Atoosa Maleki

Poly(lactide-co-glycolide)-rifampicin nanoparticles efficiently clear Mycobacterium bovis BCG infection in macrophages and remain membrane-bound in phago-lysosomes.

Summary Nanoparticles (NPs) are increasingly used as biodegradable vehicles to selectively deliver therapeutic agents such as drugs or antigens to cells. The most widely used vehicle for this purpose is based on copolymers of lactic acid and glycolic acid (PLGA) and has been extensively used in experiments aimed at delivering antibiotics against Mycobacterium tuberculosis in animal models of tuberculosis. Here, we describe fabrication of PLGA NPs containing either a high concentration of rifampicin or detectable levels of the green fluorescent dye, coumarin-6. Our goal here was twofold: first to resolve the controversial issue of whether, after phagocytic uptake, PLGA NPs remain membrane-bound or whether they escape into the cytoplasm, as has been widely claimed. Second, we sought to make NPs that enclosed sufficient rifampicin to efficiently clear macrophages of infection with Mycobacterium bovis BCG. Using fluorescence microscopy and immuno-electron microscopy, in combination with markers for lysosomes, we show that BCG bacteria, as expected, localized to early phagosomes, but that at least 90% of PLGA particles were targeted to, and remained in, low pH, hydrolase-rich phago-lysosomes. Our data collectively argue that PLGA NPs remain membrane-enclosed in macrophages for at least 13 days and degrade slowly. Importantly, provided that the NPs are fabricated with sufficient antibiotic, one dose given after infection is sufficient to efficiently clear the BCG infection after 9–12 days of treatment, as shown by estimates of the number of bacterial colonies in vitro.

Characterization of polyelectrolyte features in polysaccharide systems and mucin.

This review elucidates several aspects on the behavior of charged polysaccharides and mucin. Viscosification of dilute aqueous solutions of hyaluronan (HA) occurs in the course of time at low shear flow, whereas shear thinning as time evolves is found at moderate shear rates. Hydrogen bonds and electrostatic interaction play an important role for the emergence of these features. No time effect of the viscosity is observed for semidilute HA solutions. A degradation of HA is observed at low and high pH and this effect continues over long times, and it is only in the approximate interval 5<pH<10 that HA is stable. Small angle neutron scattering (SANS) measurements on semidilute aqueous solutions of mucin at pH=7 reveal a fractal dimension of 1.4, and the effect of temperature is insignificant on the fractal structure. This suggests that the mucin chains on a semi-local dimensional scale are rod-like. From various experimental methods on solutions of mucin it was found that at pH values around 2 (uncharged polymer), the intensive hydrophobic interactions lead to large association complexes, whereas at pH>>2 the negative charges suppress the tendency of forming associations. At pH<2, the mucin chains are compressed and they are decorated by some positive charges. In the semidilute regime, a fragmented network is developed. The intense association in semidilute solutions of mucin at pH=2 is further supported by the results from rheo-small angle light scattering measurements. Effects of ionic strength on the radius of gyration (R(g)) for dilute solutions of HA (pH=7) and positively charged hydroxyethylcellulose (HEC(+)) are studied with the aid of Monte Carlo simulations, and essential features of the polyelectrolyte effect on R(g) are captured in the computer simulation. Strong interactions are observed in aqueous mixtures of an anionic polysaccharide (HEC(-)) and an oppositely charged surfactant (cetyltrimethylammonium bromide; CTAB); this gives rise to extensive associations and macroscopic phase separation is approached. The massive association complexes are disclosed in the SANS experiments by a pronounced upturn in the scattered intensity at low values of the wave vector.

Effect of polyethylene glycol (PEG) length on the association properties of temperature-sensitive amphiphilic triblock copolymers (PNIPAAMm-b-PEGn-b-PNIPAAMm) in aqueous solution

Effects of temperature on the association behavior in aqueous solutions of a series of thermosensitive poly(N-isopropylacrylamide)-block-poly(ethylene glycol)-block-poly(N-isopropylacrylamide) triblock copolymers (PNIPAAMm-b-PEGn-b-PNIPAAMm) with the length of the PNIPAAM block fixed (m ≈ 67) and with different lengths of the PEG block (n = 23, 34, 77, and 165) have been studied with the aid of turbidity, dynamic light scattering (DLS), and Monte Carlo simulations. The turbidity results show that the sharp transition to high turbidity values is shifted to higher temperatures when the length of the PEG spacer is increased from 23 to 77, whereas no transition is observed for the longest PEG block. These findings are consistent with the DLS results, which suggest the formation of large association structures at temperatures well above the cloud point. The simulation results indicate that a long PEG-block portion separating the PNIPAAM blocks in the triblock copolymer reduces the tendency of the polymer forming interchain associations at elevated temperatures. Simulation shows that for a long PEG spacer, the copolymer moiety is rather extended even at high temperatures, whereas for copolymers with a short PEG length the thermoresponsive block copolymer undergoes a transition from an extended to a compact conformation.

Intramolecular and Intermolecular Association during Chemical Cross-Linking of Dilute Solutions of Different Polysaccharides under the Influence of Shear Flow

Effects of shear flow on intramolecular and intermolecular associations of dilute aqueous alkali solutions of dextran, hydroxyethylcellulose (HEC), and a hydrophobically modified analogue (HM-HEC) in the presence of a chemical cross-linker agent were characterized with the aid of viscometry and rheo-small-angle light scattering (rheo-SALS) methods. The picture that emerges at short times in the course of cross-linking of the polymer solutions under the influence of a constant shear rate is that HEC coils contract because of intramolecular cross-linking, whereas the HM-HEC species show an incipient association and the dextran molecules are unaffected. At longer times, interchain cross-linking of the polymers promoted the growth of large flocs, which were disrupted by shear forces when they were sufficiently large. These findings are novel, and both the building up of aggregates and disaggregation are well substantiated by the SALS results.

Temperature-Induced Aggregation Kinetics in Aqueous Solutions of a Temperature-Sensitive Amphiphilic Block Copolymer

Time effects for the temperature-induced association complexes in solutions of the thermoresponsive poly(N-isopropylacrylamide)-block-poly(ethylene glycol)-block-poly(N-isopropylacrylamide) (PNIPAAM(69)-b-PEG(23)-b-PNIPAAM(69)) copolymer that exhibit a lower critical solution temperature were studied by means of turbidimetry and dynamic light scattering (DLS). The DLS results clearly show that at temperatures below the cloud point (CP) unimers coexist with intermicellar structures, which contract as the CP is approached. At this stage, no time effect was detected. At temperatures above the CP, large association structures are formed, and these aggregates dominate the decay of the correlation functions. A novel time-dependent growth of the aggregates was observed over several hours. The growth of the clusters is strengthened as the temperature rises, and this feature is supported by the turbidity results and the reduced scattered intensity experiments. For a low polymer concentration, an initial growth of the clusters is observed, whereas at longer times the apparent hydrodynamic radius from DLS is virtually constant. The results from this work stress the importance to check possible time effects in solutions of thermosensitive copolymers as the cloud point is approached.