Chitosan Mw Research Articles

Formation and stability assessment of self-assembled nanoparticles from large Mw chitosan and sulfobutylether-β-cyclodextrin

Stable self-assembled nanoparticles were prepared by mixing aqueous solutions containing chitosan and either sulfobutylether β-cyclodextrin or sulfobutylether γ-cyclodextrin at pH 4. The nanoparticle formation was monitored by UV-VIS spectroscopy operated at 700 nm. The stability of the nanoparticle system was tested under different conditions, for example upon storage, upon dilution, at elevated temperatures and upon salt addition. The effect of media pH on the formation and stability of the nanoparticles was also tested. Stability studies were followed by dynamic light scattering measurements and showed that the nanoparticles are stable at all conditions tested except in basic media. The nanoparticles were investigated by transmission electron microscopy which confirmed that the diameter of the nanoparticles is between 100 and 200 nm. Drug release from the particles was studied using hydrocortisone as a model drug.

Changes in blood aggregation with differences in molecular weight and degree of deacetylation of chitosan

Because the molecular weight (Mw) and degree of deacetylation (DDA) of chitosan can vary depending on the purification method, the identification of appropriate chitosan structures is important for developing more effective hemostatic agents. In this study, the influence of varying Mw and DDA of chitosan on blood aggregation was characterized by 10 types of chitosan with different Mw and DDA, including oligomers. The highest aggregation of whole blood, washed erythrocytes and platelets in platelet-rich plasma (PRP) were observed in chitosan with Mw of 8.6 kDa or more and with DDA of 75 to 88%. However, chitosan with too high Mw (247 kDa) inhibited the aggregation of whole blood, washed erythrocytes and PRP at high chitosan concentration. At certain concentrations, chitosan with 75–85% DDA and 50–190 kDa and chitosan with 87.6% DDA and 247 kDa both aggregated proteins in PRP. Chitosan oligomer did not affect blood aggregation. The results suggested that the aggregation by chitosan depended on the interaction of positively charged chitosan with negatively charged erythrocytes, platelets and plasma protein. It seemed that a suitable balance of positive charge in chitosan and negative charge in hemocytes and some kinds of proteins was important. To develop a hemostatic with effective blood aggregation, the chitosan should not be limited to a single Mw and a single DDA but may be a mixed chitosan with wide range of Mw (8.6–247 kDa) and DDA of 75 to 88%.

Impact of acidity and metal ion on the antibacterial activity and mechanisms of β- and α-chitosan.

This study investigated the effects of acidity and metal ion on the antibacterial activity of α- and β-chitosan at different molecular weights (Mw, 22-360kDa) against Escherichia coli and Listeria innocua through agar well diffusion assay. Spectrophotometric, electrophoretic, and confocal fluorescence microscopy analysis were further employed to evaluate the antibacterial mechanisms probably involved. Increasing pH from 4.0 to 5.0 weakened the antibacterial ability of chitosan as shown by the decreased bacteria growth inhibition zone (BGIZ) from 0.63 to 0.57cm for β-chitosan (61kDa) and from 0.62 to 0.57cm for α-chitosan (30kDa) against E. coli. All β- and α-chitosan samples showed antibacterial activity against L. innocua, in which 22kDa β-chitosan and 30kDa α-chitosan at pH4.0 had the highest antibacterial activity with BGIZ of 1.22 and 0.98cm, respectively. Interactive effect between pH and Mw on the antibacterial activity of β-chitosan was observed, but not of α-chitosan. Adding Co(2+) and Ni(2+) significantly improved the antibacterial activity of chitosan, while adding K(+), Na(+), and Li(+) significantly weakened the antibacterial activity of some β- and α-chitosan samples (P < 0.05), and different Mw and forms of chitosan showed different metal ion absorption capacities. Results indicate that chitosan might insert into the groove of bacterial DNA double helix structure to induce DNA degradation and permeate through bacteria cell membranes and combine with genomic DNA to induce its dysfunction, providing evidences for the antibacterial mechanisms of chitosan.

Development and characterization of alginate coated low molecular weight chitosan nanoparticles as new carriers for oral vaccine delivery in mice.

In the present study, nanoparticles of low MW chitosan (CS) were formulated in which measles antigen was entrapped and subsequently coated with sodium alginate. The size and surface properties of the nanoparticle can be tuned with different MW of CS. In vitro release studies showed initial burst release followed by extended release, best fitted in the Makoid-Banakar model (R(2)>0.98). SDS-PAGE assay revealed that alginate coating could effectively protect antigen in acidic condition for at least 2h. Cell viability was assessed using MTT assay into HT 29 cell line. Formulations were orally administered to mice and immunological responses were evaluated using ELISA method. Obtained results showed that measles antigen-loaded CS nanoparticles induced strong immune response and significant correlation was observed between the immune response with CS MW. Protecting ability of antigen in gastric environment, sustained release kinetics, systemic and mucosal immune responses and low cytotoxicity observed for the alginate coated nanoparticles demonstrated that LMW CS could be promising platform for oral vaccine delivery.

Study on gamma-irradiation degradation of chitosan swollen in H₂O₂ solution and its antimicrobial activity for E. coli

Degradation of chitosan in swollen state with hydrogen peroxide solution (5% w/v) by γ-irradiation was investigated. Molecular weight (Mw) of irradiated chitosan was determined by gel permeation chromatography (GPC). Fourier transform infrared (FT-IR) and ultraviolet-visible (UV-vis) spectrawere analyzed to study the structure changes of degraded chitosan. The results showed that the chitosan of low Mw ~30-45 kDawas efficiently prepared by γ-irradiation of chitosan swollen in hydrogen peroxide solution at low dose less than 20kGy. The main structure as well as the degree of deacetylation of the degraded chitosan was almost no significant change. Furthermore, the radiation degradation yield (Gs) was remarkably enhanced by the presence of H2O2. The obtained low Mw chitosan revealed high antimicrobial activity for E. colithat can be used for food preservation and other purposes as well.

Physicochemical and morphological properties of size-controlled chitosan–tripolyphosphate nanoparticles

Chitosan–tripolyphosphate nanoparticles have been extensively studied during the last decade because of their numerous applications. In this study, we describe conditions to optimize chitosan nanoparticles as potential nano-fillers in edible films. The ionic cross-linking between the cationic amino groups on the chitosan (CS) chain and the anionic phosphate groups of sodium tripolyphosphate (TPP) was verified via FTIR. Particle size, polydispersity index (PDI) and surface ζ-potential were controlled by chitosan's Mw and concentration, CS:TPP mass ratio, and external conditions such as pH and salinity of the initial chitosan solution. Post-processing methods such as centrifugation and ultra-sonication were used to further control particle size. We show that particle size can be controlled by selecting appropriate conditions. Particles with sizes below 120 nm were produced at different CS:TPP mass ratios depending on the CS concentration. Dilute NaCl was the optimal solution ionic composition that decreased the size by 25% and also resulted in a narrow particle size distribution. We show using UV–vis spectrophotometry that particles of different size, separated by centrifugation had different phosphorus content. Ultra-sonication can be used to reduce the size by 50% but long time caused fragmentation of the nanoparticles. Transmission electron microscopy (TEM) revealed the differences in the morphology of chitosan nanoparticles under various fabrication conditions.

Effect of molecular weight of chitosan on antimicrobial properties and tissue compatibility of chitosan-impregnated bacterial cellulose films

Bacterial cellulose-chitosan (BC-C) films were developed by immersing purified BC pellicles in 1.5 ~ 2.0% (w/v) acetic acid solutions containing chitosan of varying molecular weights. Effects of different molecular weight of chitosan on physical, biological and antimicrobial properties of the composite films were investigated. The cumulative chitosan absorption capacities with Mw of 141,000, 199,000, and 263,000 were 38.43, 24.65, and 23.89 mg/cm3 of dry BC film, respectively. The cumulative release profiles of chitosan from the films strongly depended on molecular weight of chitosan and pH of solution. The order of release of chitosan from the BC-C films was dependent on molecular weight as follows: Mw 141,000 > Mw 199,000 > Mw 263,000. All BC-C films showed the antimicrobial abilities against Staphylococcus aureus and Aspergillus niger but had no inhibitory effect on the growth of Escherichia coli. The BC-C films supported for adhesion, spreading and proliferation of both human skin keratinocytes and fibroblasts. The antibacterial activity against S. aureus of the BC-C with the highest Mw chitosan (263,000) was higher than those of the others. On the other hand, the BC-C films with the lowest Mw chitosan (141,000) promoted the growth of human skin cells more than those of the others.

Combined effect of chitosan and water activity on growth and fumonisin production by Fusarium verticillioides and Fusarium proliferatum on maize-based media

The objectives of the present study were to determine the in vitro efficacy of chitosan (0.5, 1.0, 2.0 and 3.0mg/mL) under different water availabilities (0.995, 0.99, 0.98, 0.96 and 0.93) at 25°C on lag phase, growth rate and fumonisin production by isolates of Fusarium verticillioides and Fusarium proliferatum. The presence of chitosan affected growth and fumonisin production, and this effect was dependent on the dose and aW treatment used. The presence of chitosan increased the lag phase, and reduced the growth rate of both Fusarium species significantly at all concentrations used, especially at 0.93 aW. Also, significant reduction of fumonisin production was observed in both Fusarium species at all conditions assayed. The present study has shown the combined effects of chitosan and aW on growth and fumonisin production by the two most important Fusarium species present on maize. Low molecular weight (Mw) chitosan with more than 70% of degree of deacetylation (DD) at 0.5mg/mL was able to significantly reduce growth rate and fumonisin production on maize-based media, with maximum levels of reduction in both parameters obtained at the highest doses used. As fumonisins are unavoidable contaminants in food and feed chains, their presence needs to be reduced to minimize their effects on human and animal health and to diminish the annual market loss through rejected maize. In this scenario post-harvest use of chitosan could be an important alternative treatment.

Study on gamma-irradiation degradation of chitosan swollen in H₂O₂ solution and its antimicrobial activityfor E. coli

Degradation of chitosan in swollen state with hydrogen peroxide solution (5% w/v) by γ-irradiation was investigated. Molecular weight (Mw) of irradiated chitosan was determined by gel permeation chromatography (GPC). Fourier transform infrared (FT-IR) and ultraviolet-visible (UVvis) spectrawere analyzed to study the structure changes of degraded chitosan. The results showed that the chitosan of low Mw ~30-45 kDawas efficiently prepared by γ-irradiation of chitosan swollen in hydrogen peroxide solution at low dose less than 20kGy. The main structure as well as the degree of deacetylation of the degraded chitosan was almost no significant change. Furthermore, the radiationdegradation yield (Gs) was remarkably enhanced by the presence of H2O2. The obtained low Mw chitosan revealed high antimicrobial activity for E. colithat can be used for food preservation and other purposes as well.

The contribution of acidulant to the antibacterial activity of acid soluble α- and β-chitosan solutions and their films

This study evaluated individual contributions of dissolving acids (acetic acid, lactic acid, and hydrochloric acid) or acid solubilized chitosan to the antibacterial activity against Listeria innocua and Escherichia coli as solutions and dried films. Solutions containing chitosan showed significantly (P < 0.05) different inhibitory activity (measured as percentage of inhibition (PI), in percent) against L. innocua and E. coli, compared to equivalent acid solutions. This increase was calculated as additional inhibition (AI, in percent), which could be as high as 65% in solutions containing 300-320 kDa chitosan depending on the acid type, bacterial species, and the chitosan form (α or β). Solutions containing 4-5 kDa chitosan had lower AI and showed much greater variability among the different chitosan forms, acid types, and bacterial species. Higher molecular weight (Mw) chitosan also showed significantly higher levels of adsorption to bacterial cells than that of lower Mw samples, suggesting that the observed increase in inhibition was the result of surface phenomena. The contribution of acids to the antibacterial activity of chitosan films was assessed by comparing non-rinsed and rinsed films (rinsed in the appropriate broth to remove residual acids and active fragments formed on the dried film). Rinsing β-chitosan films has reduced PI by as much as 28% compared with non-rinsed films, indicating that part of the antibacterial activity of chitosan films is due to the presence of soluble acid compounds and/or other active fragments. Overall, both acidulant and chitosan were found to contribute to the antibacterial activity of acid solubilized α- and β-chitosan, with the exact antibacterial activity of chitosan varying based on the solution and film properties, suggesting a complex interaction.

Physicochemical, functional, antioxidant and antibacterial properties of chitosan extracted from shrimp wastes by microwave technique

Chitosan is produced from shrimp waste chitin at three particle sizes 20, 40 and 60 mesh by deacetylating with different concentrations of NaOH solution (30%, 40% and 50%) under microwave irradiation for 10min. The process describes a rapid synthesis procedure in comparison to conventional methods. The microwave-synthesized chitosan was characterized and the experimental results showed that the degree of deacetylation increased with increasing concentration of deacetylation alkali solution. A degree of deacetylation of 95.19% was achieved after irradiating chitin at 60 mesh with 50% NaOH solution in a microwave for 10min at 1400-W power. Microwave-synthesized chitosan exhibited antioxidant activities of 47.71–72.31% at 10mg/ml and showed reducing powers of 2.094–2.367 at 10mg/ml. On the other hand, at 10mg/ml, the scavenging ability of chitosan on 1,1-diphenyl-2-picrylhydrazyl radicals ranged from 43.03% to 90.48%. The antibacterial activities of microwave-synthesized chitosan were examined against two gram-negative (Escherichia coli and Salmonella typhimurium) and two gram-positive (Staphylococcus aureus and Bacillus cereus), tested chitosan markedly inhibited the growth of tested bacteria although inhibitory effects differed with molecular weight (Mw) of chitosan and the species of bacteria. Generally, the microwave technique can be very useful for synthesizing good functional properties chitosan with rapid and clean chemistry.

Effect of the Chitosan on the Proliferation of the Microencapsulated Escherichia coli

Immobilization of microencapsulated cell is a kind of membrane technology. Therefore, the characteristics of membrane have a great impact on the growth of microorganisms. This study is designed to achieve the greatest growth rate of microencapsulated cell, Escherichia coli were immobilized in AC microcapsules made with different type of chitosan and then cultured in the Luria-Bertani medium. Effect of deacetylated degree (DD), molecular weight, membrane formation time and the pH of chitosan on the swelling ratio, cell release and proliferation were investigated at 2, 4, 6, 12, 24and 48h respectively. The results showed that the DD has no obviously influence on terminal biomass concentration, but the swelling ratio of membrane reached lowest when the DD was 90%. The biomass concentration increased with the rising of chitosan Mw and membrane formation time. Especially, the terminal biomass concentration near to 6.5×106·mL in the microcapsules made with 10KDa chitosan Mw ands 15min membrane formation time. Moreover, the swelling ratio closed to lowest point as the chitosan solution pH was 6.3.

Microwave-assisted degradation of chitosan for a possible use in inhibiting crop pathogenic fungi

Degradation of chitosan by H2O2 under microwave irradiation was investigated. The oxidative degradation of chitosan was highly accelerated by microwave irradiation under the condition of low temperature and low concentration of H2O2. The degraded chitosans with low molecular weight (Mw) were characterized by gel permeation chromatography, Fourier-transform infrared spectroscopy, ultraviolet–visible spectroscopy, X-ray diffraction and elemental analysis. The decrease of Mw led to transformation of crystal structure and increase of water solubility, whereas no significant chemical structure change in the backbone of chitosan was observed. Antifungal activities of chitosans with different Mw against crop pathogenic fungi Phomopsis asparagi, Fusarium oxysoporum f. sp. Vasinfectum and Stemphylium solani were investigated at the concentrations of 100, 200 and 400mg/L. All degraded chitosans with low Mw exhibited enhanced antifungal activity compared with original chitosan and the chitosan of 41.2kDa showed the highest activity. At 400mg/L, the chitosan of 41.2kDa inhibited growth of P. asparagi at 89.3%, stronger than polyoxin and triadimefon, the inhibitory effects of which were found to be 55.5% and 68.5%. All the results indicated that oxidative degradation under microwave irradiation was a promising technique for large-scale production of low Mw chitosan for use in crop protection.

Chitosan-based nanocapsules: physical characterization, stability in biological media and capsaicin encapsulation

This study addresses the effect of the degree of N-acetylation (DA ~1.4–56 %) of chitosan (CS) of low and medium molecular weight (M w ~ 9.5–13.2 and ~122–266 kDa, respectively) on the biophysical properties, colloidal stability and encapsulation efficiency of capsaicin (a lipophilic drug currently used in pain therapy) of CS-based nanocapsules (NCs). The average diameter of the NCs varied within the range of ~150–200 nm, but it was slightly higher for NCs comprising high M w CS than for those with low M w CS. The zeta potential (ζ) was highly positive and clearly dependent on the M w of CS, exhibiting a monotonic decreasing trend concomitant with the increase in DA. Both results suggest that M w and DA of CS have an effect on the disposition of the polysaccharide onto the phospholipidic surface. Synchrotron SAXS studies revealed a monotonic increase in Bragg interplanar distances (from ~55 up to ~67 A) as a function of the DA, a finding that agrees with previous results that are consistent with the capacity of hydrophobic domains to disturb the crystalline state of gelled phospholipidic membranes. Colloidal stability studies carried out in cell-culture biological media revealed the determinant role of hydration forces, a short-range repulsive interaction, on the stability of the NCs. Finally, M w and DA of CS both influenced the encapsulation efficiency of capsaicin, thus showing the effect of the harnessed NCs shell on its capacity to encapsulate lipophilic drugs.

Effectiveness of chitosan on the inactivation of enteric viral surrogates

Chitosan is known to have bactericidal and antifungal activity. Although human noroviruses are the leading cause of non-bacterial gastroenteritis, information on the efficacy of chitosan against foodborne viruses is very limited. The objective of this work was to determine the effectiveness of different molecular weight chitosans against the cultivable human norovirus and enteric virus surrogates, feline calicivirus, FCV-F9, murine norovirus, MNV-1, and bacteriophages, MS2 and phiX174. Five purified chitosans (53, 222, 307, 421, ∼1150 kDa) were dissolved in water, 1% acetic acid, or aqueous HCl pH = 4.3, sterilized by membrane filtration, and mixed with equal volume of virus to obtain a final concentration of 0.7% chitosan and 5 log10 PFU/ml virus. Virus-chitosan suspensions were incubated for 3 h at 37 °C. Untreated viruses in PBS, in PBS with acetic acid, and in PBS with HCl were tested as controls. Each experiment was run in duplicate and replicated at least twice. Water-soluble chitosan (53 kDa) reduced phiX174, MS2, FCV-F9 and MNV-1 titers by 0.59, 2.44, 3.36, and 0.34 log10 PFU/ml respectively. Chitosans in acetic acid decreased phiX174 by 1.19–1.29, MS2 by 1.88–5.37, FCV-F9 by 2.27–2.94, and MNV-1 by 0.09–0.28 log10 PFU/ml, respectively. Increasing the MW of chitosan corresponded with an increasing antiviral effect on MS2, but did not appear to play a role for the other three tested viral surrogates. Overall, chitosan treatments showed the greatest reduction for FCV-F9, and MS2 followed by phiX174, and with no significant effect on MNV-1.

Biophysical properties of chitosan/siRNA polyplexes: Profiling the polymer/siRNA interactions and bioactivity

Chitosans are naturally occurring polymers widely used in life science to mediate intracellular uptake of nucleic acids such as siRNA. Four chitosans of fungal origin (Agaricus bisporus; molecular weights MW=44, 63, 93 and 143kDa) were used in this study and profiled for size, viscosity and hydrodynamic radius using gel permeation chromatography (GPC). Polyplexes made of these chitosans and siRNA were developed and optimized for transfection efficacy in vitro. The characteristics of these polyplexes were low chitosan:siRNA ratios (4–8; N:P) similar positive zeta potential (20–30mV) and comparable particle sizes (about 150nm). Endogenous luciferase reporter gene down-regulation in human epithelial H1299 cells at nanomolar concentrations (37.5–150nM) was significantly stronger for the lower molecular weight chitosans. The impact of these low N:P polyplexes on the cellular viability was minimal also at 150nM. To help develop an understanding of these differences, an energetic profile of the molecular interactions and polyplex formation was established by isothermal titration calorimetry (ITC). The four polyplexes exhibited strong binding enthalpies delta Hbind(−84 to −102kcal/mol) resulting in nanomolar dissociation constants. Intracellular trafficking studies using rhodamine labeled siRNA revealed that polyplexes made from smaller MW chitosans exhibited faster cellular uptake kinetics than their higher MW counterpart. Transmission electron microscopy and small angle X-ray scattering studies (SAXS) revealed that the 44kDa derived polyplexes exhibited regular spherical structure, whereas the 143kDa chitosan polyplex was rather irregularly shaped. With regards to adverse effects these low N:P chitosan/siRNA formulations represent an interesting alternative to so far reported chitosan polyplexes that used vast N:P excess to achieve similar bioactivity.

Preparation of water-soluble chitosan by hydrolysis with commercial glucoamylase containing chitosanase activity

Water-soluble chitosan (WSC) was prepared by hydrolyzing chitosan with commercial glucoamylase. Factors affecting the enzymatic hydrolysis of chitosan were investigated, and the optimum hydrolysis conditions were as follows: time, 24 h; pH, 4.5; temperature, 55 °C; amount of glucoamylase, 40 mg/500 mL of reaction mixture (26.8 U of chitosanase activity). Under these conditions, the weight-averaged molecular weight (Mw) of chitosan decreased to 1,700 Da. The hydrolysate was neutralized with 1 M NaOH, filtrated, concentrated to 16% (w/v), and precipitated with 5 volumes of ethanol. The product of WSC was obtained by drying the precipitate at 60 °C for 3 h. The content of WSC in the product and the yield of WSC were 95.3% (w/w) and 94.2% (w/w), respectively. The structure of WSC was characterized by Fourier transform infrared spectroscopy (FTIR).

Features of crystallization of chitosan with different molecular mass and its mixtures with pluronic F-127 according to atomic force microscopy and X-ray diffraction data

The structure of chitosan with different molecular mass and its mixtures with pluronic F-127 (triblock copolymer of ethylene and propylene oxide) crystallized from aqueous solutions was analyzed by atomic force microscopy and X-ray diffraction methods. It was established that the low-molecular-mass chitosan (Mw = 20000) had a highly ordered macrocrystalline structure, whereas the high-molecular-mass chitosan (Mw = 250000) was characterized by a poorly ordered quasi-crystalline structure. It was shown that, after evaporation of the aqueous solution of high-molecular-mass chitosan and pluronic F-127, chitosan formed a continuous film on the mica surface and pluronic formed dendritic structures on the chitosan surface. At the same time, the evaporation of the solution of the low-molecular-mass chitosan and pluronic F-127 mixture resulted in the formation of interpenetrating dendrites of chitosan and pluronic on the mica surface. In the low-molecular-mass chitosan-pluronic systems, a pluronic crystalline phase was formed at a lower pluronic content than in the case of high-molecular-mass chitosan-pluronic systems.

Low-molecular-weight chitosans: Preparation and characterization

The chitosan depolymerization products prepared by enzymatic hydrolysis, oxidative reaction or microwave irradiation were characterized by FT-IR, MALDI TOF MS, solid state 13C CP/MAS NMR and GPC. Enzymatic hydrolysis resulted in two fractions of low-molecular-weight (LMW) chitosans with Mw 34kDa (3.9wt%) and 14.6kDa (8.5wt%). In the oxidative reaction at the hydrogen peroxide and acetic acid molar ratio equal to 1.7, two LMW chitosans with Mw 9 and 10kDa (the deacetylation degree 69% and 94%, respectively) were isolated with the total yield 86wt%. In microwave irradiation, the Mw of chitosan (175.5kDa) decreased at most in two times. Two chitosan fractions with Mw 80 and 120kDa (total yield 70wt%) were obtained. In all cases, LMW chitosans served as fortifiers for chitosan oligomers, which were retained with the former through multiple hydrogen bonds. Their content in LMW chitosans was roughly 20–30wt%.

What are parameters affecting Leu-enkephalin loading and release from poly(isobutylcyanoacrylate) nanoparticles coated with thiolated chitosan?

This work aimed to investigate the loading and the release of Leu-enkephalin from chitosan and thiolated chitosan-coated poly(isobutylcyanoacrylate) nanoparticles by modifying different parameters : chitosan molecular weight (Mw 30, 85 and 145 kg/mol), its degree of thiolation and incorporation of poly(methylmethacrylate) in the nanoparticle core. According to the results, the main parameter controlling the loading and the release of Leu-enkephalin was the composition of the nanoparticle shell. Indeed, it was found that loading was higher and the release was most delayed when chitosan Mw was low. Furthermore, there was a clear trend to a better control of Leu-enkephalin release with low thiolated-chitosan proportion. As demonstrated by using isothermal titration microcalorimetry (ITC) experiments, low thiolated-chitosan proportion corresponds to a higher amount of functional amino-groups able to interact with the negative charge found on Leu-enkephalin. In contrast, the incorporation of poly(methylmethacrylate) in the nanoparticle core did not slow-down the release of Leu-enkephalin but enhanced the burst release effect.