- Email: [email protected]
Anticancer Activities of Tumor-killing Nanorobots
Box 1. Crop Breeding as an Analog for the Inoculant Industry? Major advances in crop yields from breeding have been accomplished through capturing hybrid vigor; changing plant architecture to support larger seed masses and higher planting density; selecting for optimal response to inputs such as inexpensive chemical fertilizers; and identifying genotypes best suited to regional climatic variables such as average heating degree days (time to maturity) and precipitation [6,7]. Crop breeding has been successful not by generating the optimal plant for each individual field, but by developing plants with broadly useful genotypes and phenotypes that perform across wide regions. Breeding programs weigh the benefits of wide versus specific crop adaptation, but this is targeted to large areas (e.g., Southwest Canada) [8]. On a case-by-case basis, trials for field- or farm-specific varieties would be simple to conduct, as yield is the only output to measure, and plant genotypes are the only variable being evaluated; however, the expense could never be recovered by either the seed company or farmers, given the narrow economic margins for both. Instead producers compare competing commercial hybrids or varieties on their own. For microbial inoculants, this might be even more complex, particularly since tracking product survival and function is substantially more involved than crop yield assessment.
multifactorial field trials for thousands of microbial strains across hundreds of soil– climate combinations, with multiple crops and cropping systems may be feasible over time for large industry, but is impractical for individual farms or fields.
Complicating Farmer Adoption of Products The rapid influx of new microbial products and the unpredictability of product efficacy create huge challenges for costeffective implementation by farmers. In the USA, farmers often look to university research and extension programs, or exchange knowledge with peers, to get recommendations on how to best use such products. In an industry that has battled comparisons to snake oils, complicating this process with even more products and conditions to consider adds barriers to farmer trust, understanding, and adoption. The notion that farmers at large are willing and able to conduct their own on-farm research and identify optimal combinations of individual microbial products for each field and crop, all while implementing appropriate controls, is not practical. Farmers do not expect silver bullet solutions, but simple, cost-effective strategies that are easily incorporated into existing production systems. Customizable field-scale microbial inoculants, if effective, might be successful as a fee-for-service industry where consultants and field-scouts
conduct the legwork and advise farmers on application strategies (similar to fertilizer consultants). Such an approach would still require a limited set of products for a limited number of conditions, to allow both consultants and farmers to master the requisite knowledge for implementation.
Summary
2 Department of Ecosystem Science and Management, The Pennsylvania State University, University Park, PA, USA
*Correspondence: [email protected] (T.H. Bell). https://doi.org/10.1016/j.tibtech.2019.02.009 © 2019 Published by Elsevier Ltd.
References 1. Kaminsky, L.M. et al. (2019) The inherent conflicts in developing soil microbial inoculants. Trends Biotech. 37, 140–151 2. Lauber, C.L. et al. (2009) Pyrosequencing-based assessment of soil pH as a predictor of soil bacterial community structure at the continental scale. Appl. Environ. Microbiol. 75, 5111–5120 3. Ramirez, K.S. et al. (2012) Consistent effects of nitrogen amendments on soil microbial communities and processes across biomes. Glob. Change Biol. 18, 1918–1927 4. Bach, E.M. et al. (2010) Soil texture affects soil microbial and structural recovery during grassland restoration. Soil [50_TD$IF]1Biol. Biochem. 42, 2182–2191 5. Peukert, S. et al. (2016) Spatial variation in soil properties and diffuse losses between and within grassland fields with similar short-term management. Eur. J. Soil Sci. 67, 386–396 6. Duvick, D.N. (2005) The contribution of breeding to yield advances in maize (Zea mays L.). Adv. Agron. 86, 83–145 7. Borlaug, N.E. (2002) The green revolution revisited and the road ahead. In NobelPrize.org. http://www.biologyjunction. com/green%20revolution.pdf 8. Annicchiarico, P. (2002) Adaptation and yield stability. In Geno-
We agree with Dr Awasthi that a product type x Environment Interactions - Challenges and Opportunities for Plant Breeding and Cultivar Recommendations. http:// development strategy that only considers www.fao.org/docrep/005/y4391e/y4391e05.htm opportunities related to ‘one formulation for all fields’ is unwise. In addition, we agree that certain aspects of precision farming, when affordable, could identify Forum areas of a farm that might benefit from Product A rather than Product B. In general, we hedge towards development models that aim to broaden potential inoculant ranges, whether that involves creating multistrain consortia with redun- Suping Li,1,2,4 Qiao Jiang,1,2,4 dant abilities and/or selecting for more Baoquan Ding ,1,2,* and widely adaptive organisms. In our view, Guangjun Nie 1,2,3,* developing a small number of robust and reliable products will allow this industry to Pharmaceutical uses of cancer truly cut into the synthetic additive therapeutics, such as intravenous market.
Anticancer Activities of Tumor-[72_TD$IF]killing Nanorobots
Acknowledgments This work was supported by the USDA National Institute of Food and Hatch Appropriations under Project #PEN04651 and Accession #1016233. 1
Department of Plant Pathology and Environmental Microbiology, The Pennsylvania State University, University Park, PA, USA
thrombin to elicit blood coagulation, have been hampered by lack of tumor specificity. Based on rapid progress in DNA origamibased machines capable of transporting molecular payloads, DNA nanorobots have been constructed to specifically deliver Trends in Biotechnology, June 2019, Vol. 37, No. 6
573
therapeutic agents into tumor DNA origami structure is constructed by a vascularized lung tumors), the exposed long scaffold single-stranded DNA mole- thrombin in the unrolled nanorobot activessels. Biological Activities and Therapeutic Application of Thrombin Thrombin is a key enzyme in the blood coagulation cascade. As the main effector protease, thrombin cleaves plasma fibrinogen into fibrin monomers [1], which can spontaneously form insoluble polymers. Thrombin also activates the clotting factors VIII (to VIIIa) and V (to Va) and activates platelets. Together, these hemostasis mechanisms form thrombi that comprise platelet plugs and the self-polymerizing fibrin network [2]. Thrombin’s action is not confined to the coagulation event: it also has a critical function in the wound healing process by stimulating ‘mitogenic’ events through interaction with cell surface receptors [2]. In view of the unique and potent biological activities of thrombin, it has been used as a therapeutic with a long history extending back more than [74_TD$IF]seven decades [3]. In particular, thrombin has been widely used as a spray-applied, topical hemostatic agent to stop residual bleeding, a resource as common as saline in the operating theater [4]. Furthermore, thrombin is applied by local percutaneous injection for the treatment of femoral pseudoaneurysms by inducing the formation of intravascular clots [4]. Despite these frequent uses of thrombin as a therapeutic drug, the potential systemic side effects caused by leakage into the circulation, such as elevated blood coagulation parameters and development of ischaemia in normal tissue, pose a clinical obstacle. To fully exploit the utility of thrombin as a safe therapeutic, it is critical to develop a technique for specifically targeting thrombin to particular tissues or cell types.
cule folded into an arbitrary architecture using hundreds of staple strands that fix the scaffold’s conformation [5–7]. The DNA origami method enables a rational design and production of DNA nanostructures with well-defined homogenous geometries, precise spatial addressability, and marked biocompatibility. DNA origami is a blank slate that can contain multiple therapeutic cargoes and tumortargeting ligands with rationally designed numbers and patterns anywhere on the entire addressable nanostructure. These unique advantages of DNA origami structures have conferred them various biomedical applications. For example, a dynamic hexagonal origami barrel DNA nanostructure was created to carry and precisely transport gold nanoparticles and antibody Fab fragments to target cells. In a programmed manner, this impressive DNA nanodevice demonstrated the capability of DNA origami to sense cell surface inputs and trigger cellular activation in vitro (Figure 1A) [8]. Several DNA origami-based drug delivery systems have been used for loading chemotherapeutic drugs, nanoparticles, or genes to inhibit tumors both in vitro and in vivo (Figure 1B,C) [9–11]. The static or responsive origami systems can be further advanced to realize more intelligent functions.
Based on the burgeoning developments of DNA origami technique, a tube-shaped DNA nanorobot (19 nm 90 nm) was tailored to selectively deliver thrombin into tumor vessels to induce thrombosis for tumor therapy (Figure 2) [12]. The thrombin was positioned inside the inner cavity of the nanorobot, protecting the highly reactive molecular cargo from interfering with (and interference from) the external DNA Nanorobot-[75_TD$IF]based Thrombin environment. When applied to tumorDelivery to Tumor Vessels bearing mouse models (both highly vasThe DNA origami technique was intro- cularized melanoma, breast cancer, ovarduced by Rothemund in 2006. A desired ian, and liver tumors and poorly 574
Trends in Biotechnology, June 2019, Vol. 37, No. 6
vated localized coagulation to selectively occlude tumor blood vessels, inducing the tumor to ‘starve to death’. This new technological development not only brings the first safe use of intravenously injectable nano-thrombin for effective tumor therapy but also widens the potential applicable range of thrombin for other diseases such as hemorrhagic diseases of the internal organs. More importantly, although DNA origami carriers have been used to deliver chemotherapeutics in vivo in previous literature, the intelligent thrombin delivery system further extends the potential utility of DNA origami-based delivery strategy by conferring origami nanostructures a triggered, intratumor release mechanism.
Advantages of DNA Nanorobot-[75_TD$IF][1based Tumor Infarction Therapy The induction of tumor vascular infarction is considered one of the most efficient ways to inhibit tumor growth due to the abundance of capillaries associated with tumor angiogenesis and the prothrombotic state of tumors [13]. Moreover, as an acute blood event, vessel infarction requires a much shorter duration of treatment than many other therapies, and there is less possibility of developing drug resistance. In addition, cutting off the tumor blood supply elicits a potentiation effect since a single blood vessel nourishes hundreds of tumor cells. Tumor infarction-based therapy was first proposed in the 1980s by Yamada and coauthors [14] for the treatment of highly vascularized hepatocellular carcinoma (HCC) patients who were not surgical candidates. This approach is now an efficient, first-line therapy for such patients [15]. Although the intra-arterial embolization elicits a clinically significant therapeutic effect in HCC, in conjugation with chemotherapy agents, the particles currently used for vascular infarction,
(A)
(B)
Dox@triangle
Dox@tube
3.0nm
3.0nm
100nm
100nm
(C)
200nm
–1.0nm
200nm
–1.0nm
TODP
100 nm
(See figure legend on the bottom of the next page.) Trends in Biotechnology, June 2019, Vol. 37, No. 6
575
(A)
(B)
M13 DNA
Staples
Rectangular origami sheet
Thrombin-loaded origami sheet
Nucleolin
Targeng strands
Thrombin-DNA conjugate
Fastener
Tubular nanorobot
Open state of nanorobot
Figure 2. Design and Characterization of the DNA Nanorobot. (A) The construction of the thrombin-loaded DNA nanorobot and reconfiguration of the tubular nanorobot into a rectangular DNA sheet in response to nucleolin binding. The single stranded M13 phage genomic DNA is linked by predesigned staple strands, leading to the formation of a rectangular DNA sheet. Thrombin is loaded onto the surface of the rectangular structure by hybridization of poly-T oligonucleotides conjugated to thrombin molecules with poly-A sequences that extend from the surface of the rectangular DNA sheet. The addition of the fasteners and targeting strands results in the formation of thrombin-loaded, tubular DNA nanorobots with additional targeting aptamers at both ends of the tube. The tube nanorobot opens in response to the presence of recombinant human nucleolin to expose the encapsulated thrombin. (B) The nanorobot (top) and thrombin-loaded rectangular sheet (bottom) were examined by atomic force microscopy (AFM), and representative images are shown. Scale bars denote 200 nm. Reprinted, with permission, from [5].
including gelfoam, gelatin, or polyvinyl alcohol spheres, are nontargeting and occlude tumor vessels by self-amplified tumor accumulation. Because of this, there is the potential for off-target systemic toxicity and/or insufficient occlusion. Thus, the development of a thrombin-containing nanorobot is poised to offer a valuable treatment pathway for tumor infarction with more adequate specificity and effectiveness.
highly stable and reproducible. More importantly, owing to the rapid progress in DNA nanotechnology in recent years, the large scale (milligram-scale) production of DNA nanostructures for biological application is feasible, with an acceptable time schedule and high reproducibility. Thus, the development of thrombin-containing nanorobots is poised to offer a clinically promising tumor infarction strategy.
The demonstration of successful occlusion of tumor vasculature by a thrombincontaining DNA nanorobot [5] promotes further developments in coagulationbased cancer therapy, with more adequate tumor specificity and effectiveness. In addition, due to the controllable and tailored design, the nanorobot structure is
Future Perspectives Thrombin has a central role in blood coagulation and represents a fascinating class of topical hemostatic drugs. However, thrombin’s utility typically suffers from fast degradation of the protein by proteases present in serum, with a halflife of only 25 s [1]. Moreover, potent
procoagulant activity severely hampers intravenous applications of thrombin, limiting the range of diseases for which the drug may be applied in clinical settings. A recently developed tube-shaped DNA origami nanostructure realized the targeted delivery of thrombin to tumor vessels for vascular infarction in tumor-bearing mice by tail vein injection. The immobilized thrombin inside the nanorobot is protected from degradation by proteases in the circulation. In addition to potentially filling the unmet need for a safe occluding agent to cut off tumor blood supply, the nanorobot may prove effective in extending the in vivo availability of other reactive and/or toxic biochemical functional molecules that are not amenable as conventionally administered therapeutics because of extremely high bioactivity or toxicity.
Figure 1. Examples of DNA Origami Structures for Cargo Delivery In Vitro and In Vivo. (A) Autonomous barrel-like DNA nanorobot for the targeted delivery of molecular payloads into cells to manipulate cell signaling in vitro [7]. (B) DNA origami nanostructure engineered to carry doxorubicin (Dox) and circumvent drug resistance in MCF-7 breast cancer cells [8]. (C) DNA origami nanokite as a carrier for the codelivery of a p53 expression vector and Dox in vivo [11]. The panels are reprinted with permission from the indicated references.
576
Trends in Biotechnology, June 2019, Vol. 37, No. 6
Although this and other recent studies clearly demonstrate the in vivo therapeutic potential of intelligent DNA nanorobots, translation of this technology into the clinic still faces several roadblocks. For example, detailed in vivo characteristics of DNA origami-based nanomaterials including the stability, circulating half-life, pharmacokinetics, and clearance mechanisms are needed to be addressed to further advance DNA materials toward clinically therapeutic applications. A potential immune response of DNA origami architectures is another concern that should also be intensely investigated. Furthermore, advancing the chemical synthesis techniques and biotechnological production methods of DNA nanostructures to reduce the cost of DNA production is also an important factor for moving nanorobots into clinical practice.
Nova Program Interdisciplinary Cooperation Project (Z181100006218136), Beijing Municipal Science and Technology Commission (Z161100000116036), Key Research Program of Frontier Sciences, CAS (QYZDB-SSW-SLH029), and the CAS Interdisciplinary Innovation Team. 1
Key R&D Program of China (2018YFA0208900, 2016YFA0201601), the National Natural Science Foundation
of
China
(31730032,
81871489,
31661130152, 31700871, and 21573051), Beijing
4. Lundblad, R.L. et al. (2004) A review of the therapeutic uses of thrombin. Thromb. Haemost. 91, 851–860 5. Rothemund, P.W.K. (2006) Folding DNA to create nanoscale shapes and patterns. Nature 440, 297–302 6. Kuzuya, A. and Komiyama, M. (2010) DNA origami: fold, stick, and beyond. Nanoscale 2, 309–321 7. Hong, F. et al. (2017) DNA origami: scaffolds for creating higher order structures. Chem. Rev. 117, 12584–12640
CAS Key Laboratory for Biomedical Effects of Nanomaterials [73_TD$IF]& Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology of China, Beijing 100190, China 2 Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences,
8. Douglas, S.M. et al. (2012) A logic-gated nanorobot for targeted transport of molecular payloads. Science 335, 831–834
Beijing 100049, China Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane QLD 4072, Australia 4 These authors contributed equally to this work
10. Liu, J. et al. (2018) A DNA-based nanocarrier for efficient gene delivery and combined cancer therapy. Nano Lett. 18, 3328–3334
9. Jiang, Q. et al. (2012) DNA origami as a carrier for circumvention of drug resistance. J. Am. Chem. Soc 134, 13396–13403
3
*Correspondence: [email protected] (B. Ding) and [email protected] (G. Nie). https://doi.org/10.1016/j.tibtech.2019.01.010 © 2019 Elsevier Ltd. All rights reserved.
Acknowledgments This work was supported by grants from the National
3. Mannucci, P.M. (1998) Hemostatic drugs. N. Engl. J. Med. 339, 245–253
References 1. Coughlin, S.R. (2000) Thrombin signalling and proteaseactivated receptors. Nature 407, 258–264 2. Peters, R. and Harris, T. (2018) Advances and innovations in haemophilia treatment. Nat. Rev. Drug Discov. 17, 493–508
11. Liu, J. et al. (2018) A tailored DNA nanoplatform for synergistic RNAi-/chemotherapy of multidrug-resistant tumors. Angew. Chem. Int. Ed. Engl. 57, 15486–15490 12. Li, S. et al. (2018) A DNA nanorobot functions as a cancer therapeutic in response to a molecular trigger in vivo. Nat. Biotechnol. 36, 258–264 13. Narazaki, M. and Tosato, G. (2005) Targeting coagulation to the tumor microvasculature: perspectives and therapeutic implications from preclinical studies. J. Natl. Cancer Inst. 97, 705–707 14. Yamada, R. et al. (1983) Hepatic-artery embolization in 120 patients with unresectable hepatoma. Radiology 148, 397–401 15. Habib, A. et al. (2015) Transarterial approaches to primary and secondary hepatic malignancies. Nat. Rev. Clin. Oncol. 12, 481–489
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577
multifactorial field trials for thousands of microbial strains across hundreds of soil– climate combinations, with multiple crops and cropping systems may be feasible over time for large industry, but is impractical for individual farms or fields.
Complicating Farmer Adoption of Products The rapid influx of new microbial products and the unpredictability of product efficacy create huge challenges for costeffective implementation by farmers. In the USA, farmers often look to university research and extension programs, or exchange knowledge with peers, to get recommendations on how to best use such products. In an industry that has battled comparisons to snake oils, complicating this process with even more products and conditions to consider adds barriers to farmer trust, understanding, and adoption. The notion that farmers at large are willing and able to conduct their own on-farm research and identify optimal combinations of individual microbial products for each field and crop, all while implementing appropriate controls, is not practical. Farmers do not expect silver bullet solutions, but simple, cost-effective strategies that are easily incorporated into existing production systems. Customizable field-scale microbial inoculants, if effective, might be successful as a fee-for-service industry where consultants and field-scouts
conduct the legwork and advise farmers on application strategies (similar to fertilizer consultants). Such an approach would still require a limited set of products for a limited number of conditions, to allow both consultants and farmers to master the requisite knowledge for implementation.
Summary
2 Department of Ecosystem Science and Management, The Pennsylvania State University, University Park, PA, USA
*Correspondence: [email protected] (T.H. Bell). https://doi.org/10.1016/j.tibtech.2019.02.009 © 2019 Published by Elsevier Ltd.
References 1. Kaminsky, L.M. et al. (2019) The inherent conflicts in developing soil microbial inoculants. Trends Biotech. 37, 140–151 2. Lauber, C.L. et al. (2009) Pyrosequencing-based assessment of soil pH as a predictor of soil bacterial community structure at the continental scale. Appl. Environ. Microbiol. 75, 5111–5120 3. Ramirez, K.S. et al. (2012) Consistent effects of nitrogen amendments on soil microbial communities and processes across biomes. Glob. Change Biol. 18, 1918–1927 4. Bach, E.M. et al. (2010) Soil texture affects soil microbial and structural recovery during grassland restoration. Soil [50_TD$IF]1Biol. Biochem. 42, 2182–2191 5. Peukert, S. et al. (2016) Spatial variation in soil properties and diffuse losses between and within grassland fields with similar short-term management. Eur. J. Soil Sci. 67, 386–396 6. Duvick, D.N. (2005) The contribution of breeding to yield advances in maize (Zea mays L.). Adv. Agron. 86, 83–145 7. Borlaug, N.E. (2002) The green revolution revisited and the road ahead. In NobelPrize.org. http://www.biologyjunction. com/green%20revolution.pdf 8. Annicchiarico, P. (2002) Adaptation and yield stability. In Geno-
We agree with Dr Awasthi that a product type x Environment Interactions - Challenges and Opportunities for Plant Breeding and Cultivar Recommendations. http:// development strategy that only considers www.fao.org/docrep/005/y4391e/y4391e05.htm opportunities related to ‘one formulation for all fields’ is unwise. In addition, we agree that certain aspects of precision farming, when affordable, could identify Forum areas of a farm that might benefit from Product A rather than Product B. In general, we hedge towards development models that aim to broaden potential inoculant ranges, whether that involves creating multistrain consortia with redun- Suping Li,1,2,4 Qiao Jiang,1,2,4 dant abilities and/or selecting for more Baoquan Ding ,1,2,* and widely adaptive organisms. In our view, Guangjun Nie 1,2,3,* developing a small number of robust and reliable products will allow this industry to Pharmaceutical uses of cancer truly cut into the synthetic additive therapeutics, such as intravenous market.
Anticancer Activities of Tumor-[72_TD$IF]killing Nanorobots
Acknowledgments This work was supported by the USDA National Institute of Food and Hatch Appropriations under Project #PEN04651 and Accession #1016233. 1
Department of Plant Pathology and Environmental Microbiology, The Pennsylvania State University, University Park, PA, USA
thrombin to elicit blood coagulation, have been hampered by lack of tumor specificity. Based on rapid progress in DNA origamibased machines capable of transporting molecular payloads, DNA nanorobots have been constructed to specifically deliver Trends in Biotechnology, June 2019, Vol. 37, No. 6
573
therapeutic agents into tumor DNA origami structure is constructed by a vascularized lung tumors), the exposed long scaffold single-stranded DNA mole- thrombin in the unrolled nanorobot activessels. Biological Activities and Therapeutic Application of Thrombin Thrombin is a key enzyme in the blood coagulation cascade. As the main effector protease, thrombin cleaves plasma fibrinogen into fibrin monomers [1], which can spontaneously form insoluble polymers. Thrombin also activates the clotting factors VIII (to VIIIa) and V (to Va) and activates platelets. Together, these hemostasis mechanisms form thrombi that comprise platelet plugs and the self-polymerizing fibrin network [2]. Thrombin’s action is not confined to the coagulation event: it also has a critical function in the wound healing process by stimulating ‘mitogenic’ events through interaction with cell surface receptors [2]. In view of the unique and potent biological activities of thrombin, it has been used as a therapeutic with a long history extending back more than [74_TD$IF]seven decades [3]. In particular, thrombin has been widely used as a spray-applied, topical hemostatic agent to stop residual bleeding, a resource as common as saline in the operating theater [4]. Furthermore, thrombin is applied by local percutaneous injection for the treatment of femoral pseudoaneurysms by inducing the formation of intravascular clots [4]. Despite these frequent uses of thrombin as a therapeutic drug, the potential systemic side effects caused by leakage into the circulation, such as elevated blood coagulation parameters and development of ischaemia in normal tissue, pose a clinical obstacle. To fully exploit the utility of thrombin as a safe therapeutic, it is critical to develop a technique for specifically targeting thrombin to particular tissues or cell types.
cule folded into an arbitrary architecture using hundreds of staple strands that fix the scaffold’s conformation [5–7]. The DNA origami method enables a rational design and production of DNA nanostructures with well-defined homogenous geometries, precise spatial addressability, and marked biocompatibility. DNA origami is a blank slate that can contain multiple therapeutic cargoes and tumortargeting ligands with rationally designed numbers and patterns anywhere on the entire addressable nanostructure. These unique advantages of DNA origami structures have conferred them various biomedical applications. For example, a dynamic hexagonal origami barrel DNA nanostructure was created to carry and precisely transport gold nanoparticles and antibody Fab fragments to target cells. In a programmed manner, this impressive DNA nanodevice demonstrated the capability of DNA origami to sense cell surface inputs and trigger cellular activation in vitro (Figure 1A) [8]. Several DNA origami-based drug delivery systems have been used for loading chemotherapeutic drugs, nanoparticles, or genes to inhibit tumors both in vitro and in vivo (Figure 1B,C) [9–11]. The static or responsive origami systems can be further advanced to realize more intelligent functions.
Based on the burgeoning developments of DNA origami technique, a tube-shaped DNA nanorobot (19 nm 90 nm) was tailored to selectively deliver thrombin into tumor vessels to induce thrombosis for tumor therapy (Figure 2) [12]. The thrombin was positioned inside the inner cavity of the nanorobot, protecting the highly reactive molecular cargo from interfering with (and interference from) the external DNA Nanorobot-[75_TD$IF]based Thrombin environment. When applied to tumorDelivery to Tumor Vessels bearing mouse models (both highly vasThe DNA origami technique was intro- cularized melanoma, breast cancer, ovarduced by Rothemund in 2006. A desired ian, and liver tumors and poorly 574
Trends in Biotechnology, June 2019, Vol. 37, No. 6
vated localized coagulation to selectively occlude tumor blood vessels, inducing the tumor to ‘starve to death’. This new technological development not only brings the first safe use of intravenously injectable nano-thrombin for effective tumor therapy but also widens the potential applicable range of thrombin for other diseases such as hemorrhagic diseases of the internal organs. More importantly, although DNA origami carriers have been used to deliver chemotherapeutics in vivo in previous literature, the intelligent thrombin delivery system further extends the potential utility of DNA origami-based delivery strategy by conferring origami nanostructures a triggered, intratumor release mechanism.
Advantages of DNA Nanorobot-[75_TD$IF][1based Tumor Infarction Therapy The induction of tumor vascular infarction is considered one of the most efficient ways to inhibit tumor growth due to the abundance of capillaries associated with tumor angiogenesis and the prothrombotic state of tumors [13]. Moreover, as an acute blood event, vessel infarction requires a much shorter duration of treatment than many other therapies, and there is less possibility of developing drug resistance. In addition, cutting off the tumor blood supply elicits a potentiation effect since a single blood vessel nourishes hundreds of tumor cells. Tumor infarction-based therapy was first proposed in the 1980s by Yamada and coauthors [14] for the treatment of highly vascularized hepatocellular carcinoma (HCC) patients who were not surgical candidates. This approach is now an efficient, first-line therapy for such patients [15]. Although the intra-arterial embolization elicits a clinically significant therapeutic effect in HCC, in conjugation with chemotherapy agents, the particles currently used for vascular infarction,
(A)
(B)
Dox@triangle
Dox@tube
3.0nm
3.0nm
100nm
100nm
(C)
200nm
–1.0nm
200nm
–1.0nm
TODP
100 nm
(See figure legend on the bottom of the next page.) Trends in Biotechnology, June 2019, Vol. 37, No. 6
575
(A)
(B)
M13 DNA
Staples
Rectangular origami sheet
Thrombin-loaded origami sheet
Nucleolin
Targeng strands
Thrombin-DNA conjugate
Fastener
Tubular nanorobot
Open state of nanorobot
Figure 2. Design and Characterization of the DNA Nanorobot. (A) The construction of the thrombin-loaded DNA nanorobot and reconfiguration of the tubular nanorobot into a rectangular DNA sheet in response to nucleolin binding. The single stranded M13 phage genomic DNA is linked by predesigned staple strands, leading to the formation of a rectangular DNA sheet. Thrombin is loaded onto the surface of the rectangular structure by hybridization of poly-T oligonucleotides conjugated to thrombin molecules with poly-A sequences that extend from the surface of the rectangular DNA sheet. The addition of the fasteners and targeting strands results in the formation of thrombin-loaded, tubular DNA nanorobots with additional targeting aptamers at both ends of the tube. The tube nanorobot opens in response to the presence of recombinant human nucleolin to expose the encapsulated thrombin. (B) The nanorobot (top) and thrombin-loaded rectangular sheet (bottom) were examined by atomic force microscopy (AFM), and representative images are shown. Scale bars denote 200 nm. Reprinted, with permission, from [5].
including gelfoam, gelatin, or polyvinyl alcohol spheres, are nontargeting and occlude tumor vessels by self-amplified tumor accumulation. Because of this, there is the potential for off-target systemic toxicity and/or insufficient occlusion. Thus, the development of a thrombin-containing nanorobot is poised to offer a valuable treatment pathway for tumor infarction with more adequate specificity and effectiveness.
highly stable and reproducible. More importantly, owing to the rapid progress in DNA nanotechnology in recent years, the large scale (milligram-scale) production of DNA nanostructures for biological application is feasible, with an acceptable time schedule and high reproducibility. Thus, the development of thrombin-containing nanorobots is poised to offer a clinically promising tumor infarction strategy.
The demonstration of successful occlusion of tumor vasculature by a thrombincontaining DNA nanorobot [5] promotes further developments in coagulationbased cancer therapy, with more adequate tumor specificity and effectiveness. In addition, due to the controllable and tailored design, the nanorobot structure is
Future Perspectives Thrombin has a central role in blood coagulation and represents a fascinating class of topical hemostatic drugs. However, thrombin’s utility typically suffers from fast degradation of the protein by proteases present in serum, with a halflife of only 25 s [1]. Moreover, potent
procoagulant activity severely hampers intravenous applications of thrombin, limiting the range of diseases for which the drug may be applied in clinical settings. A recently developed tube-shaped DNA origami nanostructure realized the targeted delivery of thrombin to tumor vessels for vascular infarction in tumor-bearing mice by tail vein injection. The immobilized thrombin inside the nanorobot is protected from degradation by proteases in the circulation. In addition to potentially filling the unmet need for a safe occluding agent to cut off tumor blood supply, the nanorobot may prove effective in extending the in vivo availability of other reactive and/or toxic biochemical functional molecules that are not amenable as conventionally administered therapeutics because of extremely high bioactivity or toxicity.
Figure 1. Examples of DNA Origami Structures for Cargo Delivery In Vitro and In Vivo. (A) Autonomous barrel-like DNA nanorobot for the targeted delivery of molecular payloads into cells to manipulate cell signaling in vitro [7]. (B) DNA origami nanostructure engineered to carry doxorubicin (Dox) and circumvent drug resistance in MCF-7 breast cancer cells [8]. (C) DNA origami nanokite as a carrier for the codelivery of a p53 expression vector and Dox in vivo [11]. The panels are reprinted with permission from the indicated references.
576
Trends in Biotechnology, June 2019, Vol. 37, No. 6
Although this and other recent studies clearly demonstrate the in vivo therapeutic potential of intelligent DNA nanorobots, translation of this technology into the clinic still faces several roadblocks. For example, detailed in vivo characteristics of DNA origami-based nanomaterials including the stability, circulating half-life, pharmacokinetics, and clearance mechanisms are needed to be addressed to further advance DNA materials toward clinically therapeutic applications. A potential immune response of DNA origami architectures is another concern that should also be intensely investigated. Furthermore, advancing the chemical synthesis techniques and biotechnological production methods of DNA nanostructures to reduce the cost of DNA production is also an important factor for moving nanorobots into clinical practice.
Nova Program Interdisciplinary Cooperation Project (Z181100006218136), Beijing Municipal Science and Technology Commission (Z161100000116036), Key Research Program of Frontier Sciences, CAS (QYZDB-SSW-SLH029), and the CAS Interdisciplinary Innovation Team. 1
Key R&D Program of China (2018YFA0208900, 2016YFA0201601), the National Natural Science Foundation
of
China
(31730032,
81871489,
31661130152, 31700871, and 21573051), Beijing
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3
*Correspondence: [email protected] (B. Ding) and [email protected] (G. Nie). https://doi.org/10.1016/j.tibtech.2019.01.010 © 2019 Elsevier Ltd. All rights reserved.
Acknowledgments This work was supported by grants from the National
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