Synthesis and characterization of radiopharmaceutical based on superparamagnetic nanoparticles and the effect of dual therapy on an experimental model of colon adenocarcinoma

Radionuklidna terapija (RT) predstavlja selektivnu isporuku zračenja tumorskom tkivu, primjenom jednog ili više radioizotopa (α, β, Ožeov elektron emitera) u sastavu radiofarmaceutika, čime se inhibira rast tumorskih ćelija. Zbog nedovoljno dugog zadržavanja radionuklida i hipoksičnih uslova unutar tumora, RT ima svoja ograničenja u smislu efikasnosti i poštede okolnog zdravog tkiva. Napredak u nanomedicini je doveo do razvoja nanomaterijala, koji bi trebalo riješe taj problem. Superparamagnetne nanočestice (SMNČ) na bazi magnetita (Fe3O4) i maghemita (γ-Fe2O3), pokazale su značajnu prednost u odnosu na druge nanomaterijale zbog svoje hemijske stabilnosti, niske toksičnosti, povoljnih magnetnih osobina (visoka magnetna saturacija) i niske cijene proizvodnje. SMNČ su našle svoju primjenu kao kontrastni agensi za magnetnu rezonancu, kao nosači različitih lijekova kao i u lokalnoj terapiji magnetnom hipertermijom (MHT). Magnetna hipertermija predstavlja oslobađanje toplote od strane različitih medijatora pod uticajem spoljašnjeg naizmjeničnog magnetog polja (SMP). Ova metoda je pokazala veliki potencijal u terapiji teško dostupnih tumora i dokazano povećava efikasnost RT. Oblaganje SMNČ odgovarajućom oblogom povećava koloidnu stabilnost, omogućava vezivanje radionuklida za različite funkcionalne grupe na površini SMNČ i obezbjeđuje in vivo stabilnost. Vezivanjem radionuklida za SMNČ dobija se multifunkcionalni terapijski agens koji kombinovanim efektom hipertermije i radionuklidne terapije može imati veći potencijal u terapiji malignih tumora. Cilj ovog istraživanja je sinteza i oblaganje SMNČ, ispitavanje mogućnosti upotrebe obloženih SMNČ u MHT, zatim optimizacija uslova obilježavanja monoklonskog antitijela CC49 (CC49 mAt) sa 131I kao i ispitivanje uslova konjugacije 131I-CC49 mAt sa obloženim SMNČ. Takođe cilj je i ispitavanje biodistribucije, retencije i efekta dualne terapije potencijalnog radiofarmaceutika 131I-CC49-SMNČ u in vivo uslovima. SMNČ su sintetisane metodom taloženja iz rastvora (metoda koprecipitacije) i solvotermalnom metodom u poliolima (poliol metodom). Jedinjenja mezo-2,3-dimerkaptosukcinska kiselina (DMSA) i aminosilan (APTES) korišćena su za oblaganje SMNČ. Karakterizacija neobloženih i obloženih SMNČ je urađena pomoću elektronske mikroskopije, rendgenske difrakcije na prahu, Furijeove transformacione infracrvene spektroskopije, SQUID magnetometrije, dinamičkog rasejanja svjetlosti. Karakterizacijom obloženih SMNČ utvrđen je uticaj funkcionalnih grupa (karboksilne i amino) na modifikaciju površine SMNČ-a, a određivanjem specifične apsorpcije SMNČ-a je ispitana njihova potencijalna primjena u magnetnoj hipertermiji. Obložene SMNČ koje su imale veću vrijednost specifične apsorcije su se dalje koristile u konjugaciji sa obilježenim antitijelom. Za obilježavanje CC49 mAt sa 131I korišćena je metoda sa hloraminom-T, dok je karbodiimidna metoda korišćena za konjugaciju 131I -CC49 mAt sa obloženim SMNČ. In vitro stabilnost 131I-CC49-SMNČ je ispitana u fiziološkom rastvoru i humanom serumu određivanjem radiohemijske čistoće koristeći tankoslojnu hromatografiju. Rezultati biodistribucije, retencije i terapijskog efekta 131I-CC49-SMNČ, dobijeni su ispitivanjima na imunodeficijentnim (NOD-SCID) miševima kojima su subkutano injektovane LS174T ćelije kancera, ćelijska linija humanog adenokarcinoma kolona. Biodistribucija i retencija su praćene u određenim vremenskim intervalima pomoću BRUKER®In-Vivo Xtreme II uređaja i mjerenjem raspodjele radioaktivnosti po organima. Efekat hipertermije, radionuklidne terapije i njihovog dualnog dejstva su ispitani izračunavanjem zapremine tumora svaki drugi dan nakon intratumorskog injektovanja 131I-CC49-SMNČ. Patohistološka ispitivanja su urađena u cilju određivanja toksičnosti i terapijskog efekta 131I-CC49-SMNČ. Dobijeni rezultati ispitivanja in vitro stabilnosti zajedno sa rezultatima biodistribucije, retencije i terapijskog efekta 131I-CC49-SMNČ upućuju na zaključak da je korišćenje 131I-CC49-SMNČ u dualnoj radionuklidno-hipertermijskoj terapiji obećavajući pristup za efikasno liječenje karcinoma.Radionuclide therapy (RT) represents the selective delivery of radiation to tumor tissue, using one or more radioisotopes (α, β and Auger electron emitters) in the form of radiopharmaceuticals, thereby inhibiting the growth of tumor cells. Due to insufficient retention of radionuclides and hypoxic conditions inside the tumor, RT has its limitations in terms of efficiency and sparing of the surrounding healthy tissue. Advancement in nanomedicine has led to the development of nanomaterials, which should solve the problem. Superparamagnetic nanoparticles (SPMN) based on magnetite (Fe3O4) and maghemite (γ-Fe2O3) have shown a significant advantage over other nanomaterials because of their chemical stability, low toxicity, favorable magnetic properties (high magnetic saturation) and low production cost. SPMN have found their application as magnetic resonance contrast agents, as drug carriers as well as in local magnetic hyperthermia therapy (MHT). Magnetic hyperthermia represents the release of heat by various mediators under the influence of an external alternating magnetic field (AMF). This method has shown great potential in the treatment of hard-to-reach tumors and has been proven to increase the effectiveness of RT. The proper coating of the SPMN increases their colloidal stability, facilitates the binding of radionuclides to different functional groups on the SPMN surface and provides in vivo stability. Binding radionuclides to SPMN a multifunctional therapeutic agent is obtained, which with the combined effect of hyperthermia and radionuclide therapy, can have a great potential in the treatment of malignant tumors. The aim of this study is to synthesize and coat SPMN, to examine the possibilities of using coated SPMN in MHT, then to optimize the labeling conditions of monoclonal antibody CC49 (CC49 mAb) with 131I and to examine the conditions of conjugation of 131I-CC49 mAb with coated SPMN. Also, the aim is to investigate the biodistribution, retention and effect of dual therapy of the potential 131I-CC49-SPMN radiopharmaceutical in vivo. SPMN were synthesized using coprecipitation method from solution (coprecipitation method) and solvothermal method in polyols (polyol method). Meso-2,3-dimercaptosuccinic acid (DMSA) and aminosilane (APTES) were used to coat SPMN. Characterization of uncoated and coated SPMN was performed using electron microscopy, X-ray powder diffraction, Fourier transform infrared spectroscopy, SQUID magnetometry and dynamic light scattering. Characterization of functionalized SPMN determined the influence of functional groups (carboxylic and amino) on the SPMN surface modification, while specific absorption determination investigated their potential use in magnetic hyperthermia. Coated SPMN that had a higher specific absorption value were further used in conjugation with the radiolabeled antibody. Labeling of CC49 mAb with 131I was performed using chloramine-T method, while the carbodiimide method was used for the conjugation of 131I-CC49 mAb with coated SPMN. The in vitro stability of 131I-CC49-SPMN was examined in saline and human serum by determination of radiochemical purity using thin layer chromatography. Data regarding the biodistribution, retention, and therapeutic effect of 131I-CC49-SPMN were obtained from studies in immunodeficient (NOD-SCID) mice injected subcutaneously with LS174T cancer cells, human colon adenocarcinoma cell line. Biodistribution and retention data were obtained at defined time intervals using a BRUKER®In-Vivo Xtreme II device and by measuring radioactivity distribution per organ. The effect of hyperthermia, radionuclide therapy, and their combined effects was examined by calculating tumor volume every other day after intratumoral injection of 131I-CC49-SPMN. Pathohistological studies were performed in order to determine toxicity and the therapeutic effect of 131I-CC49-SPMN. The obtained in vitro stability results together with the results of biodistribution, retention and therapeutic effect of 131I-CC49-SPMN refer to a concluson that the use of 131I-CC49-SPMN in dual radionuclide-hyperthermic therapy is a promising approach for effective cancer treatment