Nanomedicine: de novo design of nanodrugs.

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Nanomedicine: de novo design of nanodrugs Cite this: Nanoscale, 2014, 6, 663

Zaixing Yang,†a Seung-gu Kang†b and Ruhong Zhou*abc Phenomenal advances in nanotechnology and nanoscience have been accompanied by exciting progress in de novo design of nanomedicines. Nanoparticles with their large space of structural amenability and excellent mechanical and electrical properties have become ideal candidates for high efficacy nanomedicines in both diagnostics and therapeutics. The therapeutic nanomedicines can be further categorized into nanocarriers for conventional drugs and nanodrugs with direct curing of target diseases. Here we review some of the recent advances in de novo design of nanodrugs, with an

Received 25th August 2013 Accepted 23rd October 2013

emphasis on the molecular level understanding of their interactions with biological systems including key proteins and cell membranes. We also include some of the latest advances in the development of nanocarriers with both passive and active targeting for completeness. These studies may shed light on a

DOI: 10.1039/c3nr04535h

better understanding of the molecular mechanisms behind these nanodrugs, and also provide new

www.rsc.org/nanoscale

insights and direction for the future design of nanomedicines.

1. Introduction a

School of Radiation Medicine and Protection, Medical College of Soochow University & Collaborative Innovation Center of Suzhou Nano Science and Technology, Jiangsu Provincial Key Laboratory of Radiation Medicine and Protection, Soochow University, Suzhou 215123, China. E-mail: [email protected]

b

Computational Biology Center, IBM Thomas J. Watson Research Center, Yorktown Heights, NY 10598, USA

c

Department of Chemistry, Columbia University, NY 10027, USA

Feynman's vision toward a “smaller world” given in 1959 is being actively realized in recent years with the advances in nanotechnology and nanoscience.1 Over the past decade, nanotechnology has emerged into a central hub in connecting almost all elds of science and technology. Among these elds, bio-nanotechnology is also becoming one of the core subdisciplines, as witnessed by the signicant increase in the scientic output and annual research budgets. In the United

† These two authors contributed equally.

Zaxing Yang is currently an Associate Professor at the Institute of Quantitative Biology and Medicine, School for Radiological and Interdisciplinary Sciences (RAD-X), Soochow University. He received his Ph.D. in physics from Zhejiang University in 2011. From 2011 to 2013, he was a postdoctoral associate at Zhejiang University, in the Department of Engineering Mechanics, and So Matter Research Center. His current research interests include nanoparticle–protein interactions and related molecular mechanisms of nanotoxicity and nanomedicine, protein folding, misfolding, and aggregation. He has achieved ten journal publications.

This journal is © The Royal Society of Chemistry 2014

Seung-gu Kang received his Ph.D. in chemistry from University of Pennsylvania in 2009 under Prof. Jeffery G. Saven, where he was mostly dedicated to developing computational protein design algorithms based on statistical mechanics, and applying them in designing de novo proteins conjugated with non-biological molecules. He is currently working as a postdoctoral researcher in Computational Biology Center of IBM Thomas J. Watson Research Center under Prof. Ruhong Zhou. His recent research interest is in applying and developing large scale molecular dynamics simulations into various interfacial phenomena including nano–bio interaction, protein–protein association and surface-assisted biomolecular self-assembly. He has 17 publications, which includes papers related to nanomedicine and nanotoxicity.

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States alone, the National Nanotechnology Initiative (NNI) has proposed a $1.9 billion budget in 2013, remarkably expanding almost 400% from $493 million when it rst started in 2001.2 The NIH roadmap sets a denition of nanomedicine as to pursue “highly specic medical intervention at the molecular scale of curing and repairing damaged tissues”,3 which is pushing forward both ends of basic sciences and clinical applications. Nanomedicines can be roughly categorized into diagnostics and therapeutics depending on their general application purposes. The pharmacokinetic role to diseases further distinguishes the therapeutic nanomedicines into nanocarriers for conventional drugs and nanodrugs with direct curing for target diseases. It should be noted that some very recent clinical applications are getting more advanced by combining multiple components to generate “smarter” nanomedicines with multiple functions, e.g., theragnostics (therapeutics + diagnostics).4,5 Therefore, the above rough classication is rather simplied and for description purposes only. In this article, we review some of the recent advances in the de novo design of nanodrugs, including the metallofullerenol Gd@C82(OH)22 for anti-cancer therapy,6 graphene and graphene oxide for antibacterial “green” band-aids,7 lanthanide- and alumina-based nanoparticles for effective antophagy,8,9 and other nanodrugs assisted by external triggers.10,11 Since the approval of Doxil (for HIV-related Kaposi's sarcoma)12,13 and Abraxane (for breast cancer and non-small-cell lung cancer)14 by the FDA, nanocarriers for drug delivery have been studied extensively with many excellent reviews already published.5,15–18 Hence, only a brief summary of new advances is provided here for completeness. In general, the targeted delivery of nanomedicines can be approached via two major strategies: passive and active targeting. In the passive mode, the drug delivery mostly depends on the enhanced permeation and retention (EPR) effect,19 where nanoparticles are likely extravasated through irregularly vascularized leaky blood vessels in

Ruhong Zhou is a Research Scientist at the Computational Biology Center, IBM Research, and an Adjunct Professor at Chemistry Department, Columbia University. He received his Ph.D. in chemistry from Columbia University in 1997. His current research interests include protein folding, protein–nanoparticle interactions, ligand– receptor binding, and methodology developments for computational biology and bioinformatics. He has more than 130 journal publications and 18 patents, and has delivered 150+ invited talks worldwide. He is part of the IBM Blue Gene team who won the 2009 National Medal on Technology. He also won the IBM Outstanding Technical Achievement Award in 2012, 2008 and 2005. He was elected to AAAS Fellow and APS Fellow in 2011.

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tumor tissue, and accumulated at the tumor site by the decreased drainage activity of the tumor lymphatics.20,21 While the passive targeting largely depends on the diffusion along the blood circulation system, the active targeting relies on specic interactions between the nanoparticles and target cells (or intracellular compartments) by tagging the nanoparticles with antibodies, small molecules, or oligonucleotides that can recognize specic targets, particularly those over-expressed in the malignant cells.22–24 This review is organized as follows. Section 2 describes the molecular mechanism of the metallofullerenol Gd@C82(OH)22 interacting with the enzyme MMP9 for its inhibition of tumor growth. Section 3 illustrates the interaction of graphene and graphene oxide nanosheets with the E. coli cell membranes for their anti-bacterial activity. Sections 4 and 5 summarize the recent progresses of nanodrugs for effective autophagy and nanodrugs assisted by external triggers, respectively. Section 6 discusses new advances in nanocarriers of conventional drugs. Sections 2 and 3 will be described in greater detail as case studies, with the aim of providing a better understanding of the underlying molecular mechanisms.

2. Anti-tumor nanodrug metallofullerenols Carbon-based nanomaterials have attracted much attention as potential platforms for nanotherapeutics due to their unique mechanical, thermal, and electrochemical properties. In addition, their high resistance to degradable biological environments endows versatile opportunities as de novo drug carriers or clinical therapeutics. Various allotropes are employed based on their geometric specicities, including carbon nanotubes (CNTs), graphenes, fullerenes, carbon nanohorns, and nanodiamonds.5,25 These nanostructures are viewed as potential highly potent drug carriers or nanodrugs because of their relatively large surfaces and unique structures. However, well-devised surface modications via either covalent or non-covalent passivations are oen required to make these nanosystems water soluble and biocompatible. Fullerenes and their derivatives are probably the most extensively studied nanoparticles for biomedical applications.26–28 Appropriate surface modications of these nanoparticles not only demonstrated promises in de novo therapeutics, but also showed reduced potential cytotoxicity. For example, hydroxyl functionalized C60 protects mitochondria from superoxide, hydroxyl and lipid radicals,29 and enhances mitochondrial enzyme activities of superoxide dismutase and glutathione peroxidase by effectively reducing oxidative stress.30 Water soluble immunoconjugate C60 effectively targets the gp240 antigen, demonstrating fullerene immunotherapeutics for inhibiting cancer proliferation.31 In particular, endohedral metallofullerene derivatives were very recently utilized in biomedical applications for cancer diagnostics and therapeutics.32 Owing to their rigid carbon cages, encapsulated magnetosensitive metals are safely secured when serving as high contrast agents for magnetic resonance


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imaging (MRI). Among these metals, Gadolinium (Gd) is mostly employed due to its high proton relaxivity.33 Mikawa et al. showed that Gd@C82(OH)x enhances in vitro water proton relaxivity by 20-fold as compared to the commercial Gd-based contrast agent, magnevist (Gd-DTPA).34 More recently, Zhao and coworkers found that Gd@C82(OH)22 displays high efficacy as a potential anti-cancer nanodrug.35 In mice models xenograed with hepatoma cells (H22) and human breast cancer cells, Gd@C82(OH)22 was shown to effectively inhibit tumor metastasis with a very low concentration compared to conventional antineoplastic drugs such as cyclophosphamide (CTX) and cisplatin (CDDP). Meanwhile, inherited from the intrinsic property of fullerenes, Gd@C82(OH)22 functions as an effective scavenger for ROS, thus protecting normal cells and sub-cellular compartments.36 Subsequent further studies showed that Gd@C82(OH)22 inhibits multiple angiogenic factors at both RNA expression and protein activity levels, exhibiting prominent reduction of over 40% microvessel density and blood supply to tumor tissues.37 Gd@C82(OH)22 also promotes antitumoral immune responses, which is validated with phenotypic and functional maturation of dendritic cells in tumor-bearing hosts.38 Despite all this progress made in experimental labs, the molecular mechanism of Gd@C82(OH)22 was not known for quite some time until very recently (more below).35 Tumor angiogenesis suppressed by the nanodrug Cancer progress is accompanied with chaotic irregular blood vessel generation to draw nutrients and oxygen into tumor tissues, while accumulating malignant mutations, followed by degradation of the extracellular matrix (ECM) which connes the stromal mass of solid tumors. Very recently, Zhou and coworkers revealed how specically Gd@C82(OH)22 nanoparticles inhibit the important angiogenic factors MMP-2/-9 in molecular detail using an in vivo mice model, in vitro cellular and biochemical assays, as well as in silico molecular dynamics simulations.6 In the mice model xenograed with human pancreatic tumors, the tumor volumes were markedly reduced in the metallofullerenol Gd@C82(OH)22-treated group (e.g. 50.1%) as compared to the control group with saline. From a direct observation of the pancreatic carcinoma using the environmental scanning electron microscope (ESSM) and uorescence image with uorescein isothiocyanate (FITC)-labeled dextran, far fewer blood vessel were found with the Gd@C82(OH)22 treatment, while much larger and denser vessels were populated in the saline-treated carcinoma, thus displaying a rougher and disordered surface morphology. The reduction of microvessel density (MVD) is an important indicator due to its relevance to cancer prognosis.6 This anti-angiogenic activity of Gd@C82(OH)22 was further investigated for matrix metalloproteinases, such as MMP-2 and MMP-9, due to their essential role in angiogenesis and proteolysis of ECM,6 which eventually promotes tumor metastasis. Both the expressions and activities are signicantly down-regulated, especially for MMP-9, in the Gd@C82(OH)22 group compared to the control groups with saline or C60(OH)226 (see Fig. 1).


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Molecular mechanism of the tumor growth inhibition These experimental observations provide a reasonable hypothesis on the inhibitory effect of Gd@C82(OH)22 on the relevant proteins; however, it is still not clear how they disrupt the proteolytic activity of MMPs. Understanding detailed binding modes of these nanoparticles on the bio-molecules would be a critical step for de novo nanodrug design with enhanced drug efficacy as well as target specicity. In this regard, the inhibitory mechanism of Gd@C82(OH)22 on MMP-9 has been further investigated with atomic level details using large scale molecular dynamics simulations in explicit solvent (with an aggregate of multi-microseconds).6 Experiment has shown that Gd@C82(OH)22 nanoparticles favor aggregated interaction with MMP-9 which has been recognized as one of the distinctive characteristics of Gd@C82(OH)22. This was also shown in molecular dynamics simulations where Gd@C82(OH)22 molecules make clustered interaction with MMP-9, even though they were initially in separation, which supports the experimental observations. The aggregation of Gd@C82(OH)22 is attributed to the large carbon surface of the fullerene cage C82, where the hydrophobic interactions are still dominant even aer multiple hydroxylations on the cage for better solubility in aqueous media. Distinct from traditional molecular drugs, a large surface area from aggregation of Gd@C82(OH)22 has been proposed to endow a wide range of interactions with multiple pro-angiogenic factors including MMPs at the mRNA level, and to more effectively block chaotic blood vessels in cancer tissues rather than in normal tissues under tight regulation, which may synergistically inhibit tumor metastasis.37 The clustered interactions of Gd@C82(OH)22 hardly affect the overall folding of MMP-9. This is very different from many previous observations where large protein structural deformations might occur due to their strong hydrophobic interactions with nanomaterials such as CNTs and graphenes through their surface exposed aromatic residues,39–41 thus potentially resulting in their malfunctioning. In this case, the inhibitory capability of Gd@C82(OH)22 is therefore not through large protein structural disruptions, but rather through specic bindings to the key MMP-9 binding sites. The binding free energy and sitespecic residue contacts revealed that in addition to the Zncatalytic site of MMP-9, Gd@C82(OH)22 mainly interacts with its ligand specicity S10 loop that facilitates the ligand binding and leads the ligand to its Zn2+-coordinated binding pocket (see Fig. 2). The tight binding of Gd@C82(OH)22 at the S10 loop thus blocks its function as the ligand mediator, leading to the inhibition of MMP-9 function. It should be mentioned that MMPs have been highly investigated as potential targets for anti-metastatic matrix metalloproteinase inhibitors (MMPi), mostly aiming at a “direct” blockage on the Zn-catalytic site. Nevertheless, most of them failed in obtaining high target specicity among possible MMPs, even with high binding affinity.42 This is largely due to the close similarity of the active site structures among MMPs. By contrast, the S10 loops have the least sequence similarities in the catalytic domain of all available MMPs, which play an important role in recognizing the native ligands. The favorable interaction of Gd@C82(OH)22

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Fig. 1 The influences of Gd@C82(OH)22-treatment on the protein expression and mRNA levels of MMP-2 and MMP-9 in tumor tissues. (a) The immunofluorescence results of MMP-2 and MMP-9 in JF305 pancreatic tumors (200). (b) Semiquantitative reverse transcriptase-PCR analysis of the genes MMP-2 and MMP-9. Protein levels of MMP-2 (c) and MMP-9 (d) in the tumor of mice treated with saline, C60(OH)22 and Gd@C82(OH)22 nanoparticles quantified by ELISA. Results are expressed as mean S.D. *P < 0.05 and **P < 0.01, significantly different from the saline group. (e) The combined activity of MMP-9/MMP-2 was determined by a MMP enzyme activity assay. Gd@C82(OH)22 effectively inhibits MMP-9/-2 activity while C60(OH)22 has little effect on them compared to control. (Reprinted from ref. 6. Copyright 2012, Proceedings of the National Academy of Sciences USA).

on the ligand specicity S10 loop thus proposes an alternative strategy to target specic inhibition of MMP-9 via an “indirect” exocite interaction, implying that Gd@C82(OH)22 can interfere with the ligand binding on MMP-9 by allosterically modulating the ligand specicity S10 loop, in addition to its binding to the Zn-catalytic site. While the ligand specicity loop of MMP-9 was found as a characteristic site for metallofullerenol Gd@C82(OH)22, the control simulations with normal fullerenols C82(OH)22 and C60(OH)22 appeared to have less specic interaction with MMP9. Instead, surface residues from both upper and lower rims of MMP-9 participated in the contacts with the fullerenols (Fig. 2). This was consistent with the experimental results, where C60(OH)22 has no noticeable effect on the activities of MMP-2/9. The detailed analysis showed that the specic binding of Gd@C82(OH)22 is guided by sophisticated interplay between non-bonding interactions. In the early stage, long range electrostatic interaction is a dominant force for Gd@C82(OH)22 to make initial contacts with MMP-9, in which Gd@C82(OH)22 has more frequent interactions with positively charged residues. This is due to negative surface charges induced by the encaged Gd3+ ion (i.e., Gd3+@[C82(OH)22]3). The iso-electric surface of MMP-9 showed a large negative electrostatic shield around the active site, potentially avoiding the direct approach of the negatively charged metallofullerenols. Nevertheless, the intrinsic hydrophobic character of the carbon cage of Gd@C82(OH)22 is able to facilitate the interaction with the hydrophobic patch near the ligand specicity S10 loop, by which the metallofullerenol moves from a hydrophobic patch to the

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S10 loop. Finally, Gd@C82(OH)22 forms a stable binding between the S10 and Sc loops via more specic hydrophobic and hydrogen bonding interactions. This demonstrates that drug efficacy of particulated Gd@C82(OH)22 on MMP-9 is quite different from conventional molecular based drugs. In short, the specic binding of Gd@C82(OH)22 is attributed to its unique amphiphilic surface characters, with both hydrophobic fullerene aromatic carbon rings, and hydrophilic hydroxyl groups in addition to induced charges by the encaged Gd metal ion. Both the suppression of MMPs expression and specic binding mode make Gd@C82(OH)22 a potentially more effective nanodrug for cancers, such as breast cancer and pancreatic cancer, than the traditional drugs which usually target the proteolytic sites directly without selective inhibition. The current ndings with a new exocite binding mode at the ligand specicity S10 loop provide new insights and directions for future de novo design of nanomedicine for fatal diseases such as pancreatic cancer.

3. Anti-bacterial graphene and graphene-oxide As applications of nanoparticles in nanomedicine become more signicant and important, there is also rapidly growing interest in understanding their interactions with cells, especially how they might affect the integrity of cell membranes.43,44 Recent studies have shown that graphitic nanomaterials such as zerodimensional fullerenes45,46 and one-dimensional CNTs47,48 can enter cells either through direct penetration49,50 (usually for


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Fig. 2 Binding free energy landscapes and residue specific contacts on MMP-9, as well as representative binding modes and pathway of Gd@C82(OH)22 on MMP-9. (a) Binding free energy surface for fullerenol C82(OH)22 on MMP-9 shows a non-specific binding mode (left) and almost all surface residues of MMP-9 contribute to contacts with C82(OH)22 (right). (b) Metallofullerenol Gd@C82(OH)22 interacts with MMP-9 along a specified binding mode (left) and contacts with only a specific set of residues near the ligand specificity S10 loop and SC loop (right). Residue was assigned to be in contact when any atom in the residue is within 5.0 A ˚ of any atom of Gd@C82(OH)22 (or C82(OH)22). The site participation was presented by the total number of frames of each residue in contact normalized by all frames and trajectories. (c) (left) A representative binding mode (a solid ball) shows that Gd@C82(OH)22 binds between the S10 ligand specificity loop (green ribbon) and the SC loop (purple ribbon) leading to the ligand binding groove. An alternative mode with a gray ball shows that Gd@C82(OH)22 can bind at the back entrance of the S10 cavity leading into the active site (ball and stick for active sites and orange ball for the catalytic Zn2+). (right) A possible binding pathway: depending on major driving forces and duration time (only the first 100 ns shown), the binding dynamics is characterized with three different phases. Phase-I: a diffusion-controlled non-specific electrostatic interaction, phase-II: a transient non-specific hydrophobic interaction, and phase-III: a specific hydrophobic and hydrogen-bonded stable binding. (Reprinted from ref. 6. Copyright 2012, Proceedings of the National Academy of Sciences USA).

small-sized nanoparticles) or endocytosis.47,51 However, due to their intrinsically higher complexity, graphene–cell interactions are much less studied. Recently, graphene (a two-dimensional (2D) single-atom-thick nanomaterial) and graphene-oxide (GO) nanosheets have been shown to display strong antibacterial activity to both gram-negative and grampositive bacteria.52–55 This cytotoxicity of graphene nanosheets is hypothesized to originate from direct interactions between graphene and bacteria cell membranes that cause serious physical damages to the membranes. 52–54 The cytotoxicity is reduced signicantly when these nanosheets are surrounded by proteins56 such as serum proteins. Therefore, although it is lethal to bacteria, it is less of a threat to human or other mammalians, and so can be potentially used as effective novel antibiotics.


In a very recent study, Zhou and coworkers7 revealed the underlying molecular mechanism of this important graphene– membrane interaction process, using a combined approach with both experimental and theoretical techniques. They rst used graphene oxide (for water solubility) in transmission electron microscopy (TEM) experiments, followed by large scale molecular dynamics simulations using both graphene and graphene oxide for comparison with the experimental results. Fig. 3 shows the TEM images of the cell morphology of E. coli incubated with 100 mg mL1 graphene oxide nanosheets at 37 C. Roughly three stages (Stage I, II, III) of E. coli cell morphology were observed during the 2.5 hour incubation process.7 In Stage I, the bacteria E. coli cells could initially tolerate GO nanosheets for some short period of time, particularly under low concentrations. Fig. 3a represents the initial

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Morphology of E. coli exposed to graphene oxide nanosheets. (a–f) Transmission electron microscopy images showing E. coli undergoing changes in morphology after incubation with 100 mg ml1 graphene oxide nanosheets at 37 C for 2.5 h. Three stages of destruction can be seen: (a) Initial morphology of E. coli (control or Stage 1; two individual TEM images (inset and main image) are shown, the scale bar applies to both). (b and c) Partial damage of cell membranes with some bacteria showing lower density of surface phospholipids (Stage II). Arrows indicate Type B mechanism, where graphene nanosheets extract phospholipids from the cell membrane. (d–f) Three representative images showing the complete loss of membrane integrity, with some showing “empty nests” and missing cytoplasm (Stage III). (d and f) Representative images showing Type A mechanism, where graphene nanosheets cut off large areas of membrane surfaces. (Reprinted from ref. 7. Copyright 2013, Nature Nanotechnology). Fig. 3

morphology of E. coli (control run or Stage I). In Stage II, E. coli cell membranes were partially damaged, with some exhibiting lower surface phospholipid density, i.e. sparser lipids but no obvious cuts yet (see those cells marked with “Type B” in Fig. 3b and c). In the nal stage (Stage III), E. coli cells were found to lose their cellular integrity, with their membranes severely damaged, and some even lost all their cytoplasm, i.e. “empty nests” (see those cells marked with “Type A” in Fig. 3d–f).7 Although it is difficult to determine the exact timing of each stage due to the resolution limit and diversity in each individual cell, these rough but representative stages still provide some insights into the dynamical process at the cellular level for this GO-induced degradation of E. coli cell membranes. Similar results were observed in additional experiments with increasing GO lateral sizes and concentrations. Using repeated oxidation processes with the Hummers' method, various GO nanosheets with different lateral sizes, such as 500 nm (GO1), 200 nm (GO2), and 50 nm (GO3) were generated. Aer the same 2.5 h incubation, larger GO nanosheets were seen to display a much stronger antibacterial activity than the smaller ones, with 90.9%, 51.8% and 40.1% for GO1, GO2 and GO3, respectively, under the same 100 mg mL1 concentration.7 The increase of GO1 concentration also resulted in an increase in the antibacterial activity, with 54.3%, 71.4%, 90.9% for 25, 50 and 100 mg mL1, respectively.7 Similar ndings were reported by Liu et al.57 in another recent experiment. Two types of molecular mechanisms Molecular dynamics simulations were then performed to investigate the detailed interactions of both graphene and

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graphene-oxide nanosheets with E. coli membranes. Both the outer and inner membranes of E. coli were simulated with allatom lipid models in explicit solvent. Zhou and coworkers rst started with graphene nanosheets to model an earlier experiment by Akhavan and Ghaderi,53 where graphene nanosheets were deposited on stainless steel substrates by electrophoretic deposition. Akhavan and Ghaderi53 showed that the sharpened edges of these nanosheets may act like “blades”, which can insert and cut through bacteria cell membranes,53 similar to the phenomenon observed in the above Stage III TEM images (Fig. 3). The unbiased simulations then showed that the graphene nanosheet “suspended” above the membranes (mimicking the experiment) can insert into both the outer and inner E. coli membranes very quickly. During this spontaneous entrance of the graphene nanosheet into the two membranes (Fig. 4), three distinguishable modes were observed. First, the Swing Mode, where the graphene nanosheet with its initially unbiased orientation underwent a swing motion, rocking back and forth, around the restrained atom, for a short period. Second, the Insertion Mode, in which the tail end of the graphene nanosheet was eventually trapped and pulled by the membranes, due to strong van der Waals (vdW) attractions from the membrane lipids and hydrophobic interactions. Once the tail end started to enter, it took only a few nanoseconds for the graphene nanosheet to cut into the lipid membranes. This direct insertion/cutting is referred to as the “Type A” mechanism by Zhou and coworkers (see cells marked with “Type A” in Fig. 3). Interestingly, this insertion (Type A mechanism) was also observed by Gao and coworkers58 in another very recent study with three different cell types.


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Fig. 4 Graphene nanosheet insertion and lipid extraction. (a) Graphene nanosheet insertion and lipid extraction in the outer membrane (Pure POPE); and (b) in the inner membrane (3 : 1 mixed POPE–POPG). Water is shown in ice-blue, and the phospholipids in tan lines with hydrophilic charged atoms in color spheres (hydrogen in white, oxygen in red, nitrogen in blue and phosphorus in orange). The graphene sheet is shown as a yellow-bonded sheet with a large sphere marked at one corner as the restrained atom in simulations. Those extracted phospholipids are shown in larger spheres with hydrogen in white, oxygen in red, nitrogen in blue, carbon in cyan and phosphorus in orange. (Reprinted from ref. 7. Copyright 2013, Nature Nanotechnology).

Third, the Extraction Mode, where the graphene nanosheet started to vigorously extract phospholipid molecules from the lipid bilayers onto its own surfaces. The disruptive extraction of phospholipid molecules eventually led to the loss of cell membrane integrity caused by the strong dragging forces from the graphene nanosheet. This surprising lipid extraction is named the “Type B” mechanism; it was rst revealed in the molecular dynamics simulations, and subsequently validated by careful examination of the staged TEM images (see cells marked with “Type B” in Fig. 3). This strong extraction-induced deformation might also help to explain the membrane wrapping in endocytosis59 of various nanoparticles.51,60 Further analysis of the interaction energy proles between the graphene nanosheet and the two E. coli membranes clearly demonstrates the three distinguishable observed modes. The “Swing Mode” of the graphene nanosheet in bulk water is associated with an initial high-energy plateau. Subsequently, the “Insertion Mode” is depicted by a sharp energy collapse, which corresponds to further enhancement in the interaction from graphene's continuous


dragging on the membrane and direct extraction of lipid molecules. This exceptionally strong dispersion interaction mainly comes from the graphene's unique 2D-structure with all sp2carbons, which is so powerful that it can overcome the selfattraction among the lipid molecules within the membrane. As for the “Insertion Mode”, it can be shown by the signicant changes of phospholipid membranes in both the thickness (increase) and area per lipid (decrease), with both E. coli outer and inner membranes displaying similar deformations overall. However, there is no signicant change in the phospholipid tail order parameters, indicating the acyl chain orientations are not much affected as one would expect.

Robust lipid extraction by graphene and GO nanosheets As described above, during the course of simulations, a novel “Type B” mechanism which leads to the thinning of the lipid densities and eventually the loss of cell membrane integrity was rst discovered. This could be the key mechanism to demonstrate the applicability of graphene and GO nanosheets as an anti-bacterial “green” band-aid. To further verify that this lipid Nanoscale, 2014, 6, 663–677 | 669

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extraction is indeed for real and not just due to some kinetic effects, an additional graphene “docking” simulation was performed using the outer membrane (pure POPE) as an example.7 The conguration for the simulation was intentionally set up so that it is very hard for the lipid extraction to occur. Namely, the entire graphene nanosheet was restrained in space, with its plane oriented perpendicular to the membrane surface and its tail barely touching the membrane surface to ensure there is no kinetic effects. At the beginning, the lipid membrane displayed some adaptive motions to adjust to the penetration of the graphene. Shortly aer that, larger uctuations started to appear in nearby phospholipid molecules, perturbing the seemingly smooth membrane surface. Then, some phospholipids began to climb up along the surfaces of the graphene nanosheet as a consequence of the strong attraction from the graphene. Soon aer that, many other phospholipids also joined in this “climbing” activity. Interestingly, this phospholipid climbing seems to be highly cooperative due to the collective movements of their hydrophobic tails at the water–graphene interface. Also, multi-layer climbing of phospholipid molecules was observed and the lipid extraction took place simultaneously on both sides of the graphene nanosheet. A few hundred nanoseconds later, serious membrane deformations became noticeable. During the pulling process, the hydrophobic tails of these extracted phospholipids tend to spread out evenly onto the entire graphene nanosheet to maximize their contacts with the hydrophobic graphene surface, while their hydrophilic head groups prefer to be solvated in bulk water. To further demonstrate the robustness of this lipid extraction,7 the GO nanosheets were also simulated to compare directly with the TEM experiments (which was done with GO for water-dispersibility). The GO nanosheets were based on the Lerf–Klinowski GO structural model with a molecular formula of C10O1(OH)1(COOH)0.5, which represents typical outcomes from the standard oxidation process.61–63 As expected, both POPE and POPG phospholipids were found to be extracted from the membranes by the GO nanosheets because of the strong vdW attractions between the GO nanosheet and membrane lipids. Similar to the case of the graphene nanosheet, once extracted, the hydrophobic interactions also play a dominant role through nanoscale dewetting. Again, the lipid hydrophobic tails tend to spread out, mainly in the unoxidized hydrophobic regions, while the hydrophilic head groups prefer to contact polar oxide groups via favorable electrostatic interactions. It is known that large unoxidized residual graphene-like regions (sp2-domains) can exist on GO nanosheets61,64,65 and such regions have been utilized experimentally to achieve the oxidative cutting and unraveling of carbon nanotubes.66 Therefore, the above discussions on graphene nanosheets can also be applied to GO nanosheets largely. Other evidences include the nding of Gomez-Navarro et al. that upon oxidation, isolated highly oxidized areas (few nm in size) are formed, while at least 60% of the surface remains undisturbed.64 In addition, further UV-vis spectra data also show that the maximum absorption peak of graphene, resulting from the sp2-domain of carbon atoms, displays an obvious blue-shi upon repeated

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heavy oxidation due to the presence of oxygen and increased number of sp3 bonds.7 Another insight gained from these ndings is that water plays a signicant role in the lipids structure and orientation at the graphene–water interface. At the beginning, all these complicated and collective movements of phospholipids on the graphene nanosheet started from seemingly short-ranged vdW attractions between the graphene (sp2-domains in GO) and lipid molecules. However, once extracted, the strong hydrophobic interactions between the graphene and lipid tails played another important role through nanoscale “dewetting” (i.e. expelling water from the graphene surface). This strong hydrophobic packing is similar to the hydrophobic collapses found in many biomolecular self-assemblies, such as cell membrane formation and protein folding, where many recent studies67–69 have shown that nanoscale dewetting can provide signicant driving forces for the collapse speed and system stability. These strong interactions provide the underlying driving force that causes the newly discovered “Type B” mechanism for graphene's anti-bacterial capability. In short, experimental and theoretical approaches have been combined to investigate the molecular mechanisms for the graphene-induced degradation of E. coli cell membranes. This study reveals two types of mechanism: one by severe insertion and cutting, and the other by destructive extraction of lipid molecules. This surprising extraction of phospholipids directly out of lipid membranes was rst observed in computer simulations, and then validated by TEM imaging. The graphene's unique 2D-structure with all sp2-carbons is the source that facilitates exceptionally strong dispersion interactions between the graphene and lipid molecules which cause the surprisingly robust destructive lipid extraction. Even though these ndings are from E. coli, similar mechanisms should also apply to other types of bacteria. 53,55 These ndings have implications in the design of novel antibiotics and other future clinical applications. In particular, graphene might become a new type of “green” antibacterial material for everyday use with little bacterial resistance due to its “physical damage”-based bacterial killing mechanism, as indicated in a recent attempt to use graphene-coated cotton fabric for band-aid.70

4. Nanodrugs through induced autophagy It is only recently that researchers have begun to show that nanoparticles can also elicit the autophagy process promoting destructive cell deaths, which can be used as another type of potential nanodrug for cancer therapy and vaccination.8,9,71–75 Autophagy, or cellular self-digestion, has gained considerable attention in the past decade due to its importance in human health and disease such as tumorigenesis, neurodegeneration and metabolic syndrome. It is a highly regulated process by which the cell can degrade damaged organelles, long-lived nonfunctional proteins and intracellular pathogens, as well as recycle cytoplasmic material for maintenance of cellular


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homeostasis. The process of autophagy involves several sequential steps. First, double-membrane vesicles, or autophagosomes, are formed by rearrangement of subcellular membranes to the sequestered portions of the cytoplasm. Then, the autophagosomes fuse with lysosomes to generate the autolysosomes. Next, the contents within the autolysosomes are degraded through the action of hydrolytic enzymes. Finally, the degradation products are recycled back to maintain macromolecular synthesis and energy production76,77 by the cell. A normal basal level of autophagy is essential for survival, differentiation, development, and homeostasis in virtually all cells.76,77 However, abnormal autophagy has been implicated in the pathology of numerous human diseases. Several studies have reported that applications of various nanomaterials can elicit an autophagic response in the cell culture systems tested. These nanomaterials include quantum dots,78 fullerene and its derivatives,72 lanthanide oxide and other rare-earth metal oxide nanocrystals,9,71,73 a-alumna nanoparticles,8 gold and iron-core–gold-shell nanoparticles.74 One very recent study shows that lanthanide-based nanoparticles, such as Nd2O3 nanocrystals, can be safely used for therapeutic purposes by coating these nanoparticles with small peptides.9 A short synthetic peptide, RE-1 with a sequence of ACTARSPWICG, identied by means of phage display on Nd2O3 nanocrystals, shows a strong preference to the series of lanthanide oxides and upconversion nanocrystals. The addition of an arginine–glycine–aspartic acid (RGD) motif to RE-1 enhances autophagy of lanthanide oxides and upconversion nanocrystals through the interaction with integrins, where integrins are over-expressed in angiogenic endothelial cells. Thus, these lanthanide-based nanoparticles with proper peptide coating can be used as potential antiangiogenic cancer therapeutics. Another potential application of these nanoparticles in nanomedicine is vaccination. Dedicated antigen presenting cells, such as dendritic cells, are capable of presenting exogenous antigens to cytotoxic T lymphocytes – a crucial process known as cross-presentation for the development of adaptive immunity to tumors and most infectious pathogens.79 Since the magnitude of T cell expansion is regulated primarily by antigenpresentation and activation, maximizing the efficiency of crosspresentation is likely the rst key step for the successful development of vaccines for cancers.80 Cross-presentation is a complex process that involves antigen internalization, protein degradation, and loading of antigen-derived peptides into major histocompatibility complex (MHC) molecules of antigen presenting cells.81 Optimal immune responses to most antigens require antigens to be administrated with an adjuvant.82 Currently, the microparticle precipitate of aluminum compounds (also known as Alum) is the only licensed adjuvant in the United States. However, the mechanisms of Alum antibody response enhancement are poorly understood, and furthermore, it has very limited ability to enhance crosspriming of cytotoxic T lymphocytes.83 A very recent study by Hu and coworkers8 showed that a-Al2O3 nanoparticles can act as an efficient antigen carrier, by which the amount of antigens is reduced to activate T cells. The


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antigens are delivered to autophagosome in dendritic cells, followed by antigen presentation to T cells through autophagy. Immunization of mice with a-Al2O3 nanoparticles that are conjugated to either a model tumor antigen or autophagosomes derived from tumor cells resulted in tumor regression.8 These results suggest that alumina nanoparticles might be a promising adjuvant in the development of therapeutic cancer vaccines.

5. De novo nanodrugs assisted by external triggers Another major category of nanodrugs requires external triggers to be fully effective. Photodynamic therapy and thermoablative therapy are two such major applications. Due to the requirements of an electromagnetic response for material function, these nanodrugs are usually composed of metallic or inorganic nanostructures such as metals, metal-coordinated complexes, metal oxides, and carbon nanostructures. Pharmacological action of photodynamic therapy (PDT) relies on photogenerated reactive oxygen species, specically singlet oxygen (1O2), and subsequent damage to cellular components such as DNA, protein, membrane, and so on, which ultimately induce apoptosis or necrosis to cell death.84,85 Similar to conventional molecule-based PDT, the nanoparticlebased PDT generates singlet oxygen by energy or electron transfer,86 in which the photosensitizing activity of these nanoparticles are critical. Recent studies have demonstrated that passivation density and energy coupling with conjugated surface molecules on quantum dots (e.g., CdSe and CdTe) can affect 1O2 generation and cancer ablation efficacy 87,88. Compared to traditional PDT, nanoparticle-based PDT can be a more effective and versatile tool due to higher photostability and structural amenability for various targets. Of course, the relatively short lifetime and ranges of 1O2 and the use of heavy metals require more delicate designs for effective localization of large-sized nanoparticles near the target sites.89 Thermoablative therapy induces death of dysfunctional cells by generating heat localized near the target tissue. Depending on applied external elds, the heat generation mechanism is differentiated with photothermal and magnetothermal pathways. Photothermal hyperthermia depends on the plasmonic properties of the metallic nanoparticles,90,91 in which absorbed photon energy is efficiently dissipated into the surrounding solution via vibrational coupling to raise the temperature.92 Metallic nanostructures, such as Au nanostructures, have been extensively employed, including nanoparticles,93 nanorods,94 and nanoshells.95 A variety of other nanomaterials have also been investigated for photothermal therapy including carbonbased nanomaterials,96–98 Pd nanosheets,99 plasmonic quantum dots,100 etc. The corresponding cell death is generally explained with necrosis caused by membrane melting, although an apoptotic pathway via inducing abnormality in cell functions is also proposed.90 Meanwhile, magnetic hyperthermia relies on thermal energy released by spin relaxation (Neel process) of magnetic

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nanomaterials under alternating magnetic elds, or generated by nanoparticle rotation (Brownian process) under rotating magnetic elds.11 The majority of research is based on magnetic iron oxides (i.e., Fe3O4 and g-Fe2O4).101 Nevertheless, attempts are going on by varying bio-compatible surface chemistry102 as well as adjusting material composition to enhance the magnetization, such as superparamagnetic iron oxides.103 For example, Cheon and coworkers demonstrated the versatility of magnetic nanoparticles,11 where the thermal induction rate was signicantly enhanced by tuning the magnetically hard core and so shells. One optimized superparamagnetic nanostructure (i.e., [email protected]) exhibited a signicant increase in heat conversion efficiency about 34 times larger than that of Feridex, a conventional iron oxide nanoparticle. A recent study with an in vivo mice model xenograed with human brain cancer cells clearly demonstrated the power of hyperthermia of the exchange-coupled core–shell iron oxide nanoparticles.104

6. New advances in nanocarriers of conventional drugs As mentioned above, there are extensive studies and excellent reviews on nanocarriers for drug delivery. For completeness, here we only summarize some of the latest advances, based on our likely biased judgment. In general, a stable and long circulation is the critical factor to deliver and accumulate drugloaded nanoparticles in the target tissue. A notable example is Doxil, a rst-generation liposome-based nanovector for doxorubicin.12,13 While Doxil hasn't been conjugated with a special moiety or device for targeting or controlled release, it provides a moderate success especially in treating ovarian cancer and AIDS-related Karposi's sarcoma thanks to a typical EPR effect. Although the passive mode is effective for densely-vascularized solid tumors of fenestrae, the targeting would not be that efficacious in case of small clusters of metastasized cells with poorvasculature in general.105 Nevertheless, nanoparticle trafficking, as mostly relying on diffusion in the blood circulation, is still an inevitable step to draw the nanomedicines near a target organ or tissue. New advances in passive targeting Recent designs become more diversied in mechanical properties, surface electrostatics, surface chemistry as well as administration routes for the enhanced nanoparticle localization interconnected with physicochemical, physiological and anatomical characteristics of the target. The permeability of nanoparticles depends on cancer types and particle size.106 The clinically approved Doxil and Abraxane (albumin-stabilized paclitaxel nanocarrier) with diameters of 100 nm are effective in highly vascularized tumors like Kaposi's sarcoma12 and breast cancer,107 while nanovectors of diameters larger than 100 nm showed a limited permeation such as in hypovascular and hypopermeable tumors like pancreatic cancer.108 Cabral et al. showed that the nanoparticle uptake is highly restricted by particle size.109 With a set of polymer-based nanoparticles of 30,

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50, 70, and 100 nm diameter, only 30 nm nanoparticles were permeable and effectively accumulated in the pancreatic cancer, albeit all sizes were able to inltrate into tumors of dense vasculature. Surface modication with tumor growth factor b (TGF-b) inhibitor, however, improved the permeability of larger-sized nanoparticles at the poorly permeable cancer tissue. The particle size is also critical in targeting diseases in the brain. Although inltrating into the brain is one of the most challenging steps in brain therapy due to the limited access through the blood–brain barrier (BBB),110 nanoparticles are permeable through the disrupted BBB at the brain lesion.111 Even so, only small size nanoparticles under 12 nm are effectively translocated with the particle uptake dramatically reduced as the particle size increases.112–114 Meanwhile, in the lung a large-sized particle over 300 nm showed a prolonged residence115 as being entrapped in the intertwingled capillary beds of pulmonary alveoli.116 Surface electrostatics have been shown to affect the domain targeting ability of nanoparticles. Choi et al. demonstrated that surface charges can determine the trafficking to lymph nodes of inhaled nanoparticles,117 where the lymph nodes could be the rst metastatic targets, and also serve as the disseminating points of cancer cells all over the body. Within the particle size window between 6 and 34 nm, polar and neutral nanoparticles (i.e., zwitterionic cysteine and polar PEG ligand coatings) rapidly accumulated in the mediastinal lymph node preventing serum protein adsorption. Nanoparticles modied with netcharged groups ended up with different fates, although a similar migration path was expected by interaction with endogenous proteins in the lung.118 On the other hand, cationic lipid complexes have received much attention as effective nanocarriers for small interfering RNA (siRNA) therapeutics.119,120 Although lipid-based nanocarriers are regarded as an appropriate platform for nucleic acid drugs due to their superior protection from the nuclease degradation and targeting ability by effectively mediating anionic oligonucleotides and cell membranes,121,122 the positively charged surface was reported to induce the opsonization, immune responsive phagocytosis, which could thus bring about dose-escalation.123 Polach and coworkers showed that covalent conjugation on lipopolyamine with methoxypolyethylene glycol (mPEG) could reduce toxic effects by avoiding serum protein adsorption, while effectively delivering siRNA to the lung endothelium with a prolonged retention.124 The mechanism for the residence time increase still needs to be claried, although the volume expansion by binding to erythrocytes and serum proteins was proposed to increase particle entrapment in the lung.125,126

Active targeting enhancement Despite advances in nanoparticle trafficking toward the target sites, a more specic strategy is needed for more discernable targeting to malignant cells or intracellular compartments. The active targeting generally relies on a specic recognition for molecules (e.g., receptors, factors and/or endogenous proteins


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different from the neighbor) over-expressed on the target cell surface. Nanoparticles are thus elaborated with selected ligand molecules, such as antibodies, short peptides, nucleotides and small organic molecules, that can bind to the receptors of interest with a higher binding specicity and affinity. Antibody-based nanocarrier design might be one of the representative approaches in searching for cancer cells in the primary tumor site and disseminated clusters of malignant cells. For instance, tositumomab, a murine IgG2a monoclonal antibody, labeled with a radioactive 131I can be used in curing follicular B cell lymphoma in the metastatic phase III and IV, usually incurable stages by a standard radioactive therapy.22 Despite expectations of targeted delivery and immunotherapeutic activity, immunoconjugates may pose undesired effects of decreased immunoreactivity and poor solubility.127 Patri et al. demonstrated that dendrimer-based nanocarriers can solve these problems using a monoclonal antibody J591,128 which targets the prostate-specic membrane antigen (PSMA), a 100-kD, type II membrane glycoprotein highly expressed on prostate cancer cells, as well as nonprostatic tumor vasculature and vascular endothelium of virtually all solid sarcoma and carcinoma.129 Recent clinical tests showed that the antiPSMA antibody J591 has an anti-angiogenic efficacy for patients of advanced solid tumors expressing PSMA on the neovasculature.130 Small molecules also constitute another end of the targeted delivery. Folic acid is frequently conjugated with therapeutics for various malignant tumors, owing to their effective targeting on folate receptors.131–133 The receptors are exclusively expressed on tumor cells in various organs including brain, breast, lung, colon, ovary, and myelogenous leukemias, while rarely found in the normal cells.134,135 A nanomolar level sensitivity holds for the receptor binding for the folic acid moiety even aer being tethered with drugs or nanoparticles.136 More specically, conjugated nanoparticles with a payload can be internalized by receptor-mediated endocytosis,137 thus facilitating drug delivery into sub-cellular organelles. The small molecular coating is not limited in aiming the direct targeting of the intended cells. Carbohydrates, such as dextran have been shown to indirectly induce nanoparticle uptake in lymph nodes. Harisinghani et al. demonstrated that dextran-coated superparamagnetic iron-oxide can be used in diagnostics and therapeutics of the metastasized cancer in lymph nodes. As in the normal immune response, the dextran layer resembling lypopolysaccharides on bacterial surfaces can be recognized and uptaken by macrophages in blood circulation, followed by translocation to lymph nodes by means of the interstitial lymphatic-uid transport.138,139 Besides mining efforts for new ligand–receptor systems, recent nanocarriers are more exquisitely engineered to gain benets from both passive and active targeting modes in one kit. Inspired by the communicated network found in human immune-cell trafficking,140 Bhatia and coworkers devised a nanotherapeutic strategy compartmentalized with a passive targeting-based “signaling” unit and active targeting-based “receiving” unit.141 The signaling nanoparticles (i.e., tumortargeted plasmonic Au nanonodes or tumor-targeted tissue


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factor protein) detect tumors and locally activate the coagulation cascade by extrinsic and intrinsic disturbance. The receiving units (i.e., magnetouorescent iron-oxide nanoworms or doxorubicin-loaded liposomes) in circulation are effectively recruited in the tumor sites via targeting the blood coagulation, where the accumulation increased by >40 times over than the non-communicating control with a conventional direct targeting strategy. Despite needs for a thorough validation on their safety at a system level, multiple component approaches with both active and passive targeting agents deserve further investigation as a highly promising nanomedicines.

7. Conclusion and future implications In this feature paper, we have reviewed some of the recent advances in de novo design of nanodrugs, including metallofullerenol Gd@C82(OH)22 for anti-cancer therapy, graphene and graphene oxide for anti-bacterial “green” band-aids, lanthanide-based nanoparticles for effective autophagy, and other nanodrugs assisted by external triggers for photodynamic therapy and thermoablative therapy, with an emphasis on the molecular level understanding of their interactions with biological systems including key proteins and cell membranes. We have also reviewed the latest progress in nanocarriers with advances in both passive and active targeting. While the rst generation of nanocarriers, represented by Doxil and Abraxane, achieve their delivery function mainly by the EPR effect, recent designs have become more advanced by conjugating multiple active targeting elements from physicochemical factors in diffusive circulation, to physiological factors for the specic interaction with the target disease domain. The future trends include de novo design of more effective nanodrugs and more efficient nanocarriers, as well as development of “nanodevices” which combine both nanocarriers and nanodrugs as a new generation of nanomedicines. These promising progresses in nanomedicine raise hope for enhanced drug efficacy with high disease selectivity. However, the complication in nanomedicine formulation still imposes several important issues to be resolved before wider applications. Future studies will be more specied on the effects of nanomedicines on both spatial and temporal domains of pathogenesis of diseases. The molecular level understanding, with both experimental and theoretical approaches (such as molecular dynamics simulations), on nanomedicine interactions with various biological building blocks (e.g., proteins, nucleotide, carbohydrates and membranes) will be of greater importance. This deeper, systematic, molecular level understanding will lead us to a better picture at cellular and tissue levels.142 These collective future studies will be important not only in the designing of de novo nanomedicines with high target efficacy and selectivity, but also in addressing their potential biosafety issues (i.e. nanotoxicity) before any meaningful clinical trails. This nanotoxicity concern is oen less obvious and much trickier to assess due to complicated interactions with various nonspecic targets at molecular, cellular and tissue levels, with

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oen indirect and latent effects. In addition, compared to traditional drugs, the pharmacokinetics of nanodrugs are much less studied, which also partly explains the deterrence of FDA approvals even with high therapeutic efficacy.143 Therefore, molecular level understanding, as presented in the two case studies here, will be in great demand for the development of nanomedicine, which will also be accompanied with advances in the multi-scale simulation and modeling at larger scales.

Acknowledgements We would like to thank Jingyuan Li, Yuliang Zhao, Chunhai Fan, Bruce Berne and Chunying Chen for helpful discussions. This work was partially supported by the National Natural Science Foundation of China (NSFC) under Grant no. 11374221 and 21320102003, the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, and the IBM Blue Gene Science Program.

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