Amine end-functional poly(N-vinylpyrrolidone) as amMacroinitiator for L-lysine N-carboxyanhydride polymerization - towards the preparation of pH-responsive micelles for drug delivery
Date
2015-03
Authors
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Publisher
Stellenbosch : Stellenbosch University
Abstract
ENGLISH ABSTRACT: Cancer is a notorious affliction that knows no age, gender, ethnic, racial, or species bounds and is
responsible for over 14% of annual worldwide human deaths. There is no universal cure and the
treatments that exist have poor probabilities of success. Chemotherapy is often considered the
staple for cancer treatment as it can enter areas of the body that are unsafe for surgery and can
treat tumors that are too small to be detected, even with modern imaging techniques. However,
chemotherapy can induce many harmful and fatal side-effects. It can also lose its therapeutic effect
if the cancer mutates to become multi-drug resistant. These shortcomings can be linked to the poor
selectivity and pharmacokinetics of conventional chemotherapy drugs. Modern research focusses on
improving these aspects of existing chemotherapy regimens through the incorporation of drug
delivery principles. This dissertation focusses on the development of a novel, polymeric, pHresponsive
drug delivery system for chemotherapy that incorporates the chemotherapeutic drug as
well as a cell-penetrating peptide in a prodrug formulation. The system was designed to inhibit the
release of its components into healthy tissues while selectively accumulating, through the enhanced
permeability and retention effect, and releasing its payload, through reversible hydrolysis of imine
bonds, within tumor tissues. Poly(L-lysine) was chosen as the cell-penetrating peptide since it is able
to form imine bonds through its ε-amine functional groups on its residues. It was prepared by the
primary amine-initiated ring-opening polymerization of Nε-(benzyloxycarbonyl)-L-lysine Ncarboxyanhydride
at 0 °C and pressures lower than 1 mbar as these conditions allow for a
controlled, living polymerization to occur. The benzyloxycarbonyl end-group was removed by
acidolysis with HBr in a mixture of dichloromethane and 1,4-dioxane at 0 °C. The initiator used for
the ring-opening polymerization was poly(N-vinylpyrrolidone) with a primary amine end-group,
prepared by RAFT-mediated polymerization with O-ethyl-S-(phthalimidylmethyl)xanthate. This RAFT
agent was shown to display slow pre-equilibrium kinetics which was linked to the lower relative
stability of the phthalimidylmethyl radical compared to the poly(N-vinylpyrrolidone) propagating
radical. The pre-equilibrium and main equilibrium kinetics of the RAFT polymerization were
optimized by performing the polymerization in semi-batch mode. During the semi-batch
polymerization, the monomer to RAFT agent ratio could be controlled by adjusting the monomer
feed. This ratio was shown to be inversely proportional to the probability of radical transfer from the
propagating radical to the phthalimidylmethyl radical. The phthalimide end-group could be
converted to a primary amine both by reacting with hydrazine in methanol as well as reduction with
sodium borohydride in water followed by hydrolysis in 1 M HCl at 60 °C. Doxorubicin and benzaldehyde could be conjugated, via imine bonds, to the poly(N-vinylpyrrolidone-block-L-lysine)
copolymer spontaneously in methanol. Both types of conjugates would self-assemble into micelles
when dispersed in water. However, the Doxorubicin conjugates were unstable, precipitating out of
solution within 24 hours. The benzaldehyde conjugates were stable in water for over 24 hours. This
suggested that a formulation of Doxorubicin and benzaldehyde conjugated to the block copolymer
may be sufficiently stable under the physiological conditions of blood plasma.
AFRIKAANSE OPSOMMING: Kanker is 'n berugte toestand wat geen ouderdom, geslag, etniese, ras- of spesiegrense ken nie en is verantwoordelik vir meer as 14% van die jaarlikse wêreldwye menslike sterftes. Daar is geen universele kuur vir hierdie siekte nie en die behandelings wat bestaan het swak waarskynlikhede van sukses. Chemoterapie is handig want dit behandel tumore waar operasies nie as veilig beskou word nie of as dit te klein is om te spoor, selfs met moderne beeldingstegnieke. Chemoterapie kan egter skadelike en dodelike newe-effekte tot gevolg hê. Dit kan ook die terapeutiese effek verloor as die kanker muteer en multi-middelweerstandig word. Hierdie tekortkominge kan gekoppel word aan die swak selektiwiteit en farmakokinetika van konvensionele chemoterapiemiddels. Moderne navorsing probeer om hierdie probleme op te los deur die inlywing van geneesmiddel-leweringsbeginsels. Hierdie proefskrif fokus op die ontwikkeling van 'n nuwe, polimeriese, pH-reaktiewe geneesmiddelleweringstelsel vir chemoterapie wat die chemoterapeutiese middel sowel as 'n sel-indringende peptied in 'n progeneesmiddel formulering inkorporeer. Die stelsel is ontwerp om die ophoping of vrystelling van die medikasie en die peptied in gesonde weefsel te inhibeer. Terselftertyd word die medikasie deur middel van gevorderde deurlatings en terughoudings in die tumor vrygestel deur omgekeerde hidrolise van die imienbindings. Poli (L-lisien) is gekies as die sel-indringerpeptied omdat dit imienbindings kan vorm deur middel van die ε-amien funksionele groep en ook omdat dit bioafbreekbaar is. Die reaksie is daargestel deur die primêre amien-geïnisieerde ring-openingpolimerisasie van Nε-(bensieloksikarboniel)-L-lisien N-karboksianhidried. Die temperatuur is by 0 °C en die druk onder 1 mBar gehou aangesien hierdie omstandighede gekontroleerde lewende polimerisasie toelaat. Die bensieloksikarboniel eindgroep is verwyder deur middel van HBr in 'n mengsel van dichlorometaan en 1,4-dioksaan by 0 °C. Die inisieerder wat gebruik word vir die ringopeningpolimerisasie is poli(N-vinielpirrolidoon) met 'n primêre amien eindgroep. Dit is berei deur RAFT-bemiddelde polimerisasie van N-vinielpirrolidoon met O-etiel-S-(ftalimidielmetiel)xantaat. Hierdie RAFT-agent het stadige voorewewig kinetika getoon as gevolg van die laer relatiewe stabiliteit van die ftalimidielmetielradikaal in vergelyking met die poli(N-vinielpirrolidoon) voortsettingsradikaal. Die voorewewig en hoofewewig kinetika van die RAFT-polimerisasie is geoptimaliseer deur die polimerisasie in ‘n semi-bondel manier uit te voer.
AFRIKAANSE OPSOMMING: Kanker is 'n berugte toestand wat geen ouderdom, geslag, etniese, ras- of spesiegrense ken nie en is verantwoordelik vir meer as 14% van die jaarlikse wêreldwye menslike sterftes. Daar is geen universele kuur vir hierdie siekte nie en die behandelings wat bestaan het swak waarskynlikhede van sukses. Chemoterapie is handig want dit behandel tumore waar operasies nie as veilig beskou word nie of as dit te klein is om te spoor, selfs met moderne beeldingstegnieke. Chemoterapie kan egter skadelike en dodelike newe-effekte tot gevolg hê. Dit kan ook die terapeutiese effek verloor as die kanker muteer en multi-middelweerstandig word. Hierdie tekortkominge kan gekoppel word aan die swak selektiwiteit en farmakokinetika van konvensionele chemoterapiemiddels. Moderne navorsing probeer om hierdie probleme op te los deur die inlywing van geneesmiddel-leweringsbeginsels. Hierdie proefskrif fokus op die ontwikkeling van 'n nuwe, polimeriese, pH-reaktiewe geneesmiddelleweringstelsel vir chemoterapie wat die chemoterapeutiese middel sowel as 'n sel-indringende peptied in 'n progeneesmiddel formulering inkorporeer. Die stelsel is ontwerp om die ophoping of vrystelling van die medikasie en die peptied in gesonde weefsel te inhibeer. Terselftertyd word die medikasie deur middel van gevorderde deurlatings en terughoudings in die tumor vrygestel deur omgekeerde hidrolise van die imienbindings. Poli (L-lisien) is gekies as die sel-indringerpeptied omdat dit imienbindings kan vorm deur middel van die ε-amien funksionele groep en ook omdat dit bioafbreekbaar is. Die reaksie is daargestel deur die primêre amien-geïnisieerde ring-openingpolimerisasie van Nε-(bensieloksikarboniel)-L-lisien N-karboksianhidried. Die temperatuur is by 0 °C en die druk onder 1 mBar gehou aangesien hierdie omstandighede gekontroleerde lewende polimerisasie toelaat. Die bensieloksikarboniel eindgroep is verwyder deur middel van HBr in 'n mengsel van dichlorometaan en 1,4-dioksaan by 0 °C. Die inisieerder wat gebruik word vir die ringopeningpolimerisasie is poli(N-vinielpirrolidoon) met 'n primêre amien eindgroep. Dit is berei deur RAFT-bemiddelde polimerisasie van N-vinielpirrolidoon met O-etiel-S-(ftalimidielmetiel)xantaat. Hierdie RAFT-agent het stadige voorewewig kinetika getoon as gevolg van die laer relatiewe stabiliteit van die ftalimidielmetielradikaal in vergelyking met die poli(N-vinielpirrolidoon) voortsettingsradikaal. Die voorewewig en hoofewewig kinetika van die RAFT-polimerisasie is geoptimaliseer deur die polimerisasie in ‘n semi-bondel manier uit te voer.
Description
Thesis (PhD)--Stellenbosch University, 2015.
Keywords
Cancer, Tumour, Phthalimidomethyl, RAFT, Doxorubicin, Polymerization, UCTD