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134 Pages·2017·4.89 MB·English

by  Maurizio Roveri

ETH Library
Liposome-mediated delivery of
vincristine to rhabdomyosarcoma
Doctoral Thesis
Author(s):
Roveri, Maurizio
Publication date:
2017
Permanent link:
https://doi.org/10.3929/ethz-a-010871360
Rights / license:
In Copyright - Non-Commercial Use Permitted
This page was generated automatically upon download from the ETH Zurich Research Collection.
For more information, please consult the Terms of use.
DISS. ETH NO. 24189

Liposome-mediated delivery of vincristine
to rhabdomyosarcoma

A thesis submitted to attain the degree of
DOCTOR OF SCIENCES of ETH ZURICH
(Dr. sc. ETH Zurich)

Presented by
Maurizio Roveri
MSc in Pharmaceutical Sciences, ETH Zurich
Born on 18.09.1987
Citizen of Lugano, TI, Switzerland

Accepted on the recommendation of
Prof. Jean-Christophe Leroux, examiner
Prof. Stefanie-Dorothea Krämer, co-examiner
Prof. Paola Luciani, co-examiner
PD Dr. Michele Bernasconi, co-examiner

2017

Le terre emerse
Là, dove nidificheranno molti uccelli.
Insisti nello scrutare a lungo il mare
diffidando del tuo sguardo disabile.
No, niente di maestoso, per fortuna.
Piuttosto una nuova calma, una diversa
geometria della spuma.
Si vorrebbe raggiungerle
proprio nei giorni peggiori, quando le onde
sembrano ghiaccio azzurro, il cielo pesa
più grigio, e unico scampo
rimane l’improbabile.
Se ci sono,
se brillano sotto il pelo
dell’acqua, inconosciute
eppure attese, fuori vista,
saranno lastre verdi di sasso,
lievemente inclinate.
L’emersione
si addebita alle forze
e alle frizioni che sconvolgono in assenza
di ogni altra possibilità.
Un lunghissimo periodo di mestizia
si può considerare inevitabile.
Avranno freddo anche loro, intirizzite,
e forse pioverà, ci sarà il vento.
dovremo accoglierle bene, riconoscerle,
scostare adagio il buio dai loro brividi,
convincerle dolcemente a rimanere.
La geografia e tutte le coordinate
cambieranno da sole, senza fretta;
ci vorrà un po’ di tempo per capire.
E poi non devi illuderti: vedremo
al massimo l’inizio,
la timida colonia dei molluschi, un po’ di bava
d’alga bagnata nelle scanalature,
la sosta di un gabbiano, un grido roco
che sembra senza senso o troppo fragile,
eppure si propaga si moltiplica.
I fiori, l’erba e le altre cose bellissime
verranno forse dopo, ma ci basta.
Fabio Pusterla

Summary .............................................................................................................. 1
Riassunto .............................................................................................................. 3
Chapter 1 - Background and purpose ............................................................... 5
1.1. Cancer and chemotherapy .............................................................................................. 5
1.2. Cancer in children ........................................................................................................... 7
1.3. Sarcomas and rhabdomyosarcoma ................................................................................. 7
1.4. Proprotein convertases – Furin ....................................................................................... 8
1.5. Cancer nanomedicine ................................................................................................... 10
1.6. Scope of the thesis ........................................................................................................ 15
Chapter 2 - Peptides for tumor-specific drug targeting: state of the art and
beyond ................................................................................................................. 17
2.1. Introduction .................................................................................................................. 18
2.2. Screening of peptidic ligands ....................................................................................... 19
2.3. Stability- and exposure-enhancing strategies for peptides ........................................... 22
2.4. Cancer receptors and peptide-decorated NCs for tumor-targeted drug delivery .......... 27
2.5. Summary and future perspectives ................................................................................. 36
Chapter 3 - In vitro characterization of RMS-specific peptides ................... 39
3.1. Introduction .................................................................................................................. 40
3.2. Materials and methods .................................................................................................. 41
3.3. Results and discussion .................................................................................................. 45
3.4. Conclusion .................................................................................................................... 53
Chapter 4 - Formulation of liposomes for the RMS-targeted delivery of
VCR .................................................................................................................... 55
4.1. Introduction .................................................................................................................. 56
4.2. Materials and methods .................................................................................................. 57
4.3. Results and discussion .................................................................................................. 63
4.4. Conclusion .................................................................................................................... 75
Chapter 5 - General conclusion and outlook .................................................. 77
Appendix ............................................................................................................ 83
References .......................................................................................................... 91
List of Abbreviations ....................................................................................... 117
Curriculum Vitae ............................................................................................ 121
Scientific contributions ................................................................................... 123
Acknowledgements .......................................................................................... 125
Nano-sized drug carriers (NCs) hold the potential to increase antitumor efficacy of
chemotherapeutics and to decrease their side effects. This is in part due to the presence of fenestrations
in the new angiogenic tumor blood vessels, which allow extravasation of the NCs. Additionally, one
approach to improve NCs’ cellular uptake is the use of ligands on their surface that recognize specific
receptors over-expressed on the surface of tumor cells or tumor endothelial cells. Ligand-decorated NCs
recognize and bind target cells through ligand-receptor interactions, and are subsequently internalized
into cells where the drug is released. Rhabdomyosarcoma (RMS), the most common pediatric soft tissue
sarcoma, is currently treated with a combination of cytotoxic agents (i.e., vincristine (VCR),
actinomycin D and cyclophosphamide) followed by radiotherapy or surgery. However, because of the
rapid clearance of VCR and its consequent low accumulation in tumors, as well as the long-term side-
effects caused by chemo- and radiotherapy (e.g., hearing loss and secondary malignancies), novel
approaches are required to improve current RMS treatment options. RMS-P3 is a cyclic peptide which
was previously selected by phage display for its specificity towards RMS cells, and has been proposed
as a mechanism for targeting furin, a proprotein convertase (PC), consistently expressed in RMS. In this
context, the goal of this Ph.D. thesis was to explore the possible advantage of using a peptide-mediated
furin targeting system for improved VCR delivery.
Chapter 1 provides information about the incidence of cancers, with special attention to
pediatric tumors and RMS, as well as about the current status of conventional cancer therapies.
In Chapter 2, the concept of anticancer nanomedicines is introduced, with focus on peptide-
targeted systems. Furthermore, tumor receptors for which targeting peptides have been identified are
presented, and their targeting potential is considered. Various chemical modifications of peptides are
thoroughly described, and the performance of targeting peptides as well as peptide-functionalized NCs
is critically discussed. The limitations of active targeting NCs are evaluated in detail, and an outlook on
the potential of tumor-targeting peptides and their clinical applications is provided.
Chapter 3 reports the in vitro characterization of RMS-specific peptides. The previously
identified peptide was modified to ensure water solubility, and its interaction with furin (i.e., binding
affinity and furin activity inhibition) was characterized with the intent to understand if the chosen
peptide could be used as an active targeting system for RMS. The modification did not affect the
1
Summary

interaction with furin and made the peptide hydrosoluble at the tested concentrations. Studies with
PEGylated peptides proved that the presence of poly(ethylene glycol) (PEG) did not affect the
interaction with furin. The localization of furin on the surface of RMS cells under normoxic and hypoxic
conditions was investigated to evaluate the function of furin as a RMS target protein. No clear
relocalization of furin at the cell surface of Rh30 cells was detectable under the tested conditions.
Chapter 4 describes the formulation of (targeted) PEGylated sphingomyelin-based liposomes.
The uptake of these liposomal formulations was assessed in vitro on RMS cells and was shown to be
moderate for the liposomes decorated with the peptide. Furthermore, the model drug VCR was
encapsulated in PEGylated liposomes with an efficiency of 90%, either in the presence or absence of
the RMS-specific peptide on the surface. The pharmacokinetic and biodistribution profiles of these
liposomes were characterized in RMS-bearing mice. Intravenous injection of liposomal VCR in RMS-
bearing mice guaranteed a high drug concentration in plasma over 24 h and provided higher drug
accumulation in RMS tumors compared to free VCR. The presence of the peptide had no effect on the
tumor accumulation.
Chapter 5 provides a discussion of the key findings of this doctoral thesis and covers the
perspectives of liposome-based RMS targeting.
2
Le nanoparticelle per il trasporto di farmaci hanno la capacità di aumentare l’efficacia delle
molecole chemoterapeutiche e di diminuire i loro effetti secondari. Ciò è dovuto all’irregolare
architettura dei vasi sanguigni tumorali che presentano un endotelio discontinuo che permette
l’extravasazione dei nanovettori. Un metodo per aumentare ulteriormente la quantità di farmaco nelle
cellule tumorali consiste nel decorare le particelle con ligandi che riconoscono recettori sovraespressi
unicamente sulla superficie dei tumori o dalle cellule endoteliali tumorali. Di conseguenza, le particelle
riconoscono le cellule cancerose, possono essere internalizzate e rilasciare il farmaco che trasportano. Il
rabdomiosarcoma (RMS) è il più comune dei sarcomi dei tessuti molli ed è attualmente trattato con una
combinazione di agenti citotossici (vincristina (VCR), actinomicina D e ciclofosfamide) seguita da
sessioni di radioterapia o rimozione chirurgica. La rapida eliminazione della VCR dal sangue comporta
però una bassa concentrazione del farmaco nel tumore. Inoltre, gli effetti secondari causati dalla chemio-
e radioterapia (come ad esempio la perdita dell’udito), rendono necessarie nuove e migliori terapie per
il RMS. RMS-P3 è un peptide ciclico precedentemente identificato mediante “phage display” e con
specificità per le cellule di RMS. Ricerche precedenti hanno identificato la furina, una proproteina
convertasi espressa in RMS, come possibile target del peptide. In questo contesto, lo scopo di questa
tesi di dottorato era di esplorare i possibili vantaggi dell’utilizzo di peptidi per il riconoscimento
specifico della furina e di generare un sistema per migliorare il trasporto della VCR al tumore.
Il Capitolo 1 fornisce informazioni sull’incidenza dei tumori, con una particolare attenzione ai
tumori pediatrici e all’RMS. Lo stato attuale delle terapie antitumorali convenzionali è descritto.
Nel Capitolo 2 vengono introdotti il concetto di nanomedicina e i modelli di target peptidici.
Vengono quindi descritti i ricettori tumorali per i quali sono stati identificati specifici peptidi e il loro
potenziale come target per terapie viene brevemente discusso. Segue una descrizione approfondita di
diverse modifiche chimiche per peptidi e un’analisi critica dei nanovettori funzionalizzati con peptidi
specifici per ricettori tumorali. I limiti del targeting attivo sono illustrati in dettaglio e, infine, le
prospettive dei peptidi specifici per tumori e le loro applicazioni cliniche sono riportati.
Il Capitolo 3 spiega le caratterizzazioni in vitro dei peptidi specifici per il RMS. Il peptide
identificato in uno studio precedente è stato modificato per garantirne la solubilità acquosa e le sue
interazioni con la furina (affinità di legame e inibizione dell’attività di furina) sono state caratterizzate
3