DIAGRAMA DE FLUJO

To develop a new drug delivery construct, PNIPAM-co-AM/MNP nanogels were synthesized and characterized to be functional and controlled in a regulated manner. It has been shown that we can synthesize citric acid-coated Fe3O4 nanoparticles, have them encapsulated by a thermoresponsive copolymer of PNIPAM-co-AM with a VPTT behavior of around 45 C. With these nanogels, we were able to show a large burst release of Doxorubicin at a temperature higher than the VPTT, and very limited release under this temperature. When these nanogels were exposed to healthy and cancerous cells, we showed that cells remained highly viable, thus showing controllability of release at body temperature. We were also able to show that these nanogels can release DOX into surrounding cells even after incorporation into a crosslinked gelatin-based hydrogel.

Although the construct would only be injected into already existing or excised tumor sites, if any of the drug-loaded nanogels come loose due to simple physical fixation, the localized external application of AMF is hypothesized to prevent release from elsewhere in the body. In order to mitigate this possibility, nanogels could be functionalized and chemically attached to the macroscopic gel. Finally, our nanocomposite was exposed to an AMF like one presented by an MRI and a preliminary study showed a distinct increase in DOX release versus a nanocomposite not exposed to an AMF.

In the future, looking at changing AMF intensity and frequency and increasing sample size would be beneficial to optimizing drug delivery out of the already implanted construct. Commercial MRIs found in many patient hospitals produce a field strength

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ranging from 0.05 to 3 T. Our calculations showed that we were below this range, which allows the ability for us to continue working under the premise that this nanocomposite will work for biomedical applications and gives us the potential to expose the nanocomposite construct with a higher field for faster release. Collaboration with a laboratory or clinic with an MRI machine would allow us to compare the results from our induction heating system with a commercial standard in providing external magnetic stimuli process to get maximal drug delivery from our construct.

Although addition of 5 mg nanogel to the 1 mL solution of 5 wt% gel showed no true difference in rheological characteristics, It is well documented that addition of nanoparticles alters these properties, so future studies would be done in order to test optimal release profiles in a more realistic tumor model where more cells would be present and release from a larger system would vary. [52] Therefore, altering weights of nanogels exposed to cells and altering number of cells can give us a better picture of the amount of nanogels required to have sufficient coverage of a quantifiable tissue area.

We also recognize that this drug delivery system limits our potential market size to those without metallic implants due to the risks that an AMF has on metallic compounds. However, we feel that the market is sizeable enough that this gives us a decent entry point if this construct is pushed forward to further testing for market approval. A full market analysis of patients that this construct can help can also be performed in the future if needed. Instead of limiting our market to just patients with cancer, this nanocomposite construct can be loaded with any drug or combination of drugs to treat various medical

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pathologies. Having potential to apply growth factors to this construct to regenerate tissue, we believe that this system could hold a future in the biomedical industry.

43 REFERENCES

[1] Society AC. Cancer Facts & Figures 2015. Atlanta, Ga: American Cancer Society;

2015.

[2] Tacar O, Sriamornsak P, Dass CR. Doxorubicin: an update on anticancer molecular action, toxicity and novel drug delivery systems. Journal of Pharmacy and Pharmacology.

2013;65:157-70.

[3] Bawa P, Pillay V, Choonara YE, du Toit LC. Stimuli-responsive polymers and their applications in drug delivery. Biomed Mater. 2009;4:15.

[4] Zhao X, Ding X, Deng Z, Zheng Z, Peng Y, Long X. Thermoswitchable electronic properties of a gold nanoparticle/hydrogel composite. Macromolecular Rapid Communications. 2005;26:1784-7.

[5] De SK, Aluru NR, Johnson B, Crone WC, Beebe DJ, Moore J. Equilibrium swelling and kinetics of pH-responsive hydrogels: Models, experiments, and simulations. J Microelectromech Syst. 2002;11:544-55.

[6] Kwok CS, Mourad PD, Crum LA, Ratner BD. Self-assembled molecular structures as ultrasonically-responsive barrier membranes for pulsatile drug delivery. J Biomed Mater Res. 2001;57:151-64.

[7] Klouda L, Mikos AG. Thermoresponsive hydrogels in biomedical applications.

European Journal of Pharmaceutics and Biopharmaceutics. 2008;68:34-45.

[8] Guardia P, Di Corato R, Lartigue L, Wilhelm C, Espinosa A, Garcia-Hernandez M, et al. Water-soluble iron oxide nanocubes with high values of specific absorption rate for cancer cell hyperthermia treatment. ACS Nano. 2012;6:3080-91.

[9] Lu A-H, Salabas EL, Schüth F. Magnetic nanoparticles: synthesis, protection, functionalization, and application. Angewandte Chemie International Edition.

2007;46:1222-44.

[10] Wang YX. Superparamagnetic iron oxide based MRI contrast agents: current status of clinical application. Quantitative Imaging in Medicine and Surgery. 2011;1:35-40.

[11] Hayashi K, Nakamura M, Sakamoto W, Yogo T, Miki H, Ozaki S, et al.

Superparamagnetic nanoparticle clusters for cancer theranostics combining magnetic resonance imaging and hyperthermia treatment. Theranostics. 2013;3:366-76.

44

[12] Kim JI, Lee BS, Chun C, Cho J-K, Kim S-Y, Song S-C. Long-term theranostic hydrogel system for solid tumors. Biomaterials. 2012;33:2251-9.

[13] Georgiadou V, Kokotidou C, Le Droumaguet B, Carbonnier B, Choli-Papadopoulou T, Dendrinou-Samara C. Oleylamine as a beneficial agent for the synthesis of CoFe2O4 nanoparticles with potential biomedical uses. Dalton Transactions. 2014;43:6377-88.

[14] Sun SH, Zeng H, Robinson DB, Raoux S, Rice PM, Wang SX, et al. Monodisperse MFe2O4 (M = Fe, Co, Mn) nanoparticles. Journal of the American Chemical Society.

2004;126:273-9.

[15] Hariri G, Wellons MS, Morris WH, 3rd, Lukehart CM, Hallahan DE. Multifunctional FePt nanoparticles for radiation-guided targeting and imaging of cancer. Annals of biomedical engineering. 2011;39:946-52.

[16] Maiti D, Saha A, Devi PS. Surface modified multifunctional ZnFe2O4 nanoparticles for hydrophobic and hydrophilic anti-cancer drug molecule loading. Physical Chemistry Chemical Physics. 2016;18:1439-50.

[17] Manukyan KV, Chen Y-S, Rouvimov S, Li P, Li X, Dong S, et al. Ultrasmall α-Fe2O3 superparamagnetic nanoparticles with high magnetization prepared by template-assisted combustion process. The Journal of Physical Chemistry C. 2014;118:16264-71.

[18] Subhankar B, Wolfgang K. Supermagnetism. Journal of Physics D: Applied Physics.

2009;42:013001.

[19] Gupta AK, Gupta M. Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications. Biomaterials. 2005;26:3995-4021.

[20] Ghosh S, Kumar SRP, Puri IK, Elankumaran S. Magnetic assembly of 3D cell clusters: visualizing the formation of an engineered tissue. Cell Prolif. 2016;49:134-44.

[21] Tang M, Wang Q, Jiang M, Xu L, Shi Z-G, Zhang T, et al. Magnetic solid-phase extraction based on methylcellulose coated-Fe3O4–SiO2–phenyl for HPLC–DAD analysis of sildenafil and its metabolite in biological samples. Talanta. 2014;130:427-32.

[22] Kılınç E. Fullerene C60 functionalized γ-Fe2O3 magnetic nanoparticle: Synthesis, characterization, and biomedical applications. Artificial Cells, Nanomedicine, and Biotechnology. 2016;44:298-304.

[23] Mroz P, Pawlak A, Satti M, Lee H, Wharton T, Gali H, et al. Functionalized fullerenes mediate photodynamic killing of cancer cells: Type I versus Type II photochemical mechanism. Free Radical Biology and Medicine. 2007;43:711-9.

45

[24] Tóth É, Bolskar RD, Borel A, González G, Helm L, Merbach AE, et al. Water-soluble gadofullerenes:  toward high-relaxivity, pH-responsive MRI contrast agents. Journal of the American Chemical Society. 2005;127:799-805.

[25] Chen C-L, Kuo L-R, Lee S-Y, Hwu Y-K, Chou S-W, Chen C-C, et al. Photothermal cancer therapy via femtosecond-laser-excited FePt nanoparticles. Biomaterials.

2013;34:1128-34.

[26] Sahu NK, Gupta J, Bahadur D. PEGylated FePt-Fe3O4 composite nanoassemblies (CNAs): in vitro hyperthermia, drug delivery and generation of reactive oxygen species (ROS). Dalton transactions (Cambridge, England : 2003). 2015;44:9103-13.

[27] Salunkhe AB, Khot VM, Thorat ND, Phadatare MR, Sathish CI, Dhawale DS, et al.

Polyvinyl alcohol functionalized cobalt ferrite nanoparticles for biomedical applications.

Appl Surf Sci. 2013;264:598-604.

[28] Kim JI, Chun C, Kim B, Hong JM, Cho J-K, Lee SH, et al. Thermosensitive/magnetic poly(organophosphazene) hydrogel as a long-term magnetic resonance contrast platform.

Biomaterials. 2012;33:218-24.

[29] Barrena R, Casals E, Colón J, Font X, Sánchez A, Puntes V. Evaluation of the ecotoxicity of model nanoparticles. Chemosphere. 2009;75:850-7.

[30] Karlsson HL, Gustafsson J, Cronholm P, Möller L. Size-dependent toxicity of metal oxide particles—A comparison between nano- and micrometer size. Toxicology Letters.

2009;188:112-8.

[31] Jaiswal MK, Mehta S, Banerjee R, Bahadur D. A comparative study on thermoresponsive magnetic nanohydrogels: role of surface-engineered magnetic nanoparticles. Colloid and Polymer Science. 2011;290:607-17.

[32] Han HD, Shin BC, Choi HS. Doxorubicin-encapsulated thermosensitive liposomes modified with poly(N-isopropylacrylamide-co-acrylamide): Drug release behavior and stability in the presence of serum. European Journal of Pharmaceutics and Biopharmaceutics. 2006;62:110-6.

[33] Lo C-L, Lin K-M, Hsiue G-H. Preparation and characterization of intelligent core-shell nanoparticles based on poly(d,l-lactide)-g-poly(N-isopropyl acrylamide-co-methacrylic acid). Journal of Controlled Release. 2005;104:477-88.

[34] Li Z, Shen J, Ma H, Lu X, Shi M, Li N, et al. Preparation and characterization of sodium alginate/poly(N-isopropylacrylamide)/clay semi-IPN magnetic hydrogels.

Polymer Bulletin. 2011;68:1153-69.

46

[35] Gant RM, Abraham AA, Hou Y, Cummins BM, Grunlan MA, Coté GL. Design of a self-cleaning thermoresponsive nanocomposite hydrogel membrane for implantable biosensors. Acta Biomaterialia. 2010;6:2903-10.

[36] Baeza A, Guisasola E, Ruiz-Hernández E, Vallet-Regí M. Magnetically triggered multidrug release by hybrid mesoporous silica nanoparticles. Chemistry of Materials.

2012;24:517-24.

[37] Sivakumaran D, Maitland D, Hoare T. Injectable microgel-hydrogel composites for prolonged small-molecule drug delivery. Biomacromolecules. 2011;12:4112-20.

[38] Tan H, Ramirez CM, Miljkovic N, Li H, Rubin JP, Marra KG. Thermosensitive injectable hyaluronic acid hydrogel for adipose tissue engineering. Biomaterials.

2009;30:6844-53.

[39] Yang J, Yamato M, Shimizu T, Sekine H, Ohashi K, Kanzaki M, et al. Reconstruction of functional tissues with cell sheet engineering. Biomaterials. 2007;28:5033-43.

[40] Hong SW, Kim DY, Lee JU, Jo WH. Synthesis of polymeric temperature sensor based on photophysical property of fullerene and thermal sensitivity of poly(N-isopropylacrylamide). Macromolecules. 2009;42:2756-61.

[41] Yang C-C, Tian Y, Jen AKY, Chen W-C. New environmentally responsive fluorescent N-isopropylacrylamide copolymer and its application to DNA sensing. Journal of Polymer Science Part A: Polymer Chemistry. 2006;44:5495-504.

[42] Wang N, Guan YP, Yang LR, Jia LW, Wei XT, Liu HZ, et al. Magnetic nanoparticles (MNPs) covalently coated by PEO-PPO-PEO block copolymer for drug delivery. Journal of Colloid and Interface Science. 2013;395:50-7.

[43] Valverde-Aguilar G, Pérez-Mazariego J, Marquina V, Gómez R, Aguilar-Franco M, Garcia-Macedo J. Effect of PEO-PPO-PEO triblock copolymers in the synthesis of magnetic nanoparticles embedded in SiO and TiO matrices by sol-gel method. Journal of Materials Science. 2015;50:704-16.

[44] Kang GD, Cheon SH, Song SC. Controlled release of doxorubicin from thermosensitive poly(organophosphazene) hydrogels. International journal of pharmaceutics. 2006;319:29-36.

[45] Jaiswal MK, De M, Chou SS, Vasavada S, Bleher R, Prasad PV, et al.

Thermoresponsive magnetic hydrogels as theranostic nanoconstructs. ACS Applied Materials & Interfaces. 2014;6:6237-47.

47

[46] Principles of cancer therapy: surgery. In: Mark H. Beers M, Robert Berkow M, editors. The Merck Manual of Diagnosis and Therapy. Whitehouse Station, NJ: Merck Research Laboratories; 1999.

[47] Hayashi K, Nakamura M, Sakamoto W, Yogo T, Miki H, Ozaki S, et al.

Superparamagnetic nanoparticle clusters for cancer theranostics combining magnetic resonance imaging and hyperthermia treatment. Theranostics. 2013;3:366-76.

[48] Hayashi K, Nakamura M, Miki H, Ozaki S, Abe M, Matsumoto T, et al. Magnetically responsive smart nanoparticles for cancer treatment with a combination of magnetic hyperthermia and remote-control drug release. Theranostics. 2014;4:834-44.

[49] Schultz N. Leakier tumor vessels enhance drug delivery. MIT Technology Review2010. p. 2.

[50] McDonald DM, Baluk P. Significance of blood vessel leakiness in cancer. Cancer research. 2002;62:5381-5.

[51] Weissleder R, Stark DD, Engelstad BL, Bacon BR, Compton CC, White DL, et al.

Superparamagnetic iron oxide: pharmacokinetics and toxicity. AJR American journal of roentgenology. 1989;152:167-73.

[52] Gaharwar AK, Avery RK, Assmann A, Paul A, McKinley GH, Khademhosseini A, et al. Shear-thinning nanocomposite hydrogels for the treatment of hemorrhage. ACS Nano. 2014;8:9833-42.

[53] Van Den Bulcke AI, Bogdanov B, De Rooze N, Schacht EH, Cornelissen M, Berghmans H. Structural and rheological properties of methacrylamide modified gelatin hydrogels. Biomacromolecules. 2000;1:31-8.

[54] Nichol JW, Koshy ST, Bae H, Hwang CM, Yamanlar S, Khademhosseini A. Cell-laden microengineered gelatin methacrylate hydrogels. Biomaterials. 2010;31:5536-44.

[55] Koshy ST, Desai RM, Joly P, Li JY, Bagrodia RK, Lewin SA, et al. Click-crosslinked injectable gelatin hydrogels. Adv Healthc Mater. 2016;5:541-7.

[56] Loessner D, Meinert C, Kaemmerer E, Martine LC, Yue K, Levett PA, et al.

Functionalization, preparation and use of cell-laden gelatin methacryloyl-based hydrogels as modular tissue culture platforms. Nat Protoc. 2016;11:727-46.

[57] Chilkoti A, Dreher MR, Meyer DE, Raucher D. Targeted drug delivery by thermally responsive polymers. Advanced Drug Delivery Reviews. 2002;54:613-30.

48

[58] Nigam S, Barick KC, Bahadur D. Development of citrate-stabilized Fe3O4 nanoparticles: Conjugation and release of doxorubicin for therapeutic applications.

Journal of Magnetism and Magnetic Materials. 2011;323:237-43.

[59] Zhang J, Chen H, Xu L, Gu Y. The targeted behavior of thermally responsive nanohydrogel evaluated by NIR system in mouse model. Journal of Controlled Release.

2008;131:34-40.

[60] Fairbanks BD, Schwartz MP, Bowman CN, Anseth KS. Photoinitiated polymerization of PEG-diacrylate with lithium phenyl-2,4,6-trimethylbenzoylphosphinate: polymerization rate and cytocompatibility. Biomaterials.

2009;30:6702-7.

[61] Rudnev V. Handbook of induction heating. New York :: Marcel Dekker; 2003.

[62] Legendre JY, Szoka FC. Delivery of plasmid dna into mammalian-cell lines using ph-sensitive liposomes - comparison with cationic liposomes. Pharm Res. 1992;9:1235-42.

[63] Lee RJ, Low PS. Folate-mediated tumor cell targeting of liposome-entrapped doxorubicin in vitro. Biochimica et Biophysica Acta (BBA) - Biomembranes.

1995;1233:134-44.

[64] Constantin M, Cristea M, Ascenzi P, Fundueanu G. Lower critical solution temperature versus volume phase transition temperature in thermoresponsive drug delivery systems. Express Polym Lett. 2011;5:839-48.

[65] Paul A, Hasan A, Kindi HA, Gaharwar AK, Rao VT, Nikkhah M, et al. Injectable graphene oxide/hydrogel-based angiogenic gene delivery system for vasculogenesis and cardiac repair. ACS Nano. 2014;8:8050-62.

[66] Monteux C, Mangeret R, Laibe G, Freyssingeas E, Bergeron V, Fuller G. Shear surface rheology of poly(N-isopropylacrylamide) adsorbed layers at the air−water interface. Macromolecules. 2006;39:3408-14.

[67] Mason BN, Califano JP, Reinhart-King CA. Matrix stiffness: a regulator of cellular behavior and tissue formation. In: Bhatia KS, editor. Engineering Biomaterials for Regenerative Medicine: Novel Technologies for Clinical Applications. New York, NY:

Springer New York; 2012. p. 19-37.

[68] Shah SA, Asdi MH, Hashmi MU, Umar MF, Awan S-U. Thermo-responsive copolymer coated MnFe2O4 magnetic nanoparticles for hyperthermia therapy and controlled drug delivery. Materials Chemistry and Physics. 2012;137:365-71.

49

[69] Dong Z, Wang Q, Du Y. Alginate/gelatin blend films and their properties for drug controlled release. Journal of Membrane Science. 2006;280:37-44.

[70] Liu TY, Hu SH, Liu KH, Shaiu RS, Liu DM, Chen SY. Instantaneous drug delivery of magnetic/thermally sensitive nanospheres by a high-frequency magnetic field.

Langmuir. 2008;24:13306-11.

[71] Maiorano G, Sabella S, Sorce B, Brunetti V, Malvindi MA, Cingolani R, et al. Effects of cell culture media on the dynamic formation of protein−nanoparticle complexes and influence on the cellular response. ACS Nano. 2010;4:7481-91.