A new non-toxic chemically cross-linked ELP hydrogel for in situ protein delivery

Proteins play an essential role in all biological processes and have been attracting more and more attention as drug candidates due to their high specificity and activity. However, their use as therapeutics has been seriously held back given that their ability to be effectively and conveniently delivered is problematic. Indeed, simple oral administration is ineffective since proteins are degraded in the digestive tract. Until now, these therapeutic agents were delivered by infusions or frequent injections. But this causes serious problems, as the compound does not get delivered in a prolonged continuous manner. Additionally, injections are also problematic as patients are reluctant to use needles unless there illness is life threatening. That is why focus has grown over the last years on developing novel delivery systems for proteins. Amongst the various types of delivery systems that exist, hydrogel based ones are very promising, as they possess remarkable biomimetic properties. The hydrogels that are chemically cross-linked are stronger than their counterparts, the physical hydrogels. An appealing approach to protein delivery that is more and more sought is to form the hydrogel in situ from an injected aqueous polymer solution and to use the formed gel as a depot for the sustained release of the protein. Oxygen sensitive gellation is an interesting stimulus to form these gels, but has not been successfully developed, as the gelling kinetics are too slow. Therefore, research groups have turned to the use of toxic chemicals to speed up the reaction. Elastin-like poylpeptides (ELP) are artificial biopolymers that are composed of a pentapeptide repeat. This repeat consists of Val-Pro-Gly-Xaa-Gly, where Xaa denotes a guest residue. An interesting characteristic of ELPs is that they are thermally responsive. Meaning, that when in solution they go through a lower critical solution temperature (LCST) transition. Below their LCST they are soluble in water and above their LCST they are insoluble. ELPs are well suited for drug delivery applications for many reasons such as their biocompatibility. The ELP that is studied is ELP9 Gel. Its guest residues are Val, Ala and Cys in a ratio of 1:14:1 respectively. As this ELP has cysteines it has the capacity to form a gel via cross-linking of the thiols groups by oxidation. The rheological tests that were carried out showed that this ELP9 Gel has all the characteristics of a gel. Furthermore, its gelling kinetics were greatly increased by heating it, but still remain too long for in situ applications. This suggests that the LCST behaviour of ELPs seems to play some role in favouring the cross-linking. But further controls remain to be done to confirm this hypothesized tandem process. A delivery strategy that involved this ELP9 Gel and Blue Fluorescent Proteins (BFP) with added cysteines was not successful as the molecular biology to obtain the modified proteins failed. However, the elaboration of a fusion peptide between Glucagon-Like-Peptide-1 (GLP-1) and ELP9 Gel was accomplished. The preliminary activity assay showed a reduced activity in comparison to the GLP-1 peptide alone. Unfortunately this ELP9 Gel has a setback due to its slow gelling time. Therefore, before continuing the research on the delivery strategies, it would be wiser to create a better ELP. If a new ELP with a higher cysteine concentration and lower transition temperature can be created, its gelling kinetics may be favourable for in situ protein delivery applications and thus, a chemically cross-linked hydrogel that does not require any toxic cross-linker would be available. This would be a big step in creating a very robust hydrogel for protein delivery applications but also for tissue regeneration

Analysis of microbial community structures and functions in heavy metal-contaminated soils using molecular methods

Fabienne Gremion

The contamination of agricultural land and groundwater by heavy metals is essentially linked to human activities. A major problem with heavy metals is that they cannot be biodegraded and therefore reside in the environment for long periods of time if they are not removed. Thus, depending of the kind and depth of contamination, different remediation techniques were developed. One of these methods, called "phytoextraction", uses the ability of so-called hyperaccumulating plants to extract high amounts of heavy metals. Accumulation of heavy metals in the environment is a serious concern for animal and human health. At the microscopic scale, heavy metals may have also deleterious effects on bacteria which are the key-players of the different nutrient turnovers in soils. Consequently, ecosystem functioning can be seriously perturbed and the long-term soil fertility may be threatened. The recent development of molecular biology greatly contributed to the discovery of the microbial diversity and its function in the soil. However, to date only a small number of studies used molecular methods to investigate the impacts of heavy metals on the bacterial community. In this thesis, a pot experiment was conducted under controlled conditions with one hyperaccumulating plant (Thlaspi caerulescens) grown in two different soils, a long-term and an artificially heavy metal-contaminated soil. The impact of heavy metals on the microbial community was then investigated with several molecular methods. Moreover, the decrease of the bioavailable heavy metals concentrations in soil due to plant uptake allowed to study the consequences on bacterial community structure and function. Based on the 16S ribosomal RNA and the corresponding gene (16S rDNA), four clone libraries were constructed to retrieve information on the structure of the microbial community and the potentially active part of the microbial community in the rhizosphere of Thlaspi caerulescens grown during three months in a long-term contaminated soil. The data obtained with the two clone libraries (rRNA and rDNA) from the rhizosphere of Thlaspi caerulescens were compared with the bulk soil data to identify any effect of the plant on the soil microbial community structure. Partial sequence analysis of 282 clones revealed that most of the environmental sequences in both soils affiliated with five major phylogenetic groups, the Actinobacteria, α-Proteobacteria, β-Proteobacteria, Acidobacteria and the Planctomycetales. The taxa dominating the bacterial community structure in the bulk soil also dominated the rhizosphere community, indicating that the plant did not exert a major influence on the overall bacterial diversity. However, all dominant taxa, with the exception of the Actinobacteria, were relatively less represented in the rRNA libraries as compared to the rDNA libraries. On the contrary, sequences belonging to the Actinobacteria dominated both bulk and rhizosphere soil libraries derived from rRNA. Seventy per cent of these clone sequences were related to two subgroups of the Rubrobacteria, which was an indication that this group of bacteria was probably metabolically active in heavy metal-contaminated soils. Fluorescence in situ hybridization (FISH) was used for the in situ detection and quantification of selected bacterial groups previously detected in the clone libraries from the rhizosphere of Thlaspi caerulescens. By applying the most general probe EUB338, only 20% of the total rhizosphere microbial community could be detected. Based on this result, it was difficult to conclude with certitude which were the most dominant bacteria in the rhizosphere. However, despite this low detection rate, it was possible to detect the major groups present in the rhizosphere clone libraries using group-specific oligonucleotide probes. As part of our sequences were affiliated to two emerging bacterial groups, the Acidobacteria and the Rubrobacteria, two new probes were designed, Acido228 (specific for the subgroup 1 of the Acidobacterium division) and Rubro198 (specific for the all Rubrobacteria subclass), for the detection of these microorganisms. These two probes were first checked for their specificity with pure cultures and finally applied in the rhizosphere soil allowing for the first time the detection of these organisms in situ. Finally, the impact of heavy metals and a subsequent one-year phytoextraction with Thlaspi caerulescens on the soil microbial community was investigated in an artificially heavy metal-contaminated soil. All the different molecular and culture-dependent techniques used, denaturing gradient gel electrophoresis (DGGE), community level physiological profile (CLPP) and potential ammonium-oxidation measurement showed that the heavy metal addition induced drastic changes in the bacterial community. Moreover, the analysis of the different bacterial DGGE patterns (Bacteria, β-Proteobacteria and ammonia-oxidising bacteria) obtained during this experiment showed that one-year phytoremediation was not sufficient to recover the initial community present in the non-contaminated soil. However, with the CLPP analysis, it was possible to detect a stimulating effect of the plant on a part of the microbial community in both contaminated and non-contaminated soils. The most obvious result was obtained in the contaminated soil where the number of substrates metabolised increased significantly in the presence of the plant as compared to the unplanted contaminated samples. The measurement of the potential ammonium-oxidation was used as a criterion for soil quality. Although this test showed that the ammonia-oxidising bacteria were significantly stimulated in the planted non-contaminated soil samples, the positive effect of the plant on these bacteria was not sufficient to overcome the inhibition induced by the presence of the heavy metals in the contaminated soil, even after one year phytoremediation. To conclude, molecular methods in combination with culture-dependent techniques have proven in this study to be very useful for the detection of the changes induced by the heavy metals in the structure and the function of the microbial community. Moreover, the molecular techniques contributed to the identification of bacteria which could be potentially used for the bioremediation of contaminated soils thus offering new perspectives of investigation and technology development.

EPFL2003

Exploiting immunofunctional nanoparticles as vaccines and synthetic pathogens

Sai Reddy

The emergence of recombinant DNA technology has shifted research towards developing subunit vaccines, which use protein or peptide antigens instead of live/attenuated pathogens. Recent research has also shown that the best way to get sustained immunity is to deliver an antigen directly to antigen-presenting cells (APCs), specifically dendritic cells (DCs). However there are two difficulties to overcome in targeting DCs for use in subunit vaccines: (1) DCs are present in a low concentration in peripheral tissue (where typical vaccines are delivered) compared to secondary lymphoid organs such as lymph nodes. Thus delivering antigen to DCs in lymph nodes offers a powerful alternative for vaccine technology. (2) Activating DCs requires co-delivering an adjuvant also sometimes known as a molecular "danger signal" which matures DCs and initiates the subsequent adaptive immune response. Adjuvants are needed since most protein and peptide antigens are insufficiently immunogenic. Current danger signals being explored in experimental vaccine formulations mimic bacterial components (e.g., lipopolysaccharide (LPS), unmethylated CpG DNA) and often signal through a class of pattern recognition receptors knows as Toll-like receptors (TLRs), however limitations exist in that these often cause toxic side effects by inducing high levels pro-inflammatory cytokines. To address the need to target DCs, we used a nanoparticle delivery approach. We investigated the delivery of 20, 45, and 100 nm diameter poly(ethylene glycol)-stabilized poly(propylene sulfide) (PPS) nanoparticles to DCs in the lymph nodes. These nanoparticles consisted of a cross-linked rubbery core of PPS surrounded by a hydrophilic corona of poly(ethylene glycol). The PPS domain is capable of carrying hydrophobic drugs and degrades within oxidative environments. 20 nm particles were most readily taken up into lymphatics following interstitial injection, while both 20 and 45 nm nanoparticles showed significant retention in lymph nodes, displaying a consistent and strong presence at 24, 72, 96 and 120 h post-injection. Nanoparticles were internalized by up to 40-50% of lymph node DCs (and APCs) without the use of a targeting ligand, and the site of internalization was in the lymph nodes rather than at the injection site. Finally, an increase in nanoparticle-containing DCs (and other APCs) was seen at 96 h vs. 24 h, suggesting an infiltration of these cells to lymph nodes. Thus, PPS nanoparticles of 20-45 nm showed potential for vaccine delivery specifically targeting DCs in lymph nodes. Next we exploited our nanoparticle technology as a vaccine platform. We engineered antigen-bearing nanoparticle vaccines with two novel features: lymph node-targeting and in situ complement activation. Following intradermal injection, ultra-small nanoparticles (25 nm) were readily transported by interstitial flows into lymphatic capillaries, which leads to their accumulation in lymph nodes resident DCs. We further designed the nanoparticle surface chemistry to activate the complement cascade, thus spontaneously generating a danger signal in situ for DC activation. With ovalbumin as a model antigen conjugated to the nanoparticles, we demonstrated humoral and cellular immunity in mice. Finally we exploited nanoparticles as "synthetic pathogens" to isolate the effect of free surface thiols on complement activation and the subsequent immunological response induced. We found that surfaces with both hydroxyl and thiol groups activated complement to significantly higher levels than hydroxl groups alone. We then demonstrated that nanoparticles that induced thiol-associated complement activation were potent danger signals that were able to mature dendritic cells to express high levels of costimulatory molecules and the Th1 promoting cytokine interuleukin-12. Moreover, this dendritic cell maturation was dependent on a Toll-like receptor 4 (TLR4) signaling pathway and independent of the adaptor protein myeloid differentiation protein 88 (MyD88). Thus we uncover a potential direct link between complement and TLR4 signaling that is dependent on the specific activation of complement by thiol-expressing surfaces. In conclusion, this thesis develops a novel vaccine methodology, which is at the interface of nanotechnology and biotechnology, by harnessing lymphatic transport to target DCs and complement activation to activate adaptive immunity; we demonstrated an elegant vaccine technology platform for applications that include global health. Finally we exploit our nanoparticles as synthetic pathogens to discover fundamental mechanisms connecting the complement system and TLR signaling.

EPFL2008

Exploiting immunofunctional nanoparticles as vaccines and synthetic pathogens

Sai Reddy

EPFL2008

Analysis of microbial community structures and functions in heavy metal-contaminated soils using molecular methods

Fabienne Gremion

EPFL2003