Development of polymeric drug delivery systems for biotech products

Since the early 80’s the forward steps in genetics and proteomics, have led a particular interest to biotech products, such as DNA and proteins. Although difficult, their large-scale production enabled the therapeutic use of this compounds. Proteins and DNA sequences can be very interesting therapeutic molecules owing to their high selectivity/affinity for the receptor or the specific site of action. Unfortunately, some issues still limit their pharmaceutical use, such as the susceptibility to enzymatic degradation, rapid renal clearance and immunogenicity. To overcome these limitations, many researchers are seeking solutions in the field of drug delivery systems (DDSs). In this respect, many systems have been developed and conjugation with PEG (polyethylene glycol) can be considered one of the leading approaches. PEGylation brings to the conjugated molecule great solubility and stability to proteolytic digestion, furthermore it reduces the tendency to aggregate and reduces the immunogenicity. Thanks to these advantages and the particular characteristics of PEG, to date, there are on the market 12 pegylated compounds: 9 are proteins, one peptide, one aptamer and a liposomal formulation, containing doxorubicin. The improvements in the pharmacokinetic profile of these drugs, thanks to the use of drug delivery systems, can be also applied in the field of tissue engineering, where the same issues are of fundamental importance for the development of scaffolds for cells capable of releasing growth factors. In the last years various polymers have been studied by many research groups to find an alternative to PEG, but its excellent biocompatibility and the know-how in its use has not brought any polymer to be truly competitive against PEG. Nevertheless, PEG presents some limits such as its non-biodegradability and in some case there are reports of antibodies against PEG. Therefore, there is an increased need for a PEG substitute. In the first section of this work hyaluronic acid (HA) has been studied as a candidate polymer for bioconjugation of proteins (HAylation). HA, being biodegradable can compensate this limit of PEG. HA, is also present in humans and is metabolized by hyaluronidase. Moreover, HA has the advantage of a high loading compared to PEG, thanks to the presence of repetitive functional groups in each monomer. This part of the work was focused on the study of HA conjugation (HAylation) to two model enzymes, trypsin and Ribonuclease A, and then to an interesting protein in pharmaceutical field, insulin. In order to avoid cross-linking phenomena, only a fraction of all carboxyl groups of the polymer has been modified to aldehyde allowing the conjugation with the amino groups of the protein models. Furthermore, by modulating the pH of reaction two protein-HA conjugates were obtained, selective N-terminal (pH 6) or random (pH 8), this taking advantage of the different pKa values of the amino groups in the proteins. The first products obtained with the enzymes Ribonuclease A and trypsin were tested verifying the residual activity compared to the native proteins. All conjugates, in particular those obtained by N-terminal selective conjugation, maintain a good activity on small substrates (30% decrease); only the HA-derived trypsin retains about 60% of residual activity against the substrate with a high weight molecular. Furthermore, enhanced stability over time was found for HA-trypsin respect to the free enzyme (45% on average) and also susceptibility to hyaluronidase was confirmed for both conjugates. Polymer validation as potential protein carrier was then evaluated by preparing conjugates with bovine insulin, as an example of pharmacologically active protein. Two conjugates were synthesized by N-terminal selective conjugation starting from polymers with different degree of aldehyde derivatization, 4% and 21%, yielding products with a protein loading of 17% and 32% (w/w), respectively. The therapeutic efficacy of the conjugates in comparison with insulin was tested in Sprague Dawley rats with induced diabetes. The conjugate with a lower protein loading was more effective and with a longer pharmacodynamic effect on the reduction on blood glucose level. The second section of the work was focused on an innovative strategy of enzymatic PEGylation of oligonucleotides. Briefly, the method investigated on model oligonucleotides is composed of two steps: the first consists in the chemical conjugation of a short oligonucleotide to a PEG chain, the second step is the enzymatic-mediated conjuagation of the PEGylated oligonucleotide with a DNA sequence by the DNA T4 ligase. To study the enzymatic PEGylation, 4 oligo sequences have been prepared as ligation model: two complementary pairs ending with sticky-ends in turn complementary (18-mer + 21-mer and 16-mer + 19-mer). The 18-mer has a thiol group in 5’-ending, in order to perform the coupling with PEG. Applying some modifications to ligation classical protocols, excellent results were obtained: PEGylated portion completely ligate the other ds-DNA and no undesired products were found. To further confirm the effective ligation, the ligated and PEGylated sequence was restricted with EcoRI. Indeed, the EcoRI recognized a sequence that is present only the ligated DNA. Complete restriction was found in absence and even in the presence of the polymer, further confirming the successes of ligation. Furthermore it was investigated if a reduced number of bases coupled to PEG can still preserve the requirements for the ligase enzyme activity. Thus, the pair of the complementary sequences then coupled to PEG has been reduced to half (9-mer + 12-mer). Even with a shorter PEGylated sequence a complete ligation was obtained. In conclusion in this thesis it has been demonstrated that HA can be a valid alternative to PEG for protein conjugation. In the field of oligonucleotide delivery an enzymatic approach of oligonucleotide conjugation can open new horizons that so far have not been completely explored

Enzymatic Formation of PEGylated Oligonucleotides

Gene therapy, siRNA, and therapeutic aptamers attract great interest owing to their versatility to treat a wide range of diseases and their potential high selectivity. Unfortunately, oligonucleotide-based therapeutics suffer rapid degradation by nucleases, scarce cell internalization, and fast kidney clearance. To address these limitations, the covalent attachment by mild chemical reactions of an activated polyethylene glycol (PEG) is widely used to obtain PEGylated nucleic acids showing a more favorable pharmacokinetic profile. We describe here a method for the enzymatic formation of PEGylated nucleic acids employing T4 DNA ligase: the ligation protocol was set up and optimized allowing the complete achievement of PEGylated oligonucleotides amenable to further enzymatic reactions. The feasibility of this approach for bioconjugation was demonstrated employing a set of PEG-donors and oligonucleotide acceptors, differing in the chemical link between PEG and the oligonucleotide donor, and in the length, sequence, and structure of the oligonucleotides employed. The ligase reaction allowed us to obtain double-stranded as well as single-stranded oligonucleotides, thus demonstrating the applicability of the method to a variety of substrates suitable for diagnostic and therapeutic applications

Conjugation of hyaluronan to proteins

Polymer conjugation has been widely exploited to prolong half-life and reduce immunogenicity of therapeutic proteins. Here, the potentials of hyaluronic acid (HA) have been investigated by studying the conjugates with two model enzymes, trypsin and RNase A, and with insulin. As the direct coupling of proteins to the HA\u2019s carboxylic groups can cause cross-linking problems, a hyaluronan-aldehyde derivative has been synthesized for N-terminal site-selective conjugation. HA conjugation, termed HAylation, preserved the activities of enzymes and their thermal stabilities. Insulin HAylation was studied by preparing two conjugates with different peptide loadings (32% and 17%, w/w). Noticeably, the conjugate with the lower loading showed the greater effect on blood glucose level. The 17% HA-insulin conjugate showed a lowering effect on blood glucose level for up to 6 h, while free insulin exhausted its action after 1 h. This study highlights the potentials of hyaluronan-aldehyde for protein delivery

Toll like receptor-2 regulates production of glial-derived neurotrophic factors in murine intestinal smooth muscle cells

Gut microbiota-innate immunity axis is emerging as a key player to guarantee the structural and functional integrity of the enteric nervous system (ENS). Alterations in the composition of the gut microbiota, derangement in signaling of innate immune receptors such as Toll-like receptors (TLRs), and modifications in the neurochemical coding of the ENS have been associated with a variety of gastrointestinal disorders. Indeed, TLR2 activation by microbial products controls the ENS structure and regulates intestinal neuromuscular function. However, the cellular populations and the molecular mechanisms shaping the plasticity of enteric neurons in response to gut microbes are largely unexplored. In this study, smooth muscle cells (SMCs), enteric glial cells (EGCs) and macrophages/dendritic cells (M\u3a6/DCs) were isolated and cultured from the ileal longitudinal muscle layer of wild-type (WT) and Toll-like receptor-2 deficient (TLR2-/-) mice. Quantification of mRNA levels of neurotrophins at baseline and following stimulation with TLR ligands was performed by RT-PCR. To determine the role of neurotrophins in supporting the neuronal phenotype, we performed co-culture experiments of enteric neurons with the conditioned media of cells isolated from the longitudinal muscle layer of WT or TLR2-/- mice. The neuronal phenotype was investigated evaluating the expression of \u3b2III-tubulin, HuC/D, and nNOS by immunocytochemistry. As detected by semi-quantitative RT-PCR, SMCs expressed mRNA coding TLR1-9. Among the tested cell populations, un-stimulated SMCs were the most prominent sources of neurotrophins. Stimulation with TLR2, TLR4, TLR5 and TLR9 ligands further increased Gdnf, Ngf, Bdnf and Lif mRNA levels in SMCs. Enteric neurons isolated from TLR2-/- mice exhibited smaller ganglia, fewer HuC/D+ve and nNOS+ve neurons and shorter \u3b2III-tubulin axonal networks as compared to neurons cultured from WT mice. The co-culture with the conditioned media from WT-SMCs but not with those from WT-EGCs or WT-M\u3a6/DCs corrected the altered neuronal phenotype of TLR2-/- mice. Supplementation of TLR2-/- neuronal cultures with GDNF recapitulated the WT-SMC co-culture effect whereas the knockdown of GDNF expression in WT-SMCs using shRNA interference abolished the effect on TLR2-/- neurons. These data revealed that by exploiting the repertoire of TLRs to decode gut-microbial signals, intestinal SMCs elaborate a cocktail of neurotrophic factors that in turn supports neuronal phenotype. In this view, the SMCs represent an attractive target for novel therapeutic strategie

Osteogenic properties of a short BMP-2 chimera peptide

Bonemorphogenetic proteins (BMPs) play a key role in bone and cartilage formation. For these properties, BMPs are employed in the field of tissue engineering to induce bone regeneration in damaged tissues. To overcome drawbacks due to the use of entire proteins, synthetic peptides derived from their parent BMPs have come out as promising molecules for biomaterial design. On the structural ground of the experimental BMP-2 receptor complexes reported in the literature, we designed three peptides, reproducing the BMP-2 region responsible for the binding to the type II receptor, ActRIIB. These peptides were characterized by NMR, and the structural features of the peptide–receptor binding interface were highlighted by docking experiments. Peptide– receptor binding affinities were analyzed bymeans of ELISA and surface plasmon resonance techniques. Furthermore, cellular assays were performed to assess their osteoinductive properties. A chimera peptide, obtained by combining the sequence portions 73–92 and 30–34 of BMP-2, shows the best affinity for ActRIIB in the series and represents a good starting point for the design of new compounds able to reproduce osteogenic properties of the parent BMP-2