Inflammation is a non-specific immune response in vascularized tissues to harmful stimuli. Its role is to recruit leukocytes and plasma proteins to the site of damage, helping eliminate pathogens, repair tissue, and restore homeostasis. In chronic diseases, prolonged inflammation can lead to tissue deterioration and contribute to conditions such as rheumatoid arthritis and gout. Gout is a common form of inflammatory arthritis caused by the accumulation of monosodium urate crystals when blood uric acid levels exceed the solubility threshold. This activates the NLRP3 inflammasome, leading to overproduction of IL-1β, which recruits neutrophils and other immune cells, resulting in acute and painful inflammation. The mannose receptor (CD206 or MR), a transmembrane glycoprotein expressed by macrophages and other immune cells, has been studied for its involvement in inflammatory processes. The mannose receptor is formed by three distinct domains: a C-type lectin-like domain, a fibronectin type II domain, and a cysteine-rich domain that can bind 3-O-sulfo-galactosylated polymers (SO4-3-Gal)n. These polymers can bind to MR, trigger receptor endocytosis and block its recycling, potentially reducing interactions with pro-inflammatory molecules. This study focused on testing the hypothesized mechanism for (SO4-3-Gal)240 polymers anti-inflammatory activity previously observed in a gout model and to develop micro- and nanoparticles designed for oral and parenteral administration, aimed at prolonging the polymer half-life and at possibly co-encapsulating additional drugs by generating a new drug delivery system with intrinsic anti-inflammatory properties. First, the monomer was synthesized starting from b-D-galactose pentaacetate, which was modified with hydroxyethyl acrylamide to introduce a vinyl group necessary for the polymerization reaction. After deacetylation, D-galactopyranosyloxyethyl acrylamide was selectively sulfated at the 3-position to obtain 3-O-sulfo-D-galactopyranosyloxyethyl acrylamide. The monomer was polymerized through reversible addition-fragmentation chain transfer (RAFT) technique, resulting in the formation of a polymer bearing 240 unit (SO4−3−Gal)240. The polymer was used as it is for the in vitro studies with the gout model induced by cells incubation with monosodium urate crystals. THP-1 cells were differentiated into macrophages by incubation with phorbol 12-myristate 13-acetate. Proper cell differentiation was assessed through flow cytometric analysis of the lineage markers F4/80, CD80 and the target CD206. Cells pre-treatment with or without (SO4−3−Gal)240 followed by incubation with MSU in the presence or absence of NF-κB inhibitor suggested that (SO4−3−Gal)240 might reduce the production of IL-1β by interacting with the NF-κB pathway, since the pre-treatment with the glycopolymer + NF-κB inhibitor did not enhance the response as compared to cells treated with the NF-κB inhibitor alone, thus ruling out the possibility that a second factor is involved. In parallel, polymer with different degree of polymerization (DP), in the range 30-240, were synthesized and functionalized with succinic anhydride to introduce a reactive carboxyl group available for the cross-linking via diamines, to induce particle formation. By using (SO4−3−Gal)240 cross-linked with hexamethylenediamine particles with a size of 1123 nm were generated. To this aim, a block copolymer (SO4−3−Gal)30−(hydroxypropyl acrylate)90 was also synthesized. It consists of a hydrophilic segment composed of SO4−3−Gal monomer and a hydrophobic segment derived from hydroxypropyl acrylate, designed to self-assemble into particles when dispersed in aqueous solution. Future tests will assess drug encapsulation in the particles and conduct in vitro studies to verify their increased half-life compared to linear polymers.
L'infiammazione è una risposta immunitaria non specifica dei tessuti vascolarizzati a stimoli dannosi. Il suo ruolo primario è quello di reclutare leucociti e proteine plasmatiche nel sito danneggiato, contribuendo all'eliminazione dei patogeni, alla riparazione dei tessuti e al ripristino dell'omeostasi. Nelle malattie croniche, l'infiammazione prolungata può portare al deterioramento dei tessuti e contribuire a patologie come la gotta. La gotta è una comune forma di artrite infiammatoria causata dall'accumulo di cristalli di urato monosodico (MSU). Questo attiva l'inflammasoma NLRP3, portando a una sovrapproduzione di IL-1β provocando un'infiammazione acuta e dolorosa. Il recettore del mannosio (CD206 o MR), una glicoproteina transmembrana espressa da macrofagi e altre cellule immunitarie, è stato studiato per il suo coinvolgimento nei processi infiammatori. Il recettore del mannosio è formato da tre domini distinti: un dominio lecitinico di tipo C, un dominio fibronectinico di tipo II e un dominio cisteinico, che può legarsi a polimeri galattosilati solfatati 3-O (SO4-3-Gal)n. Questi polimeri possono legarsi a MR, innescare l'endocitosi del recettore e bloccarne il riciclo, riducendo potenzialmente le interazioni con molecole proinfiammatorie. Lo scopo di questo progetto è stato testare il meccanismo ipotizzato per l'attività antinfiammatoria dei polimeri (SO4-3-Gal)240, precedentemente osservato in un modello di gotta e sullo sviluppo di micro- e nanoparticelle progettate per la somministrazione orale e parenterale, con l'obiettivo di prolungare l'emivita del polimero e co-incapsulare farmaci antinfiammatori, generando un nuovo sistema di rilascio di farmaci con proprietà antinfiammatorie intrinseche. Il monomero è stato sintetizzato partendo da β-D-galattosio pentaacetato, modificato con idrossietil acrilammide per introdurre un gruppo vinilico necessario per la polimerizzazione. Dopo la deacetilazione, il monomero D-galattopiranosilossietil acrilammide è stato solfatato selettivamente in posizione 3 per ottenere 3-O-sulfo-D-galattopiranosilossietil acrilammide. Successivamente è stato sintetizzato attraverso la tecnica RAFT (reversible addition-fragmentation chain transfer) un polimero di 240 unità (SO4-3-Gal)240. Il polimero è stato utilizzato per studi in vitro su un modello di gotta indotto dall'incubazione cellulare con cristalli di MSU. Le cellule THP-1 sono state differenziate in macrofagi mediante incubazione con forbolo 12-miristato 13-acetato. Il corretto differenziamento cellulare è stato valutato tramite analisi citofluorimetrica dei marcatori F4/80, CD80 e del recettore target CD206. Il trattamento cellulare con o senza (SO4-3-Gal)240 seguito da incubazione con MSU in presenza o assenza di inibitore di NF-κB ha suggerito che il polimero potrebbe ridurre la produzione di IL-1β interagendo con la via NF-κB. Infatti trattamento con il glicopolimero e l'inibitore di NF-κB non ha migliorato la risposta rispetto alle cellule trattate solo con l'inibitore. Parallelamente sono stati sintetizzati polimeri con diverso grado di polimerizzazione (DP) compreso tra 30 e 240, successivamente funzionalizzati con anidride succinica per introdurre un gruppo carbossilico reattivo disponibile per il cross-linking con diammine. Il cross-linking con esametilendiammina ha permesso di generare microparticelle di dimensioni pari a 1123 nm. Inoltre è stato anche sintetizzato un copolimero a blocchi (SO4-3-Gal)30-(idrossipropil acrilato)90. Il polimero costituito da un segmento idrofilo composto da monomeri SO4-3-Gal e un segmento idrofobico derivato dall’idrossipropil acrilato, è progettato per autoassemblarsi in particelle quando disperso in soluzione acquosa. Test futuri valuteranno l'incapsulamento di farmaci nelle particelle e condurranno studi in vitro per verificare l'aumento dell'emivita delle particelle rispetto ai polimeri lineari.
Studio sull'effetto antinfiammatorio di un glicopolimero solfatato su un modello di gotta e la sua modifica per generare particelle
BROJANIGO, SARA
2023/2024
Abstract
Inflammation is a non-specific immune response in vascularized tissues to harmful stimuli. Its role is to recruit leukocytes and plasma proteins to the site of damage, helping eliminate pathogens, repair tissue, and restore homeostasis. In chronic diseases, prolonged inflammation can lead to tissue deterioration and contribute to conditions such as rheumatoid arthritis and gout. Gout is a common form of inflammatory arthritis caused by the accumulation of monosodium urate crystals when blood uric acid levels exceed the solubility threshold. This activates the NLRP3 inflammasome, leading to overproduction of IL-1β, which recruits neutrophils and other immune cells, resulting in acute and painful inflammation. The mannose receptor (CD206 or MR), a transmembrane glycoprotein expressed by macrophages and other immune cells, has been studied for its involvement in inflammatory processes. The mannose receptor is formed by three distinct domains: a C-type lectin-like domain, a fibronectin type II domain, and a cysteine-rich domain that can bind 3-O-sulfo-galactosylated polymers (SO4-3-Gal)n. These polymers can bind to MR, trigger receptor endocytosis and block its recycling, potentially reducing interactions with pro-inflammatory molecules. This study focused on testing the hypothesized mechanism for (SO4-3-Gal)240 polymers anti-inflammatory activity previously observed in a gout model and to develop micro- and nanoparticles designed for oral and parenteral administration, aimed at prolonging the polymer half-life and at possibly co-encapsulating additional drugs by generating a new drug delivery system with intrinsic anti-inflammatory properties. First, the monomer was synthesized starting from b-D-galactose pentaacetate, which was modified with hydroxyethyl acrylamide to introduce a vinyl group necessary for the polymerization reaction. After deacetylation, D-galactopyranosyloxyethyl acrylamide was selectively sulfated at the 3-position to obtain 3-O-sulfo-D-galactopyranosyloxyethyl acrylamide. The monomer was polymerized through reversible addition-fragmentation chain transfer (RAFT) technique, resulting in the formation of a polymer bearing 240 unit (SO4−3−Gal)240. The polymer was used as it is for the in vitro studies with the gout model induced by cells incubation with monosodium urate crystals. THP-1 cells were differentiated into macrophages by incubation with phorbol 12-myristate 13-acetate. Proper cell differentiation was assessed through flow cytometric analysis of the lineage markers F4/80, CD80 and the target CD206. Cells pre-treatment with or without (SO4−3−Gal)240 followed by incubation with MSU in the presence or absence of NF-κB inhibitor suggested that (SO4−3−Gal)240 might reduce the production of IL-1β by interacting with the NF-κB pathway, since the pre-treatment with the glycopolymer + NF-κB inhibitor did not enhance the response as compared to cells treated with the NF-κB inhibitor alone, thus ruling out the possibility that a second factor is involved. In parallel, polymer with different degree of polymerization (DP), in the range 30-240, were synthesized and functionalized with succinic anhydride to introduce a reactive carboxyl group available for the cross-linking via diamines, to induce particle formation. By using (SO4−3−Gal)240 cross-linked with hexamethylenediamine particles with a size of 1123 nm were generated. To this aim, a block copolymer (SO4−3−Gal)30−(hydroxypropyl acrylate)90 was also synthesized. It consists of a hydrophilic segment composed of SO4−3−Gal monomer and a hydrophobic segment derived from hydroxypropyl acrylate, designed to self-assemble into particles when dispersed in aqueous solution. Future tests will assess drug encapsulation in the particles and conduct in vitro studies to verify their increased half-life compared to linear polymers.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12608/80620