Intracellularly Actuated Quantum Dot-Peptide-Doxorubicin Nanobioconjugates for Controlled Drug Delivery via the Endocytic Pathway.

Nanoparticle (NP)-mediated drug delivery (NMDD) has emerged as a novel method to overcome the limitations of traditional systemic delivery of therapeutics, including the controlled release of the NP-associated drug cargo. Currently, our most advanced understanding of how to control NP-associated cargos is in the context of soft nanoparticles (e.g., liposomes), but less is known about controlling the release of cargos from the surface of hard NPs (e.g., gold NPs). Here we employ a semiconductor quantum dot (QD) as a prototypical hard NP platform and use intracellularly triggered actuation to achieve spatiotemporal control of drug release and modulation of drug efficacy. Conjugated to the QD are two peptides: (1) a cell-penetrating peptide (CPP) that facilitates uptake of the conjugate into the endocytic pathway and (2) a display peptide conjugated to doxorubicin (DOX) via three different linkages (ester, disulfide, and hydrazone) that are responsive to enzymatic cleavage, reducing conditions, and low pH, respectively. Formation of the QD-[peptide-DOX]-CPP complex is driven by self-assembly that allows control over both the ratio of each peptide species conjugated to the QD and the eventual drug dose delivered to cells. Förster resonance energy transfer assays confirmed successful assembly of the QD-peptide complexes and functionality of the linkages. Confocal microscopy was employed to visualize residence of the QD-[peptide-DOX]-CPP complexes in the endocytic pathway, and distinct differences in DOX localization were noted for the ester linkage, which showed clear signs of nuclear delivery versus the hydrazone, disulfide, and amide control. Finally, delivery of the QD-[peptide-DOX]-CPP conjugate resulted in cytotoxicity for the ester linkage that was comparable to free DOX. Attachment of DOX via the hydrazone linkage facilitated intermediary toxicity, while the disulfide and amide control linkages showed minimal toxicity. Our data demonstrate the utility of hard NP-peptide bioconjugates to function as multifunctional scaffolds for simultaneous control over cellular drug uptake and toxicity and the vital role played by the nature of the chemical linkage that appends the drug to the NP carrier.

[1]  T. Fukami,et al.  The emerging role of human esterases. , 2012, Drug metabolism and pharmacokinetics.

[2]  S. Inoue,et al.  Cellular Delivery of Doxorubicin via pH-Controlled Hydrazone Linkage Using Multifunctional Nano Vehicle Based on Poly(β-L-Malic Acid) , 2012, International journal of molecular sciences.

[3]  Juan B. Blanco-Canosa,et al.  Cellular uptake and fate of PEGylated gold nanoparticles is dependent on both cell-penetration peptides and particle size. , 2011, ACS nano.

[4]  Mauro Comes Franchini,et al.  Hard and soft nanoparticles for image-guided surgery in nanomedicine , 2015, Journal of Nanoparticle Research.

[5]  Vladimir Torchilin,et al.  Tumor delivery of macromolecular drugs based on the EPR effect. , 2011, Advanced drug delivery reviews.

[6]  J. Frangioni.  In vivo near-infrared fluorescence imaging. , 2003, Current opinion in chemical biology.

[7]  Atsushi Harada,et al.  Design of environment-sensitive supramolecular assemblies for intracellular drug delivery: polymeric micelles that are responsive to intracellular pH change. , 2003, Angewandte Chemie.

[8]  Igor L. Medintz,et al.  PEGylated Luminescent Gold Nanoclusters: Synthesis, Characterization, Bioconjugation, and Application to One‐ and Two‐Photon Cellular Imaging , 2013 .

[9]  Serena Mazzucchelli,et al.  Tumour homing and therapeutic effect of colloidal nanoparticles depend on the number of attached antibodies , 2016, Nature Communications.

[10]  Shawn J. Tan,et al.  Surface-Ligand-Dependent Cellular Interaction, Subcellular Localization, and Cytotoxicity of Polymer-Coated Quantum Dots , 2010 .

[11]  Igor L. Medintz,et al.  A New Family of Pyridine-Appended Multidentate Polymers As Hydrophilic Surface Ligands for Preparing Stable Biocompatible Quantum Dots , 2014 .

[12]  P. Trail,et al.  (6-Maleimidocaproyl)hydrazone of doxorubicin--a new derivative for the preparation of immunoconjugates of doxorubicin. , 1993, Bioconjugate chemistry.

[13]  Igor L. Medintz,et al.  Selecting improved peptidyl motifs for cytosolic delivery of disparate protein and nanoparticle materials. , 2013, ACS nano.

[14]  Igor L. Medintz,et al.  Cytotoxicity of quantum dots used for in vitro cellular labeling: role of QD surface ligand, delivery modality, cell type, and direct comparison to organic fluorophores. , 2013, Bioconjugate chemistry.

[15]  Igor L. Medintz,et al.  Multifunctional compact zwitterionic ligands for preparing robust biocompatible semiconductor quantum dots and gold nanoparticles. , 2011, Journal of the American Chemical Society.

[16]  Weiwei Guo,et al.  Glutathione-mediated release of functional plasmid DNA from positively charged quantum dots. , 2008, Biomaterials.

[17]  R. Havel,et al.  Acid hydrolases in early and late endosome fractions from rat liver. , 1991, The Journal of biological chemistry.

[18]  Igor L. Medintz,et al.  Modulation of Intracellular Quantum Dot to Fluorescent Protein Förster Resonance Energy Transfer via Customized Ligands and Spatial Control of Donor–Acceptor Assembly , 2015, Sensors.

[19]  G. Balendiran,et al.  The role of glutathione in cancer , 2004, Cell biochemistry and function.

[20]  Mi Ae Kim,et al.  Two distinct modes of cell death induced by doxorubicin: apoptosis and cell death through mitotic catastrophe accompanied by senescence-like phenotype , 2005, Oncogene.

[21]  M. Prabaharan,et al.  Amphiphilic multi-arm-block copolymer conjugated with doxorubicin via pH-sensitive hydrazone bond for tumor-targeted drug delivery. , 2009, Biomaterials.

[22]  Ali Akbar Pourfatollah,et al.  Various methods of gold nanoparticles (GNPs) conjugation to antibodies , 2016 .

[23]  Philip E. Dawson,et al.  Nanoparticle cellular uptake by dendritic wedge peptides: achieving single peptide facilitated delivery. , 2017, Nanoscale.

[24]  Duane E. Prasuhn,et al.  Combining chemoselective ligation with polyhistidine-driven self-assembly for the modular display of biomolecules on quantum dots. , 2010, ACS nano.

[25]  Juan B. Blanco-Canosa,et al.  Recent progress in the bioconjugation of quantum dots , 2014 .

[26]  W. Stone,et al.  Identification of oxidized protein hydrolase as a potential prodrug target in prostate cancer , 2014, BMC Cancer.

[27]  Jin-Zhi Du,et al.  Tailor-made dual pH-sensitive polymer-doxorubicin nanoparticles for efficient anticancer drug delivery. , 2011, Journal of the American Chemical Society.

[28]  Michael J Sailor,et al.  Hybrid Nanoparticles for Detection and Treatment of Cancer , 2012, Advanced materials.

[29]  S. Kannan,et al.  Poly(amidoamine) dendrimer-drug conjugates with disulfide linkages for intracellular drug delivery. , 2009, Biomaterials.

[30]  Yan Wang,et al.  Up-conversion nanoparticle assembled mesoporous silica composites: synthesis, plasmon-enhanced luminescence, and near-infrared light triggered drug release. , 2014, ACS applied materials & interfaces.

[31]  Duane E. Prasuhn,et al.  Polyvalent display and packing of peptides and proteins on semiconductor quantum dots: predicted versus experimental results. , 2010, Small.

[32]  Igor L. Medintz,et al.  Delivering quantum dot-peptide bioconjugates to the cellular cytosol: escaping from the endolysosomal system. , 2010, Integrative biology : quantitative biosciences from nano to macro.

[33]  P. Low,et al.  Folate-mediated tumor cell targeting of liposome-entrapped doxorubicin in vitro. , 1995, Biochimica et biophysica acta.

[34]  Duane E. Prasuhn,et al.  The controlled display of biomolecules on nanoparticles: a challenge suited to bioorthogonal chemistry. , 2011, Bioconjugate chemistry.

[35]  Igor L. Medintz,et al.  In vitro interaction of colloidal nanoparticles with mammalian cells: What have we learned thus far? , 2014, Beilstein journal of nanotechnology.

[36]  J. F. Stoddart,et al.  Esterase- and pH-responsive poly(β-amino ester)-capped mesoporous silica nanoparticles for drug delivery. , 2015, Nanoscale.

[37]  Sung Eun Kim,et al.  Disulfide-cross-linked PEG-poly(amino acid)s copolymer micelles for glutathione-mediated intracellular drug delivery. , 2008, Chemical communications.

[38]  Igor L. Medintz,et al.  Spatiotemporal multicolor labeling of individual cells using peptide-functionalized quantum dots and mixed delivery techniques. , 2011, Journal of the American Chemical Society.

[39]  M. Bawendi,et al.  Renal clearance of quantum dots , 2007, Nature Biotechnology.

[40]  Igor L. Medintz,et al.  Self-assembled quantum dot-peptide bioconjugates for selective intracellular delivery. , 2006, Bioconjugate chemistry.

[41]  Xiu Shen,et al.  In vivo renal clearance, biodistribution, toxicity of gold nanoclusters. , 2012, Biomaterials.

[42]  Yitao Wang,et al.  Polymeric micelles drug delivery system in oncology. , 2012, Journal of controlled release : official journal of the Controlled Release Society.

[43]  S. Sahoo,et al.  PLGA nanoparticles containing various anticancer agents and tumour delivery by EPR effect. , 2011, Advanced drug delivery reviews.

[44]  J. Perez,et al.  Quantum Dot-Based OFF/ON Probe for Detection of Glutathione , 2009 .

[45]  N. Zurgil,et al.  Intracellular esterase activity in living cells may distinguish between metastatic and tumor-free lymph nodes , 2008, Clinical & Experimental Metastasis.

[46]  Peixun Liu,et al.  Passing through the renal clearance barrier: toward ultrasmall sizes with stable ligands for potential clinical applications , 2014, International journal of nanomedicine.

[47]  Igor L. Medintz,et al.  Intracellular delivery of quantum dot-protein cargos mediated by cell penetrating peptides. , 2008, Bioconjugate chemistry.

[48]  R. Scheller,et al.  Oxidizing potential of endosomes and lysosomes limits intracellular cleavage of disulfide-based antibody-drug conjugates. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[49]  Igor L. Medintz,et al.  Examining the Polyproline Nanoscopic Ruler in the Context of Quantum Dots , 2015 .

[50]  J. Bennett,et al.  Resveratrol prevents doxorubicin cardiotoxicity through mitochondrial stabilization and the Sirt1 pathway. , 2009, Free radical biology & medicine.

[51]  S. Kent,et al.  Constructing proteins by dovetailing unprotected synthetic peptides: backbone-engineered HIV protease. , 1992, Science.

[52]  Fanbo Zeng,et al.  Increase of doxorubicin sensitivity for folate receptor positive cells when given as the prodrug N-(phenylacetyl) doxorubicin in combination with folate-conjugated PGA. , 2006, Journal of pharmaceutical sciences.

[53]  R. Mumper,et al.  Nanomedicinal strategies to treat multidrug-resistant tumors: current progress. , 2010, Nanomedicine.

[54]  Harald Ittrich,et al.  Exceedingly small iron oxide nanoparticles as positive MRI contrast agents , 2017, Proceedings of the National Academy of Sciences.

[55]  T. Minko,et al.  Tumor targeted quantum dot-mucin 1 aptamer-doxorubicin conjugate for imaging and treatment of cancer. , 2011, Journal of controlled release : official journal of the Controlled Release Society.

[56]  Francesco Stellacci,et al.  Effect of surface properties on nanoparticle-cell interactions. , 2010, Small.

[57]  K. Ulbrich,et al.  Polymeric micellar pH-sensitive drug delivery system for doxorubicin. , 2005, Journal of controlled release : official journal of the Controlled Release Society.

[58]  Kristofer J. Thurecht,et al.  Nanoparticle-Based Medicines: A Review of FDA-Approved Materials and Clinical Trials to Date , 2016, Pharmaceutical Research.

[59]  Pavel Zrazhevskiy,et al.  Quantum dots as a platform for nanoparticle drug delivery vehicle design. , 2013, Advanced drug delivery reviews.

[60]  Xiaohu Gao,et al.  Designing multifunctional quantum dots for bioimaging, detection, and drug delivery. , 2010, Chemical Society reviews.

[61]  A. Nishikawa,et al.  Histochemical determination of stereoselectivity of esterases in normal pancreas and pancreatic tubular adenocarcinoma of hamsters. , 1998, Biotechnic & Histochemistry.

[62]  Arthur G Erdman,et al.  The big picture on nanomedicine: the state of investigational and approved nanomedicine products. , 2013, Nanomedicine : nanotechnology, biology, and medicine.

[63]  Duane E. Prasuhn,et al.  Site-specific cellular delivery of quantum dots with chemoselectively-assembled modular peptides. , 2013, Chemical communications.

[64]  Igor L. Medintz,et al.  Controlling the actuation of therapeutic nanomaterials: enabling nanoparticle-mediated drug delivery. , 2013, Therapeutic delivery.

[65]  V. Torchilin,et al.  "SMART" drug delivery systems: double-targeted pH-responsive pharmaceutical nanocarriers. , 2006, Bioconjugate chemistry.

[66]  Hapipah Mohd Ali,et al.  Synthesis, characterization and apoptotic activity of quinazolinone Schiff base derivatives toward MCF-7 cells via intrinsic and extrinsic apoptosis pathways , 2015, Scientific Reports.

[67]  Joyce C Breger,et al.  Multifunctional nanoparticle composites: progress in the use of soft and hard nanoparticles for drug delivery and imaging. , 2017, Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology.

[68]  Joyce C Breger,et al.  Controlled actuation of therapeutic nanoparticles: an update on recent progress. , 2016, Therapeutic delivery.

[69]  Kazunori Kataoka,et al.  Preparation and biological characterization of polymeric micelle drug carriers with intracellular pH-triggered drug release property: tumor permeability, controlled subcellular drug distribution, and enhanced in vivo antitumor efficacy. , 2005, Bioconjugate chemistry.

[70]  Ganesh Gollavelli,et al.  Magnetic and fluorescent graphene for dual modal imaging and single light induced photothermal and photodynamic therapy of cancer cells. , 2014, Biomaterials.

[71]  Dai Fukumura,et al.  Multistage nanoparticle delivery system for deep penetration into tumor tissue , 2011, Proceedings of the National Academy of Sciences.

[72]  Igor L. Medintz,et al.  Monitoring of enzymatic proteolysis on a electroluminescent-CCD microchip platform using quantum dot-peptide substrates , 2009 .

[73]  Hak Soo Choi,et al.  Renal Clearance of Nanoparticles , 2009 .

[74]  Igor L. Medintz,et al.  Quantum Dot-Peptide-Fullerene Bioconjugates for Visualization of in Vitro and in Vivo Cellular Membrane Potential. , 2017, ACS nano.

[75]  S. Nie,et al.  In vitro study of a pH-sensitive multifunctional doxorubicin–gold nanoparticle system: therapeutic effect and surface enhanced Raman scattering , 2015 .

[76]  Mauro Ferrari,et al.  Principles of nanoparticle design for overcoming biological barriers to drug delivery , 2015, Nature Biotechnology.

[77]  Zhuxian Zhou,et al.  Synthesis of an esterase‐sensitive degradable polyester as facile drug carrier for cancer therapy , 2016 .

[78]  S. Choi,et al.  Mechanisms of drug release in nanotherapeutic delivery systems. , 2015, Chemical reviews.

[79]  I. Prior,et al.  Conserved effects and altered trafficking of Cetuximab antibodies conjugated to gold nanoparticles with precise control of their number and orientation. , 2017, Nanoscale.