Biocompatibility studies of intravenously administered ionic-crosslinked chitosan-BSA nanoparticles as vehicles for antitumour drugs.

In this study, a new alternative of ionic crosslinked nanoparticles (NPs) based on chitosan (C) and bovine serum albumin (A; BSA) was evaluated as drug delivery system for antitumour compounds (doxorubicin hydrochloride as a model). The different responses to the pH of the medium were determined by the electrostatic interactions induced by each polymeric combination (C50/A50; C80/A20; C20/A80). NPs revealed a nanoscale size (167-392 nm) and a positive net charge (12-26 mV), modulated by doxorubicin (DOX) loading. Drug loading capacity was higher than 5.2 ±â€¯1.8 µgDOX/mgNP (Encapsulation efficiency = 34%), and an initial burst release was followed by a sustained delivery. Cellular uptake assays confirmed the entry of NPs in three human tumor cells (MCF7, T47D and Hela), triggering antioxidant responses (superoxide dismutase, catalase, glutathione reductase and total glutathione content) in those cells. This was also consistent with the decreased in IC50 values observed after the incubation of these cells with C20/A80-DOX and C50/A50-DOX NPs (1.90-3.48 µg/mL) compared with free DOX (2.36-6.025 µg/mL). In vivo results suggested that the selected proportions of chitosan-BSA created nonhemolytic and biocompatible stable NPs at the selected dose of 20 mg/kg. Despite the different formulations, this study demonstrated that these NPs could serve as safe drug carriers in further in vivo investigations.

Biocompatibility evaluation of pH and glutathione-responsive nanohydrogels after intravenous administration.

Nanotoxicology has emerged as an important subdiscipline of nanotechnology due to the new healthy risks associated with the use of nanosystems for therapy and diagnostic. The biocompatibility of four stimuli-responsive nanohydrogel (NG) formulations based on different proportions of N-isopropylacrylamide (NIPA), N-hydroxyethyl acrylamide (HEAA) and 2-acrylamidoethyl carbamate (2AAECM), and cross-linked with N,N-cystaminebisacrylamide (CBA) or N-methylenebisacrylamide (NMBA) has been evaluated after intravenous injection in Wistar rats. All nanohydrogels were pH-sensitive, and those with CBA were also glutathione-responsive. Haematological and coagulation parameters revealed most nanogel formulations did not cause modification, only the NHA 80/15/5-CBA formulation induced a transitory light increase in platelets. Prothrombin time was in the reference normal range, there were no modifications of fibrinogen concentration and an increase in antithrombin III was observed on the last day of the study. Blood biochemical parameters such as AST, ALT, ALP, BUN, and creatinine were in the standard range for rats. The activity of enzyme antioxidant defences (SOD, CAT and GSSG-R) and total glutathione were evaluated in liver, kidney and spleen samples. Nanohydrogels cross-linked with the disulphide reducible CBA-cross-linker caused a decrease in GSSG/GSH content and an increase in GSSG-R activity in the spleen. The antioxidant response is also reflected by modifications of SOD activity in liver and kidney of NHA 80/15/5-CBA and NHA 80/10/10-NMBA groups. Histology showed no tissue damage, inflammation or morphological change in liver, kidney and spleen. Overall, the results demonstrated modifications of antioxidant defences; however, no acute or very significant changes in biomarkers of liver or kidney damage were observed.

Assessment of sequential combination of 5-fluorouracil-loaded-chitosan-nanoparticles and ALA-photodynamic therapy on HeLa cell line.

BACKGROUND: Natural polymers are used as components of nanoparticles (NPs) for drug delivery, as they provide targeted, sustained release and biodegradability. The purpose of this study was to increase the efficacy of the photodynamic therapy (PDT) by the combination of 5-aminolevulinic acid (ALA) with 5-fluorouracil-loaded-chitosan-nanoparticles (5-Fu-CNPs). METHODS: Nanoparticles based on chitosan (CNPs) were synthesized by the ionic crosslinking method via the TPP addition. 5-Fluorouracil (5-Fu), a first-line anticancer drug, was loaded into these 5Fu-CNPs, and they were assayed as controlled delivery formulation. HeLa cells were incubated in the presence of 5Fu-CNPs for 24h, next ALA was added to the culture medium and 4h later, to complete the PDT, light irradiation took place. Analysis of cell viability, reactive oxygen species (ROS) production, observation of the apoptosis by fluorescence microscopy followed by analysis of caspase-3 activity were carried out. RESULTS: Spherical 5Fu-CNPs with a mean diameter of 324±43nm, were successfully synthesized and characterized by TEM and DLS. 5-Fu incorporation was achieved successfully (12.3µg 5Fu/mg CNP) and the maximum 5-Fu release took place at 2h. The combined administration of 5Fu-CNPs and PDT mediated by ALA (ALA-PDT) led to an improved efficacy of the antineoplastic treatment by generation of great cytotoxicity inducted through an increased ROS production. HeLa cells were destroyed by apoptosis through activation of caspase pathway. CONCLUSIONS: This study proves that combination therapy (photodynamic "ALA"+chemical "5-Fu"+immunoadjuvant "chitosan") may be an effective approach for the treatment of cancer.

Bioresponsive nanohydrogels based on HEAA and NIPA for poorly soluble drugs delivery.

Environmentally sensitive hydrogels have gained considerable attention in recent years as one of the most promising drug delivery systems. In the present study, two new formulations of pH and temperature stimuli-responsive nanogels (NGs) based on poly-N-isopropylacrylamide (NIPA), N-hydroxyethyl acrylamide (HEAA) and tert-butyl 2-acrylamidoethyl carbamate (2AAECM) were synthesized and evaluated for passive targeting of paclitaxel (PTX). Nanogels were prepared by microemulsion polymerization method using N-methylenebis(acrylamide) (NMBA) as crosslinking agent. TEM images and DLS results showed nanosized spherical hydrogels. FTIR spectra confirmed the synthesis of nanogels by radical polymerization among vinyl groups of monomers. The PTX loading capacity, encapsulation efficiency and in vitro release were analyzed by HPLC. The cumulative release profile of the PTX-loaded nanohydrogels within 144h showed a faster drug release at acid pH (pH 5), similar to those observed at lysosome compartment, whereas a fewer PTX amount was released from NGs at pH similar to plasma levels. Cellular uptake assays revealed rapid penetration and intracellular accumulation of those nanogels in MCF7, HeLa and T47D cells after 48h incubation. MTT assays showed cell viability dependence on concentration and time incubation. Finally, the PTX effect on cell viability showed a G2/M cell arrest after using PTX-loaded NGs and pure PTX.

In-vivo evaluation of tamoxifen-loaded microspheres based on mixtures of poly (D,L-lactide-co-glycolide) and poly (D,L-lactide) polymers.

Microspheres of different proportions of poly-(D,L-lactide-co-glycolide) and poly-(D,L-lactide) were formulated by spray drying as a drug-delivery system for the treatment of breast cancer with tamoxifen. These systems had been evaluated previously in vitro and showed very positive results that have led to further assessment in vivo. This work evaluates the performance of these systems in an organism by carrying out a study in female Wistar rats. Microspheres were subcutaneously injected into the back of rats for the assessment of not only the biocompatibility but also the release of the drug contained and its biodistribution. As, in vitro, these systems could release the drug under physiological conditions; different plasma concentrations of tamoxifen and one of its metabolites, 4-hydroxy-tamoxifen, were achieved depending on the polymer composition. Microspheres could reduce the accumulation of the drug in different nontarget organs and presented good biocompatibility.

pH and glutathion-responsive hydrogel for localized delivery of paclitaxel.

pH and glutathion (GSH)- responsive nanogels (NGs) based on poly-N-isopropylacrilamide (NIPA), N-hydroxyethyl acrylamide (HEAA) and tert-butyl 2-acrylamidoethyl carbamate (2AAECM) were synthesized by a microemulsion polymerization method using N, N'-cystaminebisacrylamide (CBA) as a crosslinking agent and evaluated for passive targeting of paclitaxel (PTX). Physicochemical characterizations of unloaded and PTX-loaded NGs, such as particle size, morphology, encapsulation efficiency and in vitro PTX release were also assessed. Electron microscopy techniques (SEM and TEM) as well as dynamic light scattering (DLS) analysis showed nanosized spherical hydrogels. FTIR spectra confirmed the synthesis of nanogels by free radical polymerization among vinyl groups of monomers. In vitro release was analyzed by high-performance liquid chromatography (HPLC) and differences between two NG formulations were obtained. Nanogels released almost 64% of PTX after 50h at GSH concentrations equivalent to that in the cellular cytosol, whereas less PTX was released from NGs at pH and GSH levels similar to plasma. Cellular uptake and cytotoxicity were also demonstrated by using coumarin-6 and MTT assays, respectively, for three tumor cell lines (MCF7, HeLa and T47D). Cellular uptake assays revealed rapid uptake within 2h and intracellular accumulation of coumarin-6-loaded nanogels after 48 h incubation. MTT assays showed changes in cell viability at different concentrations of PTX formulations, as well as pure PTX (10 µM, 20 µM and 30 µM). To investigate PTX effect on cell viability, changes in cell cycle were examined by flow cytometry and a G2/M cell arrest was demonstrated. Overall, synthesized nanogels may be used as potential carriers for hydrophobic anticancer drugs.

Modulation of lysozyme activity by lead administered by different routes. In vitro study and analysis in blood, kidney, and lung.

Modifications in the enzyme activity of lysozyme, a protein implied in the defence barrier of the organism, can be a good biomarker of alterations in the immune system as a result of exposure to toxic metal, such as lead. The aim of this work was to evaluate the effect of a 200 ppm dose of lead on lysozyme activity in blood, kidney, and lung, and also on tissue structure. Previously, the effect of lead acetate on lysozyme activity in vitro was determined; the in vitro results indicated that lead produced a decrease in enzyme activity. The activity loss was 16 % at 200 ppm of lead. Lead acetate was administered to Wistar rats by oral and intraperitoneal injections. An increase in lysozyme activity was observed in blood when lead was administered by introperitoneal route and in kidney by the oral route. The possible immunostimulation in kidney was discarded because of the structural alterations observed in the tissue. In lung, the decrease in specific lysozyme activity, for both routes of lead exposure, seems to indicate immunosupression, which was in accordance with the structural alterations observed in this tissue.

Tamoxifen-loaded nanoparticles based on a novel mixture of biodegradable polyesters: characterization and in vitro evaluation as sustained release systems.

Nanoparticles (NP) from mixtures of two poly(D,L-lactide-co-caprolactone) (PLC) copolymers, PLC 40/60 and PLC 86/14, with poly(D,L-lactide) (PDLLA) and PCL were prepared: PLC 40/60-PCL (25:75), PLC 86/14-PCL (75:25) and PLC 86/14-PLA (75:25). Tamoxifen was loaded with encapsulation efficiency between 65% and 75% (29.9-36.3 µg TMX/ mg NP). All selected systems showed spherical shape and nano-scale size. TMX-loaded NPs were in the range of 293-352 nm. TMX release from NP took place with different profiles depending on polymeric composition of the particles. After 60 days, 59.81% and 82.65% of the loaded drug was released. The cytotoxicity of unloaded NP in MCF7 and HeLa cells was very low. Cell uptake of NP took place in both cell types by unspecific internalization in a time dependent process. The administration of 6 and 10 µm TMX by TMX-loaded NP was effective on both cellular types, mainly in MCF7 cells.

Skin laser treatments enhancing transdermal delivery of ALA.

Drug delivery across skin has been limited due to barrier properties of the skin, especially those of the stratum corneum (SC). Use of the laser radiation has been suggested for the controlled removal of the SC. The purpose of this study was to study in vitro the influence of infrared radiation from the erbium:yttrium-aluminum-garnet (Er:YAG) laser (λ = 2940 nm), and visible from the 2nd harmonic of a neodymium:yttrium-aluminum-garnet (Nd:YAG) laser (λ = 532 nm) on transdermal delivery of 5-aminolevulinic acid (ALA). Pinna skin of the inner side of rabbit ear was used for skin permeation. The light sources were an Er:YAG laser (Key III Plus KaVo) and a Q-switched Nd:YAG laser (Lotis TII SL-2132). Permeation study, morphological and structural skin examination by histology and differential scanning calorimetry (DSC) were carried out. Permeation profiles and histological observations obtained after irradiation with infrared and visible laser radiation differed due to different biophysical effects on irradiated skin. Wavelength of 2940 nm required lower energy contribution to produce the same level of permeation than visible radiation at 532 nm. Structural analysis by DSC shows a selective impact on the lipidic structure. Laser pretreatment enhanced the delivery of ALA trough the skin by SC ablation.

Tamoxifen-loaded folate-conjugate poly[(p-nitrophenyl acrylate)-co-(N-isopropylacrylamide)] sub-microgel as antitumoral drug delivery system.

Folate-conjugate poly[(p-nitrophenyl acrylate)-co-(N-isopropylacrylamide)] sub-microgel (F-SubMG) was loaded with tamoxifen (TMX) to obtain low (9.0 ± 0.4 µg TMX/mg F-SubMG) and high (112.0 ± 15.0 µg TMX/mg F-SubMG) load TMX-loaded F-SubMGs. Maximum in vitro drug release (77 ± 2% to 90 ± 2% of loaded TMX) took place between 47 and 168 h. The cytotoxicity of unloaded F-SubMGs in MCF-7 and HeLa cells was low; although it increased for high F-SubMG concentration. The administration of 10 µM TMX by TMX-loaded F-SubMGs was effective on both cellular types. Cell uptake of F-SubMGs took place in both cell types, but it was larger in HeLa cells because they are folate receptor positive. After subcutaneous administration (2.8 mg TMX/kg b.w.) in Wistar rats, F-SubMGs were detected at the site of injection under the skin, and a significant amount of them were included inside adipocytes. Signs of rejection were not observed after 60 days of injection. Pharmacokinetic study showed an increase in mean residence time of TMX and 4-hydroxytamoxifen (4-OHTMX), as well as a metabolite ratio (MR = AUC(4OHTMX) /AUC(TMX) ) nine times larger, when TMX was administered by drug-loaded F-SubMGs. Since 4-OHTMX is a more potent (at least 100-fold higher) antiestrogen than TMX, administration of TMX-loaded F-SubMGs can be considered an advantage.

Swelling properties of copolymeric hydrogels of poly(ethylene glycol) monomethacrylate and monoesters of itaconic acid for use in drug delivery.

Copolymeric hydrogels of poly(ethylene glycol) monomethacrylate (PEGMA) (P) have been synthesized for use in drug-delivery. New copolymeric hydrogels were prepared by free radical solution polymerization of PEGMA and monomethyl itaconate (MMI) or monoethyl itaconate (MEI), using ethyleneglycol dimethacrylate and tetraethyleneglycol dimethacrylate, respectively, as cross-linkers. The effect of copolymer composition on swelling behavior, thermal decomposition and drug release was studied. Three compositions of each copolymer were studied: 70P/30MMI (or MEI), 80P/20MMI (or MEI) and 90P/10MMI (or MEI). The largest equilibrium swelling degree was observed in gels containing the highest content of MMI or MEI (84.22 +/- 0.22 wt % for 70P/30MEI; 79.56 +/- 0.64 wt % for 70P/30MMI). The swelling process was in accordance with Fick's Second Law. Methotrexate (MTX), an anticancer agent used in the treatment of different hyperproliferative epithelial diseases, was chosen to be loaded in the gels. The drug was included by immersion of the copolymeric disks in an aqueous solution of the drug. The amount of MTX in the xerogels was between 5.34 +/- 0.06 mg MTX/g (90P/10MMI) and 14.94 +/- 0.91 mg MTX/g (80P/20MEI). Two stages of thermal degradation for unloaded and MTX-loaded gels were determined; the presence of the drug in the polymeric matrices decreased the temperature of the first stage of thermal degradation. MTX release was also in accordance with Fick's Second Law. The length of total drug release (340 +/- 30 min-1502 +/- 81 min) could be modulated as a function of the comonomer composition of the hydrogel.

Ketotifen-loaded microspheres prepared by spray-drying poly(D,L-lactide) and poly(D,L-lactide-co-glycolide) polymers: characterization and in vivo evaluation.

Ketotifen (KT) was encapsulated into poly(D,L-lactide) (PLA) and poly(D,L-lactide-co-glycolide) (PLGA 50/50) by spray-drying to investigate the use of biodegradable drug-loaded microspheres as delivery systems in the intraperitoneal cavity. Ketotifen stability was evaluated by HPLC, and degradation was not observed. Drug entrapment efficiency was 74 +/- 7% (82 +/- 8 microg KT/mg for PLA) and 81 +/- 6% (90 +/- 7 microg KT/mg for PLGA 50/50). PLA microspheres released ketotifen (57% of encapsulated KT) in 350 h at two release rates (221 microg/h, 15 min to 2 h; 1.13 microg/h, 5-350 h). A quicker release of ketotifen took place from PLGA 50/50 microspheres (67.4% of encapsulated KT) in 50 h (322 microg/h, 15 min to 2 h; 16.18 microg/h, 5-50 h). After intraperitoneal administration (10 mg KT/kg b.w.), microsphere aggregations were detected in adipose tissue. Ketotifen concentration was determined in plasma by HPLC. The drug released from PLA and PLGA 50/50 microspheres was detected at 384 and 336 h, respectively. Noncompartmental analysis was performed to determine pharmacokinetic parameters. The inclusion of ketotifen in PLGA and PLA microspheres resulted in significant changes in the plasma disposition of the drug. Overall, these ketotifen-loaded microspheres yielded an intraperitoneal drug release that may be suitable for use as delivery systems in the treatment of inflammatory response in portal hypertension.

5-Fluorouracil plasma levels and biodegradation of subcutaneously injected drug-loaded microspheres prepared by spray-drying poly(D,L-lactide) and poly(D,L-lactide-co-glycolide) polymers.

Microspheres (MS) of 5-fluorouracil-loaded poly(D,L-lactide) (PLA), poly(D,L-lactide-co-glycolide) 75/25 (PLGA 75/25) and poly(D,L-lactide-co-glycolide) 50/50 (PLGA 50/50) prepared by the spray-drying technique were subcutaneously injected in the back of Wistar rats in order to evaluate the 5-fluorouracil (5-FU) release and the biodegradation characteristics. Determination of plasma 5-FU concentration by HPLC with analysis of data using a non-compartmental model showed drug in plasma between 9 and 14 days after administration of drug-loaded PLGA 50/50 or PLA and PLGA 75/25 microspheres, respectively, with a maximum drug concentration of 2.4+/-0.2microg/mL at 24h (5-FU-loaded PLGA 50/50 MS), 2.5+/-0.1microg/mL at 48h (5-FU-loaded PLGA 75/25 MS), and 2.3+/-0.1microg/mL at 24h (5-FU-loaded PLA MS). Pharmacokinetically, a significant increase of AUC (up to 50 times) and MRT (up to 196 times) of 5-FU with regard to the administration of the drug in solution was observed. Scanning electron microscopy and histological studies indicated that a small fibrous capsule was observed around the microspheres in the site of injection. One month after the injection of PLGA 50/50 MS and 2 months after the injection of PLGA 75/25 and PLA MS, masses of polymers, instead of single microspheres, were observed. Close to them, macrophagic cells were present, and blood vessels were observed in the connective tissue. Total absence of fibrous capsule and injected microspheres was observed after 2 (for PLGA 50/50 MS) or 3 (PLGA 75/25 and PLA MS) months.

Degradation behaviour of microspheres prepared by spray-drying poly(D,L-lactide) and poly(D,L-lactide-co-glycolide) polymers.

Polymeric microsphere degradation must be taken into account in the design of drug delivery systems to be injected in in vivo systems, thus a prior analysis of in vitro degradation behaviour of microspheres appears to be necessary. In this study degradation characteristics of poly(lactide-co-glycolide) (PLGA) and poly(D,L-lactide) (PLA) microspheres prepared by the spray-drying technique have been examined. It was found that a slow decrease in molecular weight took place during the first stage of degradation, and the value of the rate constant decreased with the increase of the percentage of lactic acid of the polymer in a linear way. Thus, the period of time of this first stage decreased with the increase of content of glycolidyl units of the polymer, and it was the unique stage observed in PLA microspheres after 5 months of study. During this period of time, significant mass loss was not observed in the microspheres. The second stage of degradation of PLGA microspheres showed a larger rate constant, whose value increased with the content of glycolidyl units of the polymer. Mass loss was observed from number-average molecular weight about 6000. A sharp decrease of glass transition temperature (T(g)) was observed coinciding with the start of mass loss. This fact was accompanied by a physical change of the samples, fusion of microspheres to form large particles, which also fusion to form a unique mass of polymer; moment from that the degradation process was quicker.

Effect of the crosslinking degree and the nickel salt load on the thermal decomposition of poly(2-hydroxyethyl methacrylate) hydrogels and on the metal release from them.

Polymeric matrices of poly(2-hydroxyethyl methacrylate) (PHEMA) crosslinked with different percentages of ethylene glycol dimethacrylate (EGDMA) as well as different loads of nickel salt were synthesized. Nickel release from the polymeric systems, and their thermal stability were analyzed. A high percentage of the nickel loaded was released, although strong interactions between the polymeric matrices and the nickel ion must be established since a total nickel release did not take place. The values of the diffusion coefficients showed that nickel release depended on the amount of nickel salt loaded in the polymeric matrix and also on the crosslinking degree of the gels. On the other hand, the presence of nickel salt induced an evident thermal instability in the polymeric matrices, although all the polymeric systems can be considered thermally stable.