Ernesto Iii Paruli

Valentina Montagna

Mariano García-Soto

Karsten Haupt

Carlo Gonzato

A general photoiniferter approach to the surface functionalization of acrylic and methacrylic structures written by two-photon stereolithography

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Introduction

Rapid prototyping (RP) is an umbrella term that encompasses technologies that fabricate three-dimensional (3D) physical objects through a layer-by-layer additive process with the aid of digitally controlled systems. [START_REF] Chua | Rapid Prototyping of Biomaterials: Principles and Applications[END_REF][START_REF] Tofail | Additive manufacturing: scientific and technological challenges, market uptake and opportunities[END_REF] Due to its remarkable timeefficiency (i.e. few hours to few days), RP is nowadays not only used for prototyping but also for manufacturing [START_REF] Stampfl | Rapid prototyping and manufacturing by gelcasting of metallic and ceramic slurries[END_REF][START_REF] Rieger | Slurry development for lithography-based additive manufacturing of cemented carbide components[END_REF] in various fields such as aerospace,

ceramics [START_REF] Ortona | Cellular ceramics produced by rapid prototyping and replication[END_REF][START_REF] Louh | Rapid prototyping technique for ceramic mini-devices containing internal channels with asymmetrical contour[END_REF] and optics. [START_REF] Gissibl | Submicrometre accurate free-form optics by threedimensional printing on single-mode fibres[END_REF][START_REF] Headland | Analysis of 3D-printed metal for rapid-prototyped reflective terahertz optics[END_REF] The standardized terminology for RP is "additive manufacturing", as stated in ASTM 2972.

Among the different RP methods, stereolithography (SLA) is the most widely used, due to its superior accuracy and resolution. [START_REF] Martinez | 3D Printing of Pharmaceuticals[END_REF] SLA combines the use of computer-aided design (CAD) with laser technology to fabricate 3D solid objects from photosensitive precursors called "photoresins". By irradiation of a laser onto the photoresin, the monomers polymerize to assemble, layer-by-layer, into the desired 3D object. [START_REF] Bandyopadhyay | Additive Manufacturing[END_REF][START_REF] Gupta | Polymeric Gels: Characterization, Properties and Biomedical Applications[END_REF][START_REF] Melchels | A review on stereolithography and its applications in biomedical engineering[END_REF] As in any other photopolymerization reaction, SLA requires irradiation with a suitable wavelength to excite the photoinitiator and trigger the polymerization. For conventional SLA, this excitation follows the absorption of a single photon, which usually occurs in the ultraviolet (UV) region. [START_REF] Paruli | Photopolymerization and Photostructuring of Molecularly Imprinted Polymers[END_REF] In contrast, two-photon stereolithography (TPS) relies on excitation via two-photon absorption (TPA), which allows the use of lowenergy radiation, such as infrared light (IR), to match the energy provided by a single-photon at lower wavelength. To maximize the probability of TPA, photons must be "compressed" in time and in space, which is usually achieved by projecting femtosecond laser pulses through an objective with a high numerical aperture. Owing to the non-linear optical nature of TPA, the effective activation of the initiator is highly localized within the focal point of the objective, which forms the basic unit of the 3D object (i.e. the voxel) [START_REF] Spangenberg | Updates in advanced lithography[END_REF][START_REF] Wu | Two-photon polymerisation for three-dimensional micro-fabrication[END_REF][START_REF] Ovsianikov | Nanoelectronics and Photonics: From Atoms to Materials, Devices and Architectures[END_REF][START_REF] Lee | Two-photon stereolithography[END_REF] and permits the direct and precise "writing" of structures within the photoresin. This is in contrast to conventional SLA wherein polymerization is only achieved on the surface of the photoresin and the vat is continuously replenished to fabricate the next layer of the desired 3D object. 19,23 Due to its micron and even sub-micron fabrication capabilities, TPS has found diverse applications such as scaffolds for 3D cell culture, 24,25 shape-shifting 3D protein microstructures, 26 biocompatible hybrid materials, 27 tomography, [START_REF] Saha | Radiopaque Resists for Two-Photon Lithography To Enable Submicron 3D Imaging of Polymer Parts via X-ray Computed Tomography[END_REF] microelectromechanical systems (MEMS), 29-31 microsensors, [START_REF] Gomez | Rapid Prototyping of Chemical Microsensors Based on Molecularly Imprinted Polymers Synthesized by Two-Photon Stereolithography[END_REF] and optical materials (e.g. microlenses, waveguides). [START_REF] Thiele | 3D printed stacked diffractive microlenses[END_REF][START_REF] Wang | Off-Axis Holography with Uniform Illumination via 3D Printed Diffractive Optical Elements[END_REF] Despite this broad range of applications, commercial photoresins have a limited "chemical" variety as This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins most of them just play a "physical" role, except perhaps for biocompatibility, for which dedicated photoresins exist. Thus, ordinary photoresins are unsuitable for applications involving specific interactions between different chemical species, such as, for instance, molecular recognition based on molecularly imprinted polymers (MIPs). MIPs are synthetic polymers that exhibit high affinity and selectivity for a target molecule. [START_REF] Haupt | Plastic antibodies[END_REF] Also known as "plastic antibodies", MIPs surpass their natural counterparts in terms of physico-chemical stability and low cost. MIPs are synthesized through the polymerization of functional monomers and a cross-linker in the presence of the target, or a derivative thereof, which serves as a template. Functional monomers selfassemble with the template and the configuration of the resulting "complex" is "frozen" by reaction with an excess of cross-linker. Upon extraction of the template, MIPs reveal cavities that are complementary to the target molecule in terms of shape, size and position of functional groups, thus becoming capable of recognizing their target. Thanks to their properties, MIPs have been applied to many sectors such as separation science, [START_REF] Tse Sum | Toward the use of a molecularly imprinted polymer in doping analysis: Selective preconcentration and analysis of testosterone and epitestosterone in human urine[END_REF][START_REF] Xie | Magnetic Molecularly Imprinted Polymer Combined with High-Performance Liquid Chromatography for the Selective Separation and Determination of Glutathione in Various Wild Edible Boletes[END_REF][START_REF] Hroboňová | Molecularly imprinted polymer as stationary phase for HPLC separation of phenylalanine enantiomers[END_REF][START_REF] Boysen | Advances in the development of molecularly imprinted polymers for the separation and analysis of proteins with liquid chromatography[END_REF][START_REF] Sánchez-González | Development of a micro-solid-phase extraction molecularly imprinted polymer technique for synthetic cannabinoids assessment in urine followed by liquid chromatography-tandem mass spectrometry[END_REF] drug delivery, [START_REF] Kurczewska | Molecularly imprinted polymer as drug delivery carrier in alginate dressing[END_REF][START_REF] Luliński | Molecularly imprinted polymers based drug delivery devices: a way to application in modern pharmacotherapy. A review[END_REF][START_REF] Bodoki | Perspectives of Molecularly Imprinted Polymer-Based Drug Delivery Systems in Cancer Therapy[END_REF] bioimaging, [START_REF] Medina Rangel | Solid-phase synthesis of molecularly imprinted polymer nanolabels: Affinity tools for cellular bioimaging of glycans[END_REF][START_REF] Wang | Targeted imaging and targeted therapy of breast cancer cells via fluorescent double templateimprinted polymer coated silicon nanoparticles by an epitope approach[END_REF][START_REF] Panagiotopoulou | Fluorescent molecularly imprinted polymers as plastic antibodies for selective labeling and imaging of hyaluronan and sialic acid on fixed and living cells[END_REF] cosmetics, 46 catalysis [START_REF] Wang | Molecularly imprinted nanocapsule mimicking phosphotriesterase for the catalytic hydrolysis of organophosphorus pesticides[END_REF][START_REF] Mohamed | A versatile and recyclable molecularly imprinted polymer as an oxidative catalyst of sulfur derivatives: A new possible method for mustard gas and V nerve agent decontamination[END_REF][START_REF] Mathew | Design, synthesis and characterization of enzyme-analogue-built polymer catalysts as artificial hydrolases[END_REF] and (bio)chemical sensing.

50-53

In the perspective of writing MIPs by TPS, one can simply decide to prepare a dedicated photoresin; a viscous mixture including the desired monomers indeed allows the direct writing of "functional" structures, as reported by Soppera and co-workers. [START_REF] Gomez | Rapid Prototyping of Chemical Microsensors Based on Molecularly Imprinted Polymers Synthesized by Two-Photon Stereolithography[END_REF] Alternatively, one may formulate a photoresin containing a species allowing for reversible-deactivation radical-polymerisation (i.e. RDRP), in order to re-start the polymerization for tuning the surface properties, as reported for instace by Spangerberg and co-workers. [START_REF] Wu | On-Demand Editing of Surface Properties of Microstructures Made by 3D Direct Laser Writing via Photo-Mediated RAFT Polymerization[END_REF] However, these approaches are not particularly versatile for the fabrication of polymeric nano/microstructures, as every photoresin (i.e. its specific chemical composition) will require the optimization of the writing parameters (e.g. laser power and scan speed) to define the best writing conditions in terms of geometrical features and/or structural resolution. [START_REF] Jiang | Performance comparison of acrylic and thiol-acrylic resins in two-photon polymerization[END_REF] This is usually a tedious and time-consuming process that has to be repeated every time that a photoresin is modified in terms of composition, even by a slight change in molar ratio, chemical composition, solvent, etc. Thus, it would be extremely useful to have a strategy that allows "written" structures to bear different functionalities without continuously adjusting the writing parameters. We propose herein an innovative approach to turn commercially available photoresins into functional structures by following a simple scheme: 1) TPS of the desired structures, 2) coupling of a photoiniferter onto the surface of the structures, and (3) photopolymerization of the desired functional layer (Scheme 1). Hence, by taking advantage of the residual unreacted double bonds (C=C) ubiquitously present in cross-linked (meth)acrylic networks obtained by radical processes, we anchored a trithiocarbonate-based photoiniferter 55 on model TPS structures such as woodpiles and microhexagons via its addition to such C=C when irradiated with visible light (blue to green). 56,57 This allowed in turn the surface-initiated photopolymerization of either linear, rhodamine-based polymers, or MIPs against N-(carbobenzyloxy)-L-phenylalanine (Z-L-Phe) as model template, which provided the written structures with molecular recognition properties. In the case of MIPs, a minimal size increase in the nanometer range was observed, with no noticeable effect on the geometry of the honeycombs. Nonetheless, the functional structures showed affinity and enantioselectivity for their target. Due to the use of different, including commercially available, photoresins and due to its general applicability, we believe that this approach will be of interest for the polymer and material science community in terms of granting chemical functionality to arbitrary TPS-written structures.

Experimental Materials and equipment

The photoresins IP-Dip, IP-S and the fused silica (FS) substrates (3D SF DiLL, 25 mm x 25 mm x 7 mm) were from Nanoscribe GmbH, Germany. 3-(trimethoxysilyl)propyl methacrylate (TMPM) (≥ 98 %), propylene glycol methyl ether acetate (PGMEA) (≥ 99.5 %), 2-propanol (99.9 %), 4-cyano-4-((dodecylsulfanylthiocarbonyl)sulfanyl)pentanoic acid (CDTPA) (99%), N-(carbobenzyloxy)-L-phenylalanine (Z-L-Phe) (99%), methacrylic acid (99%), ethylene glycol dimethacrylate (EGDMA) (98%), 4-vinylpyridine (4-VP) (95%), methyl methacrylate (99%) (MMA), methacrylic acid (99%) (MAA), pentaerythritol triacrylate (PETA) and 2,2-dimethoxy-2phenylacetophenone (DMPA) were from Sigma-Aldrich. Methanol, glacial acetic acid, absolute ethanol and anhydrous toluene (99.8%) were from VWR Chemicals. Dansyl-Lphenylalanine (dansyl-L-Phe) was from MP Biomedicals, LLC. Methacryloxyethyl thiocarbamoyl rhodamine B (rhodamine B) was from Polysciences, Inc.

N-(carbobenzyloxy)-Dphenylalaninne (Z-D-Phe) was from Fluka Analytical. 3-((((1carboxyethyl)thio)carbonothioyl)thio)propanoic acid (BM1429) was from Boron Molecular. 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid (DoMPAT) was kindly donated by Dr. Algi Serelis, (Dulux Group, Australia). EGDMA was passed through a column of aluminum oxide to remove the polymerization inhibitor. 4-VP was freshly distilled under reduced pressure before use.

The TPS system used was a Nanoscribe Photonic Professional GT 3D printer (Nanoscribe GmbH, Germany) with a pulsed femtosecond laser source at a l center = 780 nm. Scheme 1. Functionalization of TPS-written 3D microstructures by photoiniferter coupling and subsequent functionalization with MIPs.

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Functionalization of FS substrates with TMPM

A ceramic box containing FS substrates immersed in 10 v/v% NH 3 (aq) was heated to 80°C for 15 min. The NH 3 (aq) was replaced with a freshly prepared aqueous solution of 10 v/v% H 2 O 2 and 10 v/v% HCl to be heated once more to 80°C for 15 min. The solution was removed and the FS substrates were rinsed inside the box with milliQ water (x3) and acetone (x3) and dried under a stream of N 2 . The substrates were immersed and left overnight in a 2 m/v% TMPM solution in toluene. After rinsing with acetone (x3), they were dried again with N 2 and stored at 4˚C until use.

TPS writing of microstructures

Microwoodpiles were designed in such a way that 8 stacks of 5 µm x 5 µm x 75 µm solid bars were on top of one another. The used photoresin was an in-house formulation consisting of 354.0 mg PETA (1.2 mmol), 45.6 mg DMPA (0.18 mmol) and 15 µL acetonitrile heated to 40°C until homogeneous then cooled to room temperature. The TPS was performed in air configuration using a 20X NA 0.5 objective on the same side as the formulation on a TMPM-functionalized fused silica substrate.

A basic unit of a honeycomb was designed in the XY plane on the DeScribe software such that the constituent lines were one voxel wide and a half a voxel high along the +Z direction starting from 0 (the other half-voxel in the corresponding -Z direction). The basic unit was repeated over a 50 µm x 50 µm square. 16 squares were finally assembled into a 200 µm x 200 µm honeycomb.

The writing of the honeycomb microstructures was performed using the Nanoscribe Photonic Professional (GT) printer in galvo scan mode and in the DiLL configuration with a 63X NA 1.4 objective. Either the commercial photoresin IP-Dip or IP-S was used on a TMPM-functionalized FS substrate. The scan speed (SS) used with IP-Dip was 6000 µm/s while the laser power (LP) was 20 mW. The SS and LP for IP-S was 2000 µm/s and 35 mW, respectively.

TPS-written microstructures were developed following the procedure recommended by Nanoscribe which consisted in dipping the substrate in PGMEA for 20 min then in 2-propanol for 2 min and gently drying it under a stream of air. They were visualized using scanning electron microscopy (SEM) on a QUANTA FEG 250 after gold sputter-coating. The width and height of the constituent lines were measured through the ImageJ software.

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Assessment of residual C=C on TPS-written microstructures

The presence of residual C=C on the microstructures was assessed using Raman spectroscopy on a Witec alpha300 RA (WiTec GmbH, Germany). The setup consisted of a near infrared laser from Ocean Optics (785 nm, Innovative Photonic Solutions, USA), a 20X NA 0.4 objective, a spectrometer (UHTS300, WiTec GmbH, Germany) and a back-illuminated deep-depletion charge coupled device camera (DU401A-BR-DD-352, Andor Technology, United Kingdom). Raman spectra were recorded on a 150 µm x 150 µm area including the microstructure with a final resolution of 100 pixels (integration time: 20s, number of accumulations: 10, laser intensity: 0.250).

CDTPA coupling on the TPS microstructures

A cut substrate with the microstructures on its surface was placed in an upright position inside a 4 mL glass vial. The vial was filled with 3 mL of 10 mM CDTPA in ethanol, submerging the cut substrate inside. It was sealed and purged with N 2 for 10 min away from light before exposing to 520-nm LED light (LED520-66-60, FWHM = 32 nm, Roithner LaserTechnik, Austria) at 1 mW/cm 2 for 24 h. After removing the solution, the substrate was washed with ethanol for 10 min (3x) and with dichloromethane for 10 min (3x) before letting it dry. The woodpiles were inspected under a Leica DMI6000 B fluorescence microscope (Leica Microsystems, Germany) with a TX2 filter cube (excitation filter l center = 560 nm, FWHM = 40 nm; suppression filter l center = 645 nm, FWHM = 75 nm).

Grafting MIP from CDTPA-coupled honeycombs

The MIP precursor formulation was adapted from Ref. [START_REF] Fuchs | Ultrathin selective molecularly imprinted polymer microdots obtained by evanescent wave photopolymerization[END_REF] . A mixture of the template Z-L-Phe, functional monomers MAA and 4-VP, and crosslinker EGDMA in 1 : 4 : 4: 40 molar ratio was prepared and dissolved in acetonitrile to a [C=C] = 1.80 M. The precursor of the control, i.e. non-imprinted polymer (NIP), was prepared in the same way but without Z-L-Phe. 3 mL of the precursor solution was introduced into the vial containing the CDTPA-coupled honeycomb, which was sealed and purged with N 2 for 10 min away from light. It was then exposed to 435-nm LED light at 1 mW/cm 2 for 5 h. After polymerization, the substrates were washed for 1 h each with methanol/acetic acid (9:1, v/v) (x3), ethanol (x2), methanol (x1) and dichloromethane (x3), and then left to dry.

Binding and selectivity studies on the MIP-functionalized honeycombs

A substrate containing a MIP (or NIP)-functionalized honeycomb was incubated in either 1 µM or 10 µM dansyl-L-Phe in acetonitrile for 2 h away from light under gentle magnetic stirring. Thereafter, the substrate was rinsed with 500 µL acetonitrile on each face to remove the unbound template.

After letting it dry, the honeycomb was inspected under a Leica DMI6000 B fluorescence microscope (Leica Microsystems, This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins Germany) using the A4 filter cube (excitation filter l center = 360 nm, FWHM = 40 nm; suppression filter l center = 470 nm, FWHM = 40 nm). The microscopy images were processed using the software ImageJ to quantify the fluorescence intensity of the honeycombs. The intensity was then corrected against the background using the formula:

Results and discussion

The precise and straightforward approach of TPS as a rapid prototyping technique was exploited herein to construct microstructures in the form of an array of adjacent hexagons, (i.e. a honeycomb pattern) as an elaborate model structure that would later be granted molecular recognition capabilities by growing a MIP on their surface via photoiniferter polymerization. Two commercial photoresins were used for this purpose: IP-Dip and IP-S, both supplied by Nanoscribe GmbH. According to their datasheets, [START_REF]Resins for Printing: IP-Dip[END_REF][START_REF]Resins for Printing: IP-S[END_REF] IP-Dip is acrylate-based whereas IP-S is methacrylate-based. Preliminary tests were performed by screening the SS and the LP on both photoresins in order to write the thinnest freestanding lines. This came as an arbitrary choice. The final setup consisted in SS = 6000 µm/s and LP = 20 mW for IP-Dip while SS = 2000 µm/s and LP = 35 mW for IP-S Ffigures S1 and S2). These parameters were thus used for writing the honeycombs.

The next step consisted in verifying the presence of residual C=C on the surface of the written structures to which a photoiniferter [START_REF] Montagna | RAFT coupling chemistry: A general approach for post-functionalizing molecularly imprinted polymers synthesized by radical polymerization[END_REF] would be coupled. Targeting residual C=C on highly cross-linked vinyl structures to graft a chemical species, either simple or macro, is known in literature: Zhang and coworkers reported, for instance, on the grafting of RAFTsynthesized polymers onto RAFT-synthesized MIP through a process that they termed "RAFT coupling chemistry".

Meanwhile, our group reported on the direct grafting of a RAFT agent onto MIP nanoparticles and bulk materials prepared by free-radical polymerization (FRP), which allowed their subsequent functionalization with additional polymer. [START_REF] Montagna | RAFT coupling chemistry: A general approach for post-functionalizing molecularly imprinted polymers synthesized by radical polymerization[END_REF] Both of these approaches require the presence of a radical initiator and are thermally activated. In the present work, however, we decided to couple an initiator capable of reversible-deactivation radical polymerization (RDRP) to residual C=C by a photoactivated process, with the aim to trigger the polymerization process by using light. Figure 2A-B shows the Raman spectra of both photoresins and of model structures obtained by TPS on the same photoresins. For this analysis, a cone was preferred to a honeycomb, as it allowed for an easier spot and focus under the microscope, while being absolutely equivalent to a honeycomb in terms of the concentration of residual C=C (as the writing conditions were the same). PETA was chosen as a reference of C=C bond-carrying monomer. All the photoresins featured a peak at 1635 cm -1 which is ascribed to the C=C group [START_REF] Jöhnck | Quantitative determination of unsaturation in photocured halogenated acrylates and methacrylates by FT-IR and Raman-spectroscopy and by thermal analysis[END_REF] (Figure 2A). The same peak was also present on the written structures (Figure 2B), thus confirming the possibility of targeting the residual C=C via our coupling approach. The spectrum of the IP-S-based sample suffered some autofluorescence (Figure 2B), which rendered the detection of the C=C peak difficult. Thus, in order to have clearer results, the surface of these cones was Raman imaged with respect to this peak. Figure 2D shows a brighter and homogenous color in the center of the images, which indicates the presence and a homogenous distribution of C=C on the surface of the cones.

In the case of printed structures, an obvious way of detecting the presence of a surface coating would be with respect to an optical property associated to this layer, such as for instance, fluorescence. Thus, with the aim of challenging this approach against the synthesis of fluorescent polymer brushes based on rhodamine B, we needed to verify the absence of any background emission at the selected wavelengths (i.e. Please do not adjust margins Please do not adjust margins excitation 560 nm, emission 645 nm in the case of rhodamine B as fluorophore).

However, the IP cones (both -Dip and -S) inherently featured an important fluorescence. Therefore, to circumvent this problem, we formulated a homemade photoresin based on PETA and DMPA as the photoinitiator, which arose a polymer with no detectable fluorescence upon excitation at 560 nm. The surface of its cone showed the same C=C peak, although the peak was less pronounced, with similar distribution across the surface.

Once the presence of the residual C=C was confirmed, a photoiniferter was coupled with the surface of the microstructures. We chose CDTPA as the photoiniferter since it can add to both acrylates and methacrylates upon absorption of visible light (from blue to green), [START_REF] Xu | Controlled Radical Polymerization: Mechanisms[END_REF][START_REF] Garcia-Soto | Synthesis of molecularly imprinted polymers by photo-iniferter polymerization under visible light[END_REF] which triggers the reversible dissociation of its trithiocarbonate moiety. The microstructures were then immersed in a solution of CDTPA and exposed to 520-nm light. Figure 3 shows the absorption spectrum of CDTPA: it is worth noting here that its absorbance tails at 520 nm, which only allows for a weak excitation by the green LED. This is important not only to secure the experimental conditions by reducing the probability of photobleaching, but also to assure that only a single addition to an acrylic C=C occurs, following a single-unit monomer insertion (SUMI) mechanism, 68 with no further polymerization. Indeed, upon CDTPA addition to an acrylic C=C, the absorption spectrum undergoes a blue shift (Figure 3, red dashed line) and the CDTPA can no longer be excited by the green LED. BM1429 (Figure 3) mimics the R-group of a trithiocarbonate upon insertion of an acrylic moiety, which turns the α-carbon from tertiary cyano-substituted to secondary. However, the same rationale does not apply to IP-Sbased structures, since upon coupling with a methacrylic C=C, the CDTPA absorption spectrum blue-shifts and broadens at the same time (Figure 3, brown dashed line) and can thus still be excited by the green LED. In these conditions, photobleaching cannot be excluded [START_REF] Mckenzie | Investigation into the photolytic stability of RAFT agents and the implications for photopolymerization reactions[END_REF] but the low intensity applied (i.e. 1 mW/cm 2 ) should lower it to a minimum.

In order to prove the successful CDTPA coupling and the feasibility of growing a polymer, we first run EDX analyses, searching for a sulfur peak, but without success (data not shown). The same applied for Raman spectra on CDTPA-coupled structures. We thus decided to validate the coupling step by functionalizing the written structures with a fluorescent layer and comparing the result with non-coupled structures. Since a microscale structure would be a more generic model for TPS, as well as an easier way for spotting the object under the Raman microscope, we decided to print microwoodpiles. Once CDTPA was immobilized on the resulting non-fluorescent woodpiles, linear fluorescent polymer chains were grafted from the surface by using the monomers rhodamine B and MMA. Some CDTPA was also added in the pre-polymerization solution, a condition which is reported to help getting homogeneous polymer growth. [START_REF] Li | SI-PET-RAFT: Surface-Initiated Photoinduced Electron Transfer-Reversible Addition-Fragmentation Chain Transfer Polymerization[END_REF] A similar procedure was also repeated onto bare, non-coupled woodpiles, as a negative control. As seen in figure 4, only CDTPA-coupled woodpiles exhibited strong fluorescence, in sharp contrast with the negative control. Moreover, optical images show that the woodpile geometry was unaffected by the photopolymerization (see Supporting Information, Figures S3). These results allowed to conclude that the surface-anchoring of CDTPA is essential for the subsequent surface-initiated polymerization, as the bare formulation of CDTPA in the pre-polymerization mixture (at the tested conditions) does not allow growing polymers on non-coupled IP structures (Figure 4D). This result is consistent with our previous work, wherein surface-initiated RAFT polymerization on freeradically synthesized nanoparticles was only effective upon a RAFT-coupling step.

After validating the CDTPA coupling as an advantageous step for further surface functionalization, we then moved to the synthesis of a MIP imprinted against Z-L-Phe as model template on TPS-printed honeycombs. These structures also came as an arbitrary choice, since we were looking at a flat geometry -in order to easily appreciate the effect of coating with a crosslinked layer-simultaneously bearing edges in order to also easily appreciate structural effect over the polymerization process. In this case, the "writing" was performed in repeating units such as squares, "stitching" them together as one square is written adjacent to another. This required 16 50 µm x 50 µm honeycomb squares. Under an optical microscope, it can be seen that the squares did not precisely connect together (Figure 1, entire honeycomb not shown completely) possibly because of the limited precision of the motorized stage when moving between writing fields, leaving considerable gaps between them that cannot be easily stitched especially given the thin dimensions of our structures.

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A closer look with SEM reveals that the writing parameters yielded for IP-Dip an incomplete structure, with honeycomb repeating units systematically disconnected in one direction (Figure 5A). This unexpected phenomenon can be related to the relatively high SS and low LP, which may result in a partial polymerization at the interconnection between consecutive motifs as the Nanoscribe starts to write the next line necessary to complete the hexagonal structure. On the other hand, the chosen parameters afforded for IP-S perfect honeycombs with repeating units fully written and connected to each other. SEM analyses revealed thicknesses of 171 nm and 364 nm for IP-Dip and IP-S, respectively (Figure 5).

Fluorescent binding test.

Fluorescent binding tests based on the incubation of IP-Dip-based honeycombs with the fluorescent target dansyl-L-Phe, a structural analog of Z-L-Phe, were used to assess the successful synthesis of an imprinted layer. Figure 6 shows that the MIP-functionalized honeycomb featured an increased fluorescence intensity compared to the corresponding NIP due to the target molecule captured in the binding sites of the MIP. As the control, the NIP layer was synthesized in the absence of the template, hence it is supposed to miss specific binding sites on its surface. The much weaker fluorescence observed on the NIP was thus attributed to nonspecific binding.

The ΔI f also increased when the concentration was raised tenfold, showing that the MIP layer not only responded to the presence of the analyte but also to a change in its concentration. The imprinting factor (IF), which is defined as the ratio of the MIP-bound target to the NIP-bound target, [START_REF] Haupt | Molecularly Imprinted Polymers: Antibody Mimics for Bioimaging and Therapy[END_REF][START_REF] Belbruno | Molecularly Imprinted Polymers[END_REF] was calculated to be 4.30 and 6.33 for 1 µM and 10 µM dansyl-L-Phe, respectively (Figure 6E). Similar results were also obtained on IP-S-based honeycomb microstructures, which allowed to calculate IF of 4.05 and 26.60 for 1 µM and 10 µM dansyl-L-Phe, respectively (Figure 6J).

Selectivity test. The MIP-functionalized honeycombs were further investigated for their selectivity toward their target Z-L-Phe over its D-enantiomer. Since the template and its The IP-Dip-and the IP-S-based honeycombs were incubated first in a solution containing an equal concentration (10 µM) of dansyl-L-Phe and Z-L-Phe. As seen in Figure 7, this resulted in a strong decrease of fluorescence intensity when compared to honeycombs incubated with dansyl-L-Phe alone. This was expected since Z-L-Phe was the actual template used for imprinting, meaning that the MIP's binding sites were tailored around its geometry. Likewise, the honeycombs were incubated in a solution of both dansyl-L-Phe and the enantiomer Z-D-Phe. In this case, the decrease in fluorescence was less important, meaning that Z-D-Phe was less effective than Z-L-Phe in displacing dansyl-L-Phe from the binding sites under the same conditions. The results indicate that the surface imprinting of honeycombs was effective on both IP-Dip and IP-S, with MIPfunctionalized structures capable of selectively binding their target over its enantiomer.

It has to be mentioned here that the limited height of our 2D IP structures (i.e. roughly 300 nm) is an advantage for the fluorescent measurements of this target; indeed, bigger structures, such as the IP cones (both -Dip and -S) used for Raman imaging, also suffered fluorescence emission in that region (excitation 360 nm, emission 470 nm).

Nevertheless, the main purpose of this work was to develop an approach able to surface-functionalize TPS structures with minimal effect on their size and possibly no effect on their geometry. We thus inspected the SEM images of our MIP honeycombs to elucidate the effect of the photoiniferter coupling and photopolymerization on these characteristics. As shown in Figure 8, the general honeycomb pattern of all the microstructures was conserved and no extra features were created. The IP-S-based honeycombs, in particular, revealed a negligible increase in linewidth (Z test, a = 0.05), while the linewidth of the IP-Dip-based honeycombs increased by only 20 nm. In this second case, the thickness of the MIP layer was roughly only 10 nm, which was still much below the original linewidth.

Overall, these results illustrate that the post-TPS polymerization had no detrimental effects on the resolution (few hundred nanometers) nor on the geometry of the honeycomb microstructures and can thus be envisioned as a way of solely tuning the surface properties of the written structure by introducing different chemical functionalities via surface-initiated photopolymerization.

Conclusions

As a sophisticated additive manufacturing technique, TPS has proven to be effective for the direct and precise fabrication of polymeric microstructures according to its limits of resolution. Woodpiles as well as honeycomb microstructures were fabricated using (meth)acrylate-based photoresins, both homemade and commercially available. Taking advantage of the residual C=C bonds on the surface of the microstructures revealed by Raman spectroscopy, a trithiocarbonate-based photoiniferter CDTPA was anchored upon simple irradiation with visible (green) light. This allowed in turn for the subsequent blue light-mediated, surface-initiated polymerization of fluorescent polymer brushes as well as a thin MIP layer specific and selective for Z-L-Phe as model target. Overall, the microstructures retained their original geometry, with no extrafeatures added, nor major changes in size upon functionalization with a functional polymer (i.e. MIP or NIP). Taken together, these results confirm that such post-TPS polymerization approach by coupling a photoiniferter to residual C=C on the TPS-written structure is a valuable and serves as a general strategy which allows functionalizing microstructures with minimal effect on their size and geometry.

62 CTCF=

 62 Integrated Density -(Selected Area X Mean fluorescence of background readings)where CTCF is the background-corrected fluorescence intensity. The difference between the CTCF values after and before incubation (ΔI f ) was taken to be the response of the MIP (or the NIP) layer of the honeycomb to the target dansyl-L-Phe.For the selectivity study, two kinds of solution were used based on 10 µM dansyl-L-Phe containing respectively 10 µM Z-L-Phe and 10 µM Z-D-Phe both in acetonitrile. The binding of dansyl-L-Phe on MIPs and NIPs was then assessed by fluorescence microscopy as mentioned above.

Figure 1 .

 1 Figure 1. Representative brightfield microscopy images of (A) IP-Dip-and (B) IP-Sbased honeycombs. The arrows indicate the borders of the 200 x 200 µm² area of the honeycomb.

Figure 2 .

 2 Figure 2. Raman spectra of (A) unreacted photoresins and (B) TPS-written truncated cones based on them. PETA was used as the C=C bond-containing monomer reference. (C) The brightfield image (scale bar = 60 µm) and (D) the Raman image of the top surface of the TPS-written truncated cone (scale bar = 30 µm).

Figure 3 .

 3 Figure 3. Absorption spectra of CDTPA and selected structural mimics simulating its SUMI adducts with respectively acrylic acid (BM1429) and methacrylic acid (DoMPAT). All the spectra were recorded in acetonitrile at 7.5 mM.

Figure 4 .

 4 Figure 4. The CDTPA-coupled microwoodpile after functionalization with a fluorescent polymer: (A) brightfield and (B) fluorescence microscopy images. Control (non-coupled) microwoodpile: (C) brightfield and D fluorescence microscopy images. Scale bar = 100 µm.

Figure 6 .

 6 Figure 6. Fluorescence microscopy images (enhanced) of the (A-D) IP-Dip-and (F-I) IP-S-based honeycomb microstructures: (A,B,F,G) MIP layers (A,F) before and (B,G) after incubation, and (C,D,H,I) NIP layers (C,H) before and (D,I) after incubation in 10 µM dansyl-L-Phe in acetonitrile. (E,J) Background-corrected ΔIf of the MIP and NIP layer on the (E) IP-Dip-and (J) IP-S-based honeycomb microstructure (n = 3).

Figure 7 .

 7 Figure 7. Background-corrected ΔIf of the MIP and NIP layer on (A) the IP-Dip-, and (B) the IP-S-based honeycombs after incubation in a solution of dansyl-L-Phe alone, dansyl-L-Phe with Z-L-Phe or dansyl-L-Phe with Z-D-Phe in acetonitrile (n = 3). The concentration of each compound was 10 µM.

Figure 5 . 2 .

 52 Figure 5. (A) The honeycomb design. The design was programmed to extend in an area of 200 x 200 µm 2 . Representative SEM images of TPS-written honeycomb microstructures based on (B) IP-Dip and (C) IP-S.

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 3

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 7

Acknowledgements

The authors acknowledge the Hauts-de-France Region and the European Regional Development Fund (ERDF) (CPER 2014/2020) for the funding of equipment. EP wishes to thank the Embassy of France in the Philippines and the Commission on Higher Education of the Philippines (CHED) for granting his fellowship. KH acknowledges financial support from Institut Universitaire de France. We also thank Frederic Nadaud from the Physico-chemical Analysis Group at the Université de Technologie de Compiègne for the SEM images.

Author Contributions

Conflicts of interest

There are no conflicts to declare.


Sébastien Benzekry

Mélanie Karlsen

Abdessamad Elkaoutari

Suresh Vatakuti

Petter Curle

Candice Jamois

Pharmacometrics modeling coupled with machine learning for early prediction of survival following atezolizumab monotherapy in non-small cell lung cancer

BACKGROUND••

  Treatment of advanced non-small cell lung cancer by atezolizumab-based therapy • Prediction of survival from baseline or early on-treatment data could • guide treatment decision during drug development • inform personalized health care • Current predictive biomarker: programmed death-ligand 1 (PD-L1) 1 • State of the art from baseline clinical and biological data: ROPRO score 2 • Predictive value of transcriptomic and mutation data is unclear • Tumor kinetics (TK) model parameter growth rate (KG) has important predictive power of hazard ratio (HR) 3 • Predictive value of kinetics of pharmacodynamic biomarkers is unclear OBJECTIVE To provide a digital decision-enabling tool by predicting overall survival based on early tumor size and longitudinal PD biomarker data using the strengths of pharmacometrics (PHMx) and machine learning (ML) 1 Shukuya, T. & Carbone, D. P. Predictive Markers for the Efficacy of Anti-PD-1/PD-L1 Antibodies in Lung Cancer. Journal of Thoracic Oncology 11, 976-988 (2016). 2 Becker, T. et al. An enhanced prognostic score for overall survival of patients with cancer derived from a large real-world cohort. Ann Oncol 31, 1561-1568 (2020). 3 Claret, L. et al. A Model of Overall Survival Predicts Treatment Outcomes with Atezolizumab versus Chemotherapy in Non-Small Cell Lung Cancer Based on Early Tumor Kinetics. Clin Cancer Res 24, 3292-3298 (2018). Inria Méditerranée, Centre de Recherche sur le Cancer de Marseille, Inserm, CNRS, IPC, Aix-Marseille University, Marseille, France; 2 Safety and Early Development Informatics and 3 Clinical Pharmacometrics, Pharmaceutical Sciences, Pharma Research and Early Development, Roche Innovation Center Basel, Switzerland that evaluated the efficacy and safety of antiprogrammed death-ligand 1 (PD-L1) atezolizumab in advanced NSCLC selected by tumor cell (TC) or tumor-infiltrating immune cell (IC) PD-L1 expression PD-L1 positive locally advanced or metastatic NSCLC (lines controlled trial (RCT) of atezolizumab versus docetaxel for patients with previously treated NSCLC Locally advanced or metastatic NSCLC who failed platinum therapy 134 BIRCH GO28754 Phase 2 Study of Atezolizumab in participants with PD-L1 positive locally advanced or metastatic NSCLC Locally advanced or metastatic NSCLC (lines 1, 2 or 3The combination of NLME and ML allowed to take the best of the two approaches in order to predict individual survival • NLME for longitudinal TK and PD data • ML to build multivariable models from a large number of features • A model was established based on a 26 features minimal signature: 11 baseline clinical features + longitudinal TK (3 variables) + longitudinal PD (12 variables) • C-index = 0.818 ± 0.029, AUC = 0.905 ± 0.0414 • RNAseq data did not yield substantial predictive power Perspectives • External validation on the phase 3 OAK trial • Prediction of study-level overall survival in multiple arms, from early on-study data