CALCIUM CARBONATE-BASED PARTICLES AND CAPSULES FOR THERAPEUTIC ENZYMES

Laura Van Poelvoorde Bogdan Parakhonskiy
De ladingscapaciteit, stabiliteit en de activiteit van het geladen enzym worden geëvalueerd voor calciumcarbonaat partikels. Deze wordt vergelijken met geëncapsuleerde calciumcarbonaat partikels en liposomen.

Calciumcarbonaatpartikels als oplossing voor leukemie

Cancer affects all of us, whether you’re a daughter, mother, sister, friend, co-worker, doctor or patient.”

 

Iedereen komt in contact met kanker, ofwel als patiënt ofwel door een naaste die aan de ziekte lijdt. Ongeveer 1,5% van de mannen en vrouwen met kanker worden gediagnosticeerd met leukemie en amper de helft van deze gevallen overleven de ziekte. Er bestaan echter al veel doeltreffende medicijnen, dus waar zit dan het probleem? Een van de hindernispalen om deze behandelingen te verbeteren is het efficiënt bezorgen van het geneesmiddel op de juiste plaats.

Het creëren van capsules, die het medicijn bevatten, is veelbelovend en verbetert de efficiëntie. Dat zal leiden tot een reductie in toxiciteit en het behouden van een hogere concentratie aan geneesmiddel op de doellocatie. In de praktijk worden voorlopig enkel liposomen gebruikt. Dit zijn gesynthetiseerde vesikels met inhoud omgeven door een membraan. Het is dan ook belangrijk om nieuwe partikels te vergelijken met deze liposomen.

Calciumcarbonaatpartikels

Calciumcarbonaatpartikels zijn veelbelovende nieuwe partikels door hun lage synthesekosten, simpele synthesemethode, biocompatibiliteit, lage toxiciteit, porositeit waardoor moleculen gemakkelijk worden geladen op het partikel,… In strijd tegen leukemie zou een enzym guanylaatkinase worden aangebracht op en in deze partikels. Guanylaatkinase heeft een antitumoraal effect en na injectie in het bloed zou het de kankercellen van leukemie kunnen vernietigen en de patiënt genezen.

In vorig onderzoek lag de focus op de optimalisatie van verschillende karakteristieken van het partikel, zoals de grootte, vorm, porositeit en welk polymorf van calciumcarbonaat het interessantste is voor deze toepassing. Polymorfen zijn de verschillende toestanden waarin een stof een vaste aggregatietoestand heeft. Calciumcarbonaat heeft er drie, namelijk aragoniet, vateriet en calciet. Uit deze drie vormen is vateriet het meest interassant door zijn grote porositeit. De metastabiele toestand is bovendien ook zeer interessant aangezien het vateriet bij bewaring in een oplossing zal rekristalliseren tot calciet waardoor alle enzymmoleculen worden vrijgegeven. Deze partikels kunnen niet zo gemakkelijk op zichzelf worden gebruikt door hun instabiliteit en snel rekristallisatieproces. Daarom is het nodig om deze partikels te beschermen. Dit kan onder andere met behulp van alginaatgebaseerde hydrogels die een beschermende laag vormen rond de calciumcarbonaatkern.

Guanylaatkinase is een duur enzym, dus alvorens deze te gebruiken wordt een ander goedkoper therapeutisch modelenzym, alkalische fosfatase, gebruikt. Hierbij er wordt nagegaan hoeveel enzym er op deze partikels wordt geladen, hoeveel enzym hiervan actief is en hoelang het duurt voordat de rekristallisatie plaatsvindt en de enzymmoleculen worden vrijgegeven. De experimenten zijn uitgevoerd op kleine (1,15±0,07 µm) en grote (2,3±0,3 µm) partikels, aangezien het volume en de oppervlaktegrootte een invloed zouden kunnen hebben op de resultaten.

 

Stabiliteit

Vaterietpartikels hebben een ronde vorm onder de microscoop, terwijl calcietpartikels na rekristallisatie een kubische vorm hebben. Deze rekristallisatie gebeurt binnen de 24 uur en is dus interessant indien men snel het geladen enzym wil distribueren. Dit geladen enzym heeft ook een invloed op de stabiliteit. In het geval van alkalische fosfatase zorgt dit voor een stabiliteit van enkele uren meer. Tenslotte zijn de kleine partikels langer stabiel ten opzichte van de grotere partikels.

Voor sommige toepassingen wil men dat dit langzamer gebeurt, wat door een beschermende alginaatlaag rond de calciumcarbonaatkern wordt bekomen. De stabiliteit verhoogt tot ongeveer 6 dagen. Door toevoeging van zilvernitraat of divalente kationen, zoals calcium en magnesium, kan de alginaatlaag bindingen vormen met de alginaatlaag van andere partikels en wordt er een gel gevormd. Calciumcarbonaatkernen kunnen met behulp van ascorbinezuur worden opgelost waardoor er holle capsules ontstaan waarin de geladen moleculen zich bevinden. Door afwezigheid van een calciumcarbonaatkern kan het ook niet meer rekristaliseren.

Ladingscapaciteit

De maximale ladingscapaciteit werd bepaald door een experiment uit te voeren waarbij de incubatietijd en de initiële concentratie van alkalische fosfatase worden geoptimaliseerd. De ladingscapaciteit wordt berekend door de gemeten enzymconcentratie te delen door het gewicht aan partikels. Uit de experimenten bleek een incubatietijd van 2 uur en een initiële enzymconcentratie van 10 mg/mL het beste. Hierbij werd respectievelijk 19,9% en 16,1% van de alkalische fosfatase geladen op de kleine en grote partikels. De ladingscapaciteit van liposomen was 100% als een incubatietijd van 2 uur en een initiële enzymconcentratie van 1 mg/mL werd gebruikt.

Hoeveelheid actief enzym in de partikels

Na het bepalen van de ladingscapaciteit is het interessant om te begrijpen hoeveel enzym actief is en bereikbaar is voor het substraat van het enzym. De ladingscapaciteit van de kleine partikels ligt iets hoger dan van de grote partikels. Echter het percentage aan actief enzym ligt hoger voor de grote partikels, namelijk 55.2% ten opzichte van 32.3%. Dit kan betekenen dat er een aanwezigheid is van enzym in de kleine partikels en niet enkel op het oppervlakte, waardoor het niet gemakkelijk bereikbaar is voor het substraat. Dit percentage aan actief alkalisch fosfatase ligt lager als de calciumcarbonaatpartikels worden omgeven door een beschermende laag. Dus er zal een afweging moeten worden gemaakt tussen de hoeveelheid actief enzym en tussen de stabiliteit. Bij liposomen bedraagt het percentage aan actief alkalisch fosfatase 57,5%. Dit betekent dat er 42,5% niet bereikbaar is voor het substraat. Mogelijke redenen hiervoor zijn dat enzymmoleculen de liposomen hebben gepenetreerd, maar dat het substraat hier niet in slaagt. Een andere mogelijk oorzaak is dat het enzym verkeerd gebonden is op de liposomen en het actief domein ontoegankelijk is voor het substraat.

Toekomst

Er is nog veel verder onderzoek nodig vooraleer men de partikels zal gebruiken in de praktijk, maar de resultaten in dit preliminair onderzoek zijn positief. Calciumcarbonaatpartikels hebben een lagere synthesekost en een gemakkelijkere synthesemethode ten opzichte van de liposomen, terwijl de hoeveelheid actief enzym vergelijkbaar is met deze van de liposomen. De calciumcarbonaatpartikels bieden verder ook de kans op een vertraagde vrijgave van enzym door ze te encapsuleren. Wie weet leidt dit verder onderzoek uiteindelijk tot een daling van sterfgevallen bij leukemiepatiënten?

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Universiteit of Hogeschool
Master of Science in de industriële wetenschappen: Biochemie
Publicatiejaar
2017
Promotor(en)
Andre Skirtach
Kernwoorden
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