Development of Nanoplatforms for Cancer Diagnosis and Therapy

Cancer remains one of the world's most deadly diseases; with about 12 million new cases and 7.6 million cancer death every year worldwide. Despite rapid advances in diagnostic procedures and treatments, the overall survival rate fflom cancer has not improved substantially over the past 30 years. Therefore, there is a need to develop novel approaches for the accurate detection of cancer at ari early-stage. Nanotechnology has tremendous potential to make an important contribution in cancer prevention, diagnosis, imaging and treatment. Nanoparticles delivery systems can deliver a higher amount of anticancer drugs/imaging agents to the vicinity of the tumor, thus improving therapeutic effficacy and reducing harmful non-speciffic side effects. Thus nanotechnological approaches might be most effective and effficient means to confflont early diagnosis and treatment of cancer. Tumor targetable, super paramagnetic iron oxide nanoparticles (SPIONs) capped with citric acid! 2-bromo-2-methyl propanoic acid (CA/BMPA) were developed which are compact, water dispersible, biocompatible with narrow range of size dispersity (7-10 nm) and relatively high T2 relaxivity (R2=222L.mmoF1 .sec-5. The targeting effficacy of unconjugated and folic acid (FA)-conjugated SPIONs was evaluated in folate receptor (FAR) overexpressing (MCF-7&HepG2) and negative tumor cell line (A549). FAR-positive cells incubated with FA-SPIONs showed much higher intracellular iron content without any cytotoxicity. In-vitro MRI studies on tumor cells showed better T2-weighted images in FA-SPIONs. These ffindings indicate that FACA/BMPA-SPIONs possess high colloidal stability with excellent sensitivity of imaging and can be a useful MRI contrast agent for the detection of cancer. FASPIONs were also evaluated for in-vivo pharmacokinetics, biodistribution and toxicity in mice. Bio-distribution studies revealed that presence of SPIONs in various body tissues changed with time but greater fflaction ofthe injected iron nanoparticles were observed to be localized in the liver and spleen compared to the brain, heart, kidney and lung. Changes in serum iron levels were analyzed upto 3 weeks aifier intravenous administration of SPIONs in mice and results showed a transient increase in serum iron level compare to control animal up to i week and then declinedaifierwards. FA-SPIONs did not show any toxicity at injected dose of 20mg/kg body weight in mice. Superparamagnetic iron oxide nanoparticles were also evaluated as a delivery vehicle for anticancer peptide. CAIBMPA capped SPIONs were conjugated with L-form of anticancer peptide (NuBCP-.9) without cell penetrating peptide (CPP) which kills the cancer cells overexpressing Bdl-2. The NuBCP-9-SPIONs showed effective cell killing of cancerous cells and therapeutic effficacy of this nanoformulation is 2.5-5 time higher than that of CPP conjugated NuBCP-9. Complete inhibition in proliferation was observed in cancer cells due to activation cytochrome C3 dependent pathway of apoptosis. It was established that developed SPIONs can deliver Lform of NuBCP-9 without cell penetrating peptides and cause apoptosis in Bdl-2 overexpessing cell without losing its therapeutic efficacy. In-vivo studies of tumor regression in EAT mouse model showed 50% tumor regression in animals treated with NuBCP-9-SPIONs suggesting that these nanoparticles can successfully deliver the labile L-form of the NuBCP-9 to the tumor tissue without loss in its activity. Prussian blue staining of the tumor tissue also conffirmed the presence of SPIONs. To conclude, d四elone'i n麺(na J.亜cies nos ---肥ss hoth fhe nr -------I--叩erH s ofiniwnt n-i ApIF'ipr', --------------------. --of anticancer peptide and hence can be used as theragnostic (multimodal) agents for early cancer detection and treatment with huge clinical translation capabilities. Polylactic acid (PLA) and polyethylene glycol (PEG) based diblock copolymers were synthesized and characterized by various techniques viz. NMR and GPC. Nanoparticles were prepared from these block copolymers and investigated as a carrier for delivery of Lform of anticancer peptide 倒uBCP-9) without cell penetrating peptide (CPP). NuBCP-9 loading was simultaneously achieved with particle synthesis by double emulsion precipitation technique. The effect of varied PEG chain lengths on particle size and on loading effficiency of NuBCP-9 was determined using BCA assay kit. PLA-PEG4000 showed highest loading effficiency of 65.1%while lowest encapsulation effficiency of 52.09% was observed in PLA-PEG i 000 NPs. In-vitro NuBCP-9 release studies performed under simulated conditions (with different buffers and pH) showed initial burst release followed by near ffirst order release kinetics. Maximum peptide release of about 一 90% was observed in PBS buffer at pH 7.4 while it was 68% only in pH 5.0 over a period of 60 days. In-vitro proliferation inhibition studies were performed in multiple cancer cell lines along with the normal cells. NuBCP-9 loaded PLA-PEG nanoparticles showed 100% proliferation i山ibition in cancer cell with a minimum 24 h delay while no inhibition was observed in normal cells. Therapeutic potential of NuBCP-9 loaded nanoparticles was found to be 2-3 folds higher than that ofNuBCP-9 conjugated with CPP. In-vivo toxicity studies of the peptide loaded nanoparticles showed no toxicity at a dose of i 00mg/kg body weight of the nanoformulations with 10%peptide concentration. Erlich Ascetic Tumor regression studies revealed the complete regression of tumor in 21 days in Balb-c mice treated with NuBCP-9 loaded PLA-PEG nanoparticles without reoccurrence at a dose of 20mg/kg body weight of NuBCP-9 peptide.