Nanomaterial-Enabled Cancer Therapy
Authors: Sabina Quader, Kazunori Kataoka
Journal: Molecular Therapy (2017)

While cancer remains the major cause of death worldwide, nanomaterial (NM)-based diagnosis and treatment modalities are showing remarkable potential to better tackle clinical oncology by effectively targeting therapeutic agents to tumors. NMs can selectively accumulate in solid tumors, and they can improve the bioavailability and reduce the toxicity of encapsulated cytotoxic agents. Additional noteworthy functions of NMs in cancer treatment include the delivery of contrast agents to image tumor sites, delivery of genetic materials for gene therapy, and co-delivery of multiple agents to achieve combination therapy or simultaneous diagnostic and therapeutic outcomes. Although several NM therapeutics have been successfully translated to clinical applications, the gap between the bench and the bedside remains ominously wide. Tumor heterogeneity and the disparity between pre-clinical and clinical studies have been identified as two of the major translational challenges of NM-based cancer therapies. Herein, we review a handful of recent research studies on the use of NMs in cancer therapy and imaging, with a limited discussion on the consequences of tumor heterogeneity and pre-clinical studies on translational research of NM-based delivery systems and propositions in the literature to overcome these challenges. While cancer remains the major cause of death worldwide, nanomaterial (NM)-based diagnosis and treatment modalities are showing remarkable potential to better tackle clinical oncology by effectively targeting therapeutic agents to tumors. NMs can selectively accumulate in solid tumors, and they can improve the bioavailability and reduce the toxicity of encapsulated cytotoxic agents. Additional noteworthy functions of NMs in cancer treatment include the delivery of contrast agents to image tumor sites, delivery of genetic materials for gene therapy, and co-delivery of multiple agents to achieve combination therapy or simultaneous diagnostic and therapeutic outcomes. Although several NM therapeutics have been successfully translated to clinical applications, the gap between the bench and the bedside remains ominously wide. Tumor heterogeneity and the disparity between pre-clinical and clinical studies have been identified as two of the major translational challenges of NM-based cancer therapies. Herein, we review a handful of recent research studies on the use of NMs in cancer therapy and imaging, with a limited discussion on the consequences of tumor heterogeneity and pre-clinical studies on translational research of NM-based delivery systems and propositions in the literature to overcome these challenges. Cancer remains a major public health crisis in the United States and many other parts of the globe. In the United States, cancer rates are on track to overtake heart disease as the leading cause of death. While about 595,690 Americans are expected to have died of cancer in 2016, deaths from heart disease outnumber cancer by only 20,000.1American Cancer Society. Global Cancer Facts and figures, 3rd Edition. https://www.cancer.org/content/dam/cancer-org/research/cancer-facts-and-statistics/global-cancer-facts-and-figures/global-cancer-facts-and-figures-3rd-edition.pdf.Google Scholar Globally, the number of new cancer cases is expected to reach 21.7 million by 2030, an almost 40% increase from the 14.1 million in 2012. Despite significant improvements in cancer diagnosis and treatment, cancer survival statistics have remained grim, with a 5-year relative survival rate of just 69% for all cancers diagnosed between 2005 and 2011, which is only 20% better than the 49% survival rate for cancers diagnosed between 1975 and 1977.2American Cancer Society. Cancer Facts and Figures, 2016. https://www.cancer.org/content/dam/cancer-org/research/cancer-facts-and-statistics/annual-cancer-facts-and-figures/2016/cancer-facts-and-figures-2016.pdf.Google Scholar Nevertheless, in recent years, there have been remarkable developments in clinical oncology with the potential to radically change the overall cancer scenario; treatment approaches based on a patient's genomic profile, immunotherapies, and targeted therapies are noteworthy advancements. Nanotechnology that produces, controls, and utilizes materials at the nanoscale (between one and several hundred nanometers) also has potential to revolutionize the conventional methods of cancer diagnosis and treatment.3Peer D. Karp J.M. Hong S. Farokhzad O.C. Margalit R. Langer R. Nanocarriers as an emerging platform for cancer therapy.Nat. Nanotechnol. 2007; 2: 751-760Crossref PubMed Scopus (3610) Google Scholar, 4Piktel E. Niemirowicz K. Wątek M. Wollny T. Deptuła P. Bucki R. Recent insights in nanotechnology-based drugs and formulations designed for effective anti-cancer therapy.J. Nanobiotechnology. 2016; 14: 39Crossref PubMed Scopus (5) Google Scholar Nanomaterials (NMs) are being recognized as useful in oncology, with sensitive cancer detection capabilities and effective treatment outcomes due to their small size (Figure 1). NM-based therapeutics (Figure 1B) have many advantages over traditional chemotherapy, including improved bioavailability, dose response, and targeting efficiency, with fewer side effects. NMs take advantage of an enhanced permeability and retention (EPR) effect to preferentially penetrate and accumulate in cancerous growths, which is based on the anatomical and pathophysiological characteristics of solid tumor tissues.5Matsumura Y. Maeda H. A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs.Cancer Res. 1986; 46: 6387-6392PubMed Google Scholar Besides EPR-mediated selective tumor accumulation, tumor-selective delivery and pharmacokinetic profiles of chemotherapeutic and imaging agents can be improved by carefully controlling NM size and shape,6Truong N.P. Whittaker M.R. Mak C.W. Davis T.P. The importance of nanoparticle shape in cancer drug delivery.Expert Opin. Drug Deliv. 2015; 12: 129-142Crossref PubMed Scopus (50) Google Scholar surface modification with disease-targeting ligands,7Bazak R. Houri M. El Achy S. Kamel S. Refaat T. Cancer active targeting by nanoparticles: a comprehensive review of literature.J. Cancer Res. Clin. Oncol. 2015; 141: 769-784Crossref PubMed Scopus (46) Google Scholar and engineered tumor microenvironment (TME)-triggered drug-release mechanisms.8Zhu L. Torchilin V.P. Stimulus-responsive nanopreparations for tumor targeting.Integr. Biol. 2013; 5: 96-107Crossref Google Scholar Furthermore, multi-modal NM platforms are particularly exciting, such as combination therapies9He C. Chan C. Weichselbaum R.R. Fleming G.F. Yamada S.D. Lin W. Nanomedicine for combination therapy of cancer.EBioMedicine. 2015; 2: 366-367Abstract Full Text Full Text PDF PubMed Google Scholar or "theranostic" combinations of diagnostic and therapeutic substances.10Arranja A.G. Pathak V. Lammers T. Shi Y. Tumor-targeted nanomedicines for cancer theranostics.Pharmacol. Res. 2017; 115: 87-95Crossref PubMed Scopus (1) Google Scholar The diverse applications and prospects of NMs in the biomedical field have initiated a myriad of research projects and publications, which have been reviewed, discussed, and even debated in many review articles.11Wilhelm S. Tavares A.J. Dai Q. Ohta S. Audet J. Dvorak H.F. Chan W.C.W. Analysis of nanoparticle delivery to tumours.Nat. Rev. Mater. 2016; 1: 16014Crossref Scopus (0) Google Scholar Recently, there has been debate in the NM field regarding the targeting ability and delivery efficiency of nanoparticles (NPs) in tumors.11Wilhelm S. Tavares A.J. Dai Q. Ohta S. Audet J. Dvorak H.F. Chan W.C.W. Analysis of nanoparticle delivery to tumours.Nat. Rev. Mater. 2016; 1: 16014Crossref Scopus (0) Google Scholar, 12Torrice M. Does nanomedicine have a delivery problem?.ACS Cent. Sci. 2016; 2: 434-437Crossref PubMed Google Scholar, 13Lammers T. Kiessling F. Ashford M. Hennink W. Crommelin D. Storm G. Cancer nanomedicine: is targeting our target?.Nat. Rev. Mater. 2016; 1: 16069Crossref PubMed Google Scholar In this review, we discuss NP designs and architectural strategies for efficient tumor targeting and delivery, using examples from recent research related to NMs (mostly PEGylated) for cancer therapy and imaging. While NMs have the potential to impact cancer therapy tremendously, there are fundamental challenges14Jain R.K. Stylianopoulos T. Delivering nanomedicine to solid tumors.Nat. Rev. Clin. Oncol. 2010; 7: 653-664Crossref PubMed Scopus (1078) Google Scholar, 15Hare J.I. Lammers T. Ashford M.B. Puri S. Storm G. Barry S.T. Challenges and strategies in anti-cancer nanomedicine development: an industry perspective.Adv. Drug Deliv. Rev. 2017; 108: 25-38Crossref PubMed Scopus (9) Google Scholar, 16Shi J. Kantoff P.W. Wooster R. Farokhzad O.C. Cancer nanomedicine: progress, challenges and opportunities.Nat. Rev. Cancer. 2017; 17: 20-37Crossref PubMed Scopus (28) Google Scholar in terms of design, delivery, and targeting ability that need to be addressed to ensure successful translation of this technology from the laboratory to clinical practice. Therefore, the challenges of translating NM cancer therapies to the clinic will also be discussed within the scope of this review. Broadly, the fundamental challenge in treating cancer is to destroy tumor cells while sparing healthy tissues and organs. Traditional treatment regimes often lack this specificity and are associated with frequent and severe adverse reactions. Thus, developing chemotherapeutics that can selectively target cancer cells is highly desirable. From this perspective, NM-based drug delivery systems (NM-DDSs) have already incited a paradigm shift in cancer chemotherapy by reducing toxicity while maintaining therapeutic efficacy and also improving safety and biocompatibility. In contrast to healthy tissues and organs, solid tumors generally have defective vascular architecture, impaired lymphatic drainage, and reduced uptake of interstitial fluid. Utilizing this disparity, along with a longer blood circulation profile (accomplished by introducing stealth properties, such as PEGylation), NM-DDSs selectively accumulate in tumorous lesions, a phenomenon commonly referred to as EPR-based passive targeting.5Matsumura Y. Maeda H. A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs.Cancer Res. 1986; 46: 6387-6392PubMed Google Scholar, 17Maeda H. Wu J. Sawa T. Matsumura Y. Hori K. Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review.J. Control. Release. 2000; 65: 271-284Crossref PubMed Scopus (3457) Google Scholar Various types of NM-based drug carriers have been designed to exploit this passive targeting, a number of which have successfully progressed to the clinic.15Hare J.I. Lammers T. Ashford M.B. Puri S. Storm G. Barry S.T. Challenges and strategies in anti-cancer nanomedicine development: an industry perspective.Adv. Drug Deliv. Rev. 2017; 108: 25-38Crossref PubMed Scopus (9) Google Scholar, 16Shi J. Kantoff P.W. Wooster R. Farokhzad O.C. Cancer nanomedicine: progress, challenges and opportunities.Nat. Rev. Cancer. 2017; 17: 20-37Crossref PubMed Scopus (28) Google Scholar Attempts have been made to improve tumor accumulation through active targeting by functionalization of the NM surface with ligands such as antibodies, proteins, and peptides that interact with receptors overexpressed at the tumor site, affording active targeting.7Bazak R. Houri M. El Achy S. Kamel S. Refaat T. Cancer active targeting by nanoparticles: a comprehensive review of literature.J. Cancer Res. Clin. Oncol. 2015; 141: 769-784Crossref PubMed Scopus (46) Google Scholar Aside from targeting and delivery objectives, researchers are currently focusing on additional benefits of NMs in cancer therapy, such as using NMs to overcome multi-drug resistance (MDR) in cancer,18Moon J.H. Moxley Jr., J.W. Zhang P. Cui H. Nanoparticle approaches to combating drug resistance.Future Med. Chem. 2015; 7: 1503-1510Crossref PubMed Scopus (1) Google Scholar inhibit metastasis,19Makino J. Cabral H. Miura Y. Matsumoto Y. Wang M. Kinoh H. Mochida Y. Nishiyama N. Kataoka K. cRGD-installed polymeric micelles loading platinum anticancer drugs enable cooperative treatment against lymph node metastasis.J. Control. Release. 2015; 220: 783-791Crossref PubMed Scopus (4) Google Scholar or increase the therapeutic index of molecularly targeted agents.20Ashton S. Song Y.H. Nolan J. Cadogan E. Murray J. Odedra R. Foster J. Hall P.A. Low S. Taylor P. et al.Aurora kinase inhibitor nanoparticles target tumors with favorable therapeutic index in vivo.Sci. Transl. Med. 2016; 8: 325ra17Crossref PubMed Scopus (19) Google Scholar, 21Quader S. Cabral H. Mochida Y. Ishii T. Liu X. Toh K. Kinoh H. Miura Y. Nishiyama N. Kataoka K. Selective intracellular delivery of proteasome inhibitors through pH-sensitive polymeric micelles directed to efficient antitumor therapy.J. Control. Release. 2014; 188: 67-77Crossref PubMed Scopus (23) Google Scholar Molecularly targeted therapeutics (e.g., tyrosine kinase inhibitors, proteasome inhibitors [PIs], histone deacetylase inhibitors, and angiogenesis inhibitors) have been increasingly recognized as an efficient alternative to classical cytotoxic chemotherapeutics, due to their ability to specifically interfere in the growth, progression, and spread of cancers.22Hochwald S.N. Molecular-targeted therapy for cancer and nanotechnology.Methods Mol. Biol. 2010; 624: 11-23Crossref PubMed Scopus (4) Google Scholar However, their unfavorable biodistribution upon systemic delivery hampers their use as "precision medicines." These molecularly targeted agents are either rapidly cleared from the circulatory system or deactivated before reaching the tumor site. In certain cases, the targeted drugs are rapidly deactivated inside the cancer cells and fail to display a robust therapeutic effect.23Lammers T. Hennink W.E. Storm G. Tumour-targeted nanomedicines: principles and practice.Br. J. Cancer. 2008; 99: 392-397Crossref PubMed Scopus (0) Google Scholar In this context, tumor-targeted NM-DDSs can prove useful due to their outstanding ability to target a drug to the tumor site, thereby improving tumoral drug concentrations. PIs have recently emerged as an important tool for molecularly targeted therapy24Rastogi N. Mishra D.P. Therapeutic targeting of cancer cell cycle using proteasome inhibitors.Cell Div. 2012; 7: 26-36Crossref PubMed Scopus (36) Google Scholar in oncology, and they induce apoptosis and cell cycle arrest in tumor cells. Two PIs, bortezomib (BTZ) and carfilzomib, were approved by the Food and Drug Administration (FDA) for clinical use against multiple myeloma. Although peptide aldehyde-based PIs, such as MG132, are potent inhibitors of the 20S proteasome, their clinical use is greatly limited because of their poor selectivity and specificity, which is caused by the aldehyde function of MG132. Thus, a polymeric micelle-based carrier system has been developed that covalently links MG132 to block copolymer PEG (poly(ethylene glycol))-b-poly(aspartamide) through an acid-labile hydrazone bond (Figure 2A). This protects the aldehyde group during circulation. After endocytosis, the hydrazone bond is hydrolyzed in the acidic environment of the endosomes, restoring MG132's therapeutic function.21Quader S. Cabral H. Mochida Y. Ishii T. Liu X. Toh K. Kinoh H. Miura Y. Nishiyama N. Kataoka K. Selective intracellular delivery of proteasome inhibitors through pH-sensitive polymeric micelles directed to efficient antitumor therapy.J. Control. Release. 2014; 188: 67-77Crossref PubMed Scopus (23) Google Scholar Polymer micelles loaded with MG132 have demonstrated effective in vitro proteasome inhibition, which was significantly more efficient than free MG132 (Figure 2C). In vivo, the micelles enhanced antitumor activity, while avoiding the strong side effects of free MG132 and, more importantly, significantly expanding the therapeutic window (Figure 2D). Recently, there has been significant enthusiasm to develop cancer stem cell (CSC)-targeted drug delivery strategies.25Orza A. Casciano D. Biris A. Nanomaterials for targeted drug delivery to cancer stem cells.Drug Metab. Rev. 2014; 46: 191-206Crossref PubMed Scopus (8) Google Scholar, 26Kim S.S. Rait A. Rubab F. Rao A.K. Kiritsy M.C. Pirollo K.F. et al.The clinical potential of targeted nanomedicine: delivering to cancer stem-like cells.Mol Ther. 2014; 22: 278-291Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar This is due to the realization that multiple tumor types, including leukemia and solid tumors, are sustained by a subpopulation of stem-like cells, commonly referred to as CSCs, which are associated with drug resistance, metastasis, and recurrence. Because CSCs are the "drivers" of tumor progression, therapeutically targeting CSCs is expected to inhibit tumor progression. However, tumor cell plasticity (the ability to dynamically shift between non-CSC and CSC states) limits CSC-targeted therapy.27Iseghohi S.O. Cancer stem cells may contribute to the difficulty in treating cancer.Genes Dis. 2016; 3: 7-10Crossref Scopus (0) Google Scholar Thus, the effective combination of CSC inhibitors with cytotoxic therapies that kill both CSCs and non-CSCs would be the most effective way of accomplishing a successful clinical response. An NM-DDS with a compartmentalized structure would prove useful for accommodating these two drugs, facilitating their co-delivery to the target site, and ensuring optimal therapeutic outcomes from synergistic combinations of drugs.28Kinoh H. Miura Y. Chida T. Liu X. Mizuno K. Fukushima S. Morodomi Y. Nishiyama N. Cabral H. Kataoka K. Nanomedicines eradicating cancer stem-like cells in vivo by pH-triggered intracellular cooperative action of loaded drugs.ACS Nano. 2016; 10: 5643-5655Crossref PubMed Scopus (2) Google Scholar, 29Wang T. Narayanaswamy R. Ren H. Torchilin V.P. Combination therapy targeting both cancer stem-like cells and bulk tumor cells for improved efficacy of breast cancer treatment.Cancer Biol. Ther. 2016; 17: 698-707Crossref PubMed Scopus (1) Google Scholar, 30Oak P.S. Kopp F. Thakur C. Ellwart J.W. Rapp U.R. Ullrich A. Wagner E. Knyazev P. Roidl A. Combinatorial treatment of mammospheres with trastuzumab and salinomycin efficiently targets HER2-positive cancer cells and cancer stem cells.Int. J. Cancer. 2012; 131: 2808-2819Crossref PubMed Scopus (0) Google Scholar Accordingly, a pH-sensitive polymeric micelle has been developed to incorporate the CSC inhibitor staurosporine (STS) alongside the widely used anticancer drug epirubicin (Epi) to treat an aggressive phenotype of malignant mesothelioma with an enriched CSC population (an aldehyde dehydrogenase [ALDH]-high subpopulation in mesothelioma cell line MSTO-211H, which shows CSC features).28Kinoh H. Miura Y. Chida T. Liu X. Mizuno K. Fukushima S. Morodomi Y. Nishiyama N. Cabral H. Kataoka K. Nanomedicines eradicating cancer stem-like cells in vivo by pH-triggered intracellular cooperative action of loaded drugs.ACS Nano. 2016; 10: 5643-5655Crossref PubMed Scopus (2) Google Scholar STS was incorporated in a previously developed epirubicin-micelle (Epi/m) system,31Takahashi A. Yamamoto Y. Yasunaga M. Koga Y. Kuroda J. Takigahira M. Harada M. Saito H. Hayashi T. Kato Y. et al.NC-6300, an epirubicin-incorporating micelle, extends the antitumor effect and reduces the cardiotoxicity of epirubicin.Cancer Sci. 2013; 104: 920-925Crossref PubMed Scopus (36) Google Scholar where Epi is conjugated to a PEG-PAsp-hydrazide block copolymer through a pH-sensitive hydrazone bond.32Bae Y. Fukushima S. Harada A. Kataoka K. Design of environment-sensitive supramolecular assemblies for intracellular drug delivery: polymeric micelles that are responsive to intracellular pH change.Angew. Chem. Int. Ed. Engl. 2003; 42: 4640-4643Crossref PubMed Scopus (0) Google Scholar The compartmentalized architecture of the micelles is utilized to preserve the drug combination until it reaches the intracellular space of cancer cells and CSCs, where the release of both drugs is triggered by low endosomal pH to accomplish coordinated therapeutic effects. Although STS is one of the most potent protein kinase inhibitors, its clinical translation is severely hampered due to its toxicity, lack of specificity, and poor pharmacology.33Park B.S. Abdel-Azeem A.Z. Al-Sanea M.M. Yoo K.H. Tae J.S. Lee S.H. Staurosporine analogues from microbial and synthetic sources and their biological activities.Curr. Med. Chem. 2013; 20: 3872-3902Crossref PubMed Scopus (0) Google Scholar Incorporation of STS into a micelle not only improves its safety and potency but also reverses Epi resistance by inhibiting the ATP-binding cassette (ABC) transporter (Figure 3A). The STS/Epi/m system was shown to cause cell cycle arrest and effectively eliminate the CSC population, with a convincing tumor suppression effect (Figure 3B) and improved survival time in experimental animals (Figure 3C). The STS/Epi/m system is also expected to have good potential for clinical translation, because the Epi/m system is already in the clinical pipeline.31Takahashi A. Yamamoto Y. Yasunaga M. Koga Y. Kuroda J. Takigahira M. Harada M. Saito H. Hayashi T. Kato Y. et al.NC-6300, an epirubicin-incorporating micelle, extends the antitumor effect and reduces the cardiotoxicity of epirubicin.Cancer Sci. 2013; 104: 920-925Crossref PubMed Scopus (36) Google Scholar, 34Cabral H. Kataoka K. Progress of drug-loaded polymeric micelles into clinical studies.J. Control. Release. 2014; 190: 465-476Crossref PubMed Scopus (211) Google Scholar Gene therapy, which exploits genomic materials as therapeutic entities, is already proven to have great potential for treating many severe diseases, including malignancy. Cancer is a genetic disease that is primarily caused by mutations of certain genes instigating unregulated cell proliferation; thus, taking a genetic treatment approach as an alternative or in addition to traditional cytotoxic therapy is an ingenious tactic. For the last few decades, there has been overwhelming enthusiasm in the scientific community with regard to applying gene therapy in cancer treatment.35Das S.K. Menezes M.E. Bhatia S. Wang X.Y. Emdad L. Sarkar D. Fisher P.B. Gene therapies for cancer: strategies, challenges and successes.J. Cell. Physiol. 2015; 230: 259-271Crossref PubMed Scopus (12) Google Scholar Nonetheless, in vivo delivery of genetic therapeutics into target cells poses a significant challenge, mainly due to their instability in biological fluids and inefficient cellular uptake. Several systems, broadly categorized as viral and non-viral vectors, have been developed to enable the cellular delivery of genetic materials.36Thomas C.E. Ehrhardt A. Kay M.A. Progress and problems with the use of viral vectors for gene therapy.Nat. Rev. Genet. 2003; 4: 346-358Crossref PubMed Scopus (1212) Google Scholar Favorably, a number of viral gene carriers have reached clinical practice due to their high transfection efficiency. However, severe toxicity and immunogenicity inhibits their use. In this regard, non-viral vectors are generally considered safer alternatives.37Dizaj S.M. Jafari S. Khosroushahi A.Y. A sight on the current nanoparticle-based gene delivery vectors.Nanoscale Res. Lett. 2014; 9: 252-261Crossref PubMed Scopus (27) Google Scholar Among the non-viral vectors, cationic polymer-based NMs are the most widely investigated and offer several benefits, such as non-immunogenicity, low cost, relative safety, and, more importantly, the capability of carrying larger amounts of genetic material than viruses. However, due to the presence of numerous positive charges on the surface of cationic nanocarriers, aggregation and non-specific interaction remain two major issues in their systemic application.38Sun X. Zhang N. Cationic polymer optimization for efficient gene delivery.Mini Rev. Med. Chem. 2010; 10: 108-125Crossref PubMed Scopus (78) Google Scholar, 39Jin L. Zeng X. Liu M. Deng Y. He N. Current progress in gene delivery technology based on chemical methods and nano-carriers.Theranostics. 2014; 4: 240-255Crossref PubMed Scopus (175) Google Scholar PEGylation of these carriers is considered a prerequisite for reducing non-specific interactions with biomacromolecules, such as serum proteins, and avoiding an undesirable immune response.40Nomoto T. Matsumoto Y. Miyata K. Oba M. Fukushima S. Nishiyama N. Yamasoba T. Kataoka K. In situ quantitative monitoring of polyplexes and polyplex micelles in the blood circulation using intravital real-time confocal laser scanning microscopy.J. Control. Release. 2011; 151: 104-109Crossref PubMed Scopus (74) Google Scholar, 41Li S.-D. Huang L. Gene therapy progress and prospects: non-viral gene therapy by systemic delivery.Gene Ther. 2006; 13: 1313-1319Crossref PubMed Scopus (263) Google Scholar Polyplex micelles (PMs), formulated by the self-assembly of oppositely charged PEG-polycation block copolymers and anionic plasmid DNA (pDNA), have gained significant interest due to the highly regulated folding of pDNA through polyion complexation. The outer hydrophilic PEG layer of the core-shell architecture provides a stealth effect, minimizing non-specific interactions and prolonging circulation time in vivo.42Itaka K. Kataoka K. Progress and prospects of polyplex nanomicelles for plasmid DNA delivery.Curr. Gene Ther. 2011; 11: 457-465Crossref PubMed Scopus (32) Google Scholar, 43Lächelt U. Wagner E. Nucleic acid therapeutics using polyplexes: a journey of 50 years (and beyond).Chem. Rev. 2015; 115: 11043-11078Crossref PubMed Scopus (87) Google Scholar More importantly, the adaptable size44Dirisala A. Osada K. Chen Q. Tockary T.A. Machitani K. Osawa S. Liu X. Ishii T. Miyata K. Oba M. et al.Optimized rod length of polyplex micelles for maximizing transfection efficiency and their performance in systemic gene therapy against stroma-rich pancreatic tumors.Biomaterials. 2014; 35: 5359-5368Crossref PubMed Scopus (18) Google Scholar and shape45Tockary T.A. Osada K. Chen Q. Machitani K. Dirisala A. Uchida S. Nomoto T. Toh K. Matsumoto Y. Itaka K. et al.Tethered PEG crowdedness determining shape and blood circulation profile of polyplex micelle gene carriers.Macromolecules. 2013; 46: 6585-6592Crossref Scopus (40) Google Scholar, 46Chen Q. Osada K. Pennisi M. Uchida S. Tockary T.A. Dirisala A. Li Y. Takeda K.M. Oniyanagi S. Itaka K. Kataoka K. A tadpole-shaped gene carrier with distinct phase segregation in a ternary polymeric micelle.Soft Matter. 2015; 11: 2718-2722Crossref PubMed Google Scholar of these polyplexes, along with their excellent gene expression efficiency, confirms that the packaging of pDNA into a polyion complex core surrounded by a PEG layer is a convincing approach for developing an effective non-viral gene carrier system.47Osada K. Shiotani T. Tockary T.A. Kobayashi D. Oshima H. Ikeda S. Christie R.J. Itaka K. Kataoka K. Enhanced gene expression promoted by the quantized folding of pDNA within polyplex micelles.Biomaterials. 2012; 33: 325-332Crossref PubMed Scopus (24) Google Scholar It is worth noting here that the shape (folded rod or collapsed sphere) and size of the PMs can be precisely controlled by modulating the length of the polycationic segment of the block copolymer and ultimately affects their biological activity.44Dirisala A. Osada K. Chen Q. Tockary T.A. Machitani K. Osawa S. Liu X. Ishii T. Miyata K. Oba M. et al.Optimized rod length of polyplex micelles for maximizing transfection efficiency and their performance in systemic gene therapy against stroma-rich pancreatic tumors.Biomaterials. 2014; 35: 5359-5368Crossref PubMed Scopus (18) Google Scholar, 45Tockary T.A. Osada K. Chen Q. Machitani K. Dirisala A. Uchida S. Nomoto T. Toh K. Matsumoto Y. Itaka K. et al.Tethered PEG crowdedness determining shape and blood circulation profile of polyplex micelle gene carriers.Macromolecules. 2013; 46: 6585-6592Crossref Scopus (40) Google Scholar, 46Chen Q. Osada K. Pennisi M. Uchida S. Tockary T.A. Dirisala A. Li Y. Takeda K.M. Oniyanagi S. Itaka K. Kataoka K. A tadpole-shaped gene carrier with distinct phase segregation in a ternary polymeric micelle.Soft Matter. 2015; 11: 2718-2722Crossref PubMed Google Scholar Intriguingly, rod-shaped polyplexes, formed by a quantized folding mechanism, exhibited higher gene expression than polyplexes containing collapsed globular pDNA formed by random separation of the DNA double helix, indicating that controlled packaging of pDNA into an appropriate structure is critical for achieving effective gene expression.47Osada K. Shiotani T. Tockary T.A. Kobayashi D. Oshima H. Ikeda S. Christie R.J. Itaka K. Kataoka K. Enhanced gene expression promoted by the quantized folding of pDNA within polyplex micelles.Biomaterials. 2012; 33: 325-332Crossref PubMed Scopus (24) Google Scholar Optimized PEG shielding of PMs is equally important to achieve superior biological activity after systemic administration. PEG crowdedness directly correlates with the pDNA packaging structure within PMs, which ultimately affects their size/shape and blood retention profile.45Tockary T.A. Osada K. Chen Q. Machitani K. Dirisala A. Uchida S. Nomoto T. Toh K. Matsumoto Y. Itaka K. et al.Tethered PEG crowdedness determining shape and blood circulation profile of polyplex micelle gene carriers.Macromolecules. 2013; 46: 6585-6592Crossref Scopus (40) Google Scholar, 46Chen Q. Osada K. Pennisi M. Uchida S. Tockary T.A. Dirisala A. Li Y. Takeda K.M. Oniyanagi S. Itaka K. Kataoka K. A tadpole-shaped gene carrier with distinct phase segregation in a ternary polymeric micelle.Soft Matter. 2015; 11: 2718-2722Crossref PubMed Google Scholar, 48Takeda K.M. Osada K. Tockary T.A. Diri