Biocompatibility Evaluation of Engineered Amino Acid Pairing Peptides for Drug Delivery

To ensure the effective and safe use of nanomaterials for medical applications, the biocompatibility of the materials must be tested with particular relevance to the environment in which the material is placed. In nanoparticle-based drug delivery, it is crucial to evaluate a nanoparticle’s biocompatibility to ensure minimal cytotoxicity. Of several types of nanoparticles, peptide-based nanoparticles have emerged as promising systems for targeted cancer therapy. Yet, the biocompatibility of many of these peptides and their assembled particles has not been studied. This thesis, summarizes the original contribution on the effective and safe use of the particular self/co-assembling, amino acid pairing peptides and some of their DEGylated forms (modified versions) as carriers for anticancer drug delivery application. Therefore, the biocompatibility of the self-assembling, amino acid pairing (AAP) peptides AC8, its two DEGylated forms, as well as two related peptides, EAK16-II and EK8, is systematically investigated. The toxicity of these peptides and their complexes with pirarubicin was tested against the human adenocarcinoma lung cancer cell line, A549.The biocompatibility of the peptide-drug co-assembling complexes is assessed and the potential of these five peptides as carriers for the hydrophobic anticancer drug pirarubicin is demonstrated. For the first time experimental results on cytotoxicity, haemolytic activity, red blood cell (RBC) aggregation, complement activation and anaphylotoxin activation as an end result of complement activity for these five AAP peptides is reported. AC8, the amino end DEGylated AC8 (NP-I) and EK might be strong candidates for hydrophobic drug delivery considering their lack of toxicity and the fact that they are not recognized as a foreign molecule, inducing no considerable immune reactions. These results provide a basis for in vivo experiments and predict minimal in vitro toxicity of these peptides based delivery systems.1 yea

IN VIVO EPIGENETIC STUDY OF HISTONE ACETYLATION ASSOCIATED WITH OBESITY

Post translational modifications in histone proteins are transmissible changes that are not coded for in the DNA sequence itself but have a significant affect in the control of gene expression. Eukaryotic transcription is a regulated process, and acetylation plays a major role in this regulation. Deranged equilibrium of histone acetylation can lead to alteration in chromatin structure and transcriptional dysregulation of genes that are involved in the control of proliferation, cell-cycle progression, differentiation and or apoptosis. Evidence shows that high glucose conditions mimicking diabetes can increase histone acetylation and augment the inflammatory gene expression. Recent advances also highlight the involvement of altered histone acetylation in gastrointestinal carcinogenesis or hyperacetylation in amelioration of experimental colitis. However, the role of histone acetylation under obesity conditions is not yet known. Therefore in the present study, western blot analysis in the liver of Zucker obese versus lean rats was performed to determine the pattern and level of H3 and H4 acetylation (both in nuclear and homogenate fractions) at specific lysine (K) in pathological state of hepatic steatosis The same technique was also applied in the liver of obese rats fed higher amounts of vitamin B6 (OH) versus those fed normal amounts of vitamin B6 (ON) to assess if hyper-acetylation can be a protective response to hepatic steatosis. In both experimental models, it was also of interest to elucidate the expression of anti- and pro- apoptotic factor Bcl-2 and Bax in respect to histone acetylation. It was observed that, in liver homogenate fractions in control animals (LC/OC), there was a higher level of histone H3 acetylation at (K9, K14) and H4 acetylation at K5 in the obese animals. In contrast, the nuclear level of H3 and H4 acetylation at the same lysine residues was considerably higher in the lean and lower in the obese animals. Obese animals contained lower liver preneoplastic lesions as well as liver weight as a result of higher amounts of vitamin B6, had significantly higher H3 acetylation at K9 and K14 and H4 acetylation at K5, in both homogenate and nuclear fractions. However, histone acetylation was not detected for histone H4 at lysine 12 (K12) in either control group (LC/OC) or obese with different B6 diet group (OH/ON). Nevertheless, global histone H3 and H4 acetylation in both homogenate and nuclear fractions, was slightly higher in the lean rats and obese rats fed higher amounts of B6. By using the western blot technique, the level of anti- and pro- apoptotic Bcl-2 and Bax were also evaluated. The moderately higher level expression of anti-apoptotic Bcl2 protein was found in lean animals, whereas the expression of pro-apoptotic Bax was significantly higher in obese animals. Furthermore, anti-apoptotic Bcl2 protein expression was slightly higher in the obese rats fed normal amounts of B6 diet; but, pro-apoptotic Bax was higher in the obese rats fed higher amounts of vitamin B6. This is the first study which shows that hyperacetylation of histones in liver nuclei can be correlated with amelioration of hepatic steatotis. Histone acetylation and B6 rich diet might be involved in the regulation of biological availability of key apoptotic proteins, which, in turn, can possibly modify the severity of the disease

Biocompatibility of a self-assembled crosslinkable hyaluronic acid nanogel

Hyaluronic acid nanogel (HyA-AT) is a redox sensitive crosslinkable nanogel, obtained through the conjugation of a thiolated hydrophobic molecule to the hyaluronic acid chain. Engineered nanogel was studied for its biocompatibility, including immunocompatibility and hemocompatability. The nanogel did not compromise the metabolic activity or cellular membrane integrity of 3T3, microvascular endothelial cells, and RAW 264.7 cell lines, as determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and lactate dehydrogenase release assays. Also, we didn't observe any apoptotic effect on these cell lines through the Annexin V-FITC test. Furthermore, the nanogel cell internalization was analyzed using murine bone marrow derived macrophages, and the in vivo and ex vivo biodistribution of the Cy5.5 labeled nanogel was monitored using a non-invasive near-infrared fluorescence imaging system. The HyA-AT nanogel exhibits fairly a long half-live in the blood stream, thus showing potential for drug delivery applications.The authors thank the FCT Strategic Project of UID/BIO/04469/2013 unit, the project RECI/ BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and the Project “BioHealth – Biotechnology and Bioengineering approaches to improve health quality”, Ref. NORTE-07-0124- FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER. The authors would like to acknowledge also the support of FCT for the PhD grant reference SFRH/BD/61516/2009. They would also like to thank Bioimaging department on Molecular Medicine Institute (IMM) in Lisbon, namely Dr José Rino and Dr António Temudo. Also thank the animal facilities in IMM (Lisbon), specially Dr. Dolores Bonaparte and Dr. Joana Marques. Finally, the authors thank Dr Africa Gonzalez and Mercedes Pelletero the performance of the studies on the activation of complement

Biocompatibility of a self-assembled glycol chitosan nanogel

The research of chitosan-based nanogel for biomedical applications has grown exponentially in the last years; however, its biocompatibility is still insufficiently reported. Hence, the present work provides a thorough study of the biocompatibility of a glycol chitosan (GC) nanogel. The obtained results showed that GC nanogel induced slight decrease on metabolic activity of RAW, 3T3 and HMEC cell cultures, although no effect on cell membrane integrity was verified. The nanogel does not promote cell death by apoptosis and/or necrosis, exception made for the HMEC cell line challenged with the higher GC nanogel concentration. Cell cycle arrest on G1 phase was observed only in the case of RAW cells. Remarkably, the nanogel is poorly internalized by bone marrow derived macrophages and does not trigger the activation of the complement system. GC nanogel blood compatibility was confirmed through haemolysis and whole blood clotting time assays. Overall, the results demonstrated the safety of the use of the GC nanogel as drug delivery system.Paula Pereira thanks FCT, the Ph.D. grant ref SFRH/BD/64977/2009. This work was also supported by a grant from the Spanish Ministry of Economy and Competitivity (SAF2011-30337-C02-02). We also acknowledge the European Union Seventh Framework Programme [FP7/REGPOT-2012-2013.1] under grant agreement BIOCAPS-316265. MP acknowledges fellowship from Spanish Ministry of Education (FPU predoctoral grant program)

Furin-Mediated Sequential Delivery of Anticancer Cytokine and Small-Molecule Drug Shuttled by Graphene

A cellular protease (furin)-mediated graphene-based nanosystem is developed for co-delivery of a membrane-associated cytokine (tumor-necrosis-factor-related apoptosis-inducing ligand, TRAIL) and an intracellular-acting small-molecule drug (Doxorubicin, DOX). TRAIL and DOX can be sequentially released toward the plasma membrane and nucleus, respectively

A step-by-step protocol for generating human fibroblast cell-derived completely biological extracellular matrix scaffolds

Native extracellular matrix (ECM) based scaffolds are far more superior in structural and compositional complexity than other engineered scaffolding materials such as hydrogels, electrospun fibers, and three-dimensional (3D) printed substrates. Due to the presence of native structural proteins and other macromolecules, native ECM can better restore the crucial cell-ECM crosstalk and provide a highly biomimetic microenvironment to cells. Allogenic or xenogeneic tissues have been derived by decellularization to obtain native ECM scaffolds. However, their applicability is limited by batch to batch variation, risk of pathogen transfer, undesirable immune response and scarcity of donors. Human dermal fibroblasts (hDFs) can be prescreened and maintained in a pathogen-free condition. Herein, we have described a step-by-step protocol to generate a completely biological ECM scaffold by decellularization of hDF cell sheets. Decellularization was achieved by using an anionic surfactant sodium dodecyl sulfate (SDS) and ethylene diamine tetraacetate (EDTA). The resulting ECM sheet was organized into a nanofibrous scaffold, containing major ECM structural proteins as well as other macromolecules including collagens, fibronectin, laminin and elastin. This cell-derived nanofibrous ECM is a promising scaffold material for constructing highly biomimetic functional tissues

Investigating the Pharmacokinetics and Biological Distribution of Silver-Loaded Polyphosphoester-Based Nanoparticles Using 111Ag as a Radiotracer

Purified 111Ag was used as a radiotracer to investigate silver loading and release, pharmacokinetics, and biodistribution of polyphosphoester-based degradable shell crosslinked knedel-like (SCK) nanoparticles as a comparison to the previously reported small molecule, N-heterocyclic silver carbene complex analog (SCC1) for the delivery of therapeutic silver ions in mouse models. Biodistribution studies were conducted by aerosol administration of 111Ag acetate, [111Ag]SCC1, and [111Ag]SCK doses directly into the lungs of C57BL/6 mice. Nebulization of the 111Ag antimicrobials resulted in an average uptake of 1.07 ± 0.12% of the total aerosolized dose given per mouse. The average dose taken into the lungs of mice was estimated to be 2.6 ± 0.3% of the dose inhaled per mouse for [111Ag]SCC1 and twice as much dose was observed for the [111Ag]SCKs (5.0 ± 0.3% and 5.9 ± 0.8% for [111Ag]aSCK and [111Ag]zSCK, respectively) at 1 h post administration (p.a.). [111Ag]SCKs also exhibited higher dose retention in the lungs; 62–68% for [111Ag]SCKs and 43% for [111Ag]SCC1 of the initial 1 h dose were observed in the lungs at 24 h p.a.. This study demonstrates the utility of 111Ag as a useful tool for monitoring the pharmacokinetics of silver-loaded antimicrobials in vivo