Jølck, Rasmus Irming

Funktionelle nanopartikler har tiltrukket meget opmærksomhed på grund af deres unikke egenskaber, hvilket har gjort dem egnet som sensorer, som kontraststoffer til diagnostisk brug, til vacciner eller vigtigst af alt til at transportere medikamenter frem til sygt væv. Denne PhD-afhandling omhandler nye metoder til at overfladefunktionalisere nanopartikler og efterfølgende gøre dem følsomme over interne stimuli, såsom opregulerede vævsspecifikke enzymer etc., for at facilitere frigivelse af medikamenter i det ønskede væv. En ny metode til at overfladefunktionalisere liposomer og direkte bestemme antallet af immobiliserede ligander på overfladen er blevet udviklet. Denne metode, baseret på Cu-fri Click kemi, resulterede i kvantitativ overflade funktionalisering under meget milde betingelser, som også vil kunne anvendes til at funktionalisere andre typer af nanopartikler. Antallet af overflade-ligander er en vigtig parameter, der altid bør analyseres for at minimere batch-til-batch variationer. Ydermere er antallet af overflade-ligander afgørende for at opnå nanopartiklens ønskede egenskaber. For at undersøge hvilke kemiske reaktioner der fungerer bedst på overfladen af en nanopartikel, blev et systematisk studie udført, hvor metoder til at overfladefunktionalisere liposomer blev sammenlignet med den tilsvarende reaktion i opløsning. Overfladefunktionalisering med det somatostatin 2 targeterede peptid; Octreotate, blev udført ved Michael addition, Click kemi, Cu-fri Click kemi og ved oximdannelse. Disse studier viste tydeligt, at reaktioner på en nanopartikel forløber langsommere end i opløsning og oftest er omdannelsen ikke kvantitativ. Effekten ved at overfladefunktionalisere liposomer med Octreotate og derved targetere liposomerne hen mod kræftvæv, som udtrykker somatostatin 2 receptoren blev ligeledes undersøgt. Biodistributionen af Octreotate funktionaliserede liposomer indeholdende radioaktivt 64Cu i mus inficeret med H727 kræftsvulster blev undersøgt ved hjælp af positron emissions tomografi (PET). Der blev ikke observeret en statistisk forskel i akkumuleringen af de funktionaliserede liposomer i forhold til kontrol liposomer, men forholdet mellem liposomer i kræftvæv og muskler var signifikant større for octreotate liposomerne, hvilket er en afgørende parameter for effektivt at kunne dianogstisere kræftvæv i forhold til raskt væv. Ydermere er en ny metode til at transportere medikamenter og selektivt frigive disse i kræft væv blevet udviklet. Denne metode er baseret på matrix metalloproteinaser, som er en klasse af enzymer, der oftest er udtrykt af kræft celler. To nye metoder til at syntetisere komplekse molekyler med den overordnede struktur lipid-peptid-PEG blev udviklet. Kløvning af peptidsekvensen imellem den hydrofobe komponent og PEG polymeren øgede optaget i kræftceller in vitro. Brugen af de udviklede matrix metalloproteinase følsomme liposomer og lipoplexer forøgede toksiciteten af kendte kemoterapeutiske stoffer markant, og kunne ydermere facilitere transport af gener ind i celler in vitro. Overordnet lykkedes det at udvikle nye og effektive metoder til at overfladefunktionalisere nanopartikler. Ydermere blev en ny metode til at facilitere frigivelse af lægemidler lokalt i det ønskede væv udviklet. Nye nanopartikler blev udviklet og aktivering af disse med proteaser resulterede i øget toksisiet af kemoterapeutiske stoffer. Dette system evalueres i øjeblikket in vivo for at undersøge det kliniske potentiale af den udviklede teknologi.Functional nanomaterials have attracted much attention due to the unique properties of these nanoconstructs. In recognition of the huge potential within this field, much research has been devoted to develop sophisticated nanoparticles for medical diagnostics, sensors, contrast agents, vaccines and drug delivery. The objective of this PhD thesis was to expand the field of liposomal drug delivery by developing novel methods to efficienctly functionalize and subsequently sensitize liposomes towards internal stimuli, such as matrix metalloproteinases. Initially, we investigated a novel method to post-functionalize and directly quantify the degree of conversion without prior purification. Based on Cu-free Click chemistry, quantitative conversion under very mild conditions was achieved using conditions which might be equally suited for other nanomaterials. Being able to rapidly determine ligand surface density after post-functionalization is highly important, to ensure batch-to-batch reproducibility and to ensure that the desired ligand surface density has been accomplished. A systematic study was furthermore conducted to elucidate the optimal post-functionalization chemistry, in addition to the importance of the relative position of the reactive functionalities. Surface conjugation reactions of octreotate by Michael addition, Click chemistry, Cu-free Click chemistry or oxime bond formation were investigated. From these studies it was evident that chemical reactions performed directly on the surface of functionalized liposomes were slower than the solution phase counterpart and often far from quantitative. The effect of active targeting with 64Cu octreotate liposomes targeting the somatostatin receptor 2 was evaluated to improve tumor bioimaging for diagnostic applications, using positron emission tomography. Targeted liposomes had a sufficient circulation profile to passively accumulate in tumor tissue of H727xenografts in nude mice, but no statistical difference was detected in the tumor accumulation compared to control-liposomes. Despite this, the tumor-to-muscle ratio for targeted liposomes was significantly higher than for the controlliposomes, which indicated that active targeting can improve tumor-to-muscle contrast, thus, improving bioimaging for diagnostic applications.Finally, a novel drug delivery system based on charge-triggering of matrix metalloproteinase 2/9 sensitive PEGylated lipopeptides was designed. Methods to efficiently synthesize conjugates with the lipid-peptide-PEG motif were developed, and the use of these conjugates to shield positively charged liposomeand lipoplex formulations were described. The synthesized conjugates efficiently shielded cationic charges present at the surface of the nanoconstructs, resulting in anionic nanoparticles with long circulation properties in xenograft HT1080 tumor‐bearing mice. Charge reversal by peptide hydrolysis was achieved in the presence of proteases, resulting in cationic particles which were readily internalized by cells in vitro. The use of matrix metalloproteinase sensitiveliposomes and lipoplexes dramatically enhanced the cytotoxicity of known chemotherapeutics and facilitated effective gene transfection in vitro. Concluding on the work of this PhD thesis, we managed to expand the field of functional nanomaterials by developing novel methods to conjugate and directly quantify the surface density of immobilized ligands. Furthermore, a unique drug delivery system based on charge shielding and subsequent charge triggering by matrix metalloproteinase 2 has been established. This system is currently being further investigated in vivo, in order to test the therapeutic capacity of the system

Functionalization of Self-Organized Nanoparticles for Biological Targeting and Active Drug Release

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