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Insulin is a peptide hormone with many physiological functions, besides its use in diabetes treatment. An important role of insulin is related to the wound healing process—however, insulin itself is too sensitive to the external environment requiring the protective of a nanocarrier. Polymer-based nanoparticles can protect, deliver, and retain the protein in the target area. This study aims to produce and characterize a topical treatment for wound healing consisting of insulin-loaded poly-DL-lactide/glycolide (PLGA) nanoparticles. Insulin-loaded nanoparticles present a mean size of approximately 500 nm and neutral surface charge. Spherical shaped nanoparticles are observed by scanning electron microscopy and confirmed by atomic force microscopy. SDS-PAGE and circular dichroism analysis demonstrated that insulin preserved its integrity and secondary structure after the encapsulation process. In vitro release studies suggested a controlled release profile. Safety of the formulation was confirmed using cell lines, and cell viability was concentration and time-dependent. Preliminary safety in vivo assays also revealed promising results.

Drug discovery has faced many challenges, and the diversity of natural products offers a huge number of opportunities for new drug findings. Most of the potential candidates result from plants since plants have several and interesting biological activities. However, the in vivo efficacy of such candidates is frequently limited due to their low absorption. Thus, enhancing the bioavailability of natural products through the improvement of their pharmacokinetic and biodistribution features, as well as their targeting efficacy, is a crucial step in the development of new therapeutic strategies.

Glycyrrhiza glabra L. is considered an important source of bioactive compounds. This study aimed at the development of an efficient solution for the treatment of oral candidiasis. Several extracts of Glycyrrhiza glabra L. were prepared using different solvents and their potential in vitro antifungal activity was assessed. Ethanolic extracts showed the most promising results against C. albicans. This extract was incorporated into mucoadhesive nanoparticles (PLA, PLGA and alginate), which were further included in an oral gel, an oral film and a toothpaste, respectively. The results showed that nanoparticles were successfully produced, presenting a mean size among 100–900 nm with high encapsulation efficiency. In vitro studies showed that the most bioadhesive formulation was the oral film with extract-loaded PLGA nanoparticles, followed by the toothpaste with extract-loaded alginate nanoparticles and the oral gel with extract-loaded PLA nanoparticles.

Candidiasis is by far the most common fungal infection in the oral cavity and locally antifungal drugs are generally considered the first line therapy. However, some antifungal drugs like Nystatin (Nys) have very short time in the target tissue, which generally it requires several daily applications. One of the solutions to overcome those issues could be the incorporation of Nys into small, protective and polymeric nanoparticles (NPs). In this work, unloaded NPs were prepared and fully characterized and implemented in toothpaste and an oral gel. The three-best unloaded system (smallest, stable, safe and bioadhesive) was selected for further studies. After loading, NPs presented a size range from 300 to 800 nm, high encapsulation efficiency (above 70%), prolonged release and high adhesion capacity to oral mucosa when compared with free Nys. Free Nys lost its activity when it was not encapsulated. The presented results suggested that these new bioadhesive systems could be considered as good candidates to improve the time residence of Nys in buccal mucosa, reducing the number of applications during the day, using alcohol free-formulations and easily included in the daily lives of the patients.

Pancreatic cancer is one of the most lethal cancers, with an extremely poor prognosis. The development of more effective therapies is thus imperative. Natural origin compounds isolated from Plectranthus genus, such as parvifloron D (PvD), have cytotoxic and antiproliferative activity against human tumour cells. However, PvD is a very low water-soluble compound, being nanotechnology a promising alternative strategy to solve this problem. Therefore, the aim of this study was to optimize a nanosystem for preferential delivery of PvD to pancreatic tumour cells. Albumin nanoparticles (BSA NPs) were produced through a desolvation method. Glucose cross-linking and bioactive functionalization profiles of BSA platform were elucidated and analysed using static lattice atomistic simulations in vacuum. Using the optimized methodology, PvD was encapsulated (yield higher than 80%) while NPs were characterized in terms of size (100&ndash, 400 nm) and morphology. Importantly, to achieve a preferential targeting to pancreatic cancer cells, erlotinib and cetuximab were attached to the PvD-loaded nanoparticle surface, and their antiproliferative effects were evaluated in BxPC3 and Panc-1 cell lines. Erlotinib conjugated NPs presented the highest antiproliferative effect toward pancreatic tumour cells. Accordingly, cell cycle analysis of the BxPC3 cell line showed marked accumulation of tumour cells in G1-phase and cell cycle arrest promoted by NPs. As a result, erlotinib conjugated PvD-loaded BSA NPs must be considered a suitable and promising carrier to deliver PvD at the tumour site, improving the treatment of pancreatic cancer.

Pancreatic cancer remains one of the most lethal cancers worldwide, with an extremely poor prognosis. This cancer is considered the 5th leading cause of cancer related death. The median survival after diagnosis is generally 2-8 months and five-year survival rate is less than 5%. In recent years, nanotechnology is emerging as a rising approach for drug delivery since it has opened up new landscapes in medicine through introduction of smart nanocarrier systems that can selectively deliver the therapeutic agent in a specific region and in appropriate levels, reducing the adverse side effects. This review covers the main delivery systems developed so far for anticancer drug delivery to the pancreas over a period of 20 years, from polymeric to lipidic-based nanosystems, with a particular emphasis on albumin as core material.

Candida species remain a significant cause of nosocomial bloodstream infections, associated with prolonged hospital stay in the ICU and high healthcare cost. The incidence of Candida is very high in certain risk groups of patients (AIDS, diabetes, cancer, etc.). Recent developments of nanotechnology have strongly contributed to the design of new multifunctional drug carriers that improve drug bioavailability through a controlled and prolonged release profile or even through a more specific targeted delivery of the antifungal agent. Those types of systems have strongly increased with a progressive generation of new structures, permitting the conjunction of new materials, biomolecules, physical and chemical techniques, for better outcomes. Nanotechnology shows expanded possibilities within the medical field and in the case of the yeast infections it may overcome several issues related with the fungal proliferation or higher inhibition of the pathogen causing the infection. This review covers a period of the most representative research of Candidiasis since 1993 to the present.

Aim: Parvifloron D is a natural diterpene with a broad and not selective cytotoxicity toward human tumor cells. In order to develop a targeted antimelanoma drug delivery platform for Parvifloron D, hybrid nanoparticles were prepared with biopolymers and functionalized with α-melanocyte stimulating hormone. Results/methodology: Nanoparticles were produced according to a solvent displacement method and the physicochemical properties were assessed. It was shown that Parvifloron D is cytotoxic and can induce, both as free and as encapsulated drug, cell death in melanoma cells (human A375 and mouse B16V5). Parvifloron D-loaded nanoparticles showed a high encapsulation efficiency (87%) and a sustained release profile. In vitro experiments showed the nanoparticles’ uptake and cell internalization. Conclusion: Hybrid nanoparticles appear to be a promising platform for long-term drug release, presenting the desired structure and a robust performance for targeted anticancer therapy.

Background: Photothermal response of plasmonic nanomaterials can be utilized for a number of therapeutic applications such as the ablation of solid tumors. Methods & results: Gold nanoparticles were prepared using different methods. After optimization, we applied an aqueous plant extract as the reducing and capping agent of gold and maximized the near-infrared absorption (650–900 nm). Resultant nanoparticles showed good biocompatibility when tested in vitro in human keratinocytes and yeast Saccharomyces cerevisiae. Gold nanoparticles were easily activated by controlled temperature with an ultrasonic water bath and application of a pulsed laser. Conclusion: These gold nanoparticles can be synthesized with reproducibility, modified with seemingly limitless chemical functional groups, with adequate controlled optical properties for laser phototherapy of tumors and targeted drug delivery.

Cannabinoids show promise for the treatment of various medical conditions such as emesis, anorexia, pain, cancer, multiple sclerosis, Parkinson's disease and glaucoma. However, their high lipohilicity and instability complicate their handling and dosing, and restrict their use as pharmaceuticals. The objective of the present work was to assess the feasibility of developing cannabinoid loaded poly-?-caprolactone (PCL) microparticles prepared by the oil-in-water emulsion-solvent evaporation technique as a suitable dosage form for their administration. Spherical microparticles with a size range of 20-50 ?m, and high entrapment efficiency (around 100%) were obtained. Cannabidiol (CBD) dissolved in the polymeric matrix of the microspheres was slowly released in vitro within 10 days. In vitro cell viability studies demonstrated the antitumoral activity of CBD released from microparticles. After 4 and 7 days of incubation, CBD in microspheres significantly inhibited the growth of MDA-MB-231 cells by 60% as compared to the 50% attained with free drug. The results suggest that PCL microparticles could be an alternative delivery system for long-term cannabinoid administration, showing potential therapeutic advantages over free drug.

Oral candidiasis is an important opportunistic fungal infection and polyenes and azoles are still the most used antifungal agents. However, the oral absorption resulting from most available treatments is generally poor and, consequently, a very high frequency of administrations of antifungal agents is strongly required. Therefore, the major challenge is to improve the retention of the antifungal agents in buccal mucosa, and the encapsulation into mucoadhesive systems may be considered as a possible strategy to achieve this objective. Three types of mucoadhesive polymeric nanoparticles (polylactic acid (PLA), polylactic-co-glycolic acid (PLGA) and alginate) were prepared using nystatin as model drug. The drug-loaded nanoparticles were then included in toothpaste, oral gel and oral films, respectively. The results demonstrated that the loaded nanoparticles were successfully produced, presenting a mean size between 300-900 nm and with a negative surface charge. Also, the determination of the encapsulation efficiency of all nanoparticles showed values above 70%. In terms of the in vitro mucoadhesion, the best formulation was the oral film loaded with the PLGA nanoparticles followed by the oral gel with PLA nanoparticles and thirdly the toothpaste with alginate nanoparticles. This was confirmed in an in vitro rinsing model with mucus producing HT29-MTX cells, where the percentage of nystatin retained to the cells after 40 min of simulated saliva flow was between 10-27% when formulations were used and only 4% for free nystatin. Further studies will include in vivo testing using animal models.

Topical glucocorticosteroids were incorporated into nanocarrier-based formulations, to overcome side effects of conventional formulations and to achieve maximum skin deposition. Nanoparticulate carriers have the potential to prolong the anti-inflammatory effect and provide higher local concentration of drugs, offering a better solution for treating dermatological conditions and improving patient compliance. Nanoparticles were formulated with poly-ϵ-caprolactone as the polymeric core along with stearic acid as the fatty acid, for incorporation of betamethasone-21-acetate. Oleic acid was applied as the coating fatty acid. Improvement of the drug efficacy, and reduction in drug degradation with time in the encapsulated form was examined, while administering it locally through controlled release. Nanoparticles were spherical with mean size of 300 nm and negatively charged surface. Encapsulation efficiency was 90%. Physicochemical stability in aqueous media of the empty and loaded nanoparticles was evaluated for six months. Drug degradation was reduced compared to free drug, after encapsulation into nanoparticles, avoiding the potency decline and promoting a controlled drug release over one month. Fourier transform infrared spectroscopy and thermal analysis confirmed drug entrapment, while cytotoxicity studies performed in vitro on human keratinocytes, Saccharomyces cerevisiae models and Artemia salina, showed a dose-response relationship for nanoparticles and free drug. In all models, drug loaded nanoparticles had a greater inhibitory effect. Nanoparticles increased drug permeation into lipid membranes in vitro. Preliminary safety and permeation studies conducted on rats, showed betamethasone-21-acetate in serum after 48 h application of a gel containing nanoparticles. No skin reactions were observed. In conclusion, the developed nanoparticles may be applied as topical treatment, after encapsulation of betamethasone-21-acetate, as nanoparticles promote prolonged drug release, increase drug stability in aqueous media, reducing drug degradation, and increase drug permeability through lipid membranes.

The present study describes the development of a new cyclosporine formulation based on polycaprolactone (PCL) microspheres (MS) prepared by the solvent evaporation method. Ternary phase diagrams were used to identify the domains where MS were formed. The application of central composite designs established the influence of several technological (stirring speed) and formulation factors (polymer and surfactant amounts, and organic solvent volume) on the size of PCL MS. Cyclosporine-loaded MS of a size around 2.5 microm were prepared and characterized. The stability of the systems, either alone or loaded with cyclosporine, stored at 8 degrees C and room temperature (RT) was assessed as well. Freeze-drying was evaluated as an alternative method to achieve long-term stability. The experimental design showed that the stirring speed and the organic phase volume were the only parameters significantly affecting the MS size. Experimental conditions selected to obtain CyA-loaded MS of 2.5 microm resulted in a high entrapment percentage (98.4 +/- 0.66%) with the drug dissolved or molecularly dispersed within the dense polymeric matrix of MS. After 12 months of storage at 8 degrees C and RT, PCL MS remained physically stable, although the crystallinity of the polymer increased by 35% upon storage at both temperatures. Freeze-drying studies revealed that MS could be successfully lyophilized in the absence of cryoprotectants without significant changes of the drug entrapment; however, the presence of at least 5% cryoprotectant was essential to keep the initial particle size. Therefore, a stable MS-based CyA formulation was easily prepared and characterized. This formulation offer the possibility of CyA administration through different routes.

Our experiments analyzed the uptake of free and nanoparticles (NP)-associated digitoxin (DGT) by rat glomerular mesangial cells. NP were prepared by the nanoprecipitation method using the biodegradable polyester, polycaprolactone (PCL). Prior to in vitro experiments, the systems were characterized by means of spectrofluorimetry, dynamic light scattering, and size exclusion chromatography (SEC). The loading efficiency was 80.30 +/- 1.03% of the initial DGT amount in the preparation, and the average particle size was 176 +/- 8 and 161 +/- 6 nm for DGT-NP and "empty" NP, respectively. SEC studies revealed noncovalent interactions among the different chemical compounds in the formulation. In vitro experiments were conducted at 37 degrees C and pH 7.5 by incubating "empty" NP, free DGT or DGT-NP (10 microg PCL/mL; 100 ng DGT/mL) with glomerular mesangial cells for 30 and 60 min. Uptake of DGT by the cells was favored by its incorporation into PCL-NP and showed time dependency. After 30 min of incubation, no significant differences of drug uptake were seen between free DGT (13.1 +/- 2.8%) or DGT-NP (17.4 +/- 4.9%); however, the uptake of DGT, when it was associated to the polymeric carrier, increased by approximately 2-fold (37.8 +/- 5.7%) at 60 min, whereas no significant changes were observed for free drug (20.0 +/- 6.8%). The pharmacologic activity of the drug was evaluated by measuring the planar cell surface area (PCSA). "Empty" NP, free drug, or DGT-NP did not produce significant variations on the PCSA as compared with control cells after a 30-min incubation. Nonetheless, DGT-NP reduced the PCSA to 82.51 +/- 8.42% of control values when the incubation lasted 60 min. The ability of cells to exclude the trypan blue dye and the leakage of lactate dehydrogenase into the medium revealed no signs of increased toxicity from incorporation of DGT into PCL-NP. Therefore, PCL-NP improved drug uptake by the cells without altering the pharmacologic activity and toxicity of the drug. Thus, they can be a useful approach to target drugs to the kidneys or the heart.

Therapeutic monitoring of Cyclosporine (CyA) by using area under the curve (AUC) from abbreviated kinetic profiles is of recent trend in clinical practice due to the potential improvement in transplant and clinical outcome with costs reduction in mind. Several papers describe successful use of the limited sampling strategy to predict AUCs in different transplant populations when treated with Sandimmun or Sandimmun Neoral. However, the same predictive potential is achieved for the latter formulation with lesser effort. The present paper describes the application of the limited sampling strategies to demonstrate the advantages of using CyA incorporated in polymeric nanoparticles (CyA-NP) as compared to two reference Sandimmun formulations which consisted of an emulsion of the oily solution in milk (SIM-EM) and a microemulsion (SIM-Neoral) formerly tried on rats. Two independent data batches were used: group 1 which included 36, 31 and 10 animals receiving SIM-EM, CyA-NP and SIM-Neoral, respectively, and group 2 made of nine and eight rats treated with SIM-EM and CyA-NP. Several limited sampling equations were derived for each formulation from group 1 by stepwise multiple linear regression. Statistical analysis disclosed that CyA concentrations 8 and 32 h after dose administration vouched for 88 and 69% variability in AUC (0-48 h) for CyA-NP and SIM-EM, respectively. When summed up, these two concentrations revealed nearly 97% of AUC (0-48 h) variability. CyA concentrations 8 h post-treatment with SIM-Neoral explained 89% variability in AUC (0-48 h). This value raised to 98% when a second CyA concentration (24 h) was introduced. The equations derived from group 1 were then employed to predict AUCs in group 2. CyA blood levels at 8 h post-treatment confirmed AUC for CyA-NP (r(2)=0.98) to be very precise and unbiased (error=1. 46%, interval -16.2 to 21.33%), while the results for SIM-EM obtained with the CyA concentration at 32 h were r(2)=0.93 plus error=5.71%, interval -44.33 to 105.94%. Similar results were obtained when the study period was reduced to 24 h. The use of these limited sampling models manifested the coincidence between CyA-NP and SIM-Neoral as well as the advantages of both formulations over SIM-EM when it comes to CyA monitoring.

The present paper describes the stability of poly (D, L-lactide-glycolide) nanoparticles (PLGA NP) and microspheres (MS), either alone or loaded with cyclosporine (CyA), stored at 8 degrees C and room temperature (RT). Freeze-drying of these formulations was evaluated as an alternative method to achieve long term stability. A significant polymer rupture was detected during PLGA MS preparation by solvent evaporation, which correlated with the stirring rates used for the formation of the primary emulsion. On the other hand, the polymer remained unchanged during NP formation. After 6 months of storage, PLGA NP of a size below 80 nm aggregated when stored at RT whereas no changes of particle size were observed for the remaining formulations and experimental conditions. Drug entrapment significantly increased by about 9.5% only during PLGA NP storage at RT. The PLGA molecular weight of NP dropped at RT being these changes related to the initial particle size and amount of CyA incorporated. The same effect was observed at 8 degrees C but only the particle size showed a significant influence. The drop of PLGA molecular weight observed during storage of MS was not dependent on the storage temperature but it was directly related to the molecular weights obtained after MS preparation. Freeze-drying studies revealed that it was not feasible to maintain the initial PLGA NP characteristics after reconstitution. On the other hand, MS lyophilized in the absence of cryoprotectants retained the drug initially entrapped; however, the presence of at least 5% cryoprotectant was essential to keep the initial particle size. Therefore, PLGA NP and MS show a significant instability when stored as suspensions. Freeze-drying offers a good alternative to stabilize polymeric MS but the preservation of the PLGA NP characteristics by freeze-drying needs for further investigations.

The dependency of cyclosporine (CyA) pharmacokinetics on the age (10 and 40-week-old) and the gender was studied in Wistar rats who were given 10 mg/kg dose of the drug intravenously. CyA levels in whole blood were analyzed by a specific fluorescence polarization immunoassay (Abbot TDx). Blood concentration vs time profiles were characterized by resorting to compartmental and noncompartmental methods. The first approach showed that the best model fitting experimental data was a three-compartment open model with first-order kinetics. It indicated that the drug undergoes extensive distribution in a wide variety of tissues. The mean half-lives corresponding to λ 1 and λ 2 phases coincided in all groups and lasted on average 0.22 and 4.30 h, respectively. The mean volume of distribution at steady-state depended mainly on the rats gender and age, indicating values of 2.64±0.37 l/kg for older males as compared to 2.03±0.29, 1.71±0.12, and 1.87±0.16 l/kg for young males and females and 40-week-old females, respectively. Drug elimination rates ranged from 0.019 to 0.123 h −1 and manifested a marked dependency on both, the gender and the age. The bioavailability (AUC 0−∞ ) was higher for males (81.06±8.31 and 139.62±41.34 μ g·h per ml vs 50.19±2.10 and 49.65±5.68 μ g·h per ml) while the systemic blood clearance (CL) was significantly lower for males than for females (85±19 and 109±3 ml/h per kg vs 198±9 and 205±29 ml/h per kg). No statistically significant differences were detected between compartmental and noncompartmental parameters by paired t -tests. Therefore, the results demonstrate that female rats clear CyA faster than males, probably due to differences in drug metabolism.

A rotatable central composite design (RCCD) was applied to optimize the preparation of cyclosporine-loaded poly D,L (lactide-glycolide) (PLAGA) nanoparticles (NP) and microspheres (MS) by solvent displacement and solvent evaporation techniques, respectively. The joint influence of needle gauge, polymer amount and the injection rates on the mean particle size, relative standard deviation (RSD), yield and drug encapsulation percentage in NP were evaluated. With regards to MS, the polymer amount and the stirring rate were evaluated. Scan electron microscopy of MS and NP showed spherical particles with a dense polymeric network in the first case. From the statistical analysis of data polynomial equations were generated. The mean particle size ranged from 50 to 150 nm for NP and from 1.5 to 30/pm for MS. Smallest nanoparticles (46 nm) were obtained by using the lowest polymer amounts, the highest injection rates and the lowest needle gauges (r2 = 0.9443). Under these conditions the drug entrapment percentage was maximum (85.2%), suggesting the drug might be entrapped and adsorbed on the nanoparticle surface. The relative standard deviation was only affected by the polymer amount (r2 = 0.8034) and the yield rose with the amount of PLAGA (r2= 0.9016). A very important increase in particle size (r2 = 0.9855), relative standard deviation (r2 = 0.9353) and encapsulation percentage (r2 = 0.9669) were observed for MS by decreasing emulsification stirring rates and increasing polymer amounts, the stirring rate being the most significant independent variable (α < 0.0001) in all cases. The highest experimental encapsulation value (97.69 ± 0.78%) correspond to samples prepared from 150 mg of polymer and a global stirring rate of 2000 rpm. By using response surface diagrams and the mathematical models proposed, it is possible to easily deduce experimental conditions to prepare NP and MS with the desired properties.

The solubility of cyclosporin A was determined in water and aqueous solutions of a surface active poly (oxyethylene)-poly(oxypropylene)-poly(oxyethylene) block copolymer (poloxamer 188 or pluronicR F68 (PF68)) at concentrations and temperatures ranging from 4 to 22 g/1 and from 10 to 50°C, respectively. The solubility behaviour was different between PF68 solutions and water. Solubility values indicated an exothermic heat of solution in each case except for PF68 solutions above 10 g/l at 37–50°C, where a change to endothermic heat of solution was detected. The slopes corresponding to water samples or 4g/l PF68 solutions at temperatures above 37°C do not statistically differ from zero. Aqueous solutions of the poloxamer 188 were also studied using frequency spectrum analysis (dynamic light scattering) and size exclusion chromatography. Below 40°C and 16 g/1 essentially invariant values for the hydrodynamic radius were found with broad polydispersities associated. Increasing temperature and poloxamer concentration, the hydrodynamic radius also increased and the systems showed a narrower size distribution, possibly due to micelle formation according to the closed association model.

Cyclosporin A (CyA) is a good candidate for incorporation in colloidal carriers such as nanoparticles (NP) that would diminish the adverse effects associated with its use under conventional pharmaceutical dosage forms and improve bioavailability after oral administration. In this study a composite rotational experimental design was used to evaluate the joint influence of five formulation variables: temperature of the aqueous phase, needle gauge, volume of the organic phase, and the amounts of polymer and surfactant on the micromeritic characteristics of the CyA-loaded NP obtained by the method of Fessi et al. The percentage of drug encapsulated in the NP was also evaluated for each formulation, and the yield, which was expressed as the ratio between the experimentally measured quantity of drug in the formulation and the theoretical content, was determined because CyA undergoes surface absorption. Potential variables such as stirring speed (500 rpm), final drug concentration (100 micrograms/mL), or injection rates (GRi = 0.379 mL/s) were maintained constant. The ANOVA corresponding to the experimental design showed that the amounts of polymer and surfactant, and the diameter of the needle used in the preparation of NP, significantly affected the percentage of entrapped drug (I2 = 0.8916). The mean particle size was significantly affected by all the formulation variables tested except for the amount of surfactant dissolved in the external aqueous phase (r2 = 0.9518). Neither the yield (mean value of 99.61%) nor the size distribution parameters (polydispersity and coefficient of variation) presented good correlation coefficients for the equations obtained, although some variables showed statistical significance. A second study was carried out to investigate the effects on the drug-loaded NP characteristics of varying the global injection rates (GRi) for the organic phase into the aqueous medium. The results showed a dramatic decrease in both particle size and drug incorporation in the carrier as the rate of mixing increased. From the results of both the experimental design and the second study, a theoretical model for nanoparticle formation is proposed that considers the most significant variables, and an empirical relationship to predict mean particle size is presented. Thus, particle size can be controlled by the injection rates (GRi), the needle gauge, and the polymer concentration.

Cannabinoids present an interesting therapeutic potential as antiemetics, appetite stimulants in debilitating diseases (cancer, AIDS and multiple sclerosis), analgesics, and in the treatment of multiple sclerosis and cancer, among other conditions. However, despite their high clinical potential, only few dosage forms are available to date. In this paper, the development of Δ(9)-tetrahydrocannabinol (THC) biodegradable microspheres as an alternative delivery system for cannabinoid parenteral administration is proposed. Tetrahydrocannabinol was encapsulated into biodegradable microspheres by the oil-in-water (o/w) emulsion solvent evaporation method. Several formulations were prepared using different drug:polymer ratios. The influence of antioxidant (α-tocopherol acetate) concentration on the release of THC from the microparticles was studied. Elevated process yield and entrapment efficiencies were achieved. The in vitro drug release studies showed that the encapsulated drug was released over a two week period. As THC has shown therapeutic potential as anticancer drug, the efficacy of the microspheres was tested on different cancer cell lines. Interestingly, the microspheres were able to inhibit cancer cell proliferation during the nine-day study period. All the above results suggest that the use of biodegradable microspheres would be a suitable alternative delivery system for THC administration.

Cannabinoids, the active components of marijuana and their derivatives, are currently investigated due to their potential therapeutic application for the management of many different diseases, including cancer. Specifically, Δ(9)-Tetrahydrocannabinol (THC) and Cannabidiol (CBD) - the two major ingredients of marijuana - have been shown to inhibit tumor growth in a number of animal models of cancer, including glioma. Although there are several pharmaceutical preparations that permit the oral administration of THC or its analogue nabilone or the oromucosal delivery of a THC- and CBD-enriched cannabis extract, the systemic administration of cannabinoids has several limitations in part derived from the high lipophilicity exhibited by these compounds. In this work we analyzed CBD- and THC-loaded poly-ε-caprolactone microparticles as an alternative delivery system for long-term cannabinoid administration in a murine xenograft model of glioma. In vitro characterization of THC- and CBD-loaded microparticles showed that this method of microencapsulation facilitates a sustained release of the two cannabinoids for several days. Local administration of THC-, CBD- or a mixture (1:1 w:w) of THC- and CBD-loaded microparticles every 5 days to mice bearing glioma xenografts reduced tumour growth with the same efficacy than a daily local administration of the equivalent amount of those cannabinoids in solution. Moreover, treatment with cannabinoid-loaded microparticles enhanced apoptosis and decreased cell proliferation and angiogenesis in these tumours. Our findings support that THC- and CBD-loaded microparticles could be used as an alternative method of cannabinoid delivery in anticancer therapies.

Gelatine gels and polyoxyethylene-polyoxypropylene (PluroniR) F-108 and F-127 gels were prepared at concentrations ranging between 5 and 25% (W/V), the former by dispersion at 37°C, the later by dispersion at 4°C. The viscosity, the gel-sol transition temperature and the “in vitro” release kinetics of these gels were compared as a first step for the elaboration of parented controlled release formulations. Phenolsulphonftaleine (PR) was used as a tracer.In all cases the viscosity increased with the rise in the concentration of gelatin (20 to 264 cps for 5 to 20%) or pluronic (260 and 1,520 cps for 20 and 25% F-108). The gel-sol transition temperature for gelatine gels was directly related to the concentration. On the contrary, for pluronic gels an inverse relation was observed, being the gel-sol transition temperature higher in copolymers with a large percentage of polyoxyethylene groups (30±0.2 °C for 25 % F-108). In both types of gels, a rise in pH and ionic strength decreased the gel-sol transit...

The commercially available formulations of cyclosporine (cyclosporin A, CyA) are associated with acute hemodynamic changes that result in high nephrotoxicity. Among colloidal vectors, nanoparticles (NPs) are receiving much attention as potential drug carriers that would avoid the therapeutic risks of conventional formulations. Two different mechanisms for obtaining polymeric NPs loaded with CyA were studied with regard to their preparation and physicochemical characterization. Isobutyl-2-cyanoacrylate monomer (IBCA) was polymerized, whereas poly-E-caprolactone (PCL, a preformed polymer) was precipitated; both reactions took place in an aqueous medium containing Pluronic F-68 (polyoxypropylene polyoxyethylene block copolymer) as a surface active agent. The encapsulation efficiencies were 78.49 +/- 5.87 and 84.85 +/- 5.02%, respectively, and they remained stable over a wide range of drug concentrations. The polymeric NP had average sizes of 81 +/- 25 and 95 +/- 25 nm for poly-IBCA and PCL, respectively, as confirmed by photon correlation spectroscopy. Poly-IBCA NPs were built from oligomers with molecular weights of 157 to 2644 that joined to form a polymeric nanomatrix. In vitro activity of the drug and the carrier was tested by inhibition of lymphocyte proliferation induced by Concanavalin A. Drug-loaded PCL NPs and free CyA inhibited lymphocyte proliferation by 91.40 and 86.19%, respectively. However, drug-free NPs also exhibited statistically significant (p0.05) immunosuppressive activity.

Polyoxyethylene-polyoxypropylene surface-active block copolymers (Pluronic ® ) were evaluated as a vehicle for subcutaneous administration of drugs using a phenolsulfophthalein (PR) as a tracer. The type of Pluronic ® copolymer (F108 or F127), and their concentrations, and the effect of solutes (NaOH, NaCl or PR) on gelation properties were studied. Sodium hydroxide and sodium chloride decreased the gel-sol transition temperature, whereas the opposite effect was observed with PR. The ‘in vitro’ release rates obtained for PR were inversely proportional to the concentration of Pluronic used and a zero-order release rate was observed in all preparations assayed. Pluronic ® F127/PR preparations were administered subcutaneously (SC) to Wistar rats and PR plasma levels were compared with those reached after SC or intravenous (i.v.) administration of a PR aqueous solution. The gel formulation produced a sustained plateau level within 15 min that lasted 8–9 h. In vivo data analysis was performed with the JANA and PCNONLIN computer programs. The best fittings for experimental data from Pluronic gels were obtained using a zero-order input and first-order output two-compartment model. The results obtained suggest that PF127 aqueous gels may be of practical use as a vehicle for SC administration of drugs.

Aim: The nephrotoxicity and immunosuppressive ability of cyclosporine (CyA) incorporated into polycaprolactone nanoparticles (CyA-NP) was assessed in vitro and in vivo and compared to the effects caused by free drug (Sandimmun®). Methods: The in vivo study included four groups (12 Wistar rats each) receiving oral CyA (10 mg/kg/day for 3 days) as an emulsion of Sandimmun in whole milk or CyA-NP and equivalent doses of empty NP or cremophor® in milk as controls. CyA concentrations in blood, urine, liver, spleen and kidney at 24 h post-dosing were measured by fluorescence polarization immunoassay (FPIA). The nephrotoxicity induced by each drug treatment was determined by measuring creatinine plasma levels, malonyl dialdehyde production, and H2O2 and reduced glutathione contents in glomeruli. On the other hand, the immunosuppressive effect was estimated in vivo by incubating lymphocyte suspensions obtained from CyA-, CyA-NP- and control-treated rats, as well as in vitro on lymphocyte suspensions from non-treated healthy animals. Results: Significantly higher blood, urine and tissue levels were achieved with CyA-NP compared to free CyA. However, no changes in creatinine plasma levels were detected due to either CyA or CyA-NP treatment. Only the production of H2O2 in the glomeruli exhibited a significant increase as compared to control groups, but no differences could be ascribed to the different drug treatments. In vivo, the immunosuppressive activity was also comparable for both drug treatments. In contrast, CyA-NP showed a better drug uptake in vitro at concentrations above 25 μM. No immunosuppression was detected in control groups. Conclusion: NP improve the oral bioavailability of CyA and its uptake by lymphocytes in vitro above 25 μM. On the contrary, specific immunosuppression and adverse effects were not simultaneously increased. Further studies are needed to clarify the results.

The present study was geared at identifying the conditions to stabilize poly (D,L-lactic-glycolic) (PLGA) and polycaprolactone (PCL) nanoparticles (NP) by freeze-drying with several cryoprotective agents. Differential scanning calorimetry and freeze-thawing studies were used to optimize the lyophilization process. These studies showed that all samples were totally frozen at -45 degrees C and evidenced the necessity of adding sucrose, glucose, trehalose or gelatine to preserve the properties of NP regardless of the freezing procedure. However, only 20% sucrose and 20% glucose exerted an acceptable lyoprotective effect on PLGA and PCL NP, respectively. Nonetheless, the final to initial size ratios ( approximately 1.5) indicated that particle size was slightly affected in both cases. In vivo studies with CyA-loaded PCL NP whose sizes matched those obtained after NP preparation (100 nm) and after being lyophilized (160 nm) showed that the changes of particle size might have some relevance on drug pharmacokinetics. The MRT was significantly (P0.05) modified after an oral CyA dose of 5 mg/kg and the treatment with 160-nm sized CyA-loaded NP produced a higher drug partition into the liver of Wistar rats potentially affecting the toxic and immunosuppressive profile of the drug. Therefore, although the particle size changes induced by NP lyophilization were slight, they need to be carefully evaluated and cannot be neglected.

Cyclosporine (CyA) was incorporated into polycaprolactone nanoparticles (PCL-NP) in order to increase its oral bioavailability and to control drug distribution, thereby potentially reducing its toxicity. Prior to in vivo studies, the carrier was optimized and characterized by using different techniques. Light scattering (LS) and transmission and scanning electron microscopy (TEM and SEM) indicated the NP were spherical in shape with a mean size of approximately 100 nm. The influence of the solvent evaporation conditions and the polymer and drug amounts on CyA incorporation was established in order to optimize drug loading. When acetone and excess water were removed at constant temperature, no aggregation phenomena were observed. A value of 180 mg PCL was the minimum polymer amount necessary to encapsulate 95% of the drug initially added to the preparation. Under these conditions, HPLC analysis revealed that approximately 130 microg CyA per mg PCL were incorporated for a total CyA concentration of 2.5 mg/ml, being part of the drug adsorbed onto the particle surface. No structural changes or instability of the components during NP preparation were detected by gel permeation chromatography (GPC) and differential scanning calorimetry (DSC). However, GPC studies showed a competition between poloxamer and CyA for adsorption onto the carrier. In addition, DSC results suggested that at least part of the drug associated to NP remained in its crystal form. Therefore, CyA-loaded NP were easily manufactured and characterized and allow for the administration of therapeutic drug doses to experimental animals.

In a previous study we demonstrated the dependency of cyclosporine (CyA) pharmacokinetics on the age and gender of Wistar rats given 10 mg/kg intravenously. The present study has been conducted under the same experimental conditions (10 mg/kg as a single intravenous dose) to identify the mechanisms behind such differences. On the one hand, drug distribution was studied by measuring the CyA levels in blood, liver, kidney, spleen, adipose tissue, skin and muscle at 48 h post-treatment by using a specific fluorescence polarization immunoassay (m-FPIA, Abbott Laboratories). Drug blood and tissue levels in male rats were significantly higher than the female counterparts except for adipose tissue where the concentrations were 2-fold higher in females. In males, the highest CyA concentrations were observed in the liver, followed in rank order by kidney and spleen, fat, skin, muscle, then blood. On the contrary, females showed the highest drug levels in fat, followed by liver, kidney, spleen, skin, muscle and blood. Age exerted a significant influence on CyA tissue levels in males but no effect was observed in females. The potential differences in drug metabolism were established by measuring (HPLC) the amounts of CyA and its metabolites accumulated in faeces after hepatic biotransformation and biliary excretion. The amounts of circulating metabolites in blood as well as those accumulated and excreted in the liver and urine were also estimated by using specific (m-FPIA) and non-specific fluorescence polarization immunoassay (p-FPIA, Abbott Laboratories), respectively. The analysis of faeces revealed that AM9 was the major identified metabolite with females excreting lower amounts of unchanged CyA than males. In addition, the comparison of the AUC values corresponding to parent CyA and total CyA derivatives suggested that blood concentrations of CyA metabolites were higher in females indicating higher biotransformation rates. Therefore, both CyA distribution and metabolism are responsible for the sex-associated differences in drug pharmacokinetics previously found in rats.

The aim was to evaluate the long-term stability of cyclosporin A-loaded nanoparticle suspensions, stored at 8 and 25 degrees C. The stability of freeze-dried samples was also investigated. Nanoparticles (NP) of poly-sigma-caprolactone (P sigma CL), a biodegradable polymer, were obtained by a modified nanoprecipitation method. A central composite experimental design was used to investigate the simultaneous effect of technological factors (temperature of the aqueous phase and needle gauge) and formulation variables (volume of acetone and the amount of polymer and surfactant). The effect of these variables on the stability of the 100-220 nm particles obtained was evaluated. The percentage of cyclosporin A (CyA) encapsulated in the NP suspensions stored at 8 and 25 degrees C for at least 3 months remained unaltered. Moreover, there was no change in the size of NP. After 4 months storage, the physical stability of the preparation was affected. NP aggregates could be observed by light microscopy. Reconstituted freeze-dried preparations showed a mean increase of 1% in the incorporated drug and also a considerable increase in mean size and size distribution. Additional experiments investigated the effect of freezing temperature (-70 and -196 degrees C) and of 5, 10 and 20% (w/v) cryoprotector (mannitol, sorbitol, glucose and threalose) on 100 nm particles. The addition of glucose and threalose at concentrations > 10% permitted adequate reconstitution of the freeze-dried product with conservation of the encapsulated CyA.