Cervadoro A, Palomba R, Vergaro G, Cecchi R, Menichetti L, Decuzzi P, Emdin M and Luin S (2018) Targeting Inflammation With Nanosized Drug Delivery Platforms in Cardiovascular Diseases: Immune Cell Modulation in Atherosclerosis. Front. Bioeng. Biotechnol. 6:177. doi: 10.3389/fbioe.2018.00177 Atherosclerosis (AS) is a disorder of large and medium-sized arteries; it consists in the formation of lipid-rich plaques in the intima and inner media, whose pathophysiology is mostly driven by inflammation. Currently available interventions and therapies for treating atherosclerosis are not always completely effective; side effects associated with treatments, mainly caused by immunodepression for anti-inflammatory molecules, limit the systemic administration of these and other drugs. Given the high degree of freedom in the design of nanoconstructs, in the last decades researchers have put high effort in the development of nanoparticles (NPs) formulations specifically designed for either drug delivery, visualization of atherosclerotic plaques, or possibly the combination of both these and other functionalities. Here we will present the state of the art of these subjects, the knowledge of which is necessary to rationally address the use of NPs for prevention, diagnosis, and/or treatment of AS. We will analyse the work that has been done on: (a) understanding the role of the immune system and inflammation in cardiovascular diseases, (b) the pathological and biochemical principles in atherosclerotic plaque formation, (c) the latest advances in the use of NPs for the recognition and treatment of cardiovascular diseases, (d) the cellular and animal models useful to study the interactions of NPs with the immune system cells.

Keywords: atherosclerosis, inflammatory diseases, smart nanomaterials, drug delivery, nanomedicine, imaging and theranostics, immune cells, cardiovascular diseases

### ATHEROSCLEROSIS AND INFLAMMATION

Atherosclerotic disease, or simply atherosclerosis (AS), is initially characterized by the formation of fatty streaks, with the accumulation of lipids [primarily cholesterol, but also triglycerides (Goldberg, 2018)] in the intima and inner media of arterial wall, especially in regions with abnormal flow patterns (Chistiakov et al., 2017). Fatty streaks may then evolve into soft, lipid-rich plaques, and eventually into thick cap-calcified lesions and/or unstable plaques characterized by inflammatory infiltration and sustained oxidative processes. Stable fibrocalcific atheroma is characterized by calcium deposits, small lipid deposits, slight lumen reduction, and, often, by poor functional relevance. Vulnerable atheroma, which is more prone to rupture, is characterized by a large lipid-rich necrotic core, thin fibrous cap (<65µm), neovascularization, spotty calcifications, inflammatory cells, and positive remodeling (Moore and Tabas, 2011; Hansson et al., 2015). The possibility of plaque regression has been reviewed in Chistiakov et al. (2017) focusing on animal models, and in Fisher (2016) also considering the dynamic changes in lipid and immune cells distributions.

AS is currently considered an inflammatory disorder, characterized by the infiltration into sub-endothelial space of various immune cells (ICs), especially circulating monocytes (MCs) that subsequently differentiate into macrophages (M8s) and then into foam cells (FCs), going along with plaque formation and evolution (Moore et al., 2013; Zhang et al., 2017). Such simplified picture is complicated by the heterogeneity of the cells within and close to lesions during evolution [MCs, M8s, and FCs, but also neutrophils, dendritic cells (Butcher and Galkina, 2012), T cells (Taleb, 2016), and possibly others]. In the cited reviews particular attention is given to the markers of the various IC phenotypes, although cells can present more than one function and can express a continuum of markers of different subtypes (Butcher and Galkina, 2012).

A key example regards M8s, the characterizing cells in AS: their different subtypes can have antithetic roles. Initially classified only as classically activated M1 (pro-inflammatory) or alternatively activated M2 (anti-inflammatory), evidences have brought to the definitions of additional subtypes (e.g., Mox, M4, Mhem) and even "sub-subtypes" (e.g., M2a, M2b, M2c); there could actually be a continuum of specializations, and M8s could even convert into each other (Butcher and Galkina, 2012; Leitinger and Schulman, 2013). It must be noted that also smooth muscle cells (SMCs) can assume some of the functions usually assigned to M8s (efferocytosis, internalization of lipids or cholesterols), and may transform in foam cells (Chistiakov et al., 2017).

In any case, cells with phenotypes closer to Mox or M1 cells are the most active in internalizing lipids (particularly cholesterol), especially if agglomerated within oxidized low density lipoprotein (oxLDL). Upon this process, efferocytosis efficiency of cells is reduced, they transform into foam cells, and are easily subjected to imbalances in cholesterol influx/efflux process and hypoxia. The final outcome is most often apoptosis or necrosis, which resolve in local accumulation of the lipid content, the major constituent of the inner (possibly necrotic) core of the atherosclerotic plaque (Moore and Tabas, 2011). At the same time, the cell's residual components promote further inflammation signals and generation of oxidative species, in a persistent cycle of recruitment of intimal macrophages and their polarization toward pro-inflammatory subsets (Libby et al., 2014). Moreover, both monocytes and macrophages have been shown to contribute to the increase of the gamma-glutamyl transferase enzyme (γGT) in AS lesions, increasing the oxidative character of these zones (Pucci et al., 2014; Belcastro et al., 2015).

This self-sustained state of inflammation (similar to what happens in chronic wound environments) is characteristic of the vulnerable plaque. The presence of activated macrophages leads to the secretion of matrix-components degrading enzymes (such as matrix metalloproteinases-MMPs), inhibits the production of collagen by the SMCs, and finally contributes to vascular wall reshaping. These mechanisms are useful in early lesions, supporting resolution of disrupted endothelial layers and avoiding accumulation of toxic species, as well as for the reduction of atherosclerotic plaques; but, at the same time, they induce fragility in late plaques, with the possible final consequence of plaque rupture (Libby et al., 2014; Hansson et al., 2015; Martinez and White, 2018). Inflammation also modulates the clinical consequences of the thrombotic complications of AS, while its inhibition could attenuate progression, mitigate the risk of plaque rupture, and even promote regression of AS (Awan and Genest, 2015; Bäck and Hansson, 2015; Bäck et al., 2015; Kamaly et al., 2016).

### ATHEROSCLEROSIS TREATMENTS

Current therapeutic approaches to AS aim at reducing promoting factors, including hypertension, smoke, and dyslipidemias; considered drugs (e.g., inhibitors of cholesterol hepatic synthesis like statins) particularly affect production and transport of cholesterol (or other lipids) to the arterial walls (Coomes et al., 2011; Duivenvoorden et al., 2014; Tsujita et al., 2015). Different approaches have been investigated, such as blocking absorption of cholesterol in the intestine (e.g., by ezetamibe) or controlling its reverse transport in ICs, e.g., by using an agonist for the liver X receptor (LXR) like GW3965, or administering artificial forms of high density lipoproteins (HDL; Duivenvoorden et al., 2014; Tsujita et al., 2015; Pulakazhi Venu et al., 2017; Goldberg, 2018; Mueller et al., 2018).

Controlling inflammation is another promising strategy; in particular, the interleukin-1 pathway has been identified as a possible therapeutic target. Canakinumab, a monoclonal antibody targeting interleukin-1β, was tested in the CANTOS (Canakinumab ANti-inflammatory Thrombosis Outcomes Study) trial (Ridker et al., 2011, 2017). Moreover, the chemotherapeutic drug methotrexate (MTX), largely used as an immunomodulatory and anti-inflammatory (AI) drug, was described to lower the risk for total cardiovascular diseases in patients with chronic inflammation (Popkova et al., 2015; Gomes et al., 2018), and has been observed to inhibit atherogenesis and macrophage migration to the intima in animal models (Bulgarelli et al., 2012). Although its mechanisms of action are not fully clear, this molecule is capable of reducing the secretion of pro-inflammatory cytokines and the expression of adhesion molecules on both immune and endothelial cells (Coomes et al., 2011).

While promising, systemic administration of AI drugs is often limited by a narrow therapeutic index and by severe adverse effects, including bone marrow suppression, neutropenia and immunodepression. Blockade and stimulation of the mechanisms involved in inflammation can hold positive, null, or even detrimental results depending on the phase of the atherogenic process (Aluganti Narasimhulu et al., 2016). Based on these premises, it is authors' belief that a rational modulation of the functions of ICs is promising for an efficient treatment and hopefully comes with fewer adverse effects.

Ideally, it would be desirable to identify an effective strategy for all of the AS phases; e.g., in (Libby et al., 2014) Resolvin E1 is indicated as a mediator that reverses all the advanced lesions associated processes, while contributing to the resolution of the plaque also in earlier phases. Another promising possibility is controlling release or activation of compounds used to alleviate or cure AS symptoms/consequences. Possible approaches include: the use of drugs modified with a glutamyl in order to exploit the increased concentration of γGT in AS lesions (Belcastro et al., 2015); developing methods exploiting high cholesterol concentrations; tackling pathways leading the transformation process from monocyte to pro-inflammatory M8s and finally to foam cells (Rousselle et al., 2013).

Due to their ambivalent role of inducing inflammation and regulating tissue regeneration, macrophages have been the first candidates addressed in order to control AS. Vannella and Wynn presented an interesting review describing macrophages behavior in different tissues, the mediators of the various mechanisms involved, and finally several possible ways to modulate them (Vannella and Wynn, 2017). It would be ideal to force their egress from AS lesions as transformation into foam cells is occurring; to this end, a suggestive idea could be activating or releasing a "macrophage migration-enhancement factor" (Weisbart et al., 1974; Ueno et al., 1997; Nunami et al., 1998), or interfering with cell surface adhesion molecule only in overabundancy of cholesterol or other AS markers, e.g. by stopping the phosphorylation of CD44 (Qin, 2012).

### TOWARD TARGETED THERANOSTICS NPs

As hinted above, spatial and temporal control of drug activity can reduce collateral short and long term effects; moreover, it can result in more convenient administration methods. These premises strongly call for the development of efficient, targeted delivery strategies for AI molecules, just like the ones based on their encapsulation into NPs (Costa Lima and Reis, 2015). Several kinds of NP have been considered for drug delivery; characteristics and production protocols for some of these are reviewed in Allen et al. (2016), Ulbrich et al. (2016), Cheraghi et al. (2017), and Matoba et al. (2017). There are still limitations and drawbacks for the clinical use of many NPs; these could arise from a not-yet perfect control of the final fate of many formulations, since they often accumulate also in the organs of the reticuloendothelial system (RES), from polydispersion and/or poor reproducibility in their preparation, or from the often difficult scale-up and high cost for their production, especially when multifunctional capabilities are added (Cheng et al., 2012; Ulbrich et al., 2016). However, NP physicochemical properties can be finely tailored during their synthesis and this allows optimizing drug loading and NP target specificity (Allen et al., 2016; Pentecost et al., 2016).

In the clinical setting of atherosclerotic disease, NPs loaded with anti-inflammatory drugs can be a powerful tool to hit inflammatory targets at the plaque level, preventing systemic side effects (Jokerst and Gambhir, 2011; Di Mascolo et al., 2013). Drug-loaded NPs targeting macrophages and other immune cells could control their pro-inflammatory activities and thus prevent, attenuate, and possibly reverse related disorders (i.e., increased atherosclerosis, but also altered adipocyte function and insulin resistance). At the same time, NPs could also be loaded with imaging agents allowing the detection of vulnerable atherosclerotic plaques; similar theranostic strategies already showed potential for detection and treatment of other diseases (including cancer and neurodegenerative disorders), exploiting a number of imaging modalities, among which optical imaging (OI), magnetic resonance imaging (MRI), ultrasound and photoacoustic (US-PA), computed tomography (CT), and nuclear imaging based on single photon and positron emission tomography (SPECT, PET; Xie et al., 2010; Kim et al., 2014; Weissleder et al., 2014; Atukorale et al., 2017; Stigliano et al., 2017; Zhang et al., 2017).

There is evidence that NPs can segregate into the plaque in preclinical models of AS via a "passive" targeting mechanism (Weissleder et al., 2014); while such a relative selectivity has not been completely understood, some hypotheses have been proposed. First, the abnormal hemodynamic forces where plaques develop may favor NPs deposition (Hossain et al., 2015); second, the endothelium appears discontinuous with openings for sufficiently small objects (Kim et al., 2014); third, there can be an active role of immune cells in vehiculating or accumulating the nanoparticles in inflamed districts (Moore et al., 2017).

Other strategies focused on specific targeting, e.g., toward inflammatory factors, dysfunctional endothelial cells, or specific macrophage receptors involved in cholesterol transport. Examples are drug carriers functionalization with selectin ligands or antibodies directed to CAMs (sialyl-Lewis X, PSFL-1, ICAM and VICAM ligands, anti-ICAM, anti-VCAM; Robbins et al., 2010) or anti-oxLDL receptor (Li et al., 2010). Li et al. used liposomes decorated with LOX-1 antibodies, Indium (111In) or Gadolinium (Gd), and DiI fluorescence markers to image atherosclerotic plaques in ApoE−/<sup>−</sup> mice. Alternatively, it has been proposed to target collagen IV, which is present on the vascular basement and exposed when vascular permeability increases (Chan et al., 2011; Chen et al., 2013; Kamaly et al., 2016; Meyers et al., 2017). Also common strategies to target macrophages are based on functionalizing nanoparticles with dextran sulfate coating or peptides mimicking low-density lipoproteins (LDL) such as apolipoprotein A1 (ApoA-1), whose receptors (Class A Scavenger Receptor 1 MSR-1, and class B Scavenge Receptor CD36) are expressed on macrophages cell membrane (Canton et al., 2013). Further works focusing on visualization of macrophages distribution using nanoparticles are well reviewed in Weissleder et al. (2014).

Active targeting was achieved also using a "biomimetic" approach: NPs can derive from, or have properties similar to, aggregates like LDL or HDL, naturally accumulating in AS plaques (Allijn et al., 2013; Duivenvoorden et al., 2014; Gomes et al., 2018). More specific tissue or cell targeting can be found testing nanoparticle libraries in-vitro or in-vivo (Kamaly et al., 2016; Tang et al., 2016), but the mechanisms of the found specificity should be understood, also in order to ensure it is preserved downhill of modifications of nanoconstructs or in different biological environments. Libraries are often tested preliminarily in cell cultures, but in-vivo tests are necessary at least to evaluate the impact of the different biological media.

Indeed, upon entry in an organism, NPs are usually coated by a protein corona (PC), changing their biological identity. PC formation (or "opsonisation") is often the first step toward the sequestration of NPs by the RES. Various approaches have been considered to avoid such phenomenon. Recent strategies are based on controlling nanoconstruct stiffness, since it was found that deformable particles are less subject to uptake by macrophages in RES in off-target tissues (Key et al., 2015; Palomba et al., 2018). Other methods exploit different coatings and functionalization developed for controlling the PC, e.g., by using polymers like PEG. This is thought to be an antifouling agent, but it actually seems to modulate the PC (Schöttler et al., 2016); moreover, it has been shown to be immunogenic, requiring the use of additional functionalizing moieties (Mima et al., 2017). Other possible coating molecules are based on peptides: examples comprise zwitterionic ones, for limiting serum-protein adsorption (Ranalli et al., 2017), or aptamer-likes, for enriching the PC with specific molecules present in biological fluids, which act as targeting moieties when properly oriented (Santi et al., 2017). All these approaches easily grant nanoconstructs with extended circulation half-life.

An advantage that will play important role consists in NPs potential multimodality: not only they can contain more than one drug and/or imaging agent, but it could also be possible to implement a trigger for drug release/activation. This could either be intrinsic (provided by the pathological environment) or exogenous (ultrasound, light, oscillating magnetic fields).

Especially under this view, an interesting development can arise from a synergy between nanotechnology and personalized medicine (Mendes et al., 2018). Early screening of the most suited bioactives (Tang et al., 2016; Risum et al., 2017), real time monitoring of local accumulations (Zavaleta et al., 2018), observing early feedbacks to the treatment (Qiao et al., 2017), and predicting patient responses (Sykes et al., 2016) are all possible aspects of personalized medicine. We believe that the rational application of nanotools in all these steps will embody a fundamental role in the upcoming clinical and pre-clinical research (Mura and Couvreur, 2012; Polyak and Ross, 2017; Yordanova et al., 2017).

### NPs FOR AS TREATMENT AND DIAGNOSIS

The use of NPs for treatment of AS and visualization of plaques up to 2015 has been elegantly reviewed in Zhang et al. (2017); we further selected more recent works not reported there (**Table 1**). In this session, we describe some of these and other relevant works.

Often, intrinsic properties of NPs lead to the exploitation of drug delivery strategies together with photothermal- and radiofrequency-mediated triggering effects; e.g., Johnston proposed the possibility to use photothermal destruction of macrophages using iron oxide NPs with thin gold and dextran coating, excited by a laser pulse at 755 nm. In this manuscript the particles were used for MRI and in vitro photothermolysis (Ma et al., 2009). A similar approach has been tested in the NANOM-FIM trial (Kharlamov et al., 2015); here, NPs composed by silica shells containing gold and eventually magnetic nanoparticles were delivered on AS plaques by a bioengineered on-artery patch or using a magnetic navigation system; detonation of NPs using a NIR laser caused a significant final reduction of the total atheroma volume.

A different, interesting strategy consists on preventively acting on the selective recruitment of monocytes from the precursors of pro-inflammatory macrophages M1 (Nakashiro et al., 2016; Matoba et al., 2017). The authors proposed polymeric NPs loaded with Pioglitazone, an agonist of the receptor PPARγ shown to be able to influence macrophage polarization. The formulation was tested in ApoE−/<sup>−</sup> mice fed a high fat diet (HFD) and infused with angiotensin II, promoting inflammation driven by monocytes/macrophages. 2 days post injection the ratio between peripheral pro- and non-inflammatory monocytes subsets decreased substantially, and tissue macrophage polarity was regulated toward the noninflammatory phenotype M2, with consequent suppression of EMMPRIN/MMP pathway and reduction of plaque destabilization risk.

In a similar approach, Stigliano et al. (2017) confirmed the potentiality of MTX in preventive-oriented treatments by loading it into NPs. The authors demonstrated specific accumulation of NPs into macrophages residing within lipid-rich plaques along the arterial tree. In the aortic arch of ApoE−/<sup>−</sup> mice on HFD treated with MTX-NPs, 50% less coverage of plaques was found in comparison to the control group. Importantly, this result was obtained by injecting a dose four times lower than reported in literature (Leite et al., 2015; Gomes et al., 2018).

MTX has also been investigated in synergy with different bioactive compounds. The group of Serrano Jr. (Gomes et al., 2018) tested the combined effect of injecting Paclitaxel (PTX) loaded LDL-mimicking NPs and MTX-loaded NPs. In New Zealand white rabbits on atherogenic diet treated with both the NPs formulations, the regression of the lesion area and the intimal width reduction corresponded to 17 and 63%, respectively, compared to the group treated with PTX-NPs alone. The authors speculate that this athero-regression is mostly due to the macrophage reduction effect and not to an inhibition of SMCs migration into the intima.

Other possible applications of nanomedicine in the treatment of AS are based on gene regulation. Majmudar et al injected siRNA-containing polymeric NPs to silence the expression of C–C chemokine receptor type 2 (CCR-2), a key player in recruiting inflamed monocytes to atherosclerotic plaques; they observed reduced PET signals from <sup>89</sup>Zr-labeled dextran nanoparticles in aortic root when compared to mice treated with an irrelevant siRNA (Majmudar et al., 2013). Another example is the downregulation of the tissue inhibitor of metalloproteinase 3 by the use of miR-712, delivered by cationic lipid NPs targeting VCAM1. The treatment was performed in ApoE−/<sup>−</sup>

Frontiers in Bioengineering and Biotechnology | www.frontiersin.org

diet; ApoE, apolipoprotein

 E; w, week(s); NIRF, near infrared fluorescence;

 IL, interleukin; EMMPRIN, extracellular

 matrix

metalloproteinase

 inducer; Ldlr, low density lipoprotein receptor.

(Continued)

TABLE

2

Selected

reviews.

**121**

mice and was able to significantly attenuate the development of atherosclerotic plaques (Kheirolomoom et al., 2015).

### TOOLS AND IDEAS FOR ADDITIONAL RESEARCH

As reviewed above, fundamental and pre-clinical researches on AI-drugs-loaded NPs against AS are beginning to bloom, and there are also clinical trials for the (separated) use of NPs and AI drugs for regression or consolidation of AS plaques. More fundamental and translational research on the mechanisms underlying their action could inspire and motivate future more efficient clinical trials using AI-drug loaded NPs against AS.

Important future steps for the development of new NP-based theranostics strategies can arise from the study of interaction mechanisms between NPs and immune cells, with particular regard to what fate NPs and their components will face upon site deposition and cellular uptake. Investigations on these directions will remarkably improve the development of NPs rational designs aimed at specific accumulation and cargo smart activation.

Indeed, where, how and in which proportion NPs are internalized in the various subtypes of ICs is still not clear, also due to the different internalization routes observed even in close phenotypes (Lunov et al., 2011), nor it is clear how or if they are exocytosed (Oh and Park, 2014). In particular, are NPs mostly internalized by circulating monocytes that enter the lesions, or by resident macrophages in lesions, where NPs enter because of the enhanced permeability of inflamed endothelium?

Immortalized cell lines can be used for preliminary experiments. The most used are murine macrophage-like RAW 264.7 and J774, and human monocyte-like THP-1 and U937 (Luster et al., 1995; Qin, 2012; Andreu et al., 2017) reviews in particular the use of THP-1, unchanged or differentiated toward a M8 phenotype (especially M1), but also cites other human monocyte/macrophage models. Following these models, even though there are evident parallelisms, care must be taken for intrinsic differences: between cells originating from different organisms (Ingersoll et al., 2010; Matoba et al., 2017; Zhang et al., 2017); between immortalized cell lines and primary cells (Andreu et al., 2017); even between possible different differentiations ICs may undergo during regular cultures. Primary cells should be used in final tests, but in this case the last type of unwanted differentiations are even more critical [e.g., monocytes are extremely sensible and may be activated just by sole adhering on surfaces (Belcastro et al., 2015)]. For these reasons, a thoughtful characterization of their phenotypes (by visual inspection or, better, by markers analysis) should be carried out before every experiment.

Together with the internalization pathway, also the biodistribution of NPs deserves attention; a nice review for both these issues, which considers MC and M8s, can be found in Pentecost et al. (2016), with interesting discussions about dependencies on NP geometry and surface chemistry, targeting, control on M8 phenotype, and imaging. In addition, medium and long-term fates of nano-construct components (in particular its bioactive cargo) should be addressed; these details can indeed impact the efficacy of the researched treatment, but also the long-term effects on the patients' health.

Finally, various animal models have been developed for the different phases of AS, as reviewed in Emini Veseli et al. (2017) and Lee et al. (2017). A recently developed model of controllable and reversible hypercholesterolemia is based on transient knockdown of the hepatic LDL receptor (LDLR) by antisense oligonucleotides in wild type C57BL/6 mice, followed by its rapid restoration (Basu et al., 2018).

### CONCLUSION

Cardiovascular events, such as acute myocardial infarction and stroke, are often associated with erosion/rupture of arterial atherosclerotic plaques and superimposed thrombosis. These can cause arterial-vessel occlusion, downstream ischemia and necrosis, with subsequent heart failure and the whole clinical spectrum of vascular encephalopathies. Being AS an important source of morbidity and mortality, intense research efforts toward precision and personalized medicine in this field are motivated.

There are several reviews for selected aspects of the highly complicated issue of AS and its theranostics with NPs (**Table 2**). Here, we shortly presented the broad background necessary to understand the use of NPs for smart delivery and activation of anti-inflammatory drugs. We tried to focus on how basic science, together with the translational and clinical aspects, can address different portions of this, aiming at providing a better

### REFERENCES

answer for the underlying mechanisms of NPs intra-plaque internalization, as well as for the molecular and cellular processes at the bases of different treatments. We stressed that a more thorough understanding of these would help foresee possible side effects, even long term ones, and also the opportunity to employ similar techniques and (nano)tools to other pathologies.

While single studies can be carried out in specialized groups, a deeper understanding of concepts, mechanisms and applicability of the developed (nano)tools needs a highly interdisciplinary environment, involving e.g. cardiologists, biologists, physicists, chemists, engineers; professionals able to talk with each other, and fostering the formation of multi-faceted scientists.

### AUTHOR CONTRIBUTIONS

SL conceived the idea. SL and AC wrote the manuscript, with major contributions from RP, RC, and GV, starting from texts written in collaboration with LM, ME, and PD. All authors discussed, edited, and contributed to the manuscript.

### FUNDING

SL acknowledges funding from Scuola Normale Superiore (university coordinated project Visualization and theranostic of atherosclerotic PLAQUEs VIA compound NANOparticles, PLAQUEviaNANO, in collaboration with Scuola Superiore Sant'Anna) and Fondazione Pisa (project Nanotechnology for tumor molecular fingerprinting and early diagnosis, RST 148/16).

contributes to b-gamma-glutamyltransferase accumulation inside atherosclerotic plaques. J. Transl. Med. 13:325. doi: 10.1186/s12967-015- 0687-6

permeable endothelium in experimental atherosclerosis. Proc. Natl. Acad. Sci. 111, 1078–1083. doi: 10.1073/pnas.1322725111

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Cervadoro, Palomba, Vergaro, Cecchi, Menichetti, Decuzzi, Emdin and Luin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Frontiers of Medical Micro/Nanorobotics: in vivo Applications and Commercialization Perspectives Toward Clinical Uses

Fernando Soto<sup>1</sup> \* and Robert Chrostowski <sup>2</sup>

*<sup>1</sup> Department of Nanoengineering, University of California, San Diego, La Jolla, CA, United States, <sup>2</sup> Texas Materials Institute, The University of Texas at Austin, Austin, TX, United States*

The field of medical micro/nanorobotics holds considerable promise for advancing medical diagnosis and treatment due to their unique ability to move and perform complex task at small scales. Nevertheless, the grand challenge of the field remains in its successful translation towards widespread patient use. We critically address the frontiers of the current methodologies for *in vivo* applications and discuss the current and foreseeable perspectives of their commercialization. Although no "killer application" that would catalyze rapid commercialization has yet emerged, recent engineering breakthroughs have led to the successful *in vivo* operation of medical micro/nanorobots. We also highlight how standardizing report summaries of micro/nanorobotics is essential not only for increasing the quality of research but also for minimizing investment risk in their potential commercialization. We review current patents and commercialization efforts based on emerging proof-of-concept applications. We expect to inspire future research efforts in the field of micro/nanorobotics toward future medical diagnosis and treatment.

### Edited by:

*Gianni Ciofani, Politecnico di Torino, Italy*

#### Reviewed by:

*Arianna Menciassi, Scuola Sant'Anna di Studi Avanzati, Italy Edoardo Sinibaldi, Fondazione Istituto Italiano di Technologia, Italy*

\*Correspondence:

*Fernando Soto<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Nanobiotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology*

Received: *29 August 2018* Accepted: *25 October 2018* Published: *14 November 2018*

#### Citation:

*Soto F and Chrostowski R (2018) Frontiers of Medical Micro/Nanorobotics: in vivo Applications and Commercialization Perspectives Toward Clinical Uses. Front. Bioeng. Biotechnol. 6:170. doi: 10.3389/fbioe.2018.00170* Keywords: nanomedicine, medical translational research, in vivo, commercialization, microrobot, nanorobot

### INTRODUCTION

Imagine a world where robots the size of cells operate inside our body. This might sound like a science fiction story written by Isaac Asimov, or a visionary speech from Richard Feynman; however, it is conceivable that micro/nanorobotics will soon play a prominent role in medicine. (Wang, 2013; Wang et al., 2013; Li et al., 2017b) We use the term medical micro/nanorobots to refer to all nano- to micron-size structures (300 nm−300µm) capable of converting power sources into kinetic energy.Three groups of powered micro/nanorobots are mainly described. Biohybrid systems integrate synthetic nanostructures with motile microorganisms as the engine of the micro/nanorobot. (Kim and Tung, 2015; Ricotti et al., 2017; Bente et al., 2018; Palagi and Fischer, 2018). Chemically powered micro/nanorobots use asymmetric catalytic engines to selectively convert chemical fuels into locomotion (Chen et al., 2016; Nourhani et al., 2017; Schattling et al., 2017). Physically powered nanorobots convert external energy inputs (e.g., magnetic, ultrasound, or light fields) into translational motion based on engine geometry and material designs (Garcia-Gradilla et al., 2013; Dai et al., 2016; Bi et al., 2018; Pal et al., 2018).

The ability of micro/nanorobots to perform different task has been demonstrated at the laboratory scale, reporting their use for diverse proof of concept applications, including targeted cargo delivery (Solovev et al., 2010; Srivastava et al., 2018), fluid mixing (Orozco et al., 2013; Singh et al., 2015) and physical manipulation of micro objects (Cappelleri et al., 2014; Schuerle et al., 2017). The clinical aspirations of medical micro/nanorobots are still beyond the current capabilities of nanotechnology and bioengineering. Nevertheless, recent engineering breakthroughs have led to the successful in vivo operation of medical micro/nanorobots, illustrating initial proofs of concept for biopsy, delivery, healing and retention, as represented by the scheme in **Figure 1A.** While promising, these technological innovations are still challenging to translate into actual clinical therapies due to the safety concerns and the complexity of operating inside the human body.

This review focuses on the recent progress in the in vivo usage of micro/nanorobotics and on the efforts to commercialize and translate laboratory results into clinical applications. While there are several micro/nanorobot reviews addressing power and actuation principles (Sánchez et al., 2014; Teo and Pumera, 2016; Tu et al., 2017; Ren et al., 2018), fabrication procedures (Lin et al., 2015; Wang and Pumera, 2015; Jurado-Sánchez et al., 2017), and applications (Guix et al., 2014; Peng et al., 2017; Kim et al., 2018; Luo et al., 2018; Safdar et al., 2018), none of these reviews have addressed the crucial emerging clinical translations and potential commercial uses. We envision medical micro/nanorobotics as the frontier in treatment and diagnosis, potentially entailing benefits to human health by opening new therapies that are otherwise impossible to achieve.

### In vivo MICRO/NANOROBOTIC APPLICATIONS

The applications of micro/nanorobots for medical purposes in animal models are still limited compared to the large number of in vitro proofs of concept. However, the increase in the number of cumulative in vivo micro/nanorobotic publications (**Figure 1B**) and the high impact factor of the journals in which they are published (**Figure 1C**), both attest to the advancement in medical applications for micro/nanorobots, and to the encouraging level of interest within the scientific community (based on reviewed articles summarized in **Table 1**). Although there are multiple methodologies to power micro/nanorobots, we identify that only biohybrid (20%), chemical (30%), and physical systems (50%) have been used within inside living animals. The in vivo studies are detailed in the following sections based on their clinical aspiration or area of study.

### Delivery of Therapeutic and Imaging Agents for Cancer Therapy

Medical micro/nanorobotics hold great potential to deliver drugs with a higher degree of precision and speed when compared to passive diffusion methods. In general, this direction has been the motivation for in vivo applications (Erkoc et al., 2018). Thus, targeted delivery has benefited by recent in vitro developments in micro/nanorobotic chemotaxis (Peng et al., 2015; Shao et al., 2017) and material research using stimuli triggered drug release (Genchi et al., 2017; Rao et al., 2018). For example, magnetically guided nanorobots were used toward the delivery of fluorouracil medication for reducing tumor growth in a mice model. The released drug was externally triggered, allowing the nanorobotic platform to distribute a high amount of the therapeutic agent in a localized area of the tumor (Hoop et al., 2018).

Biohybrid nanorobots have also been used for targeted delivery of payloads inside living animals. Listeria monocytogenes has been used to deliver attached nanoparticles containing a payload of genes and proteins within a mouse. These payloads were used to monitor gene expression through differences in the luminescence produced within the different mouse organs (Akin et al., 2007). Magnetotactic bacterias, which naturally produce magnetic iron oxide nanoparticles, have been coupled with liposomes loaded with therapeutic payloads in vitro, as shown in **Figure 2A** (Taherkhani et al., 2014). More recently, these modified bacteria were guided using an external magnetic field to deliver the drug-loaded liposomes in vivo to a mouse tumor site (**Figure 2B**) (Felfoul et al., 2016).

The use of fully bio-engineered biohybrid micro/nanorobots without any inorganic/artificial components for carrying and transporting the therapeutic cargo has become possible through recent advances in synthetic biology. The use of genetically engineered bacteria, S. Typhimurium, have been reported to locally produce a therapeutic payload (α-emolysin E, a poreforming toxin) and to trigger the payload's release upon bacterial lysis. A small number of bacteria survive the lysis event, which allows for a continuous and cyclical delivery process controlled by an activator/repressor, synchronized lysis circuit (Din et al., 2016).

## Transport and Release of Cells

Micro/nanorobots have also been used toward delivering stem cells to a damaged location for tissue restoration. Magnetically guided microrobots have been reported toward carrying and delivering live cells to targeted areas in the body. In vivo transport and proliferation of HeLa cells in a nude mouse model demonstrated that the carried cells could be spontaneously released from the microrobot to the surrounding tissues and proliferate as shown in **Figure 3** (Li et al., 2018). These applications demonstrate that micro/nanorobots could serve as platforms for regenerative medicine and cell-based therapy, potentially proving to be especially useful in the later stages of life, when organs and systems start to fail. Moreover, there are still plenty of other promising in vitro applications to be developed for biohybrid microrobots, such as using a helical structure to guide a sperm toward an egg, for assisted fertilization (Magdanz et al., 2017).

### Retention of Payloads in the Gastrointestinal Tract

The aim of medical micro/nanorobotics is not only to deliver therapeutic payloads to a specific site but also, to retain the payloads within site as long as possible. In this direction, Wang's group has proposed the use of biodegradable zinc and magnesium powered microrobots that utilize gastric and intestinal fluids as fuels to promote cargo retention in the

stomach and intestinal tissues (Gao et al., 2015; Esteban-Fernández de Ávila et al., 2017a). This retention platforms have been applied toward pH neutralization of the gastric fluid (Li et al., 2017a) and for the treatment of a bacterial infection (Helicobacter pylori) in the stomach (Esteban-Fernández de Ávila et al., 2017b). This retention of the microrobot could be explained by direct piercing the surrounding tissue, or by an improvement in mass transport and nucleation due to the gas bubbles generated as means of locomotion for the microrobot, in an effect similar to effervescence.

Magnesium-based microrobots have also been designed with built-in delay activation, by using polymeric enteric coatings that activate the microrobot motion based on their thickness or environmental pH conditions. The polymeric coating only dissolves at neutral pH conditions found in the intestinal fluids resulting in localized ignition of the microrobot. The thickness of the coating allows to selectively localize the retention of the microrobots in different target sections of the gastrointestinal tract (the duodenum, jejunum, and ileum) (**Figure 4**) (Li et al., 2016). More recently, microrobots were integrated/loaded inside a pill matrix toward streamlining their administration with existing pharmaceutical protocols (Karshalev et al., 2018). In general, the use of magnesiumbased microrobots could benefit medical applications where autonomy and simplicity are desired. However, these chemicallypropelled microrobots might be limited to operate in largescale areas, such as the digestive system, as their depletion of propellant through bubble generation results in a short lifetime that could create unexpected complications in smaller capillaries.

### Wound Healing

The human body has diverse mechanisms and biological triggers for identifying wounds and repairing them. Nevertheless, these biological mechanisms can fall short when the wound is bleeding profusely, or there are not enough localized coagulant agents in the target region (Das and Baker, 2016). In this direction, medical micro/nanorobotics aim to simulate such systems by using active delivery toward fast and effective wound healing. Chemically-propelled calcium carbonate-based microrobots have been reported for delivering thrombin to halt the bleeding of wounds in the vasculature of mouse and pig models. The distribution mechanism relied on a combination of lateral propulsion, buoyant rise and convection (Baylis et al., 2015). Another reported approach consisted in the use of locomotive microrobots toward laser-based wound sealing. The high temperature generated by the laser-microrobot interaction produced localized collagen denaturation and melting, whereby a subsequent temperature decrease allowed condensation and wound closure (He et al., 2016).

### Biopsy

Other in vivo applications with potential implications in medicine are the ones targeting biopsy/surgery. Diverse in vitro platforms have been proposed toward precision micro/nanoscale surgery but still have not been translated to in vivo models

(Nelson et al., 2010; Xi et al., 2013; Kwan et al., 2015; Soto et al., 2015). Nonetheless, micro/nanorobotics could serve as a complement to current minimally surgical procedures, allowing unprecedented access into diseased tissues for biopsy analysis or therapeutic applications.

Microrobots with star-shaped grippers, which can reach narrow conduits in the body, have been used to excise tissue samples from a pig bile duct (Gultepe et al., 2012). Additionally, an initial proof of concept based on magnetic microrobots has demonstrated controlled navigation inside the eye of a living rabbit. Although this method has not been demonstrated directly for a surgical procedure, a magnetic coil system enabled the precise navigation of the untethered magnetic microrobots in the posterior eye section (Ullrich et al., 2013; Pokki et al., 2016). Ultimately, biopsy applications are fertile ground for further micro/nanorobotics research as the success of these applications depends on the ability of the robot to physically manipulate its environment and on the ability of the robot's controller to retrieve the robot. Both these problems have been studied much less extensively than propulsion.

### Local Mixing for Enhanced Thrombolysis

The potential modes through which a micro/nanorobot can physically manipulate its environment are not limited to merely excising tissue. Another recent trend in in vivo micro/nanorobots applications is the use of mixing effects to promote blood clot dissolution. In this case, magnetically actuated nanorobots loaded with tissue plasminogen activator were intravenously injected.

The vasculature flow drove the nanorobots to the blood clot. Once at their destination, the nanorobots were rotated by an external magnetic field. Their rotation generated local flow mixing which induces an increased interaction of the tissue plasminogen activator molecule with the blood clot interface, resulting in acceleration of thrombolysis (Cheng et al., 2014).

### Real-Time Imaging

From the reviewed articles, none of the chemically propelled micro/nanorobots are supported with real-time imaging, which introduces a severe limitation for understanding their therapeutic effect. 75% of biohybrid robots are supported with real-time imaging, but fluorescence is the only technique used (Akin et al., 2007; Park et al., 2013; Din et al., 2016). Physical robots are supported with the most diverse type of imaging techniques, but only 60% of articles are supported with real-time imaging. Represented techniques include the use of endoscopy and X-rays to detect microgrippers inside the gastrointestinal tract (Gultepe et al., 2012), use of optical camera to visualize movement inside the eye (Ullrich et al., 2013; Pokki et al., 2016), and fluorescence imaging techniques to track the position of magnetically actuated helical microrobots inside the peritoneal cavity of a mouse (Servant et al., 2015) or subcutaneously (Li et al., 2018). Moreover, a dual imaging approach was used to detect biodegradable magnetic microhelix nanorobots in mice. Fluorescence imaging was used to detect the nanorobot's position inside the subcutaneous tissue, and the intraperitoneal cavity of a mouse and magnetic resonance-based imaging was used to detect the nanorobot's position inside the mouse's stomach, as shown in **Figure 5** (Yan et al., 2017). Future research such address this important parameter, a key aspect for medical micro/nanorobotic use in clinical uses will rely on individual or population tracking, with consideration of tissue background signal (Medina-Sánchez and Schmidt, 2017; Vilela et al., 2018; Wang et al., 2018a).

### Toxicity

Most of the review micro/nanorobot studies used in animal models, provide only qualitative analyses of safety and toxicology based on histological assays. Achieving accurate targeted delivery requires an understanding of how foreign materials accumulate throughout the body and how to minimize the distribution of the administered micro/nanorobots to non-target tissue, establishing their specific effect on health. Each micro/nanorobot design has different safety concerns. Biohybrids could infiltrate and proliferate in undesired ways. Chemically propelled micro/nanorobots might change the local chemical environment which could have a significant effect on the microbiome of the gastrointestinal tract. For physical micro/nanorobots, most of the materials themselves pose a danger, as they are rigid and nondegradable. Despite these differences, researchers could report more relevant parameters to address the toxicity even at this early stage. Potential parameters to be considered include: the number of motors used for treatment (number or grams) indicated the escalation of units/dosage (limit for toxicity and inefficacy), but most importantly the distribution and toxicity of the nanorobot's constitutive materials within the living system.

### Administration and Retrieval

The administration and retrieval strategy of nanorobots is not discussed in length in the reviewed articles, which present as the most common administration method injection (60%), followed by oral administration (30%), catheter (5%), and topical administration (5%). Regarding the retrieval strategy for micro/nanorobots, both biohybrid and chemical systems were considered to be biodegradable by the authors. Although, in some cases, the fate of their synthetic components was not explicitly stated or demonstrated. For physically powered robots, in most cases, the fate of the nanorobot structure was not clearly stated,

only three articles provide a retrieval mechanism and one other is biodegradable. One plausible solution for retrieval consists of the use of a magnetic catheter to enable both the deployment and retrieval of microrobots in clinical practice (Iacovacci et al., 2018). We note that recent in vitro research efforts have described micro/nanorobot systems that are fully biodegradable (Peters et al., 2015; Chen et al., 2016; Yan et al., 2017; Bozuyuk et al., 2018; Wang et al., 2018c).

### MICRO/NANOROBOTS: STEPS TOWARD CLINICAL TRANSLATION

Basic science research is fundamental for creating both breakthrough medical advances and economic growth. Nevertheless, most basic science research will not achieve clinical translation. This challenge arises, in part, from the prolonged time required to go from basic research to clinical trials. Moreover, the conclusion of most trials indicates that most of the new technologies are less effective or more harmful than the current standards of care. This is a lesson to be learned in the micro/nanorobotic community. It is essential to identify strengths/weaknesses and remain objective about their relevance for later development.

The potential of the current generation of micro/nanorobotics is based on their translational motion and mixing capabilities. The idea of "smart" nanorobots that explore their surroundings and respond to environmental stimuli, commonly described in the field, is still beyond the current capabilities of micro/nanorobotics. Crucial questions of environmental manipulation, micro/nanorobot retrieval, and toxicology still need to be addressed. Indeed, we are far away from a killer app, but this should not be considered uncommon or discouraging to current researchers in the field. As previously mentioned, medical technologies have a significant time delay from lab to the clinic/commercialization, in many cases, taking several decades.

Nevertheless, considerable progress has been achieved in the field. It took less than a decade to go from the initial proof

of concept using chemically propelled nanowires in peroxide (Paxton et al., 2004) to the recent explosion of in vivo studies over the past 3 years. In order to reach the inflection point toward clinical translation, it is necessary first to standardize the methodologies and reproducibility of micro/nanorobotics research and to actively consider their commercialization outlook. Scientists without clear monetary value proposition rarely see their discoveries materialize.

(i: control, ii: after 6 h, and iii: after 12 h of administration. Reprinted with permission from Li et al. (2016). Copyright 2016 American Chemical Society.

### Reproducibility and Standardization

Commonly, the performance of micro/nanorobotic medical technology in vitro is reported by measurements of movement, such as their velocity and directionality, or on indirect methods such as measuring the mean squared displacement of tracer

particles or comparing a clinical outcome (e.g., the percentage of dead cancer cells) vs. passive diffusion. Although these methods serve to validate early proof-of-concept studies, they are not good predictors for successful clinical translation.

The variability between currently used in vivo micro/nanorobotic platforms makes it almost impossible to provide a comparative or quantifiable analysis of their efficiency vs. current standards of care. Moreover, there cannot be a clear definition of clinical efficiency when there is no minimum standardized information for reporting micro/nanorobotic research. Therefore, we need clear information standards in reporting experimental methodology, characterization, and results, which will be key for the successful translation of multidisciplinary research into improved health outcomes.

Some journals have implemented reporting summaries as a requisite for submission (Faria et al., 2018), which has shown to increase the quality of reporting for preclinical biomedical research (Han et al., 2017). In general, an experimental finding can be considered to have a stronger case for translation when the experiments supporting it have been carried out using blinding and randomization, and when, most importantly, those findings have been replicated in other laboratories apart from the inventors' group. Currently, no study presented in this review covers these standards. In this direction, here we suggest guidelines (measures that should be reported in parenthesis) for reporting micro/nanorobotic research that should allow better comparisons, both qualitative and quantitative, of micro/nanorobot performance in addition to already established checklists for safety and toxicology. The goal of the checklist is not to propose research trends or criticize current research, as new technologies start without clear standards. Instead, the homogeneity of experimental reporting can facilitate comparison, improve experimental design, and increase reproducibility between various micro/nanorobotic platforms.

The justification of the power source used to provide energy/movement to the micro/nanorobot could help to establish the unique advantage of each method. Material characterization of the micro/nanorobot, including their size distribution (length units), average lifetime of propulsion (time unit), surface charge (zeta potential), and storage lifetime (days), can help to plan into eventual fabrication and transport in medical uses. Moreover, is important to quantify the performance of the nanorobotic platform by evaluating the therapeutic effect based on experiment dependent parameters. For drug delivery applications, an important parameter is the administered and delivered dose (% by mass). For retention studies the number of nanomotors localized in the target region (% of administered population). For biopsy applications, the micro/nanorobot force applied to a specific area of tissue (Pascal). Moreover, the micro/nanorobot therapeutic effect should be compared against the state-of-the-art treatment, not only passive diffusion. Finally, parameters covered through the review includes, imaging (real time localization), toxicity (folllowing establish guidelines), Administration route (Target Area and method of insertion), and retrival region (% of administered population) could streamline their tranlatio into exisitng therapies.

### Intellectual Property and Commercialization

Clinical efficacy alone is not enough to warrant translation to market. The transition of new technologies from lab to market requires extensive research and development costs, overcoming regulatory barriers, and most importantly, having the right commercialization potential. The potential customer for micro/nanorobotics' technology is not the general population or the physicians, but rather pharmaceutical companies and insurance providers. Therefore, micro/nanorobotics research should consider their commercialization opportunities from the research stages, in order to provide tangible therapies that reduce cost, increase throughput efficiency and productivity.

Minimizing the investment risk can be achieved by protecting the intellectual property of the new technological developments (which is one of the most valuable assets for high-tech companies) and establishing a clear business model with identifiable revenue streams. New technological developments are commonly initiated using patents filed by research groups through their university system, which are later licensed to emerging start-up companies or larger corporations. The prohibitive cost of prototyping and clinical trials is initially financed by non-dilute financing, which includes government research and industry grants, and at later stages by investment from venture capital and industry partnerships.

Tracking the filing of relevant patents, and the formation of relevant startups, provides useful insight into the progress made in identifying potential markets, competitors and consumer needs. **Figure 6** traces the accumulated United States applied and granted patents in nanorobotics, while also separating them into the categories of fabrication, actuation, imaging, and application. The patent filing data were collected manually, as keyword searches do not accurately capture the state of nanorobotic technology due to the multiple names used to refer to nanorobots in the literature (micro/nanobot, micro/nanorobot, micro/nanorocket, micro/nanoengines, micro/nanomotor, micro/nano-swimmer, active colloid). Instead, the search was conducted by reviewing the output of the most prolific nanomotor research groups in the field that have published results in animal models and by reviewing relevant Google patents.

The first patents published consisted of the fabrication methods commonly used for parallel mass fabrication using template platforms, consisting of nanorod structures (Stonas et al., 2005; Natan et al., 2007; Fan et al., 2017b; Odell et al., 2018), microcoils (Schmidt et al., 2010; Jeong et al., 2018), tubular structures (Yao et al., 2015; Wang et al., 2016a), emulsions (Percec et al., 2017), biohybrids (Schmidt and Leibniz-Institut Fuer Festkoerper-Und Werkstofffor Schung, 2017), and colloid chains (Duan et al., 2017).

The next wave of patents consists of tracking/imaging nanorobots using magnetic (Martel et al., 2015; Muntwyler et al., 2017; Odell et al., 2017), optical (Benaron et al., 2008), and ultrasound methods (Mattrey et al., 2017).

The final, most recent wave, has focused on applications for these micro/nanorobots, including for intracellular delivery by nanospearing (Cai et al., 2011) exploration of subterranean geophysical formations and oil retrieval (Kamal et al., 2015), nanomotor-based patterning of surface microstructures (Wang et al., 2016b), transporting, positioning and assembling nanorobots using electric twisers (Chien et al., 2017; Fan et al., 2017a), capturing and isolating of target biomolecules and living organisms using microrobots (Wang et al., 2018b).

There are only a few active companies working toward the commercialization of nanorobots for use in medical applications. Most notably, Nelson's group has spun off two companies. The first was Aeon Scientific, based on the electromagnetic manipulations systems developed to guide magnetic motors. However, they have recently shifted their focus to developing new manipulation systems for catheters. Indeed, it is not surprising that the technology developed around micro/nanorobots could have applications that were never envisioned at the beginning.

The second, Swiss Magnetibox formed in 2014 is based on wireless tools for actuating and imaging micro/nanomachines using magnetic fields. They primarily sell microscopy equipment coupled with magnetic actuation systems and rod shape microrobots toward expanding basic science in micro/nano robotics research. Although these systems are not specifically designed for in vivo use, they provide the necessary tools to test proof of concept applications (Schuerle et al., 2017).

Finally, Weingberg medical physics has developed an image-guided therapy for targeted delivery of magnetic micro/nanorobots that uses ultra-fast MRI to generate magnetic fields for imaging and manipulation of the magnetic nanorobots. They have demonstrated diverse in vitro applications using the mechanical force of the nanorobots to dislodge bacteria biofilm using rotating nanorobots (Mair et al., 2017) and drilling into mice's brain post-mortem (Jafari et al., 2019).

The mass fabrication of nanorobotics is one of the first challenges to be addressed toward tangible translation to market.

Reproducibility and availability of templates will be significant hurdles that must be solved with traditional micro/nanorobotic fabrication techniques. New methods based on 3D printing and two-photon lithography could allow for complex designs such as microhelices or cell carriers. Although, the cost and slow production throughput of these methods may limit the use of such intricate designs. Moreover, new methods of characterization and manipulation of individual nanorobots are necessary to ensure the quality of each batch. In this regard, nano-manipulation systems are ideal for transporting, inspecting and testing nanorobotic designs (Wang et al., 2015; Lu et al., 2017; Meng et al., 2017; Zhang et al., 2018). Once these technical capabilities are well established, the reduction in the cost to reach the market will allow new companies to spring up and provide innovative medical value propositions.

A possible path toward early adoption of micro/nanorobots in the clinic could be achieved by offering micro/nanorobotic technology as a complementary tool to existing medical procedures. For example, their integration with current oral delivery platforms such as pills, allow for dose escalation by increased retention of therapeutic payloads at the mucosae wall. Although special consideration should be placed in therapies where the primary therapeutic effect is based on systemic distribution, as in this case, the dose escalation proposed by micro/nanorobotic platforms is not desired. Moreover, micro/nanorobots used for tissue biopsy and suturing veins could be integrated with a catheter and mili surgical robotics, allowing the larger robots to reach scale ranges where their size normally does not permit them to operate. The possibilities discussed here are driven by the need to improve patients' outcomes after surgical procedures including reduced hospital stay, lower chances of infection, and minimum scarring. We should note that it is not likely that micro/nanorobots will be used for preventive care or as a chronic treatment since the sustained introduction of synthetic objects into the human body might produce unknown repercussion due to possible accumulation.

### REFERENCES

## FUTURE OUTLOOK

The field of medical nanorobotics has achieved considerable advances. However, several issues and challenges must be addressed before micro/nanorobots could have real-world clinical applications. The goal of the in vivo model is not only to evaluate the therapeutic efficiency of the platforms, but to identify the clinical risk, as evaluating off-target effects of nanorobots is as essential as evaluating efficacy. Indeed, there is a discrepancy between the aspirations of medical nanorobotics and reality, as the legacy of science fiction has set the conceptual boundaries of what to expect long before scientists could. The manufacturing of the micro/nanostructure engines must be optimized, with special consideration for material biocompatibility and degradation, to address in vivo safety concerns.

Furthermore, proper standards should be established to clarify the advantages of micro/nanorobot therapies over traditional methods which already fulfill FDA standards. Nonetheless, micro/nanorobotics might potentially improve medical diagnosis and treatment. We should also consider that the designs and aspirations of a small group of scientist and engineers could soon affect the lives of millions of people directly and profoundly, therefore it is essential to consider the economic, social, and ethical implications of the use of medical nanorobotics. These implications are likely to be on par with those of the most significant technological revolutions.

### AUTHOR CONTRIBUTIONS

FS and RC wrote and revised the manuscript and approved it for publication.

### ACKNOWLEDGMENTS

FS acknowledges the funding from the UC MEXUS-CONACYT program. RC acknowledges his funding from the Cockrell School of Engineering at The University of Texas at Austin.

microrobots for implantation: corrosion, cell culture, and in vivo animal tests. J. Biomed. Mater. Res. B Appl. Biomaterials 105, 836–845. doi: 10.1002/jbm.b.33618

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Soto and Chrostowski. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Transdifferentiating Astrocytes Into Neurons Using ASCL1 Functionalized With a Novel Intracellular Protein Delivery Technology

Meghan Robinson<sup>1</sup> , Ian Fraser 1,2, Emily McKee<sup>2</sup> , Kali Scheck <sup>3</sup> , Lillian Chang<sup>4</sup> and Stephanie M. Willerth1,2,5,6,7 \*

<sup>1</sup> Division of Medical Sciences, University of Victoria, Victoria, BC, Canada, <sup>2</sup> Biomedical Engineering Program, University of Victoria, Victoria, BC, Canada, <sup>3</sup> Biology Program, University of Victoria, Victoria, BC, Canada, <sup>4</sup> Biochemistry Program, Bates College, Lewiston, ME, United States, <sup>5</sup> Mechanical Engineering, Faculty of Engineering, University of Victoria, Victoria, BC, Canada, <sup>6</sup> Center for Biomedical Research, Faculty of Engineering, University of Victoria, Victoria, BC, Canada, <sup>7</sup> International Collaboration for Repair Discovery, University of British Columbia, Vancouver, BC, Canada

Edited by:

Gianni Ciofani, Politecnico di Torino, Italy

#### Reviewed by:

Stefania Moscato, Università degli Studi di Pisa, Italy Madoka Suzuki, Osaka University, Japan

> \*Correspondence: Stephanie M. Willerth<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Nanobiotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology

Received: 12 September 2018 Accepted: 31 October 2018 Published: 21 November 2018

#### Citation:

Robinson M, Fraser I, McKee E, Scheck K, Chang L and Willerth SM (2018) Transdifferentiating Astrocytes Into Neurons Using ASCL1 Functionalized With a Novel Intracellular Protein Delivery Technology. Front. Bioeng. Biotechnol. 6:173. doi: 10.3389/fbioe.2018.00173 Cellular transdifferentiation changes mature cells from one phenotype into another by altering their gene expression patterns. Manipulating expression of transcription factors, proteins that bind to DNA promoter regions, regulates the levels of key developmental genes. Viral delivery of transcription factors can efficiently reprogram somatic cells, but this method possesses undesirable side effects, including mutations leading to oncogenesis. Using protein transduction domains (PTDs) fused to transcription factors to deliver exogenous transcription factors serves as an alternative strategy that avoids the issues associated with DNA integration into the host genome. However, lysosomal degradation and inefficient nuclear localization pose significant barriers when performing PTD-mediated reprogramming. Here, we investigate a novel PTD by placing a secretion signal sequence next to a cleavage inhibition sequence at the end of the target transcription factor–achaete scute homolog 1 (ASCL1), a powerful regulator of neurogenesis, resulting in superior stability and nuclear localization. A fusion protein consisting of the amino acid sequence of ASCL1 transcription factor with this novel PTD added can transdifferentiate cerebral cortex astrocytes into neurons. Additionally, we show that the synergistic action of certain small molecules improves the efficiency of the transdifferentiation process. This study serves as the first step toward developing a clinically relevant in vivo transdifferentiation strategy for converting astrocytes into neurons.

Keywords: reprogramming, transcription factors, neuroscience, small molecules, drug delivery

## INTRODUCTION

Transdifferentiation converts mature cells from one specialized cell type into another by varying gene expression patterns (Tanabe et al. 2015). These expression patterns are controlled by transcription factors, regulatory proteins which bind to DNA promoter regions to activate transcription of key developmental genes. Ectopic expression of these transcription factors in somatic cells can change cell fates without the need for inducing a pluripotent state (Tanabe et al., 2015). This process avoids the risk of tumorigenesis associated with transplanting pluripotent stem cell-derived therapies in vivo, where incomplete differentiation leads to uncontrolled proliferation (Gordeeva and Khaydukov, 2017). The final genetic signatures of these transdifferentiated cells are stable and not restricted to the lineage of the original cell type, allowing cells to be sourced directly from endogenous tissue local to the site of injury (Tanabe et al., 2015). Performing in vivo transdifferentiation would eliminate the need for cell transplantation and immunosuppression depending on the target application.

Ectopic transcription factor expression can be efficiently accomplished through insertion into the genome by viral vectors encoding the amino acid sequence of the desired transcription factor. However, this method is not safe for in vivo use in clinical trials due to the risk of activating oncogenes by random DNA insertion into the host genome (Li et al., 2002; Modlich et al., 2006; Medvedev et al., 2010). Non-integrating vectors have been developed capable of transdifferentiation, but at lower efficiencies. Some wellknown examples of non-integrating methods include vectors composed of single-stranded RNA, such as the Sendai virus, circular DNA, such as plasmid vectors and minicircles, and mRNA vectors, which reside in the cytoplasm where they are translated into proteins. Despite these improvements in non-viral delivery, vector-based transdifferentiation strategies still face challenges such as transgene silencing, inflammation due to the presence of residual bacterial DNA, and poor nuclear uptake due to their physical size (Hardee et al., 2017).

The search for non-integrating, non-viral reprogramming methods has led to the discovery of transdifferentiation using microRNAs and small molecules (Ma et al., 2017). In 2011, it was determined that the microRNAs miR-124 and miR-9/9<sup>∗</sup> were sufficient to induce neurons from fibroblasts (Ambasudhan et al., 2011; Yoo et al., 2011;). Later in 2014 Zhu et al. discovered that viral-mediated expression of octamer binding transcription factor 4 (OCT4) was sufficient to transdifferentiate somatic cells into neural stem cells when combined with small molecules that promote lineage-specific signaling (Zhu et al., 2014). Morphogens and bioactive small molecules can potentially enhance the action of transcription factors by improving chromatin accessibility, targeting key signaling pathways, and regulating metabolism (Lin and Wu, 2015). These results led several groups to identify combinations of small molecules capable of transdifferentiating somatic cells such as fibroblasts, astrocytes and even glioblastoma cells into neurons (Cheng et al., 2014; Hu et al., 2015; Li et al., 2015; Zhang et al., 2015, 2016; Gao et al., 2017; Lee et al., 2018). Although the transdifferentiation methods differ, these small molecule and microRNA protocols share a common thread in that they produce mainly one neuronal subtype–medium spiny neurons in the case of microRNAs, or interneurons in the small molecules methods with the exception of the glioblastoma-transdifferentiated neurons which resulted in immature post-mitotic neurons. The inability to produce the neuronal subtypes which are lost in neurodegenerative disorders like Parkinson's Disease, Alzheimer's Disease, amyotrophic lateral sclerosis (ALS), Huntingdon's Disease, spinal cord injury and blindness, represents a major limitation in current small molecule and microRNA reprogramming protocols.

Researchers have begun to explore protein transduction domains (PTDs), also known as cell penetrating peptides (CPPs), as tools for transdifferentiation to address these limitations (Zahid and Robbins, 2012). These CPPs are small peptides that penetrate the cell membrane to deliver bioactive macromolecules. The first PTD was derived from the transactivator of transcription (TAT) protein sequence of the human immunodeficiency virus (HIV), which binds to a phospholipid component of the inner surface of the cell membrane through a cationic poly-arginine sequence to enable cellular internalization (Yang et al., 2012b; Gordeeva and Khaydukov, 2017). Other examples of PTDs include Penetratin, a cationic arginine-rich sequence taken from the Drosophila protein antennapedia homeotic transcription factor (Antp), and CADY, a sequence combining arginine residues with aromatic tryptophan, whose helical conformation within the cellular membrane favors cellular uptake (Crombez et al., 2009; van den Berg and Dowdy, 2011). PTDs have the potential to be fitted with binding domains to target specific cell populations, making them ideal tools for in vivo reprogramming of endogenous cells (van den Berg and Dowdy, 2011; Zahid and Robbins, 2015).  In 2014, Hu et al. successfully transdifferentiated neuropilin-1 expressing retinal pigmented epithelial cells into neurons using a C-end rule PTD fused to the transcription factor self-determining region Y-box2 (SOX2) (Hu et al., 2014).

Little work has been done to develop such protocols to date despite the promise of PTDs as a tool for in vivo transdifferentiation. A search using the Pubmed database revealed only one further publication using PTD delivery where Islas et al. transdifferentiated human dermal fibroblasts into immature contractile cardiomyocytes (Islas et al., 2012). They fused TAT-PTDs to two cardiac transcription factors, erythroblastosis virus E26 oncogene homolog 2 (ETS2) and mesoderm posterior 1 (MESP1). Treatment with these TAT-PTDs followed by treatment with cardiac gene-inducing morphogens activin A and BMP2 resulted in successful transdifferentiation of fibroblasts into functional cardiomyocytes. Morphogen treatment significantly improved induction of key cardiac genes, whereas addition of the morphogens alone had no effect, further illustrating the transdifferentiation potential of combining morphogens or bioactive small molecules with transcription factors.

In this work, we developed a protocol using a novel PTD to transdifferentiate somatic cells into interneurons. We hypothesized that the delivery of the neural transcription factor achaete scute homolog 1 (ASCL1) by this novel PTD design in combination with the action of bioactive small molecules would promote neuronal transdifferentiation of astrocytes, the most abundant cell type in the central nervous system. ASCL1 is a transcription factor and master regulator of neurogenesis (Vasconcelos and Castro, 2014). Its ability to bind readily to closed chromatin and promote the appearance of new regions of open chromatin allows coordinated activation of previously silenced genes (Iwafuchi-Doi and Zaret, 2014; Raposo et al., 2015). In 2015, Liu et al. showed that in vivo viral delivery of ASCL1 to mouse dorsal midbrain astrocytes was sufficient for neural transdifferentiation, with 76.8% of astrocytes adopting a neural fate by day 10 (Liu et al., 2015). ASCL1 was also chosen for its versatility. While the action of ASCL1 alone leads to an interneuron fate, it is known to induce dopaminergic, cholinergic or serotonergic neuronal subtypes when combined with dopaminergic factors LMX1B/NURR1 (Addis et al., 2011; Theka et al., 2013), cholinergic factors MYT1L/BYRN2/LHX3/HB9/ISL1/NGN2 (Son et al., 2011), or serotonergic factors FOXA2/LMX1B/FEV (Xu et al., 2016). The PTD employed for these experiments was designed by iProgen Biotech Inc. to take advantage of the cell's innate retrograde transport pathway by integrating a secretion signal peptide and a cleavage inhibition peptide to a cargo protein (Lee et al., 2013). Initially the PTD integrates itself into the cell surface membrane due to its amphipathic nature. Once internalized, the secretion signal peptide preferentially binds to its native binding partner, translocon, a complex of proteins tasked with translocating polypeptides with a targeting signal sequence into the interior space of the endoplasmic reticulum (ER). As the translocon retrogrades back through the ER into the cytoplasm, so does the cargo. This combination of secretion signal peptide and cleavage inhibition peptide, termed IPTD, shows greatly improved delivery of proteins into the cytoplasmic space over conventional PTDs such as TAT and poly-arginine. In 2016, we showed that ASCL1-IPTD could efficiently generate neurons from human induced pluripotent stem cells (hiPSCs) (Robinson et al., 2016). We show here that ASCL1-IPTD in combination with the small molecules LDN193189, SB431542, DAPT and valproic acid can rapidly reprogram astrocytes into mature GABAergic and glutamatergic interneurons with high efficiency, and that this process is unique to the transcription factor ASCL1. We also show that in the absence of these small molecules ASCL1-IPTD cannot transdifferentiate into a neuronal fate, but does generate a divergent cell fate of myoblasts. Because of its inherent versatility in generating other neuronal subtypes, this ASCL1-IPTD protocol serves as a starting point for further development through the functionalization of subtypespecific factors with IPTD to generate subtypes of neurons. Additionally, the use of IPTD allows for future adaption with specific tissue-targeting motifs. This method can be applied as novel treatment for neurodegenerative diseases or spinal cord injury by transdifferentiating astrocytes into specific neuronal populations as a way to restore function.

### MATERIALS AND METHODS

### Cell Culture

Human fetal astrocytes (ScienCell cat#1800) were cultured on poly-L-lysine coated plates in Astrocyte Media (ScienCell). Media was changed every 2 days and cells were passaged 1:6 when 90% confluent using trypsin-EDTA (ScienCell). 10% fetal bovine serum (FBS, Gibco) was added to media for 6 passages (6 weeks) as previously described to purify astrocyte cultures of neural progenitors (Zhang et al., 2015). Astrocytes were incubated for 24 h on an orbital shaker plate at 200 rpm as previously described to purify low passage (P2) astrocytes of neural progenitors (Lian et al., 2016). Mouse embryonic fibroblasts (MEFs) were purchased from MTI GlobalStem and thawed onto poly-L-ornithine (PLO)/laminin (Sigma Aldrich) substrates in high glucose DMEM (Gibco) supplemented with 10% FBS.

### Astrocyte Reprogramming

Astrocytes were grown to confluence before reprogramming. Cells were cultured on poly-L-lysine (PLO)/laminin substrates and media was changed to high glucose Dulbecco's Modified Eagle Medium (DMEM) with 2 mM Glutamax (Gibco), 1% N2/B27 (ThermoFisher Scientific), 1% Human Serum Replacement 3 (Sigma Aldrich), and 1% penicillin/streptomycin (ThermoFisher Scientific) for cellular reprogramming experiments. 15µg/mL ASCL-IPTD (iProgen Biotech) was supplemented for all 12 days, with 50% media changes every 2 days. For days 1-2, 5µM SB431542 (Stemcell Technologies) and 0.25µM LDN193189 (Stemcell Technologies) were added to cultures. For days 3–12 the following small molecules were added: forskolin (F, 10µM, Medchem Express), CHIR99021 (C, 1.5µM, Medchem Express), ISX9 (I, 20µM, Medchem Express), DAPT (D, 5µM, Medchem Express). The combinations tested were C, F, D, I, CF, CI, CD, FI, FD, ID, FCI, FCD, CID, FID, CFID. For days 3–6, 0.5µM valproic acid (Stemcell Technologies) was also added to improve chromatin accessibility.

### Fibroblast Reprogramming

MEFs were seeded 40,000 cells/cm<sup>2</sup> on PLO/laminin and cultured for 24 h in high glucose DMEM + 10% FBS to adhere. Media was then switched to high glucose DMEM, 1% N2/B27, 1% Human Serum Replacement 3, 2 mM GlutaMAX, 1% penicillin/streptomycin, 15µg/mL ASCL1-IPTD and 20 mM forskolin for 14 days, with media changes of 50% every 2nd day.

### Immunocytochemistry

Immunocytochemistry was performed as described previously (Agbay et al., 2018). Cells were fixed with 3.7% formaldehyde/phosphate buffered saline (PBS) solution for 1 h, then permeabilized in 0.1% Triton X-100 (Sigma Aldrich) in PBS for 45 min at 4◦C, then blocked with 5% normal goat serum (NGS, Sigma Aldrich) in PBS for 2 h at 4◦C. Cells were then incubated with the primary antibodies mouse anti-TUJ1 (Millipore), rabbit anti-MYH3 (Abcam), rabbit anti-ASCL1 (Abcam), rabbit anti-DCX (Abcam), rabbit anti-SOX2 (Abcam), rabbit anti-MAP2 (Abcam), rabbit anti-GLUT1 (Abcam), rabbit anti-GAD65/67 (Abcam), mouse anti-NEUN (Novus Biologicals), mouse anti-TH (Stemcell Technologies), or goat anti-CHAT (Millipore), diluted 1:500 in PBS, and incubated overnight at 4◦C in the dark. The next day cells were washed with PBS three times for 15 min each, and incubated with secondary antibodies goat anti-mouse IgG AlexaFluor488 (ThermoFisher Scientific), goat anti-rabbit IgG AlexaFluor568 (ThermoFisher Scientific), or donkey anti-goat AlexaFluor405 (Abcam) diluted 1:200 in PBS for 4 h at 4◦C. Cells were washed again three times as previously described. For cells counterstained with DAPI nucleic acid stain, a 300 nM DAPI solution (Invitrogen) in PBS was added to the cultures after the final wash and incubated for 3 min, followed by rinsing with PBS. Cells were visualized and imaged with a Cytation5 (Biotek) Imaging platform at 20 × magnification. Fluorescently labeled cells were visualized with Biotek Dapi (377/477), GFP (469/525), and Texas Red (586/647) filter cubes, using dichroic mirror-based wavelength selection.

### Flow Cytometry

Flow cytometry was performed as previously described (Montgomery et al., 2015). The cells were stained for SOX2-PE Mouse IgG2A (R&D Systems) with IgG2A-PE isotype control (R&D Systems), TUJ1-PerCP Mouse IgG2A (R&D Systems) with IgG2A-PerCP isotype control (R&D Systems), MAP2-PE Mouse IgG1 (Clone AP20, Milli-Mark) with Mouse IgG1 PE isotype control (R&D Systems), and NEUN-PE Mouse IgG1 (Clone A60) with Mouse IgG1 PE isotype control (R&D Systems). Cells were collected rinsed with PBS 3 times, followed by incubation in fixation/permeabilization buffer (R&D Systems) for 10 min, and 3 more washes with PBS. Next cells were resuspended in permeabalization/wash buffer (R&D Systems) with each antibody, diluted as per manufacturer's instructions, for 45 min at 4◦C in the dark. Cells were then washed in permeabilization/wash buffer 3 times. Data was collected using the Millipore Guava EasyCyte HT flow cytometer. Cells were gated to exclude small debris and clustered cells, and a maximum of 5,000 gated events were collected for each sample. All analysis was completed using GuavaSoft EasyCyte software.

### Quantification of Transdifferentiated Neurons

Cells were first immunostained with the neuronal cytoskeletal marker TUJ1 to quantify transdifferentiated neurons in each group of the small molecule screen. Next, a grid of 5 × 5 images was taken in each of 3 wells for each group (25 images per well, n = 3 biological replicates). Cells were visualized with a Leica DMI 3000B microscope equipped with an XCite Series 120Q fluorescent light source and QImaging RETIGA 2000R camera at 100 × magnification, and images were captured using QCapture Software 2.9.12 with a GFP filter (blue/470, Leica Systems) and a DAPI filter (400, Leica Systems) and dichroic mirrors (500, Leica Systems). Cells were counted manually if they were positive for TUJ1 and possessed a small rounded soma, with at least 2 neuritic processes. Total cells were counted by DAPI stain using ImageJ: images were made binary and a watershed was applied to separate nuclei that were touching by a single pixel width, and each cell was counted as a particle under the Analyze-Particles Menu.

### Statistical Analysis

Statistical analysis was performed by a one-way ANOVA followed by a one-tailed Student's t-test with a 95% confidence level (α = 0.05). Results are presented as the mean ± standard deviation. All experiments were carried out with a biological n = 3.

### RESULTS

### Transdifferentiation Protocol Developmental

We sought to characterize the transdifferentiation strength of ASCL1-IPTD before designing our protocol. We executed a well-known protocol where ectopic expression of ASCL1 alone converts mouse embryonic fibroblasts (MEFs) into neurons, and at lower expression levels, into skeletal myoblasts (Treutlein et al., 2016). The addition of the small molecule forskolin, an activator of cyclic adenosine monophosphate (cAMP) signaling, dramatically improves the efficiency of this protocol and so it was tested in parallel to determine if conversion efficiency could be similarly improved with ASCL1- IPTD (Shi et al., 2016). After 14 days of treatment, skeletal myoblasts were generated instead of neurons as indicated by myosin heavy chain 3 (MYH3) expression, with or without the addition of forskolin (**Figure 1)**. Increasing the concentration of ASCL1-IPTD 5-fold also had no effect in generating neurons (**Supplemental Figure 1**).

We concluded that the strength of ASCL1-IPTD was not meeting the threshold expression level necessary for neuronal transdifferentiation. Accordingly, we adopted the strategy to first lower this threshold by repressing astrocytic gene expression using small molecules to inhibit transforming growth factor β1 (TGF-β1)/SMAD and bone morphogenic protein (BMP)/SMAD signaling pathways, which are jointly responsible for astrocyte commitment and maintenance (Stipursky and Gomes, 2007; Chambers et al., 2009; Yang et al., 2013; Zhang et al., 2015). Human cerebral cortex astrocytes were primed for 2 days followed by screening of a set of small molecules known to have neuronal transdifferentiation potential: CHIR99021, an inhibitor of glycogen synthase kinase 3 (GSK3) signaling; DAPT, an inhibitor of Notch signaling; forskolin, an activator of cAMP signaling; and isoxazole 9 (ISX9), an activator of calcium-mediated signaling (Zhang et al., 2015; Gascón et al., 2016, 2017; Gao et al., 2017). For days 3–6, valproic acid, a histone deacetylase inhibitor, was added to enhance chromatin accessibility (Huangfu et al., 2008). ISX9 induced a significant degree of toxicity in cultures, and the combination of CHIR99021 and ISX9 caused all the cells to detach from the plate (data not shown). All remaining combinations produced some degree of transdifferentiation from astrocytes into neurons within 10 days, with DAPT alone being the most effective enhancer of this process (**Figure 2**).

### The Action of DAPT Does Not Translate to NGN2-Mediated Transdifferentiation

We exchanged ASCL1-IPTD with another functionalized neurogenic transcription factor, NGN2-IPTD to determine if ASCL1 could be replaced with NGN2. NGN2 also serves

FIGURE 1 | Mouse embryonic fibroblasts (MEFs) express the skeletal myoblast gene myosin heavy chain 3 (MYH3) and lack the neuronal gene beta tubulin (TUJ1) after 14 days of exposure to ASCL1-IPTD and the cAMP activator forskolin, indicating that they have adopted a myogenic cell fate rather than a neuronal cell fate. (A) Control. Scale bar is 100µm. (B) High magnification of (A). Scale bar is 10µm. (C) Forskolin only. Scale bar is 100µm. (D) High magnification of (C). Scale bar is 10µm. (E) ASCL1-IPTD only. Scale bar is 100µm. (F) High magnification of (E). Scale bar is 10µm. (G) ASCL1-IPTD with forskolin. Scale bar is 100µm. (H) High magnification of (H). Scale bar is 10µm.

as a master regulator of neural fate, and when delivered virally is sufficient to transdifferentiate astrocytes into neurons with high efficiency (71%) (Parras et al., 2002; Berninger et al., 2007). However, replacing ASCL1-IPTD with NGN2-IPTD in combination with DAPT did not generate neurons, but was seen by immunocytochemistry to generate a population of cells expressing the neuroblast gene doublecortin (DCX), suggesting that ASCL1 and NGN2 are not interchangeable in our protocol **(Figure 3).**

FIGURE 2 | Small molecule screening of combinations of forskolin, ISX9, DAPT and CHIR99021 with ASCL1-IPTD show that DAPT is the most potent inducer of neural transdifferentiation. (A) Cells express beta tubulin (TUJ1), an early neuronal marker, and ASCL1 after 2 days of priming with LDN193189 and SB431542 and ASCL1-IPTD followed by 10 days of treatment with ASCL1-IPTD and DAPT. Scale bar is 100µm. (B) High magnification of (A). Scale bar is 20µm. (C) Cell survival varied in each condition, with the DAPT alone condition being the least toxic by far (n = 3). (D) Neurons counted from each small molecule combination on day 12 (n = 3). (E) Neuronal conversion percentages. (F) Protocol timeline: 2 days of priming with SMAD inhibitors LDN193189 and SB431542 followed by 10 days of small molecule combinations. All 12 days included the addition of ASCL1-IPTD. Statistical analysis was performed by a one-way ANOVA followed by a one-tailed Student's t-test with a 95% confidence level (α = 0.05). Results are presented as the mean ± standard deviation, with a \* representing a statistically significant difference between that condition and the DAPT alone condition.

FIGURE 3 | NGN2-IPTD in place of ASCL1-IPTD does not generate neurons, but does produce cells expressing doublecortin (DCX), a marker for neuroblasts. (A) Control. Scale bar is 100µm. (B) Small molecules only. Scale bar is 100µm. (C) NGN2-IPTD plus small molecules. Scale bar is 100µm. (D) High magnification image of (C). Scale bar is 10µm. Note that TUJ1 is weakly expressed in astrocytes in addition to neurons and can be regarded as background stain in these images.

### Astrocytic Plasticity Dramatically Improves Transdifferentiation of Neurons

Both early astrocytes and astrocytes found at the site of injury possess the ability to form multi-potent neurospheres, making them highly plastic (Yang et al., 2012a; Götz et al., 2015; Sirko et al., 2015; Wang et al., 2015; Michelucci et al., 2016). We investigated the extent to which our protocol might transdifferentiate plastic astrocytes by applying it to early passage (P3) astrocytes. Indeed, analysis by flow cytometry showed that efficiency was dramatically improved to 73.09 ± 5.83% (from 1.81 ± 1.04%) by priming and DAPT exposure alone (**Figures 2G**,**4**,**6**), while the further addition of ASCL-IPTD resulted in abundant neurosphere formation, giving rise to cells expressing the mature neuronal markers MAP2 (8.48 ± 2.29%, vs. 2.56 ± 0.47% without) and NEUN (2.20 ± 1.01% with ASCL1-IPTD vs. 0.47 ± 0.05% without) by day 12 (**Figures 5**, **6**). These neurons were seen by immunocytochemistry to express both glutamatergic and GABAergic neurotransmitter genes, suggesting a mix of excitatory and inhibitory interneurons, while treatment with small molecules alone were negative for neurotransmitter markers (**Figure 5**).

## DISCUSSION

In this work, we show that ASCL1-IPTD combined with the complementary action of SMAD and Notch signaling inhibition serves an efficient inducer of neurons from astrocytes, particularly those which exhibit plasticity. This effect is important since astrocytes become reactive at the site of injury in vivo and acquire enhanced plasticity, enabling them to proliferate and de-differentiate to generate multipotent neurospheres with the potential to self-renew and give rise to astrocytes, neurons or oligodendrocytes (Yang et al., 2012a; Götz et al., 2015; Sirko et al., 2015; Wang et al., 2015; Michelucci et al., 2016). SMAD signaling regulates genes involved in astrocyte commitment and maintenance (Stipursky and Gomes, 2007; Chambers et al., 2009; Yang et al., 2013; Zhang et al., 2015), therefore we treated cells with SMAD inhibitors from days 1 to 2 to disrupt astrocyte commitment. Our screening process identified DAPT to be the most potent enhancer of ASCL1-IPTD in transdifferentiating neurons from astrocytes. Notch signaling maintains neural progenitors in a state of proliferation by repressing genes responsible for cell cycle arrest and neural differentiation such as ASCL1 and NGN2 (Dhanesh et al., 2016), thus inhibition of Notch signaling is a necessary action for the generation of mature neurons.

A unique aspect of our transdifferentiation protocol is the presence of a progenitor stage characterized by neurosphere formation. Neurospheres are 3-D clusters of neural stem cells possessing the characteristics of self-renewal and multipotency (Bez et al., 2003). Since neurospheres were absent from cultures treated with small molecules alone we attribute the progenitor stage in our protocol to the action of ASCL1-IPTD. Furthermore, only neurons arising from neurospheres expressed mature neuronal markers MAP2 and NEUN by day 12. Passing through

(G) Cells are negative for the neurotransmitter tyrosine hydroxylase expressed by dopaminergic neurons TH, the neurotransmitter glutamic acid decarboxylase expressed by inhibitory interneurons GAD65/67, and the neurotransmitter choline acetyltransferase expressed by motor neurons CHAT. Scale bar is 100µm. (H) High magnification of (G). Scale bar is 30µm. Note that some fluorescence is picked up by all the cells producing background fluorescence which can be ignored.

a progenitor stage has two advantages. First, the rapid metabolic transition that takes place during the fate switch from somatic cell to neuron puts enormous stress on the cell, leading to the formation of reactive oxygen species (ROS), known to induce toxicity and affect cell fate regulation, posing a major barrier to transdifferentiation. It stands to reason that an intermediate stage would reduce this oxidative stress, promoting a safer transition between cell fates and improving efficiency (Gascón et al., 2016). Second, the generation of neural stem cells improves the efficiency of the protocol since each neural stem cell can produce

FIGURE 5 | Plastic astrocytes generate neurospheres and mature neurons after 12 days of exposure to ASCL1-IPTD, including 2 days of priming by LDN193189 and SB431542 followed by 10 days of DAPT. (A) Cells express the early neural marker TUJ1 and the neural stem cell marker SOX2. Scale bar is 100µm. (B) High magnification of (A). Scale bar is 30µm. (C) Cells express the mature neural marker MAP2. Scale bar is 100µm. (D) High magnification of (C). Scale bar is 30µm (E) Cells express the mature neural marker NEUN and the neurotransmitter glutamate expressed by excitatory interneurons GLUT. Scale bar is 100µm. (F) High magnification of (E). Scale bar is 30µm. (G) Cells are negative for the neurotransmitter tyrosine hydroxylase expressed by dopaminergic neurons TH, and negative for the neurotransmitter choline acetyltransferase expressed by motor neurons CHAT, but do express the neurotransmitter glutamic acid decarboxylase expressed by inhibitory interneurons GAD65/67. Scale bar is 100µm. (H) High magnification of (G). Scale bar is 30µm. Note that some fluorescence is picked up by all the cells producing background fluorescence which can be ignored.

several neurons. In vivo, the effect of neural stem cells may also be to prolong the activity of neural regeneration, providing neurotrophic support and newly formed neurons for a sustained period of time (Abe, 2000; Lu et al., 2003; Chen et al., 2017).

Our transdifferentiation process was highly efficient in comparison with protocols using viral ASCL1 for similar applications. For instance, viral delivery of 3 transcription factors, ASCL1, BYRN2, and MYT1L, is reported to transdifferentiate MEFs into neurons after 22 days with an efficiency of 15–19% (Vierbuchen et al., 2010), while viral ASCL1 alone is reported to also transdifferentiate human fibroblasts into neurons by day 22, but at lower efficiencies (Chanda et al., 2014). Astrocytes have proven easier to transdifferentiate into neurons, and viral ASCL1 delivered to postnatal mouse astrocytes is reported to convert 77% into neurons by day 21 (Liu et al., 2015), and when delivered to human astrocytes (Sciencell #1800) in combination with the pluripotency transcription factor Nanog homeobox (NANOG) is reported to produce 41% neurons by day 15 (Corti et al., 2012). By comparison, our protocol generated 69% neurons from human astrocytes (Sciencell #1800) by day 12.

Small molecule protocols are reported to generate neurons from astrocytes with similarly high efficiencies, but with considerable cell death in the process, ultimately reducing total efficiency. For instance, Gao et al. used a combination of 6 small molecules (valproic acid, CHIR99021, repsox, forskolin, i-Bet151, and ISX9) to transdifferentiate human adult astrocytes into neurons in 12 days, reporting an efficiency of 70% of surviving cells, or 8% of the initial cell population (Gao et al., 2017). In contrast, Zhang et al. showed that by adding fewer small molecules in a sequential manner, cell death can be avoided in small molecule protocols. In their protocol they used a combination of 9 small molecules (LDN193189, SB431542, TTNPB, Thiazovivin, CHIR99021, DAPT, valproic acid, SAG and purmorphamine) in a step-wise manner to transdifferentiate human astrocytes (ScienCell #1800) into neurons after 14 days, with an efficiency of 67% (Zhang et al., 2015). They used the priming small molecules LDN193189 and SB431542 for days 1-2 similar to our protocol. They found by removing small molecules one at a time that the Notch signal inhibitor DAPT was the most significant enhancer of transdifferentiation, followed closely by the GSK3 signal inhibitor CHIR99021. GSK3 signaling represses canonical Wnt signaling and NGN2 expression, both key regulators of neurogenesis (Hirabayashi et al., 2004; Li et al., 2012; Valvezan and Klein, 2012), thus GSK3 inhibition can be used to promote neurogenesis. Surprisingly, our screening data showed that while either CHIR99021 or DAPT were necessary to enhance transdifferentiation with ASCL1-IPTD, when used together actually decreased transdifferentiation by ∼50%. This suggests that although efficiencies are similar, the signaling pathways involved in our protocol differ substantially from the small molecule protocol.

Cooperation between ASCL1 and Notch signal inhibition can occur naturally in astrocytes in vivo after injury (Magnusson et al., 2014). Magnusson et al. identified Notch inhibition shortly after injury as a trigger for a latent neural transdifferentiaton program in resident astrocytes. The similarities between this latent program and the protocol we developed are striking. In their study, they observed a downregulation of Notch receptors and ligands in astrocytes followed by the appearance of ASCL1 expression after a stroke. These ASCL1-positive astrocytes clustered around the lesion and multiplied, giving rise to small, round DCX-positive neural stem cells by week 2, and eventually to mature NEUN-positive neurons by week 7, some of which expressed nitric oxide synthase (nNOS), a marker for GABAergic striatal interneurons. Based on these similarities, it is possible that our protocol takes advantage of this latent program in astrocytes by first recapitulating the plastic aspects of astrocytes at the site of an injury using SMAD inhibition, followed by the concerted action of ASCL1 upregulation by IPTD and Notch signal inhibition by DAPT. By this reasoning, it can be hypothesized that in vivo application of ASCL1-IPTD may be sufficient to trigger transdifferentiation of astrocytes at a site of injury into functional neurons. Although further work will be required to verify such a strategy, it would lend itself well to in vivo translation using a version of IPTD fitted with a domain to specifically target reactive astrocytes.

### CONCLUSION

Here we have shown a novel and non-viral protocol for efficient transdifferentiation of astrocytes into neurons. This protocol requires the delivery of only one transcription factor, ASCL1, by a novel PTD, and 4 small molecules, the SMAD inhibitors LDN193189 and SB431542, the Notch inhibitor DAPT, and the histone deacetylase inhibitor valproic acid. The efficiency of this protocol is enhanced significantly by the natural plasticity of astrocytes, suggesting its suitability for application in vivo where injuries lead to increased plasticity in reactive astrocytes. Future work will determine if the small molecules are necessary in vivo, and incorporate an astrocyte-targeting motif in the IPTD design. Further work will also adapt this protocol for producing specific neuronal subtypes through cooperation of ASCL1-IPTD with other IPTD-functionalized transcription factors.

### REFERENCES

### AUTHOR CONTRIBUTIONS

MR designed the experiments, analyzed results, and wrote the manuscript. IF assisted in background research, guidance on experimental design and analysis of the small molecule screening experiment. KS assisted in executing the ASCL-IPTD experiments on astrocytes. EM assisted in executing and analysing the NGN2-IPTD experiments on astrocytes. LC assisted in executing the ASCL1-IPTD experiments on MEFs and providing background research. SW provided experimental guidance, provided help writing and editing the final manuscript.

## FUNDING

Funding for this project was provided by a Collaborative Research and Development grant (CRD) and a Discovery Grant (DG) by the Natural Sciences and Engineering Research Council (NSERC), along with the Canada Research Chair (CRC) program.

### ACKNOWLEDGMENTS

We would like to acknowledge NSERC and the CRC for funding this project, and iProgen Biotech for their support by providing the functionalized transcription factors.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fbioe. 2018.00173/full#supplementary-material

Supplemental Figure 1 | Mouse embryonic fibroblasts (MEFs) express the skeletal myoblast gene myosin heavy chain 3 (MYH3) and lack the neuronal gene beta tubulin (TUJ1) after 14 days of exposure to ASCL1-IPTD and the cAMP activator forskolin, indicating that they have adopted a myogenic cell fate rather than a neuronal cell fate. (A) Exposure to 3µg/mL ASCL1-IPTD is insufficient for transdifferentiation. Scale bar is 100µm. (B) Addition of forskolin results in transdifferentiation into myoblasts rather than neurons. Scale bar is 100µm. (C) Increasing the concentration of ASCL1-IPTD 5-fold still generates myoblasts rather than neurons. Scale bar is 100µm. (D) Increasing the concentration of ASCL1-IPTD 5-fold and adding forskolin still generates myoblasts rather than neurons. Scale bar is 100µm (E) Control. Scale bar is 100µm (F) Forskolin only control. Scale bar is 100µm.

functional neurons under defined conditions. Cell Stem Cell 9, 113–118. doi: 10.1016/j.stem.2011.07.002

**Conflict of Interest Statement:** This research was in part funded by an NSERC-CRD grant which we hold with iProgen Biotech who owns the PTD technology. This work was conducted in collaboration with iProgen Biotech who provided the functionalized transcription factors used in this work.

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