Leaf-Inspired Authentically Complex Microvascular Networks for

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Biological and Medical Applications of Materials and Interfaces

Leaf-Inspired Authentically Complex Microvascular Networks for Deciphering Biological Transport Processes Marco Elvino Miali, Marianna Colasuonno, Salvatore Surdo, Roberto Palomba, Rui Pereira, Eliana Rondanina, Alberto Diaspro, Giuseppe Pascazio, and Paolo Decuzzi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b09453 • Publication Date (Web): 14 Aug 2019 Downloaded from pubs.acs.org on August 15, 2019

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ACS Applied Materials & Interfaces

Leaf-Inspired authentically Complex Microvascular Networks for Deciphering Biological Transport Process Marco E. Mialia,e, Marianna Colasuonnob,e, Salvatore Surdoc, Roberto Palombae, Rui Pereirae, Eliana Rondaninad, Alberto Diasproc, Giuseppe Pascazioa, Paolo Decuzzie 

Dipartimento di Meccanica, Matematica e Management, DMMM, Politecnico di Bari, Via Re David, 200-70125, Bari, Italy. a

b

Sant'Anna School of Advanced Studies, Piazza Martiri della Libertà 33, 56127, Pisa, Italy

Nanophysics Department, Fondazione Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genoa, Italy c

Nanostructures, Fondazione Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genoa, Italy d

Laboratory of Nanotechnology for Precision Medicine, Fondazione Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genoa, Italy e

 corresponding author: Paolo Decuzzi Ph.D., [email protected]

KEYWORDS: Biomimicry; microfluidics; circulating tumor cells; thrombolysis; vascular transport

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ABSTRACT The vascular transport of molecules, cells and nanoconstructs is a fundamental biophysical process impacting tissue regeneration; delivery of nutrients and therapeutic agents; and the response of the immune system to external pathogens. This process is often studied in singlechannel microfluidic devices lacking the complex tri-dimensional organization of vascular networks. Here, soft lithography is employed to replicate the vein system of a Hedera elix leaf on a polydimethilsiloxane (PDMS) template. The replica is then sealed and connected to an external pumping system to realize an authentically complex microvascular network. This satisfies energy minimization criteria by the Murray’s law and comprises a network of channels ranging in size from capillaries ( 50 m) to large arterioles and venules ( 400 m). Micro-PIV (Micro – Particle Image Velocimetry) analysis is employed to characterize flow conditions in terms of streamlines, fluid velocity, and flow rates. To demonstrate the ability to reproduce physiologically relevant transport processes, two different applications are demonstrated: vascular deposition of tumor cells and lysis of blood clots. To this end, conditions are identified to culture cells within the microvasculature and realize a confluent endothelial monolayer. Then, the vascular deposition of circulating breast (MDA-MB 231) cancer cells is documented throughout the network under physiologically relevant flow conditions. Firm cell adhesion mostly occurs in channels with low mean blood velocity. As a second application, blood clots are formed within the chip by mixing whole blood with a


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thrombin solution. After demonstrating the blood clot stability, tissue plasminogen activator (tPA) and tPA-carrying nanoconstructs (tPA-DPNs) are employed as thrombolytics. In agreement with previous data, clot dissolution is equally induced by tPA and tPA-DPNs. The proposed leaf-inspired chip can be efficiently used to study a variety of vascular transport processes in complex microvascular networks, where geometry and flow conditions can be modulated and monitored throughout the experimental campaign.



INTRODUCTION Understanding the vascular transport of blood-borne agents and cells is of fundamental importance in the rational design of intravenously administered nanoconstructs, in elucidating the mechanisms regulating cancer spreading, and boosting the response of immune cells to exogenous and endogenous insults.

1

Traditionally, parallel plate flow

chamber systems 2-4 and intravital video microscopy 5-7 have been employed to replicate in vitro and document in vivo the vascular behavior of nanoconstructs, cancer cells, platelets and leukocytes. In vitro fluidic systems allow scientists to accurately, and independently, control a variety of biophysical governing parameters, such as the channel size, flow rate and surface density of adhesive molecules. As such, fluidic assays support the realization of systematic analyses and help elucidating underlying biophysical mechanisms. 8 On the other hand, in vivo experiments only can properly account for the biological and architectural complexity of a living tissue and vasculature, where multiple and different cell types are organized in a tri-dimensional microenvironment. In this context, realizing fluidic systems that could recapitulate more accurately the complex architecture of the vasculature and the three dimensional organization of endothelial cells would further advance our knowledge on the transport of cells and nanoconstructs, improve the statistical significance of the presented results, reduce costs and alleviate ethical issues.

Microfabrication techniques can be readily employed to produce single-channel fluidic assays for dissecting the mechanisms influencing the vascular behavior and adhesion of leukocytes 9-10,

cancer cells 11-12, platelets 13-14, and nano/micro-constructs. 4, 15-17 Yet, these microfluidic


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chips cannot replicate the complex branching and hierarchical organization of an authentic vascular network. To address this point, some authors have exploited the natural process of vasculogenesis whereby endothelial cells in a tissue chamber would spontaneously organize over time to form a tubular network.

18-20

This approach tends to reproduce the complex

geometry of capillary beds but it only provides vessels with a characteristic size of a few tens of microns. Also, given the complex signaling cascade and myriad of biochemical stimuli regulating vasculogenesis, the geometry and hydrodynamic conditions of these spontaneously formed capillary beds vary significantly over multiple experiments. Very recently, an alternative approach has been proposed insisting on the idea of replicating the vein system of natural leaves. 21

Cells on leaves receive nutrients through a naturally-organized network of micro-channels – the leaf’s vein system.22-23 The geometry of this vascular network generally obeys an energy minimization criterion, known as the Murray’s law. 24 This states that the cube of the radius of the parent channel should equal the sum of the cubes of the radii of the daughter channels. Several biological systems, including the vascular and respiratory system in animals 25 and the lymphatic systems in plants 26-27, do follow Murray’s law. Taking advantage of this remarkable feature, the complex vein network of a leaf can be replicated, using conventional molding techniques, and integrated into a microfluidic chip to generate an authentically complex, hierarchically-organized vascular network. This was demonstrated by the groups of Manz and Li, who proposed to use this approach for realizing microfluidic chips with diverse vascular geometries, originating from different leaves.21,

28

Subsequently, the same group

demonstrated that human melanoma cells can be cultured for several days within a leaf-



template (Tilia platyphyllos) microfluidic chip.29 Following the same strategy, another group fabricated a multi-well chip for conducting high-throughput cell proliferation and cytotoxicity assays.

22

This leaf-template strategy allows the realization of complex vascular networks

obeying basic physiological laws and presenting a hierarchical structure with a vessel caliber ranging from several hundreds to a few tens of microns. Importantly, starting from different leaves, an armamentarium of microfluidic chips with diverse geometries can be realized and experimentally employed to perform high-throughput characterizations with a strong statistical power.

In this work, the vein system of an Hedera helix leaf is replicated using molding techniques and integrated into a microfluidic chip for assessing the vascular dynamics of circulating breast cancer cells – MDA-MB-231 – over a continuum endothelial layer and the dissolution of blood clots. First, a stable, epoxy-replica of the leaf vein system is patterned on a SU-8 photoresist, which is then used as a reference for multiple polydimethylsiloxane (PDMS) replicas. The geometry of the replicated vein network is analyzed in terms of hierarchical distribution, branching symmetry, channel size and area ratios. Then, micro-particle velocimetry (-PIV) analyses are performed to characterize the hydrodynamic behavior of the network and provide specific information on flow rates and mean velocities. Before conducting biologically relevant experiments, the entire vascular network is covered by a continuous monolayer of endothelial cells (HUVECs). Finally, the vascular dynamics of circulating cancer cells and the thrombolytic activity of clinical and preclinical agents is assessed.


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MATERIALS AND METHODS Fabrication of the Leaf Microfluidic Chip. The vascular architecture of the microfluidic device was replicated using soft lithography, from a freshly collected natural ‘Hedera helix’ leaf. First, the leaf was firmly taped to the bottom of a plastic petri dish (Figure.1a). A negative fingerprint of the leaf was obtained via polydimethylsiloxane (PDMS) replica molding. Specifically, a mixture of PDMS pre-polymer and curing agent (Sylgard 182, Dow Corning), with a ratio of 10:1 (w:w), was degassed in a vacuum chamber and poured onto the leaf. PDMS curing was performed at 40 °C overnight limiting any possible damage and degradation of the leaf. The PDMS replica was then peeled off from the natural leaf and an additional curing step was performed at 80 °C for 3 h. This PDMS replica was then used to produce a more stable SU8-5 template, which precisely replicate the geometry and lymphatic architecture of the original leaf (Figure.1b). Specifically, the liquid SU8-5 resin was poured onto the PDMS replica and then the sample was heated up on a hot plate at 65 °C for 20 minutes and at 95 °C for 40 minutes in order to remove any solvent. The SU8-5 was cured under UV light (SUSS, MicroTec, λexcitation = 365 nm) for 30 minutes with a dose of 10.8 W/mm2. A post-exposure bake, first at 65 °C for 20 minutes and then at 95 °C for 30 minutes, was used to complete the polymer cross-linking. Finally, the SU8-5 leaf replica was detached from the PDMS and hardly baked at 180 °C for 4 minutes in order to increase the mechanical stiffness and chemical stability of the resin. From this more durable SU8-5 template, a new PDMS replica of the leaf was generated. The SU8-5 leaf template was replicated with a mixture of PDMS pre-polymer and curing agent with a ratio of 20:1 (w:w). After curing in the oven at 50 °C for 12 hours, the PDMS replica was peeled off from the SU8-5 template. Inlet and outlet holes were realized by using a punch with diameter of 1.5 and 0.4 mm, respectively (Figure.1c). Finally, a PDMS layer with pre-polymer and curing agent ratio of 3:1 (w:w) was



used to seal the replica. 30 The liquid PDMS was deposited on a silanized silicon (Si) wafer and spin coated at 350 rpm for 3 minutes to obtain a 110 𝜇m-thick layer. The resulting film was cured in a thermal oven at 65 °C for 1 h and bond to the bottom surface of the PDMS replica via oxygen plasma treatment (Plasma System Tucano, Gambetti) at 20 W for 20 s, at 1 mPa (Figure.1d). The sealed chip was peeled off from the Si wafer and placed in the oven at 65 °C for 4 h to enhance the chemical interaction between the two PDMS layers. The chip was finally mounted on a glass coverslip (25 mm x 60 mm) for microscopy (Figure.1e). The bonding was performed using Oxygen plasma O2 treatment, following standard procedures. This sandwiched system, comprising the PDMS replica of the leaf, the thin PDMS layer and the glass slide, was connected to an external pumping system through inlet and outlet ports (Figure.1f). Note that generating a durable SU8-5 template is instrumental for the efficient and reliable use of the leaf-microfluidic chip. Indeed, the original leaf is subjected to progressive degradation and embrittlement with time and only a limited number (

Leaf-Inspired Authentically Complex Microvascular Networks for

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