated conditions. These nanoparticle systems show promise as insulin and potentially other therapeutic polypeptides carriers.

Development and validation of a rapid reversed‐phase HPLC method for the determination of insulin from nanoparticulate systems

A reversed-phase high-performance liquid chromatographic (HPLC) method has been developed and validated for the determination of insulin in nanoparticulate dosage forms. Its application for the development and characterization of insulin-loaded nanoparticulates composed of polyelectrolytes has also been carried out. A reversed-phase (RP) C18 column and gradient elution with a mobile phase composed of acetonitrile (ACN) and 0.1% aqueous trifluoroacetic acid (TFA) solution at a flow rate of 1 mL/min was used. Protein identification was made by UV detection at 214 nm. The gradient changed from 30:70 (ACN:TFA, v/v) to 40:60 (v/v) in 5 min followed by isocratic elution at 40:60 (v/v) for a further five minutes. The method was linear in the range of 1-100 microg/mL (R2 = 0.9996), specific with a good inter-day and intra-day precision based on relative standard deviation values (less than 3.80%). The recovery was between 98.86 and 100.88% and the detection and quantitation limits were 0.24 and 0.72 microg/mL, respectively. The method was further tested for the determination of the association efficiency of insulin to nanoparticulate carriers composed of alginate and chitosan, as well as its loading capacity for this protein. Encapsulant release under simulated gastrointestinal fluids was evaluated. The method can be used for development and characterization of insulin-loaded nanoparticles made from cross-linked chitosan-alginate.

Insulin‐Loaded Calcium Pectinate Nanoparticles: Effects of Pectin Molecular Weight and Formulation pH

Insulin‐loaded calcium pectinate nanoparticles were prepared as a potential colonic delivery system by ionotropic gelation with calcium ions. The effects of pectin molecular weight (Mv) and formulation pH on the characteristics of the nanoparticles were evaluated. Commercial pectins, LM101 and LM104, with respective degrees of esterification of 36% and 28%, were depolymerized by mechanical milling to give Mv ranging from 89 to 5.6 kDa. Milled pectins did not yield nanoparticles with significantly different mean diameter and insulin association efficiency (AE) compared to nanoparticles of unmilled pectins. LM104 nanoparticles had smaller variation in mean size than the LM101 nanoparticles. Formulation pH significantly influenced the AE and stability of the nanoparticles. Increasing the pH from 2 to 3 enhanced the AE by three‐fold, from 32.76% to 93.31%, at an insulin loading concentration of 80 U/mL. This increase in AE was correlated to the charge density on the pectin molecules as a function of pH. Subsequent release of associated insulin from the nanoparticles was dependent on the extent of dilution of the nanoparticle dispersion and the pH of the dissolution medium. Cross‐flow filtration could be used to separate the nanoparticles from unassociated ions and molecules, without compromising the characteristics of the nanoparticles.

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 pKa 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.