3D Printing and In Situ Surface Modification via Type I Photoinitiated Reversible Addition-Fragmentation Chain Transfer Polymerization

Podsumowanie

The present protocol describes the digital light processing-based 3D printing of polymeric materials using type I photoinitiated reversible addition-fragmentation chain transfer polymerization and the subsequent in situ material post-functionalization via surface-mediated polymerization. Photoinduced 3D printing provides materials with independently tailored and spatially controlled bulk and interfacial properties.

Streszczenie

3D printing provides facile access to geometrically complex materials. However, these materials have intrinsically linked bulk and interfacial properties dependent on the chemical composition of the resin. In the current work, 3D printed materials are post-functionalized using the 3D printer hardware via a secondary surface-initiated polymerization process, thus providing independent control over the bulk and interfacial material properties. This process begins with preparing liquid resins, which contain a monofunctional monomer, a crosslinking multifunctional monomer, a photochemically labile species that enables initiation of polymerization, and critically, a thiocarbonylthio compound which facilitates reversible addition-fragmentation chain transfer (RAFT) polymerization. The thiocarbonylthio compound, known commonly as a RAFT agent, mediates the chain growth polymerization process and provides polymeric materials with more homogeneous network structures. The liquid resin is cured in a layer-by-layer fashion using a commercially available digital light processing 3D printer to give three-dimensional materials having spatially controlled geometries. The initial resin is removed and replaced with a new mixture containing functional monomers and photoinitiating species. The 3D printed material is then exposed to light from the 3D printer in the presence of the new functional monomer mixture. This allows photoinduced surface-initiated polymerization to occur from the latent RAFT agent groups on the surface of the 3D printed material. Given the chemical flexibility of both resins, this process allows a wide range of 3D printed materials to be produced with tailorable bulk and interfacial properties.

Wprowadzenie

Additive manufacturing and 3D printing have revolutionized material manufacturing by providing more efficient and facile routes for the fabrication of geometrically complex materials¹. Apart from the enhanced design freedoms in 3D printing, these technologies produce less waste than traditional subtractive manufacturing processes via the judicious use of precursor materials in a layer-by-layer manufacturing process. Since the 1980s, a wide range of different 3D printing techniques has been developed to fabricate polymeric, metal, and ceramic components¹. The most commonly employed methods include extrusion-based 3D printing such as fused filament fabrication and direct ink writing techniques², sintering techniques such as selective laser sintering³, as well as resin-based photoinduced 3D printing techniques such as laser and projection-based stereolithography and masked digital light processing techniques⁴. Among the many 3D printing techniques in existence today, photoinduced 3D printing techniques provide some advantages compared to other methods, including higher resolution and faster printing speeds, as well as the ability to perform solidification of the liquid resin at room temperature, which opens the possibility to advanced biomaterial 3D printing^4,5,6,7,8,9.

While these advantages have allowed the widespread adoption of 3D printing in many fields, the limited ability to independently tailor the 3D printed material properties restricts future applications¹⁰. In particular, the inability to easily tailor the bulk mechanical properties independently of the interfacial properties limits applications such as implants, which require finely tailored biocompatible surfaces and often vastly differing bulk properties, as well as antifouling and antibacterial surfaces, sensor materials, and other smart materials^11,12,13. Researchers have proposed surface modification of 3D printed materials to overcome these issues to provide more independently tailorable bulk and interfacial properties^10,14,15.

Recently, our group developed a photoinduced 3D printing process that exploits reversible addition-fragmentation chain transfer (RAFT) polymerization to mediate network polymer synthesis^15,16. RAFT polymerization is a type of reversible deactivation radical polymerization that provides a high degree of control over the polymerization process and allows for the production of macromolecular materials with finely tuned molecular weights and topologies, and broad chemical scope^17,18,19. Notably, the thiocarbonylthio compounds, or RAFT agents, used during RAFT polymerization are retained after polymerization. They can thus be reactivated to modify further the chemical and physical properties of the macromolecular material. Thus, after 3D printing, these dormant RAFT agents on the surfaces of the 3D printed material can be reactivated in the presence of functional monomers to provide tailored material surfaces^{20,21,22,23,24,25,26}. The secondary surface polymerization dictates the interfacial material properties and can be performed in a spatially controlled fashion via photochemical initiation.

The present protocol describes a method for 3D printing polymeric materials via a photoinduced RAFT polymerization process and the subsequent in situ surface modification to modulate the interfacial properties independently of the bulk material mechanical properties. Compared to previous 3D printing and surface modification approaches, the current protocol does not require deoxygenation or other stringent conditions and is thus highly accessible for non-specialists. Furthermore, the use of 3D printing hardware to perform both the initial material fabrication and the surface post-functionalization provides spatial control over the material properties and can be performed without the tedious alignment of several different photomasks to make complex patterns.

Protokół

1. Preparation of 3D printing program and 3D printer

1. Design the digital model for 3D printing following the steps below.
   1. Open a computer-assisted design program (see Table of Materials).
   2. In the x-y plane, create a rectangle centered on the origin having dimensions of 80 mm x 40 mm, then extrude along the positive z-axis for 1.5 mm to make a solid rectangular prism, called the base object.
   3. Above the base object, i.e., at z = 1.5 mm, draw the desired surface patterns (in this case, two yin-yang symbols) on the surface of the rectangular prism.
   4. Extrude the surface patterns in selected regions 0.05 mm along the positive z-axis to create a slightly raised pattern relative to the base object.
   5. Export the 3D model to provide a stereolithography file with .STL file extension.
      NOTE: In this work, dog-bone-shaped specimens were designed²⁷. For other desired models to be printed, follow steps 1.1.1-1.1.5.
   6. Open a 3D printer slicing program (see Table of Materials) to enable single-layer settings.
   7. Open the converted .STL files from the computer hard drive by clicking on File > Open then navigating to the saved .STL file.
   8. Arrange the 3D models on the build platform using the "Model Rotate" and "Model Move" buttons to fit at least 1 mm between all objects on the build stage.
   9. By entering text in the entry field boxes in the right-hand panel, change the parameters as mentioned in Table 1.
   10. Click on the blue Slice button in the bottom left-hand corner and save it as a slice file with an extension of. PWS or other 3D printer readable sliced file.
   11. Click on the Preview button once the pop-up menu appears and navigate through the sliced layers using the scroll bar on the right-hand side. Take careful note of the layer numbers for the last base layer (layer 29 in this case) and the surface pattern layer (30 in this case).
       NOTE: The first printed layer is "layer 0" not "layer 1".
   12. In the right-hand panel, select Single-layer settings, then expand the drop-down menu.
   13. Change the "Exposure Time (s)" for only the surface layer (layer 30) to 180 s, leaving all other layer exposure times as the default value.
   14. Click on Save button in the top left corner to save the sliced file to a USB.
2. Prepare the 3D printer.
   1. Insert the USB containing the sliced file into the 3D printer (see Table of Materials).
   2. Before 3D printing, level the build stage and calibrate the z-axis position to the z = 0 by following the specific 3D printer method (manual or automatic calibration following the 3D printer manual).
   3. Inspect the film of the 3D printer vat to ensure a smooth and clean surface free of defects.
   4. If the vat film appears damaged, replace it according to the manufacturer's protocol.

2. Preparation of resins

NOTE: Resins are categorized as "Bulk Resin" for the resin used to 3D print the original material (base substrate) and "Surface Resin" for the solution used to perform the surface functionalization (surface pattern).

1. Prepare the Bulk Resin.
   1. For preparing the bulk resin, weigh 0.36 g of 2-(n-butylthiocarbonothioylthio) propanoic acid (BTPA) into a clean 50 mL amber vial.
   2. Add 13.63 mL of poly (ethylene glycol) diacrylate average Mn 250 (PEGDA) to the amber vial using a micropipette.
   3. Add 14.94 mL of N, N-dimethylacrylamide (DMAm) to the amber vial using a micropipette.
   4. In a separate 20 mL clean glass vial covered with aluminum foil, add 0.53 g of diphenyl (2,4,6-trimethyl benzoyl) phosphine oxide (TPO).
   5. Using a micropipette, add 10 mL of DMAm to the 20 mL glass vial containing the TPO and seal the vial using the cap.
   6. Thoroughly homogenize the solution of TPO and DMAm by mixing using a vortex mixer for 10 s and then using a standard laboratory sonic bath (~40 kHz) to sonicate the mixture for 1 min at room temperature (Figure 1C, left).
   7. Using a glass pipette and rubber pipette bulb, transfer the solution from the 20 mL glass vial to the 50 mL amber vial and seal the vial with a cap and moldable plastic film.
   8. Gently shake the 50 mL amber vial and then place the vial in a sonic bath for 2 min at room temperature to ensure the mixture is homogeneous (Figure 1C, second from left).
   9. Place the sealed amber vial filled with the bulk resin in a fume hood for later use.
2. Prepare the Surface Resin.
   1. For preparing the surface resin, weigh 0.50 g of TPO into a clean 50 mL amber vial.
   2. Using a micropipette, add 3.56 mL of DMAm and 11.98 mL of N, N-dimethylformamide (DMF) to the 50 mL amber vial and seal the vial with a cap moldable plastic film.
   3. Gently shake the sealed amber vial and sonicate for 1 min at room temperature using a standard laboratory sonic bath (~40 kHz).
   4. To a clean 20 mL vial covered with foil, add 0.29 g 1-pyrenemethyl methacrylate (PyMMA).
   5. Add 10 mL of DMF to the 20 mL vial and seal the vial with a cap using a micropipette.
   6. Gently shake the 20 mL glass vial and sonicate in increments of 1 min at room temperature using a standard laboratory sonic bath, visually inspecting between cycles until the PyMMA appears to be dissolved entirely (Figure 1C, third and fourth from left).
   7. Using a glass pipette and rubber pipette bulb, transfer the solution from the 20 mL glass vial to the 50 mL amber vial.
   8. Gently shake the 50 mL amber vial and then place the vial in a sonic bath for 2 min at room temperature to ensure the mixture is homogeneous (Figure 1C, right and second from right).
   9. Place the sealed amber vial filled with the bulk resin in a fume hood for later use.
      ​CAUTION: Some chemicals used in this protocol may cause severe skin and eye irritation and other toxicity to humans and the environment. Ensure safety protocols are followed in line with the safety data sheet and local regulations.

3. 3D printing and surface functionalization

1. Perform 3D printing of the base substrate following the steps below.
   1. Pour the previously prepared bulk resin (step 2.1) into the 3D printer vat (see Table of Materials), ensuring that the solution completely covers the bottom film in the vat without any air bubbles or other inhomogeneities, and then close the 3D printer case.
   2. Navigate the USB using the 3D printer screen and select the sliced model file by clicking on the triangle Play button to begin the 3D printing process.
   3. By watching the 3D printer screen, take careful note of the number of layers printed, and pause the printing program by pressing the two vertical lines Pause button during 3D printing of the last layer of the base substrate (layer 29 in this case).
   4. Remove the entire build stage and gently rinse the build stage and printed material with undenatured 100% ethanol from a wash bottle for 10 s to remove residual bulk resin from the 3D printed material and the build stage.
   5. Using compressed air, gently dry the 3D printed material and build stage to remove residual ethanol and then reinsert the build stage into the 3D printer.
   6. Remove the vat from the 3D printer and pour the remaining bulk resin into an amber vial. Store the vial in a cool dark place.
   7. Using undenatured 100% ethanol from a wash bottle, carefully rinse the vat to remove any residual bulk resin.
   8. Dry the vat using a stream of compressed air to remove any residual ethanol and reinsert the vat into the 3D printer.
2. Perform surface functionalization.
   1. Pour the previously prepared surface resin (step 2.2) into the 3D printer vat, ensuring that the solution completely covers the bottom film without any air bubbles or other inhomogeneities, and then close the 3D printer case.
   2. Resume the 3D printing program by clicking on the triangle Play button to allow the predetermined surface patterning to occur.
   3. Once the printing program has been completed, remove the build stage from the 3D printer and wash for 10 s with undenatured 100% ethanol using a wash bottle to remove residual surface resin from the 3D printed material and the build stage.
   4. Using compressed air (flow rate, 30 L/min), gently dry the 3D printed material and build stage to remove residual ethanol.
   5. While still attached to the build stage, post-cure the material by inverting the entire build stage and placing it under 405 nm light for 15 min.
   6. Gently remove the surface-functionalized 3D printed material from the build stage using a thin metal plate or paint scraper.
   7. Without further adjustments, analyze the material's mechanical and surface properties.

4. Analysis of 3D printed samples

1. Perform the fluorescence analysis.
   1. Place the 3D printed, surface-functionalized material under a 312 nm UV gas discharge lamp (see Table of Materials) in a dark place, ensuring the surface-functionalized layer is facing up.
   2. Turn the lamp on to continuously irradiate the surface layer with 312 nm light and observe the fluorescent pattern. Take photographs if required.
      NOTE: This is a visual inspection step; time cannot be specified. Irradiation is continuous while observation is occurring.
   3. Place the 3D printed, surface-functionalized material into a Fluorescence imager. Using the provided software, capture digital fluorescence images of the top and bottom surfaces using the Trans-UV (302 nm) gas discharge source (see Table of Materials).
2. Perform the tensile property analysis.
   1. Measure the gauge with and thickness of the dog-bone specimens (in millimeters).
   2. Place the dog-bone-shaped specimens between the grips of a tensile testing machine, ensuring the 3D printed material is equally placed at a distance specified by the standards document, in this case, 50.3 mm.
   3. Set the tensile test program; in this case, the lift speed was set to 1.1 mm/min, the number of samples was set at 10 per second.
   4. Start the program to acquire force (N) vs. travel (mm) data.
   5. Once the sample gets prepared, stop the machine, and save the data as column-separated data with a .CSV file extension.
   6. Convert the force (N) data to stress (MPa) by dividing each point of the force column by the gauge area (mm², obtained by multiplying the gauge width by the gauge thickness).
   7. Convert the travel data to strain (%) by diving the travel data by the gauge length (50.3 mm) at every point and multiplying each result by 100.
   8. Calculate toughness (MJ/m³) using the trapezoidal rule to calculate the area under the stress-strain curve.
   9. Calculate Young's modulus (MPa) by taking the gradient of the stress (MPa) vs. strain (%) curve in the elastic region, in this work from 1%-2% elongation²⁷.

Wyniki

The general procedure for 3D printing and surface functionalization is shown in Figure 1. In this protocol, a network polymer is initially synthesized via a photoinduced RAFT polymerization process¹⁵, using a 3D printer to fabricate an object in a layer-by-layer process (Figure 1A). The bulk resin used to form the polymer network contains a photolabile initiating species (TPO), which generates radicals upon exposure to 405 nm lig...

Dyskusje

The present protocol demonstrates a process for 3D printing of polymer materials with independently tunable bulk and interfacial properties. The procedure is performed via a two-step method by 3D printing the base substrate and subsequently modifying the surface layer of the 3D printed object using a different functional resin but using the same 3D printing hardware. While the 3D printers used in this work are designed to print crosslinked materials in a layer-by-layer fashion, the surface functionalization can ...

Materiały

Name	Company	Catalog Number	Comments
1-pyrenemethyl methacrylate	Sigma-Aldrich	765120
2-(n-butylthiocarbonothioylthio) propanoic acid	Boron Molecular	BM1640
3D Printer	Photon	Mono S	light intensity at digital mask surface = 0.81 mW cm-2
3D Printing Slicing Software	Photon	Photon Workshop V2.1.19
40 kHz Ultrasonic Bath	Thermoline	UB-410
Compressed Air	Coregas	230142	Tank operating at 130 kPa
Computer Assisted Design Program	SpaceClaim	SpaceClaim Design Manager V19.1
Diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide	Sigma-Aldrich	415952
Ethanol Undenatured 100% AR	ChemSupply	EL043-2.5L-P
Ethanol Wash bottle	Rowe Scientific	AZLWGF541P
Fluorescence Imager	Bio-Rad	Gel Doc XR+	Uses a 302 nm gas discharge lamp as emission source
Light intensity power meter	Newport	843-R
Mechanical Tester	Mark–10	ESM303	1 kN force gauge M5–200
Moldable plastic film	Parafilm	PM992
N,N-dimethlacrylamide	Sigma-Aldrich	274135
N,N-Dimethylformamide HPLC	ChemSupply	LC1051-G4L
Poly(ethylene glycol) diacrylate average Mn 250	Sigma-Aldrich	475629
Post Cure Lamp	Leoway	‎B0869BY79P	60 W 405 nm
Standards document	ASTM	ASTM Standard D638-14
Tensile testing machine	Mark-10
UV Light	Fisher Scientific	11-982-30	6 W Spectroline E-Series, Gas discharge lamp
Vortex Mixer IKA Vortex 3	LabTek	3340000I