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Final Report Summary - NANOCPPS (Developing smart Coordination Polymer Nanoparticles as Biomedicine for Metastatic Neuroblastoma)

Neuroblastoma (NB) is the most common cancer in childhood and it is a neuroendocrine tumor, which displays solid cancer in nerve tissues of adrenal gland, neck, chest or spinal cord. Metastatic NB occurs when tumor cells migrate via the bloodstream to the lymph nodes or distant sites non-adjacent to the primary tumour. Tumour cells can also reach to brain, lungs, liver cortical bone, bone marrow, etc., via migration through blood vessel. About half of patients are diagnosed with evidence of haematogenous metastasis. Nowadays, the treatment is based on different types of therapies: chemotherapy, radiation therapy, retinoid therapy or immunotherapy.[1] Some of the most common chemotherapy agents are cyclophosphamide, cisplatin, carboplatin, doxorubicin, and vincristine, approved by the Food and Drug Administration (FDA), but they all show high side effects. Other limitations of conventional cancer chemotherapy include drug resistance, lack of selectivity or lack of drug solubility. These problems can easily overcome by using nanoparticles as nano-carriers. This is due to the following unique properties of nanoparticles as drug delivery vehicles, such as i) large surface-to-volume ratio, ii) nano size which allow it to pass through the lipid bilayer and cell membrane, iii) the ability to encapsulate various drugs. Interestingly, the nano-sized coordination polymer particles (CPPs) can be a good drug vehicle to overcome the limitations of ordinary chemotherapy.[2] The incorporation of metals into polymeric materials through the use of coordination chemistry offer new systems with added value in the biomedical applications. Miniaturization of coordination polymers to the nanoscale represents a novel class of highly customizable functional particles for drug delivery by combining the rich diversity, chemistry, and properties of coordination complexes with the advantages of nanomaterials. Our first approximation consist on the synthesis of Pt(IV) prodrug-based CPPs. The Pt(IV) prodrug is used as building block to construct the CPPs that can encapsulate other drugs used in combination for the NB therapy. In that manner, we can increase the payload of Pt-based therapeutic agent and keep it inactive until the degradation of the nanoparticles and subsequent reduction of Pt(IV) prodrug to generate the cytotoxic cis-platinum. Moreover Pt(IV) prodrugs have more advantages over Pt(II) such as less drug resistances, less clinical dose, less toxicity and more stability. The adverse effects of using Pt(II) drugs are haematological toxicity, gastrointestinal toxicity, and neurotoxicity; these can be overcome by using a Pt(IV) prodrug.
We have successfully designed and synthesize a novel platinum-based CPPs as promising nano-constructs for neuroblastoma therapy. In this system, a Pt(IV) prodrug, with important cytotoxic properties towards anti-neuroblastoma, is used as building-block of the coordination polymer. We report two coordination polymer nanoparticles namely [{Zn(Bix)(DSCP)(H2O)2}·EtOH]∝ (Zn-Pt-CPP) and [{Ni(Bix)(DSCP)(H2O)2}·EtOH]∝ (Ni-Pt-CPP) obtained from the extended coordination of bix and a Pt(IV) prodrug [disuccinatocisplatin or DSCP] with Zn(II) and Ni(II), respectively. We also explore the ability of CPPs to encapsulate an anticancer drug doxorubicin (DOXO), and synthesized a combination drug cargo of CPP namely Zn-Pt-CPP@DOXO having a concentration of Pt(IV) and DOXO of 21% and 2%, respectively. In fact we deliberately encapsulate DOXO within Zn-Pt-CPP, owing to possibility to utilize the single CPP cargo for combinatorial therapy. Combinatorial therapy is important to fight against drug resistance metastatic cancer like neuroblastoma. In vitro cancer cell cytotoxicity assays showed that these CPPs readily release Pt, which displays specific cytotoxicity against MCF7 cells, with an IC50 of about 100 μM. Zn-Pt-CPP showed unprecedented cytotoxic activity (IC50 = 110.5-158.4 μM) on the human neuroblastoma cell line (M17), when incubating at 37°C for 72 hour, which is three fold cytotoxic active than that of the Pt(IV) prodrug building block of CPP. We believe this CPP is a promising candidate as it can improve upon many of the drawbacks of conventional therapy, including high doses, rapid clearance, poor pharmacokinetics and strong side effects, which will be confirmed in the future investigation of its in vivo activity. More interestingly the DOXO encapsulated within the CPP matrix, kinetically support the M17 cell death which is revealed from the three fold cytotoxic effect of Zn-Pt-CPP@DOXO than that of Zn-Pt-CPP. MCF7 and Hela cells also react to the Zn-Pt-CPP in the similar way like M17 cell and showed four times more cytotoxic effect, than that of the Pt(IV) prodrug. Zn-Pt-CPP@DOXO showed enhanced cytotoxic effect to both MCF7 and Hela cells, a combined contribution effect from Pt(IV) prodrug and DOXO. We determined the combination index (CI) values of the as-synthesized nanoparticle Zn-Pt-CPP@DOXO by using the Compusyn software. From the CI values, it revealed Zn-Pt-CPP@DOXO showed synergistic and additive effect at high dosage (Fa = 0.95 and 0.96) towards MCF7 and M17 cancer cells, respectively. We also used confocal laser scanning microscopy (CLSM) to investigate the cellular uptake of the as-synthesized CPPs. After 4 h of incubation in Dulbecco’s modified eagle’s medium (DMEM) at 37°C under 5 % CO2 having a concentration of the CPP above its IC50 value. CLSM images clearly showed the internelization of of these nanoparticles inside the Hela cells, when excited with blue light (λex = 490 nm).
The fast release of the Pt and DOXO drug from Zn-Pt-CPP@DOXO, prompted us to coat the as-synthesized CPPs with a biocompatible and biodegradable polymers. We choose poly-Lactide-co-glycolide (PLGA) as the coating agent, and we successfully coated the CPPs. The resultant PLGA coated Zn-Pt-CPP@DOXO showed moderate and sustained release of drugs; Zn-Pt-CPP@DOXO showed faster burst release (within 4 hour ~ 70 % Pt release) than that of the PLGA coated Zn-Pt-CPP@DOXO (within 4 hour ~ 55 % Pt release). However the PLGA coated Zn-Pt-CPP@DOXO could not purify from the mixture of coated CPPs and PLGA NPs, despite it showed more control release of drug after five hour. An alternative system was proposed based on the synthesis of a coordination polymer using DSCP and a catechol-polyethylene glycol ligand which coordinate to iron ions, resulting in PEG coated CPPs namely Fe-Pt-PEG-Amide-CAT. From the four independent characterization techniques such as FT-IR, ICP-MS, 1H NMR and TGA, we propose the formula for Fe-Pt-PEG-Amide-CAT as [Fe6(DSCP)6(PEG-Amide-CAT)(H2O)6]∝. SEM shows the morphology of the as-synthesized CPP, as aggregated nanoparticles. DLS was used to determine the stability of CPPs in aqueous environment mimicking physiological media, which confirmed the nanoparticle having an average size distribution of 220 nm. We also could encapsulate DOXO within the CPP matrix of Fe-Pt-PEG-Amide-CAT by following our method.[3] The resultant compound namely Fe-Pt-PEG-Amide-CAT@DOXO, were characterized by various techniques. The STEM and DLS data revealed that the CPPs retain its nanoparticle aggregate morphology and size, even after encapsulation of DOXO. To determine the amount of DOXO encapsulated in the CPP matrix, we used fluorescence spectroscopy. The experimental data of fluorescence spectroscopy, revealed that around 0.832% in weight of DOXO is present in the CPPs. The in vitro drug release profile of the as-synthesised CPP showed a fast release in the first 5 h (around 65 % of Pt released during this time). In fact after the burst release, the nanoparticles show moderate and sustained release. PEG functionality present gives hydrophilicity towards the CPPs, which allows PBS solution to more easily mixed with the nanoparticle matrix and this could be the reason behind the fast release of drug molecules during the initial five hours. Finally we evaluated the cytotoxicity profile of the CPPs, Fe-Pt-PEG-Amide-CAT and Fe-Pt-PEG-Amide-CAT@DOXO. We used the NB M17 cell line and carried out MTT assay 72 hours at 37°C Dulbecco's Modified Eagle's Medium(DMEM). The IC50 values thus determined were 93.10 μM and 14.58 μM for Fe-Pt-PEG-Amide-CAT and Fe-Pt-PEG-Amide-CAT@DOXO, respectively. This data indicated that the combination drug based nano-carriers such as Zn-Pt-CPP@DOXO and Fe-Pt-PEG-Amide-CAT@DOXO are much more effective drug against the aggressive NB cancer, compare to the single drugs such as Pt(IV) prodrug (DSCP), cisplatin, doxorubicin, and the host CPPs Zn-Pt-CPP and Fe-Pt-PEG-Amide-CAT.
This project gave the student to conduct truly innovative interdisciplinary research, combining their expertise in synthetic organic, inorganic and materials chemistry, by working close with well-established colleagues in nanotechnology and biomedicine (Prof. D. Ruiz-Molina, Dr. F. Novio and Prof. J. Lorenzo) and by using unique validation models and state–of–the–art infrastructure and knowledge.
Several issues such as toxicity, dispersity, hydrophilicity and surface functionalities of the CPPs were solved by this project, and the results obtained from this project lead to the development of a novel Pt(IV) prodrug cocktailed CPP, encapsulate with a the chemotherapy drug DOXO. In chemotherapy, DOXO and cisplatin were identified as the active agents. Thus a combination of both agents in a single nano-cargo namely Zn-Pt-CPP@DOXO will give potential impact to the nanomedicine research, owing to the fact that such combination is already showed excellent response rate in phase III clinical trials.[4] Motivated from this clinical trial results, a more detailed in vivo study of the nano-cargo (Zn-Pt-CPP@DOXO) developed from this project is under progress. We are expecting excellent results from this study, and which will eventually passed for clinical research. If success, it will be a break through and which will lead to socio-economic impacts.

• References:
1. N. K. Cheung, B. H. Kushner, M. LaQuaglia, K. Kramer, S. Gollamudi, G. Heller, W. Gerald, S. Yeh, R. Finn, S. M. Larson, D. Wuest, M. Byrnes, E. Dantis, J. Mora, I. Y. Cheung, N. Rosenfield, S. Abramson and R. J. O'Reilly, Med. Pediatr. Oncol., 2001, 36, 227–230.
2. C. He, D. Liu, and W. Lin, Chem. Rev., 2015, 115 (19), 11079–11108.
3. I. Imaz, M. Rubio-Martınez, L. Garcıa-Fernandez, F. Garcia, D. Ruiz-Molina, J. Hernando, V. Puntes and D. Maspoch, Chem. Commun., 2010, 46, 4737–4739.
4. G. F. Fleming, V. L. Brunetto, D. Cella, K. Y. Look, G. C. Reid, A. R. Munkarah, R. Kline, R. A. Burger, A. Goodman and R. T. Burks, J Clin Oncol., 2004, 22(11), 2159-66.

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Ana Maria de la Fuente, (Head of European Programmes Unit)
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Record Number: 199446 / Last updated on: 2017-06-20
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