Cytotoxicity and anti-inflammatory activity of cyclosporine A loaded PLGA nanoparticles for ocular use.

Cyclosporine A loaded poly(lactide-co-glycolide) nanoparticles were prepared using the o/w emulsification solvent evaporation method and the effect of four preparation parameters on particle size and zeta potential was investigated. Release properties of the nanoparticles were examined and in vitro experiments were performed in order to evaluate the cytotoxicity and anti-inflammatory activity of the nanoparticles developed. Particle sizes varied between 191 and 303 nm depending on the different preparation parameters and all nanoparticle dispersions were monodisperse. The nanoparticles showed negative zeta potential values varying between -16 and -35 mV and 57 to 70 % of the amount of loaded cyclosporine A was released after 24 h. None of the nanoparticle formulations showed significant cytotoxicity compared to the negative control using human epithelial cells (HaCaT). Cyclosporine A incorporated in the various nanoparticle formulations retained its anti-inflammatory activity as significant suppression of interleukine-2 secretion in concanavalin A stimulated Jurkat T cells was measured. As the overall influence of the freeze-drying process on the characteristics of nanoparticles was limited, trehalose and carnitine should be preferred as cryoprotectants in ocular formulations for treatment of dry eye disease.

PLGA nanoparticles and nanosuspensions with amphotericin B: Potent in vitro and in vivo alternatives to Fungizone and AmBisome.

This paper describes the development of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) and nanosuspensions with the polyene antibiotic amphotericin B (AmB). The nanoformulations were prepared using nanoprecipitation and were characterised with respect to size, zeta potential, morphology, drug crystallinity and content. Standard in vitro sensitivity tests were performed on MRC-5 cells, red blood cells, Leishmania infantum promastigotes and intracellular amastigotes and the fungal species Candida albicans, Aspergillus fumigatus and Trichophyton rubrum. The in vivo efficacy was assessed and compared to that of Fungizone and AmBisome in the acute A. fumigatus mouse model at a dose of 2.5 and 5.0mg/kg AmB equivalents. The developed AmB nanoformulations were equivalently or more effective against the different Leishmania stages and axenic fungi in comparison with the free drug. The in vitro biological activity, and especially hemolytic activity, clearly depended on the preparation parameters of the different nanoformulations. Further, we demonstrated that the superior in vitro antifungal activity could be extrapolated to the in vivo situation. At equivalent dose, the optimal AmB-loaded PLGA NP was about two times and the AmB nanosuspension about four times more efficacious in reducing the total burden than AmBisome. The developed AmB nanomedicines could represent potent and cost-effective alternatives to Fungizone and AmBisome.

Intracellular drug delivery in Leishmania-infected macrophages: Evaluation of saponin-loaded PLGA nanoparticles.

Drug delivery systems present an opportunity to potentiate the therapeutic effect of antileishmanial drugs. Colloidal carriers are rapidly cleared by the phagocytic cells of the reticuloendothelial system (RES), rendering them ideal vehicles for passive targeting of antileishmanials. This paper describes the development of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) for the antileishmanial saponin ß-aescin. NPs were prepared using the combined emulsification solvent evaporation/salting-out technique. Confocal microscopy was used to visualise the internalisation and intracellular trafficking of fluorescein- and nile red-labelled PLGA NPs in J774A.1 macrophages infected with GFP-transfected Leishmania donovani. The in vitro activity of aescin and aescin-loaded NPs on L. infantum was determined in the axenic model as well as in the ex vivo model. The developed PLGA NPs were monodispersed with Z(ave)<300 nm, exhibited negative zeta potentials and had relatively high drug loadings ranging from 5.80 to 8.68% w/w PLGA. The fluorescent NPs were internalised by the macrophages and trafficked towards the lysosomes after 2 h in vitro incubation. Co-localisation of the NPs and the parasite was not shown. A two-fold increase in activity was observed in the ex vivo macrophage model by encapsulating ß-aescin in PLGA NPs (IC(50), 0.48-0.76 µg/mL vs. 1.55 ± 0.32 µg/mL for the free drug).

Mixture designs in the optimisation of PLGA nanoparticles: influence of organic phase composition on ß-aescin encapsulation.

The objective of this study was to enhance the encapsulation of the antileishmanial saponin aescin in poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs). We prepared the NPs by the O/W and W/O/W combined emulsification solvent evaporation/salting-out technique and investigated the influence of organic phase composition on the NPs' size, zeta potential and entrapment efficiency (EE%) using mixture designs. The obtained NPs were monodispersed with Z(ave)<300 nm and exhibited negative zeta potentials. For the single emulsion, the co-solvent concentration was shown to be the primary determinant of drug entrapment. The EE% increased from 14% to 22% by decreasing the amount of DMSO from 80% to 25% (v/v) in the organic polymer solution. For the double emulsion, EE% was 22% on average and independent of the organic phase composition. The double-emulsion technique did not enhance the aescin encapsulation as expected due to its amphiphilic nature. The optimised aescin-loaded NPs meet the requirements for further in vitro activity tests.

PLGA nanoparticles loaded with the antileishmanial saponin ß-aescin: factor influence study and in vitro efficacy evaluation.

Colloidal carriers are known to improve the therapeutic index of the conventional drugs in the treatment of visceral leishmaniasis (VL) by decreasing their toxicity whilst maintaining or increasing therapeutic efficacy. This paper describes the development of poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) for the antileishmanial saponin ß-aescin. NPs were prepared by the W/O/W emulsification solvent evaporation technique and the influence of five preparation parameters on the NPs' size (Z(ave)), zeta potential and entrapment efficiency (EE%) was investigated using a 2(5-2) fractional factorial design. Cytotoxicity of aescin, aescin-loaded and blank PLGA NPs was evaluated in J774 macrophages and non-phagocytic MRC-5 cells, whereas antileishmanial activity was determined in the Leishmania infantum ex vivo model. The developed PLGA NPs were monodispersed with Z(ave)<500 nm and exhibited negative zeta potentials. The process variables 'surfactant primary emulsion', 'concentration aescin' and 'solvent evaporation rate' had a positive effect on EE%. Addition of Tween 80 to the inner aqueous phase rendered the primary emulsion more stable, which in its turn led to better saponin entrapment. The selectivity index (SI) towards the supporting host macrophages increased from 4 to 18 by treating the cells with aescin-loaded NPs instead of free ß-aescin. In conclusion, the in vitro results confirmed our hypothesis.

In vitro evaluation of gentamicin- and vancomycin-containing minitablets as a replacement for fortified eye drops.

OBJECTIVE: Ocular bioadhesive minitablets containing gentamicin and vancomycin were developed using different powder mixtures of pregelatinized starch and Carbopol (physical or cospray-dried mixtures). METHODS: Drug content, antimicrobial activity, and radical formation of the powders used for tablet preparation were evaluated immediately and 30 days after gamma sterilization. Tablet properties and in vitro drug release from the sterilized minitablets were determined. Storage stability of vancomycin and gentamicin in sterilized bioadhesive mixtures was examined by LC-UV/MS and a microbiological assay, respectively. A bioadhesive powder mixture containing only vancomycin was irradiated by X electron-magnetic radiation to evaluate vancomycin stability following sterilization through irradiation. RESULTS: The antimicrobial activity of gentamicin against Staphylococcus epidermidis was not altered in comparison to nonsterilized formulations. Only after an overkill dose of 50 kGy, the concentration of vancomycin decreases to an extent that was pharmaceutically significant. No significant difference in radiation stability between drug substance and product (i.e., powder mixture) was observed. A shift in stability profile was not observed at 6 weeks after irradiation. All other degradation products were present only in small quantities not exceeding 1.0%. The in vitro drug release from the minitablets prepared with physical powder mixtures of pregelatinized starch and Carbopol® 974P NF (96 : 4) was faster compared to the cospray-dried mixtures of starch with Carbopol® 974P NF (ratio: 95:5 and 85:15). The electron paramagnetic resonance signals of the radicals formed during sterilization were still visible after storage for 30 days. The slug mucosal irritation test indicated mild irritation properties of the bioadhesive powder mixtures although no tissue damage was observed.

Evaluation of the in vivo behaviour of gentamicin sulphate ocular mini-tablets in ponies.

The in vivo behaviour of 5% gentamicin sulphate ocular mini-tablets (2-mm diameter, 6.525 mg weight) was compared with gentamicin eye drops in six ponies. Two mini-tablets were inserted on the bulbar conjunctiva of the right eye while a similar dose of gentamicin was administered via eye drops in the left eye. Irritation induced by the mini-tablets and the eye drops was evaluated using a visual analogue scale (0-10). Tears were sampled with ophthalmologic absorption triangles for 1 min for the determination of the concentration of gentamicin sulphate using a microbiological plate diffusion method. Irritation induced by the tablets was minor and clinically acceptable (overall median score of 1.7 +/- 1.4). Eye drops induced a sharp increase in gentamicin sulphate concentration (364.4 microg/mL after 5 min) followed by a fast decline (10.8 microg/mL after 60 min). The increase in concentration induced by the ocular mini-tablets was less pronounced (up to 56.2 microg/mL after 30 min) and followed by a gradual decrease; the concentration remained above 15 microg/mL for 8 h. Ocular 5% gentamicin sulphate mini-tablets are clinically well-tolerated in ponies, assuring a constant concentration in the tears for at least 8 h.

Influence of the concentrations of liposomes and a submicron emulsion on the rheological properties of a topical gel.

Two different kinds of liposomes and a submicron emulsion were added to a topical hydrogel consisting of alginates. The rheological characteristics of the gels after various dilutions were investigated. The various preparations studied exhibited a similar rheological behaviour. No important interactions were observed by mixing the hydrogel with liposomes or a submicron emulsion. The viscoelastic properties and gel state were preserved, only a decrease in consistency was measured.

Characterization and in vivo evaluation of ocular minitablets prepared with different bioadhesive Carbopol-starch components.

The purpose of this study was to evaluate different bioadhesive ocular formulations based on drum dried waxy maize starch (DDWM), Amioca starch and Carbopol 974P. The concentrations of Carbopol 974P in the mixtures varied between 5 and 25% (w/w). The rheological properties of the non-sterilized and gamma-irradiated physical blends of Carbopol 974P with either DDWM or Amioca were compared to those of the corresponding co-spray dried Amioca starch/Carbopol powders. Higher viscosity or consistency values were measured for sterilized co-spray dried powder mixtures containing an amount of Carbopol 974P equal or above 15% (w/w) compared to the physical blends. Sustained release minitablets (2 mm, 6 mg), consisting of sodium fluorescein as model drug and the bioadhesive powders, were manufactured at a compression force of 1.25 kN. Afterwards, the tablets were sterilized with gamma-irradiation. The amount of Carbopol in the co-spray dried powder mixtures on the one hand and gamma-irradiation on the other hand had no significant influence on the crushing strength and friability of the minitablets evaluated. However, these two factors affected the in vitro release properties of the minitablets. The slowest release was obtained with tablets containing 25% Carbopol 974P, which unfortunately possess mucosal irritating properties. By using co-spray dried Amioca with 15% (w/w) Carbopol 974P, a slower release can be achieved compared to the physical mixtures of DDWM or Amioca starch with Carbopol 974P. Moreover, this ocular formulation is very promising and is preferred, as it did not cause any mucosal irritation and released the model drug for at least 12 h, after application in the fornix.

Rheological evaluation of dispersions, prepared with different non- and gamma-irradiated bioadhesive cospray dried powder mixtures.

Various polymer mixtures were used to prepare gels: native drum dried waxy maize starch, Amioca starch, Carbopol 974P NF and powder mixtures obtained by cospray drying Amioca starch and Carbopol 974P NF, and by blending Carbopol 974P NF with Amioca starch or drum dried waxy maize starch. Oscillatory rheology was employed to investigate the influence of gamma-irradiation, but also of the different compositions (i.e. Carbopol 974P NF varied from 5 to 25% w/w in the powder mixtures tested) and the preparation procedures of the powder mixtures on the viscoelastic properties of the polymer dispersions. The rheological data showed that gamma-irradiation has a significant influence on the rheological behaviour of the polymers dispersed. More elastic properties were obtained when the concentration of Carbopol 974P NF was increased. The dispersions containing at least an amount of 15% Carbopol 974P NF (w/w) are mainly elastic. Contrary to cospray drying the powders, blending the powders leads to higher elastic and gel properties of the dispersed polymers.

Effect of different sterilisation methods on the properties of bioadhesive powders and ocular minitablets, and clinical evaluation.

The purpose of this study was to evaluate the influence of gamma-irradiation and dry heat sterilisation on the properties of a bioadhesive powder mixture containing ciprofloxacin and its corresponding ocular minitablets. The molecular weight characteristics of drum dried waxy maize starch (DDWM), employed as major component of the bioadhesive formulation, the decay kinetics of radicals, the rheological properties of the bioadhesive polymers and the microbial activity of ciprofloxacin were studied. The influence of the different sterilisation methods on the characteristics of the ocular minitablets was investigated by measuring the crushing strength, the friability, and the in vitro release of ciprofloxacin from the minitablets. Finally, the clinical value of the selected sterilised minitablets was evaluated in seven healthy volunteers. Both sterilisation methods similarly affected the properties of the bioadhesive formulation by inducing stable radicals and decreasing the molecular weight of DDWM, although no changes in the microbiological activity of ciprofloxacin were measured. An obvious influence of both sterilisation methods was observed in the in vitro release study. The crushing strength and friability of the minitablets were not significantly influenced by gamma-irradiation. Based on these data, gamma-irradiation was more adequate as sterilisation method for the bioadhesive ocular minitablets than dry heat sterilisation, because it affected the least the physical properties of the minitablets. Therefore, the gamma-sterilised minitablets were selected for an in vivo evaluation in seven volunteers. The concentration of ciprofloxacin in the tear film remained above its MIC value for the most common ocular pathogens for at least 8 h. Consequently, the gamma-irradiated minitablets containing ciprofloxacin can be considered as a promising formulation to treat bacterial keratitis and conjunctivitis.

Rheological evaluation of the influence of sterilisation on ocular gels using an experimental design.

Different rheological characterisation methods are employed to investigate the influence of the sterilisation method (autoclaving), the polymer concentration (0.50, 0.75 and 1.00%) and the dispersing medium (i.e. isotonic phosphate buffer and mannitol solution) on Carbopol 974 P NF dispersions, used as ocular gels. The evaluation of the different polyacrylic acid-based dispersions was performed using flow and oscillatory shear measurements, and herewith an experimental design was set up. The rheological data show that the choice of the dispersing medium has a significant influence on the rheological behaviour of the ocular gels prepared. The dispersions in mannitol exhibit higher elastic properties than those made in the phosphate buffer solution (PBS). Sterilisation increases only the elastic properties of the 0.50% (w/w) Carbopol 974 P NF/PBS dispersion but has no influence on the other dispersions prepared.

Characterization and in vivo evaluation of ocular bioadhesive minitablets compressed at different forces.

The influence of the compression force on the physical properties, the in vitro release and the in vivo behavior of ocular minitablets is evaluated in the present study. The bioerodible minitablets (Ø 2 mm, 6 mg) were produced at different compression forces. The crushing strength, friability, water uptake, hydration and swelling of the minitablets both in vitro as well as in vivo after application in the cul-de-sac were evaluated. The friability remained below 1% only for the minitablets made at 0.500 and 0.750 kN. The crushing strength measured was 3.53+/-0.98, 12.34+/-1.69 and 18.64+/-2.37 N for minitablets made at 0.250, 0.500 and 0.750 kN, respectively. The full hydration time equalled 20 and 30 min for minitablets compressed at 0.250 kN and 0.500-0.750 kN, respectively. Increasing the compression force resulted in a decreased swelling capacity. The in vivo release was evaluated in healthy volunteers using a non-invasive method to measure the apparent sodium fluorescein concentration in the tearfilm-cornea compartment as a function of time. The longest residence time of the fluorescent tracer at the administration site was obtained by the minitablets compressed at 0.750 kN. The in vitro release was evaluated with three different dissolution methods: the reciprocating cylinder method, vials in an oscillatory shaking bath and a static method with vials. The best correlation with the in vivo behavior of the matrix minitablets was obtained with the shaking bath method.

In vitro assessment of the interaction mechanisms between mucin and a cationic cellulosic derivative by rheological methods using an experimental design.

An oscillatory rheological method is used for the determination of the mucoadhesive properties of a cationic cellulosic derivative (JR-30M) proposed for ocular dosage forms and dry eye viscous solutions. An experimental design was set up to study the influence of the concentrations of the polymer and mucin on mucoadhesion. From the rheological data it can be deduced that the interactions between mucin and JR-30M are mainly based on physical entanglements and less on ionic interactions.