Klaas Poelstra

Cell-selective delivery of interferon gamma peptidomimetic inhibits chronic hepatic fibrosis and Tumor angiogenesis in vivo

Vanessa S.S. Gonçalves1,2,3, Liliana Rodrigues1,2, Joana Poejo1,2, Pavel Gurikov4, Ana A. Matias1,2, Irina Smirnova4 and Catarina M.M. Duarte1,2 1Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. Da República, 2780-157 Oeiras, Portugal; 2iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901, Oeiras, Portugal; 3Escuela de Ingenierías Industriales, Universidad de Valladolid, C/Dr. Mergelina s/n 47011 Valladolid, Spain; 4Institute of Thermal Separation Processes, Hamburg University of Technology, Eißendorferstr. 38, 21073 Hamburg, GermanyS main function is to protect our body from external aggression. This is mainly achieved by a strong barrier function performed by the outer layer of the skin: The stratum corneum (SC). In contrast, most skin alterations and pathologies originate in the inner layers of the skin (Epidermis, Dermis or Hypodermis) and therefore require targeted treatments. Skin penetration is one of the biggest challenges for drug delivery. Most topical ingredients do not really penetrate the SC, acting on the skin’s surface and disappearing after a single wash. Other ingredients include chemical enhancers and/or other aggressive compounds that penetrate disrupting the skin barrier and causing damage. In both cases, efficacy and safety are compromised. Formulators of are facing the challenge to create skin care products able to penetrate gently the skin, remains there and deliver their content in the targeted layers. Bicosome platform offers a solution to these challenges. Structurally, Bicosomes are made up of internal smart biocompatible structures enclosed in a lipid vesicle protecting them and boosting their effects. Active molecules of many different natures can be incorporated to this platform. The smart structures of the bicosomes are small enough to penetrate the skin and self-aggregate into the tissue and grow being retained in specific layers. This induces a reinforcement of the skin structures and a targeted delivery. This delivery strategy allows a prolonged effect of actives because bicosome components retained in specific target layers remain there until this layer is lost following the natural desquamation process of the skin. These systems open a new and disruptive strategy, in which actives directed to the target layers and are remain retained exerting their action for days.M Melanoma (MMel) is the most deadly skin cancer frequently associated with metastasis and poor survival prognosis. Systemic therapy for MMel is still too early in development to demonstrate efficacy, and chemotherapy still remains the major adjuvant treatment against MMel. Obviously, new targeted drug delivery approaches are needed to overcome toxicological problems and improve efficacy. Integrins are a family of at least 24 distinct cell surface receptors commonly over-expressed on many types of cancer cells. They are essential for tumor progression, and therefore attractive targets for selective therapeutic intervention and drug delivery. Importantly, integrins are generally recognized by the “RGD tripeptidic sequence” and therefore many peptides bearing this recognition motif have been found to be effective ligands for the selective delivery of chemotherapeutics. However, their therapeutic and targeted effectiveness was not adequately demonstrated in clinical trials and even paradoxically can enhance, rather than suppress, tumor progression. We developed new non-RGD cyclic peptide ALOS-4 which binds to a non-RGD site on integrin αvβ3. Our preliminary in-vivo studies demonstrate that ALOS4 markedly blocks murine B16F10 melanoma tumor growth and lung metastasis, dramatically increases animal survival rates and prevents cancer-related weight loss. In addition, ex vivo fluorescence imaging studies on human metastatic melanoma (WM-266-4) animal model showed the accumulation of the ALOS4-FITC only in the tumor tissue and not in the spleen or liver. The ALOS4-Drug conjugates as well as their potency in treatment of human metastatic melanoma will also be presented.T drug delivery is regarded as an important route with high potential for drug delivery to overcome limitations of oral delivery and hypodermic injections. Among various delivery methods available, microneedles have gained interest due to their ability in delivering drugs with a high efficacy compared with topical application. Microneedles are referred to microscopic needles that are capable of delivering pharmaceutical compounds, proteins and even cosmetics into the skin in a minimally invasive manner. There are 3 main categories of microneedles; hollow, solid and dissolving. Dissolving microneedles are polymeric structures fabricated over a patch that encapsulate drug and deliver it into skin upon application. However, due to stiffness properties of skin, only small portion of microneedles get inserted into skin and the rest remains on the patch. Therefore, we developed a patch-less dissolving microneedle delivery system which delivers microneedles into skin through micro-pillar structures without causing pain in less than a second. We tested this sytem using insulin on diabetic mice and results showed that our micropillar based delivery system was capable of delivering insulin with approximately 60% higher efficacy than microneedles fabricated over patch and at a similar rate as hypodermic injection. Therefore, we believe that this system have a high potential to treat people suffering from diabetis in a minimally invasive manner without the need of hypodermic injection.T work aimed at developing inhalable powders of insulin-loaded chitosan nanoparticles (INS-CS NPs), by microencapsulation method, and investigating their pulmonary absorption in-vivo. To this end, INS-CS NPs were prepared by incorporating insulin (INS) into nanoparticlulate entities (NPs), consisting of the polysaccharide chitosan (CS) and the cross-linker sodium tripolyphosphate (TPP), usnig ionotropic gelation. Afterwards, INS-CS NPs were characterized with respect to morphology, size, zeta potential and loading capacity. Next, the inhalable powders were produced by co-spray drying the suspensions of INS-CS NPs with the sugar mannitol (thermoprotectant), resulting in microstructured powders with adequate aerodynamic properties for lung deposition. In-vivo performance of INS-CS NPs spray-dried powders was assessed via monitoring plasma glucose levels, following intratracheal administration in rats. The spray-dried INS-CS NPs were successfully microencapsulated into mannitol microspheres, forming powders with appropriate aerodynamic properties for deep lung deposition. The IN-CS NPs/mannitol weight ratios as well as spray drying process parameters affected the properties of the microspheres obtained. Additionally, the NPs were easily recovered after reconstitution of the spray-dried powders in aqueous media. The in-vivo study revealed that the microencapsulated INS-CS NPs induced a more pronounced and prolonged hypoglycaemic effect, as compared to the controls, including INS-loaded mannitol microspheres, native INS solution and the suspension of INS-CS NPs. Overall, besides the advantage of non-invasive administration and the desired stability of dry formulations, when compared to their liquid counterparts, inhalable micro/nanoparticulate systems may hold promise for lung delivery of therapeutic macromolecules for systemic or local effects (e.g., Cystic fibrosis, lung cancer).B technology encompasses all of the basic and applied sciences in microbiology, biochemistry and molecular biology as well as the engineering aspects to fully exploit living systems and bring their products to the market place. To-day bioprocesses have become widely used in several fields of commercial biotechnology including in medicines and drug discovery. While our understanding of biotechnological process has rapidly and remarkably advanced in recent years, it has been in existence since prehistoric times, making it one of the oldest technology even before the discovery of the field of microbiology. The discovery of microbial enzymes and the development of bioconversion technology led to the production of new drug with high yields and cost effective. Bioconversion process is also known by the name biotransformation and refers to the use of living organisms or its extracted enzymes to carry out chemical reactions that are not feasible or costly when produced by synthetic chemistry methods. These enzymes convert a substance to a chemically modified form with multiple uses and applications including medicines. In the 1980s, the recombinant gene technology led to the production of genetically engineered insulin for diabetes as the first product manufactured with recombinant technology. This newly developed genetic engineering technology has led to the introduction of a large number of new bio drugs such as interferon, tissue plasmogen activator, erythropoietin, colony-stimulating factors, and monoclonal-antibodies.T study aims at improving the buccal delivery of vitamin B6 (VB6) as a model which is highly water-soluble and low permeable vitamin. Two main strategies were combined; first VB6 was entrapped in liposomes, which were then formulated as mucoadhesive film. Both plain and VB6-loaded liposomes (LPs) containing Lipoid S100 and propylene glycol (~200 nm) were incorporated into mucoadhesive film composed of SCMC and HPMC. Results showed prolonged release of VB6 (72.65%, T50% diss 105 min) after 6 h from LP-film compared to control film containing free VB6 (96.37%, T50% diss 30 min). Mucoadhesion was assessed both ex vivo on chicken pouch and in-vivo in human. Mucoadhesive force of 0.2 N and residence time of 4.4 h were recorded. Ex vivo permeation of VB6, across chicken pouch mucosa indicated increased permeation from LP-systems compared to corresponding controls. Interestingly, incorporation of the vesicles in mucoadhesive film reduced the flux by 36.89% relative to LP-dispersion. Meanwhile, both films provided faster initial permeation than the liquid forms. Correlating the cumulative percent permeated ex vivo with the cumulative percent released in-vitro indicated that LPs retarded VB6 release but improved permeation. These promising results represent a step forward in the field of buccal delivery of water-soluble vitamins.P represent a large and diverse class of naturally occurring hydrophobic polymers, found in almost all living organisms. These linear compounds, constructed of 5 to more than 150 isoprene units, are commonly divided into dolichols (with hydrogenated α-bond) and α-unsaturated polyprenols. Polyisoprenoid alcohols play numerous roles in cells but above all, as structural components of cellular membranes, they modulate their properties. Biophysical studies proved that polyisoprenoids act as membrane modulators by influencing their fluidity and permeability. Latest research demonstrated that semi-synthetic, cationic derivatives of polyisoprenoid alcohols (called amino-prenols, APrens) possess lipofecting properties, because they facilitate the transfer of genetic material towards the cells. Amino-prenols were obtained by chemical modification in that hydroxyl group on α end was replaced by a quaternary ammonium group. This generated specific properties, whereby these cationic lipids can influence biological membranes more easily and effectively. Hence, the idea to use amino-prenols as components of liposomal carriers of drugs and genetic material was obtained. In the first stage of our study, we tested potential toxicity of novel carriers with particular emphasis on their impact on renal function. In the next steps, we examined usefulness of newly designed carriers for liposomal delivery of various drugs. Obtained results lead us to suspect that amino-prenols can be used as components of drug carriers, not only augmenting the biodistribution of active substances, but also improving the stability and loading capacity of liposomes. In the future, we plan to further study aminoprenols in an attempt to enhance gene expression in-vivo.R Nanotubes (RNTs), a self-assembled supramolecular nanomolecule composed of fused guanine-cytosine (G^C) bases, provide a novel and innovative modality for the delivery of gene therapeutics to target cells with high efficacy. This study aims at utilizing RNTs as a nanocarrier platform for the delivery of small interfering RNA (siRNA) to knockdown oncogenic genes to facilitate the elimination of cancer from the body. By conjugating positively charged lysine functional groups to the surface of the RNTs, the nanotubes gain the ability to complex negatively charged siRNA through electrostatic interactions. Through specialized FRET-labeled siRNA and gel retardation assay, we have shown that cationic charges on the RNTs strongly affect the binding interaction and the intracellular delivery of the RNT-siRNA nanocomplex. Furthermore, we have observed higher levels of intracellular siRNA delivery utilizing the RNTs as compared to commercially available Lipofectamine through the use of fluorescently labelled siRNA. Improved gene silencing capabilities were also exhibited compared to commercially available siRNA transfection agents. These data suggest that RNTs has the potential to be both an efficient and biocompatible gene delivery platform.I diseases constitute an immense global threat, being responsible for 15 million of deaths per year worldwide. Treatment of infectious diseases caused by intracellular microorganisms, such as M. tuberculosis, M. avium, Leishmania and Plasmodium spp. are often hampered by limited access of drugs to infected cells. Over the last decades, liposomes, the most studied and successful drug delivery system allowed to improve the the pharmacologic and therapeutic properties of several molecules. One example is the case of aminoglycosides that due to inappropriate biodistribution and/or pharmacokinetic profiles render them not satisfactory for medical use. Paromomycin (PRM) is an aminoglycoside with a broad spectrum in-vitro activity against protozoa and mycobacteria. However, it is poorly absorbed into systemic circulation after oral administration and when parenterally injected undergoes rapid clearance being excreted upon glomerular filtration in urine. In the present work, the association of PRM to liposomes resulted in a huge accumulation of the antibiotic in liver, spleen and lungs, relative to free form. The in-vivo biodistribution changes of PRM liposomes were translated into an enhanced therapeutic effect in murine models infected with M. avium and Leishmania infantum with an absence of toxic effects. The obtained results demonstrate the potential of PRM liposomes as an alternative therapeutic strategy for treatment of mycobacterial and parasite infections.T formulation of pharmaceutical quality dietary supplements that have adequate physical and chemical stability as well as are safe, cost effective and technologically feasible can entail numerous challenges. In contrast to drugs which are usually well defined chemical entities, botanicals are complex ingredients containing multiple chemical components and often several classes of compounds are present in a single product. Many of these compounds are unstable to heat, light, oxygen, alkaline pH and elevated humidity. They may also have poor flow, bulk density and variable particle size distribution. Thus successful development of nutraceuticals requires knowledge of the fundamental aspects of the physical and chemical properties of the various forms of the ingredients, the use of adequate techniques of manufacturing, selection of the right excipient and the addition of suitable manufacturing overages based upon critical stability studies. Regulatory requirements also pose challenges to the development of dietary supplements. Based on the ingredients and the claims, the formula can fit into different categories in different countries. Registration complexity and timing varies greatly by category and country with ever increasing scrutiny. In this oral presentation, I will talk about the formulation of dietary supplements and how it is similar and/or different from pharmaceutical formulations. The formulation of pharmaceutical quality dietary supplements that have adequate physical and chemical stability as well as are safe, cost effective and technologically feasible can entail numerous challenges. In contrast to drugs which are usually well defined chemical entities, botanicals are complex ingredients containing multiple chemical components and often several classes of compounds are present in a single product. Many of these compounds are unstable to heat, light, oxygen, alkaline pH and elevated humidity. They may also have poor flow, bulk density and variable particle size distribution. Thus successful development of nutraceuticals requires knowledge of the fundamental aspects of the physical and chemical properties of the various forms of the ingredients, the use of adequate techniques of manufacturing, selection of the right excipient and the addition of suitable manufacturing overages based upon critical stability studies. Regulatory requirements also pose challenges to the development of dietary supplements. Based on the ingredients and the claims, the formula can fit into different categories in different countries. Registration complexity and timing varies greatly by category and country with ever increasing scrutiny. In this oral presentation, I will talk about the formulation of dietary supplements and how it is similar and/ or different from pharmaceutical formulations.R is a 30mer, arginine-rich, amphipathic peptides with both cell-penetrating and endosomolytic properties. When mixed with plasmid DNA it condenses to form small, serum-stable nanoparticles which, when administered in-vivo, are capable of transfecting cells. However, bioavailability in-vivo was limited to the lungs and liver of treated mice. In order to improve nanoparticle pharmacokinetics, polyethylene glycol (PEG) 5K was conjugated to the C-terminus of the RALA peptide and the resulting conjugate (RALA-P) was characterized in-vitro. Disappointingly, activity in-vitro was nullified following addition of PEG which lead us to adopt an alternative strategy that involved mixing of RALA-P with native RALA at various w/w ratios in an attempt to restore cellular level activity without compromising pharmacokinetic benefits instilled through the addition of PEG. The resulting nanoparticle exhibited improved salt stability at physiological concentrations of NaCL and was also capable of transfecting cells in-vitro. To test biodistribution of the modified nanoparticles, various w/w ratios of RALA/RALA-P containing plasmid fire-fly luciferase were administered to tumour-bearing mice in order to determine whether introduction of PEG in this way could enable transfection in-vivo, augment accumulation in the tumour by EPR and reduce accumulation in off-target organs. The results demonstrated a significant reduction in accumulation in the liver and lungs of treated mice and an increase in tumour expression for RALA/RALA-P nanoparticles as compared to RALA only.