Effect of surface hydrophobicity of therapeutic protein loaded in polyelectrolyte nanoparticles on transepithelial permeability.

Oral delivery of protein drugs is greatly limited by low hydrophobicity, an important determinant for intestinal epithelial permeation and bioavailability. Herein, surface properties of recombinant erythropoietin were investigated using the fluorescent dye bis-ANS to monitor relative hydrophobicity for correlation with permeabilities with Caco-2 cells. At various pHs, bis-ANS fluorescence intensity indicated different surface hydrophobicities of erythropoietin molecules. Erythropoietin incorporated in chitosan or chitosan-trimethylchitosan (CS-TMC) nanoparticles prepared by polyelectrolyte complexation and ionotropic gelation with tripolyphosphate also showed different surface hydrophobicities. Chitosan nanoparticles with erythropoietin provided the most hydrophobic surface, followed by free erythropoietin (in water) and that loaded into CS-TMC nanoparticles. Chitosan nanoparticles were more effective than CS-TMC nanoparticles for permeation of erythropoietin across Caco-2 cell monolayers; the lowest permeability was shown by erythropoietin itself. Thus, hydrophilic protein molecules complexed with polyelectrolytes can provide more hydrophobic surfaces that enhance transepithelial permeability. This bis-ANS method also provides valuable information for the design of polyelectrolyte nanoparticules for oral delivery of protein drugs.

Freeze-drying and release characteristics of polyelectrolyte nanocarriers for the mucosal delivery of ovalbumin.

Polyelectrolyte complexes (PEC) consisting of an alginate core entrapping the protein ovalbumin and the chitosan coating were prepared by the self-assembly of oppositely charged polyelectrolytes. The PEC were prepared at pH 4.0 and consisted of alginate, ovalbumin and chitosan in a concentration of 0.5, 0.5 and 0.05 mg/ml, respectively, having a particle size of around 300nm, a zeta potential of -44 mV and a protein association efficiency of 80%. The release of ovalbumin from PEC was mostly dependant on the pH of release medium and the presence of strong electrolytes contributed to higher release. Approximately 90% of the ovalbumin was released in a phosphate buffer media, pH 7.4. The release was lower in media with pH 4.0, reaching the value of app. 40% and 60% of ovalbumin released in water (pH 4.0) and NaCl solution (0.9% w/v, pH 4.0), respectively. In an acidic saline solution, pH 3.0, there was only 5% of ovalbumin release, however, increasing the pH to 6.8, approximately 70% of ovalbumin immediately released from the PEC. The PEC were freeze-dried aided by various excipients. Their efficiency on the redispersibility of the freeze-dried product was evaluated according to the mean particle diameter, polydispersity, average scattering intensity (particle concentration) and visual appearance of the PEC (Tyndal effect). In the presence of trehalose and mannitol, the aggregation and integrity of the PEC were prevented, yielding properties similar to the PEC dispersion before lyophilisation. The surface hydrophobicity of the ovalbumin either free or formulated in the nanocomplexes was determined by the bis-ANS fluorescence intensity, indicating a higher surface hydrophobicity for the PEC. The mild formulation conditions, nanometre-sized particles, high protein association efficiency, pH-dependant release, and modified surface properties are promising factors towards the development of an oral delivery system for protein made by the self-assembly of oppositely charged polyelectrolytes.

Toward effective long-term prevention of thromboembolism: novel oral anticoagulant delivery systems.

Despite intensive research in the field of oral anticoagulants over the last decade, simple and effective long-term prevention of thromboembolism is still an unmet need. In addition to drug discovery approaches, the development of novel oral drug delivery systems (DDSs) of clinically well-established anticoagulants presents an intriguing mean of improvement of anticoagulant therapy. The latter topic is therefore the focus of the present review. All relevant clinical trials with anticoagulants formulated in the oral DDS are reviewed, and selected preclinical examples of promising novel anticoagulant DDSs are also described. For greater understanding, a background on DDS and drug absorption from the gastrointestinal tract is also provided. Three leading approaches for the oral anticoagulant DDS are currently being investigated in clinical settings, all relying on coadministration of anticoagulants with specific carriers. In contrast to the clinical setting, a diverse range of possibilities for oral delivery of anticoagulant are being investigated in preclinical trials (e.g., nanotechnology), and it would be therefore interesting to examine their performance in clinical trials.

Self-assembled Polyelectrolyte Nanocomplexes of Alginate, Chitosan and Ovalbumin.

Polyelectrolyte complex (PEC) nanoparticles for delivering model protein drug ovalbumin were prepared from two polysaccharide polymers, alginate and chitosan. The parameters influencing the complex formation were characterised using colloid titration in combination with dynamic light scattering. The polyelectrolyte interactions and morphology of the formed complexes were verified by differential scanning calorimetry and scanning electron microscopy, respectively. The PEC formation was predominantly pH- and concentration-dependent. The complexation of ovalbumin with a negatively charged alginate occurred only at a pH below the isoelectric point of the ovalbumin. After the complexation, negatively charged complexes of alginate and ovalbumin were further coated with chitosan. The optimal composition of the PEC, yielding 280 nm sized particles having a zeta potential -40 mV, was determined for alginate:ovalbumin:chitosan in a mass ratio 1: 1: 0.1, respectively, giving their final concentration 0.5: 0.5: 0.05mg/ml. The loading of ovalbumin in the PEC depended on the initial amount of ovalbumin used to produce the PEC, and ranged from 7-38% for different formulations, however, the association efficiency remained pretty similar for all formulations, i.e. 80-85%. Mild formulation conditions, nanometre-sized particles, and a high protein association efficiency are promising factors towards the development of a delivery system for proteins.

Immunonanoparticles--an effective tool to impair harmful proteolysis in invasive breast tumor cells.

Breast cancer cells exhibit excessive proteolysis, which is responsible for extensive extracellular matrix degradation, invasion and metastasis. Besides other proteases, lysosomal cysteine protease cathepsin B has been implicated in these processes and the impairment of its intracellular activity was suggested to reduce harmful proteolysis and hence diminish progression of breast tumors. Here, we present an effective system composed of poly(D,L-lactide-coglycolide) nanoparticles, a specific anti-cytokeratin monoclonal IgG and cystatin, a potent protease inhibitor, that can neutralize the excessive intracellular proteolytic activity as well as invasive potential of breast tumor cells. The delivery system distinguishes between breast and other cells due to the monoclonal antibody specifically recognizing cytokeratines on the membrane of breast tumor cells. Bound nanoparticles are rapidly internalized by means of endocytosis releasing the inhibitor cargo within the lysosomes. This enables intracellular cathepsin B proteolytic activity to be inhibited, reducing the invasive and metastatic potential of tumor cells without affecting proteolytic functions in normal cells and processes. This approach may be applied for treatment of breast and other tumors in which intracellular proteolytic activity is a part of the process of malignant progression.

Targeting cancer cells using PLGA nanoparticles surface modified with monoclonal antibody.

Targeting drugs to their sites of action is still a major challenge in pharmaceutical research. In this study, polylactic-co-glycolic acid (PLGA) immuno-nanoparticles were prepared for targeting invasive epithelial breast tumour cells. Monoclonal antibody (mAb) was used as a homing ligand and was attached to the nanoparticle surface either covalently or non-covalently. The presence of mAb on the nanoparticle surface, its stability and recognition properties were tested. Protein assay, surface plasmon resonance, flow cytometry and fluorescence-immunostaining confirmed the presence of mAb on nanoparticles in both cases. However, a binding assay using cell lysate revealed that the recognition properties were preserved only for nanoparticles with adsorbed mAb. These nanoparticles were more likely to be bound to the targeted cells than non-coated nanoparticles. Both types of nanoparticles entered the target MCF-10A neoT cells in mono-culture. In co-culture of MCF-10A neoT and Caco-2 cells immuno-nanoparticles were localized solely to MCF-10A neoT cells, whereas non-coated nanoparticles were distributed randomly. Immuno-nanoparticles entered only MCF-10A neoT cells, while non-coated nanoparticles were taken up by both cell types, indicating specific targeting of the immuno-nanoparticles. In conclusion, we demonstrate a method by which mAbs can be bound to nanoparticles without detriment to their targeting ability. Furthermore, the results show the effectiveness of the new carrier system for targeted delivery of small or large active substances into cells or tissues of interest.

Intracellular delivery of cysteine protease inhibitor cystatin by polymeric nanoparticles.

Two polymers chitosan and poly(lactide-co-glycolide) copolymer (PLGA) were investigated to develop nanoparticles (NPs) for delivery of protein drug substance into tumour cells. Cystatin was selected as a model protein drug due to its high potential to inhibit cysteine proteases, known to trigger the invasive process. Ionotropic gelation of chitosan with tripolyposphate and precipitation of PLGA polymer from a double emulsion system by a solvent diffusion process were used to produce 250-300 nm in diameter NPs. The cellular uptake of NPs was tested on a transformed human breast epithelial cell line, MCF-10A neoT, characterized by an increased expression of cysteine proteases and highly invasive cell phenotype. The influence of NPs on cell viability was evaluated by MTT test showing IC50 400 microg/ml for PLGA and 5 mg/ml for chitosan NPs. As determined by fluorescence microscopy chitosan NPs did not enter the cells during 1-hour incubation whereas the same amount of PLGA NPs were in the cells already after 5 min of incubation. Cystatin delivered into MCF-10A neoT cells by PLGA NPs effectively inhibited intracellular proteolytic activity of cathepsin B, as detected by specific fluorogenic substrate Z-Arg2 cresyl violet. On the contrary, free cystatin in solution did not internalise into the cells and inhibit cathepsin B. To conclude, PLGA NPs with cystatin but not chitosan NPs were targeted through endocytosis to the lysosomal compartments that are rich of proteases enzymes. Our results suggest new strategy to inactivate intracellular tumour-associated proteases, and consequently the invasion behaviour of tumour cells, which could contribute to cancer therapy.

Nanoscale polymer carriers to deliver chemotherapeutic agents to tumours.

Nanoscale polymer carriers have the potential to enhance the therapeutic efficacy of antitumour drugs as they can regulate their release, improve their stability and prolong circulation time by protecting the drug from elimination by phagocytic cells or premature degradation. Moreover, nanoscale polymeric carriers are capable of accumulating in tumour cells and tissues due to enhanced permeability and retention effect or by active targeting bearing ligands designed to recognise overexpressed tumour-associated antigens. The diversity in the polymer structures being studied as drug carriers in cancer therapy allows an optimal solution for a particular drug to be provided regarding its delivery and efficacy, and thus the patient's quality of life. This review is focused on the different types of nanoscale polymer carriers used for the delivery of chemotherapeutic agents and on the factors that affect their cellular uptake and trafficking.

Poly(lactide-co-glycolide) nanoparticles as a carrier system for delivering cysteine protease inhibitor cystatin into tumor cells.

Cystatins are able to inhibit the tumor-associated activity of intracellular cysteine proteases cathepsins B and L and have been suggested as potential anticancer drugs. We have incorporated chicken cystatin, a model protein inhibitor of cysteine proteases, in poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) to improve its bioavailability and delivery into tumor cells. Cystatin-loaded NPs, 300-350 nm in diameter, were prepared by the double emulsion solvent diffusion method using low energy emulsification to preserve the biological activity of the protein. PLGA NPs and cystatin-loaded PLGA NPs at concentrations higher than 80 microg/ml were cytotoxic towards MCF-10A neoT cells, but not free cystatin at concentrations up to 5 microM. To visualize the uptake of cystatin into living MCF-10A neoT cells, NPs loaded with Alexa Fluor 488-labeled cystatin were added to the culture medium. They rapidly internalized into the cells, whereas the uptake of free-labeled cystatin was very slow. Cystatin, released from the NPs, effectively inhibited cathepsin B activity, as detected by degradation of specific Z-Arg-Arg cresyl violet substrate. In contrast, the same amount of free cystatin showed no inhibition of intracellular cathepsin B. Our results show that PLGA NPs are a useful carrier system for rapid delivery of protein inhibitors into tumor cells, enabling effective inhibition of intracellular proteolysis. The approach can be applied to other protein drugs active against intracellular targets.

Cystatin incorporated in poly(lactide-co-glycolide) nanoparticles: development and fundamental studies on preservation of its activity.

Preservation of biological activity is still a major challenge for successful formulation and delivery of protein drugs. Cystatin, a potential protein drug in cancer therapy, was incorporated in poly(lactide-co-glycolide) nanoparticles by the water-in-oil-in-water emulsion solvent diffusion technique. In order to preserve the biological activity of cystatin, a specific modification of the method of producing nanoparticles was introduced. The activity of cystatin was strongly influenced by the stirring rate during preparation and, to a lesser extent, by selected organic solvents. A synergistic effect of mechanical stirring and sonication, both at low energy levels, enabled nanoparticles to be formed without denaturing the cystatin. Nanoparticles produced by the optimised method ranged from 300 to 350 nm in diameter with 85% of the starting cystatin activity. The loading efficiency of cystatin depends on polymer type and ranged from 12 to 57%, representing an actual loading of 0.6-2.6% (w/w). Among various cryo-/lyoprotectants bovine serum albumin was identified as the most successful. The use of a protein protectant prior to nanoparticle formation was essential to maintaining the biologically active three-dimensional structure of cystatin. In addition, a specific type of poly(lactide-co-glycolide) polymer, particularly in terms of its functional groups, was identified to be important in retaining cystatin activity. Cystatin incorporated into nanoparticles in this way maintains its structural integrity, making it suitable for effective drug delivery.