Francisca Araújo

Establishment of a triple co-culture in vitro cell models to study intestinal absorption of peptide drugs

In vitro cell culture models for studying oral drug absorption during early stages of drug development have become a useful tool in drug discovery and development, with respect to substance throughput and reproducibility. The aim of this study was to establish an in vitro cellular model based on human colon carcinoma Caco-2, mucus-producing HT29, and Raji B cells in order to design a model that more accurately mimics the small intestinal epithelial layer. Normal oriented model was set up by seeding co-cultures of Caco-2 and HT29 cells into Transwell filters and maintained under identical conditions following addition of Raji B to the basolateral chamber. Inverted model was set up seeding Caco-2 and HT29 cells on the basolateral chamber and then transferred in the Transwell device with the epithelial cells facing the basolateral chamber following Raji B addition to the apical compartment. Morphological differences on size and thickness of cell membranes were detected between the models studied by using fluorescence microscopy. On the triple co-culture models, cell membranes were increasing in size and thickness from the Caco-2 to Caco-2/HT29 and Caco-2/Raji B. Also, the nuclei seem to be larger than in the other studied models. Insulin permeation was higher on the triple co-culture model when compared to the Caco-2/HT29 co-culture model. Also, insulin permeation as mediated by nanoparticles and insulin solution permeation was higher on the normal oriented Caco-2/HT29/Raji B model as compared to the inverted model. Overall, our results suggest that Caco-2/HT29/Raji B triple co-culture normal oriented cellular model may be reliable to obtain a more physiological, functional, and reproducible in vitro model of the intestinal barrier to study protein absorption, both in solution and when delivered by nanocarriers.

Oral Insulin Delivery: How Far are We?

Oral delivery of insulin may significantly improve the quality of life of diabetes patients who routinely receive insulin by the subcutaneous route. In fact, compared with this administration route, oral delivery of insulin in diabetes treatment offers many advantages: Higher patient compliance, rapid hepatic insulinization, and avoidance of peripheral hyperinsulinemia and other adverse effects such as possible hypoglycemia and weight gain. However, the oral delivery of insulin remains a challenge because its oral absorption is limited. The main barriers faced by insulin in the gastrointestinal tract are degradation by proteolytic enzymes and lack of transport across the intestinal epithelium. Several strategies to deliver insulin orally have been proposed, but without much clinical or commercial success. Protein encapsulation into nanoparticles is regarded as a promising alternative to administer insulin orally because they have the ability to promote insulin paracellular or transcellular transport across the intestinal mucosa. In this review, different delivery systems intended to increase the oral bioavailability of insulin will be discussed, with a special focus on nanoparticulate carrier systems, as well as the efforts that pharmaceutical companies are making to bring to the market the first oral delivery system of insulin. The toxicological and safety data of delivery systems, the clinical value and progress of oral insulin delivery, and the future prospects in this research field will be also scrutinized.

Polymer-based nanoparticles for oral insulin delivery: Revisited approaches

Diabetes mellitus is a high prevalence and one of the most severe and lethal diseases in the world. Insulin is commonly used to treat diabetes in order to give patients a better life condition. However, due to bioavailability problems, the most common route of insulin administration is the subcutaneous route, which may present patients compliance problems to treatment. The oral administration is thus considered the most convenient alternative to deliver insulin, but it faces important challenges. The low stability of insulin in the gastrointestinal tract and low intestinal permeation, are problems to overcome. Therefore, the encapsulation of insulin into polymer-based nanoparticles is presented as a good strategy to improve insulin oral bioavailability. In the last years, different strategies and polymers have been used to encapsulate insulin and deliver it orally. Polymers with distinct properties from natural or synthetic sources have been used to achieve this aim, and among them may be found chitosan, dextran, alginate, poly(γ-glutamic acid), hyaluronic acid, poly(lactic acid), poly(lactide-co-glycolic acid), polycaprolactone (PCL), acrylic polymers and polyallylamine. Promising studies have been developed and positive results were obtained, but there is not a polymeric-based nanoparticle system to deliver insulin orally available in the market yet. There is also a lack of long term toxicity studies about the safety of the developed carriers. Thus, the aims of this review are first to provide a deep understanding on the oral delivery of insulin and the possible routes for its uptake, and then to overview the evolution of this field in the last years of research of insulin-loaded polymer-based nanoparticles in the academic and industrial fields. Toxicity concerns of the discussed nanocarriers are also addressed.

Towards the characterization of an in vitro triple co-culture intestine cell model for permeability studies

Caco-2 based cell models have been the gold standard in vitro method to study intestinal drug permeability, despite the absence of many important features with major influence in the drug absorption mechanism. In the present work, a triple co-culture comprising Caco-2, HT29-MTX and Raji B cells was established to mimic in a closely way the human intestinal epithelium, presenting the main components in the process of drug absorption, namely the absorptive cells that resemble enterocytes, mucus producers cells and cells able to induce M-cell phenotype on Caco-2 cells. All the three cell lines maintained their function when cultured together with each other being, thus, an asset to new orally administrated drugs development. The seeding ratio of 90:10 between Caco-2 and HT29-MTX showed to be the best to achieve physiological proportions after cells maturation and differentiation in culture. The formation of M-cells phenotype from enterocytes was identified for the first time in a co-culture system comprising Caco-2 and HT29-MTX cells through immunocytochemical techniques. Thus, the triple co-culture model presented in the herein work is a good and reliable alternative to the in vitro methods already existents for the study of drugs permeability.

Chitosan-Coated Solid Lipid Nanoparticles for Insulin Delivery

The delivery of therapeutic proteins like insulin, exploiting routes of administration different from the traditional injectable forms, has been investigated extensively, taking advantage of the nanotechnology tools available nowadays in the massive drug delivery system pipeline. In this chapter, we describe in detail the preparation of solid lipid nanoparticles (SLN), further coated with the mucoadhesive polymer chitosan, intended for intestinal absorption of insulin after oral administration. We give special focus on the characterization of the SLN and of the biomacromolecule by itself after encapsulation, because of the intrinsic labile properties of insulin during the manufacturing process. We also describe methods to determine the in vitro intestinal permeability of insulin that solid lipid and chitosan-coated SLN can afford, as well as in vivo models to evaluate the hypoglycemic effect in diabetic animals.

In Vitro and Ex Vivo Evaluation of Polymeric Nanoparticles for Vaginal and Rectal Delivery of the Anti-HIV Drug Dapivirine

Prevention strategies such as the development of microbicides are thought to be valuable in the fight against HIV/AIDS. Despite recent achievements, there is still a long road ahead in the field, particularly at the level of drug formulation. Drug nanocarriers based on polymers may be useful in enhancing local drug delivery while limiting systemic exposure. We prepared differently surface-engineered poly(ε-caprolactone) (PCL) nanoparticles (NPs) and tested their ability to modulate the permeability and retention of dapivirine in cell monolayers and pig vaginal and rectal mucosa. NPs coated with poly(ethylene oxide) (PEO) were shown able to reduce permeability across monolayers/tissues, while modification of nanosystems with cetyl trimethylammonium bromide (CTAB) enhanced transport. In the case of coating NPs with sodium lauryl sulfate (SLS), dapivirine permeability was unchanged. All NPs increased monolayer/tissue drug retention as compared to unformulated dapivirine. This fact was associated, at least partially, to the ability of NPs to be taken up by cells or penetrate mucosal tissue. Cell and tissue toxicity was also affected differently by NPs: PEO modification decreased the in vitro (but not ex vivo) toxicity of dapivirine, while higher toxicity was generally observed for NPs coated with SLS or CTAB. Overall, presented results support that PCL nanoparticles are capable of modulating drug permeability and retention in cell monolayers and mucosal tissues relevant for vaginal and rectal delivery of microbicides. In particular, PEO-modified dapivirine-loaded PCL NPs may be advantageous in increasing drug residence at epithelial cell lines/mucosal tissues, which may potentially increase the efficacy of microbicide drugs.

A prospective cancer chemo-immunotherapy approach mediated by synergistic CD326 targeted porous silicon nanovectors

Combination therapy via nanoparticulate systems has already been proposed as a synergistic approach for cancer treatment. Herein, undecylenic acid modified thermally hydrocarbonized porous silicon nanoparticles (UnTHCPSi NPs) loaded with sorafenib and surface-biofunctionalized with anti-CD326 antibody (Ab) were developed for cancer chemo-immunotherapy in MCF-7 and MDA-MB-231 breast cancer cells. The cytocompatibility study showed no significant toxicity for the bare and antibody-conjugated UnTHCPSi (Un-Ab) NPs at concentrations lower than 200 μg·mL−1. Compared to the bare UnTHCPSi, Un-Ab NPs loaded with sorafenib reduced the premature drug release in plasma, increasing the probability of proper drug targeting. In addition, high cellular interaction and subsequent internalization of the Un-Ab NPs into the cells expressing CD326 antigen demonstrated the possibility of improving antigen-mediated endocytosis via CD326 targeting. While an in vitro antitumor study revealed a higher inhibitory effect of the sorafenib-loaded Un-Ab NPs compared to the drug-loaded UnTHCPSi NPs in the CD326 positive MCF-7 cells, there was no difference in the anti-proliferation impact of both the abovementioned NPs in the CD326 negative MDA-MB-231 cells, suggesting CD326 as an appropriate receptor for Ab-mediated drug delivery. It was also shown that the anti-CD326 Ab can act as an immunotherapeutic agent by inducing antibody dependent cellular cytotoxicity and enhancing the interaction of effector immune and cancer cells for subsequent phagocytosis and cytokine secretion. Hence, the developed nanovectors can be applied for simultaneous tumor-selective drug targeting and immunotherapy.

Nanoparticles-in-film for the combined vaginal delivery of anti-HIV microbicide drugs.

Combining two or more antiretroviral drugs in one medical product is an interesting but challenging strategy for developing topical anti-HIV microbicides. We developed a new vaginal delivery system comprising the incorporation of nanoparticles (NPs) into a polymeric film base - NPs-in-film - and tested its ability to deliver tenofovir (TFV) and efavirenz (EFV). EFV-loaded poly(lactic-co-glycolic acid) NPs were incorporated alongside free TFV into fast dissolving films during film manufacturing. The delivery system was characterized for physicochemical properties, as well as genital distribution, local and systemic 24h pharmacokinetics (PK), and safety upon intravaginal administration to mice. NPs-in-film presented suitable technological, mechanical and cytotoxicity features for vaginal use. Retention of NPs in vivo was enhanced both in vaginal lavages and tissue when associated to film. PK data evidenced that vaginal drug levels rapidly decreased after administration but NPs-in-film were still able to enhance drug concentrations of EFV. Obtained values for area-under-the-curve for EFV were around one log10 higher than those for the free drugs in aqueous vehicle (phosphate buffered saline). Film alone also contributed to higher and more prolonged local drug levels as compared to the administration of TFV and EFV in aqueous vehicle. Systemic exposure to both drugs was low. NPs-in-film was found to be safe upon once daily vaginal administration to mice, with no significant genital histological changes or major alterations in cytokine/chemokine profiles being observed. Overall, the proposed NPs-in-film system seems to be an interesting delivery platform for developing combination vaginal anti-HIV microbicides.

Oral hypoglycaemic effect of GLP-1 and DPP4 inhibitor based nanocomposites in a diabetic animal model

Glucagon-like peptide-1 (GLP-1), an incretin hormone, is used for type 2 diabetes mellitus (T2DM) treatment because of its ability to stimulate insulin secretion and release in a glucose-dependent manner. Despite of its potent insulinotropic effect, oral GLP-1 delivery is greatly limited by its instability in the gastrointestinal tract, poor absorption efficiency and rapid degradation by dipeptidylpeptidase-4 (DPP4) enzyme leading to a short half-life (~2min). Thus, a multistage dual-drug delivery nanosystem was developed to deliver GLP-1 and DPP4 inhibitor simultaneously. The system comprised of chitosan-modified porous silicon (CSUn) nanoparticles, which were coated by an enteric polymer, hydroxypropylmethylcellulose acetate succinate MF, using aerosol flow reactor technology. A non-obese T2DM rat model induced by co-administration of nicotinamide and streptozotocin was used to evaluate the in vivo efficacy of the nanosystem. The oral administration of H-CSUn nanoparticles resulted in 32% reduction in blood glucose levels and ~6.0-fold enhancement in pancreatic insulin content, as compared to the GLP-1+DPP4 inhibitor solution. Overall, these results present a promising system for oral co-delivery of GLP-1 and DPP4 inhibitor that could be further evaluated in a chronic diabetic study.

Controlled Dissolution of Griseofulvin Solid Dispersions from Electrosprayed Enteric Polymer Micromatrix Particles: Physicochemical Characterization and in Vitro Evaluation.

The oral bioavailability of a poorly water-soluble drug is often inadequate for the desired therapeutic effect. The bioavailability can be improved by enhancing the physicochemical properties of the drug (e.g., dissolution rate, permeation across the gastrointestinal tract). Other approach include shielding the drug from the gastric metabolism and targeted drug release to obtain optimal drug absorption. In this study, a poorly water-soluble model drug, griseofulvin, was encapsulated as disordered solid dispersions into Eudragit L 100-55 enteric polymer micromatrix particles, which were produced by electrospraying. Similar micromatrix particles were also produced with griseofulvin-loaded thermally oxidized mesoporous silicon (TOPSi) nanoparticles dispersed to the polymer micromatrices. The in vitro drug dissolution at pH 1.2 and 6.8, and permeation at pH 7.4 across Caco-2/HT29 cell monolayers from the micromatrix particles, were investigated. The micromatrix particles were found to be gastro-resistant, while at pH 6.8 the griseofulvin was released very rapidly in a fast-dissolving form. Compared to free griseofulvin, the permeability of encapsulated griseofulvin across the intestinal cell monolayers was greatly improved, particularly for the TOPSi-doped micromatrix particles. The griseofulvin solid dispersions were stable during storage for 6 months at accelerated conditions. Overall, the method developed here could prove to be a useful oral drug delivery solution for improving the bioavailability of poorly water-soluble or otherwise problematic drugs.