WO2025259944A1

WO2025259944A1 - 3d topological nanofabrication in hydrogel scaffold

Info

Publication number: WO2025259944A1
Application number: PCT/US2025/033478
Authority: WO
Inventors: Edward Boyden; Gaojie Yang; Quansan YANG; Peter T. So; Yuichiro Kunai; Takahiro NAMBARA
Original assignee: Fujikura Ltd; Massachusetts Institute of Technology
Current assignee: Fujikura Ltd; Massachusetts Institute of Technology
Priority date: 2024-06-14
Filing date: 2025-06-13
Publication date: 2025-12-18
Anticipated expiration: 2026-12-14

Abstract

The invention relates, in part, to methods of 3D topological nanofabrication within hydrogel scaffolds. The method including contacting a hydrogel comprising one of more photosensitizers and catalysts with light to pattern the hydrogel.

Description

3D TOPOLOGICAL NANOFABRICATION IN HYDROGEL SCAFFOLD

Related Applications

This application claims benefit under 35U.S.C.§119(e) of U.S. Provisional application serial number 63/660,001 filed June 14, 2024 the disclosure of which is incorporated by reference herein in its entirety.

Government Support

This invention was made with government support under R01EB024261 awarded by the National Institutes of Health. The government has certain rights in the invention.

Field of the Invention

The invention relates, in part, to methods of nanofabricating hydrogel scaffolds.

Background of the Invention

In recent years, various nano/micro-fabrication techniques within hydrogel matrices have been developed or proposed, yet they often face limitations in terms of material selection and structural resolution. For instance, 3D additive manufacturing requires the use of extrudable hydrogels, such as temperature-sensitive agarose, gelatin, or light-sensitive polymer precursors. These materials not only limit the choice of usable substances but also restrict the achievable structure resolution to above a hundred micrometers due to process constraints. Conversely, 3D subtractive manufacturing necessitates specially designed photosensitive polymers capable of laser ablation, which are not commercially available and require several days to modify existing polymer precursors. Such photosensitive hydrogels, though complex, restrict the choice of materials that can be 3D printed, thereby limiting application scope.

Previous methods, termed Implosion Fabrication (ImpFab, IP No.: US 2017/0081489) demonstrated the construction of porous structures on polyacrylate-based hydrogels using direct laser writing. However, prior publications have not mentioned the use of more efficient photosensitizers and catalysts to achieve micro/nano vacancy structures, resulting in the necessity for higher laser power and often compromising the smoothness and precision of the patterned edges. Additionally, this method's applicability is limited to specific types of hydrogels, further narrowing its utility. Development of better fabrication methods would largely extend the applicability of hydrogels and hydrogel scaffolds in many diverse industries.

Summary of Elements of the Invention

According to an aspect of the invention, a method for fabricating a hydrogel scaffold that includes 3D structures is provided, the method including: (a) preparing an expandable hydrogel; (b) expanding the hydrogel, wherein, exposed polymer chains are present within the expanded hydrogel; (c) contacting the expanded hydrogel with a photosensitizer; and (d) patterning the contacted expanded hydrogel with a light, wherein the patterning results in the fabricated hydrogel scaffold including one or more 3D structures. In some embodiments, the patterning of the contacted, expanded hydrogel includes a three-dimensional exposure of the hydrogel to a light. In certain embodiments, the light is a UV light. In certain embodiments, the light is a laser light. In some embodiments, the laser light is generated with a multi-photon laser. In some embodiments, the laser light is generated with a two-photon laser. In certain embodiments, the power of the laser light contacting the hydrogel is below 50mW. In certain embodiments, the power of the laser light contacting the hydrogel is between 5mW and 200mW (inclusive), 5mM and lOmW (inclusive), 5mW and 15mW (inclusive), 5mW and 20mW (inclusive), 5mW and 25mW (inclusive), 5mW and 30mW (inclusive), 5 mW and 35mW (inclusive), 5 mW and 40mW (inclusive), 5mW and 45mW (inclusive), 5 mW and 50mW (inclusive), 5 mW and lOOmW, lOmW and lOOmW (inclusive), or lOmW and 50mW (inclusive). In some embodiments, the patterning includes a three-dimensional exposure of the contacted expanded hydrogel with the laser light. In certain embodiments, the method also includes contacting the expanded hydrogel with a catalyst that increases the photosensitizer effect in the hydrogel. In some embodiments, the catalyst is an oxidizing catalyst. In some embodiments, the catalyst is hydrogen peroxide or an azide. In certain embodiments, the photosensitizer is xanthene, phenothiazine, and porphyrin. In certain embodiments, the photosensitizer is octadecyl Rhodamine B chloride (R18), Methyl Blue (MB), Rhodamine B (Rhod B), or Rhodamine 123 (R123). In some embodiments, the one or more photosensitizer includes a functionalized photosensitizer. In certain embodiments, the functionalized photosensitizer attaches to the exposed polymer chains within the hydrogel matrix. In some embodiments, the hydrogel includes one or more of: PLGA, PEG, gelatin, hyaluronic acid, agarose, collagen, and sodium alginate. In some embodiments, the patterning includes one or both of multi-layered and multi-tiered 3D hollow structures in the hydrogel. In certain embodiments, the one or more 3D hollow structures are each less than 10 microns in diameter. In certain embodiments, the one or more 3D hollow structures are each between 5 and 10 microns in diameter, between 5 and 15 microns in diameter, between 1 and 15 microns in diameter, between 5 and 20 microns in diameter. In certain embodiments, the method also includes contacting the hydrogel with one or both of ground oxygen and a hydroxyl radical prior to the patterning. In some embodiments, the patterning is not performed with the gel under deoxygenated conditions. In certain embodiments, the method also includes contacting the expanded hydrogel with a solution including isopropylamine to maintain the hydrogel’s expanded state. In some embodiments, the isopropylamine is at a 500 pM concentration in the solution. In some embodiments, the isopropylamine is at a concentration between 200 pM and 600 pM, between 300 pM and 600 pM, 400 pM and 600 pM, 400 pM and 700 pM, or 400 pM and 800 pM in the solution. In certain embodiments, the method also includes washing the hydrogel after patterning. In some embodiments, the hydrogel is washed 1, 2, 3, 4, 5, 6, 7, 8, 9, or more times. In some embodiments, the washing includes washing with distilled water. In certain embodiments, the method also includes shrinking the hydrogel after the patterning.

Brief Description of the Drawings

Fig. 1 provides a schematic illustration of mechanisms for 3D nanoscale topological engineering in scaffolds. Vacancy generation in a pre-polymerized hydrogel scaffold caused by leveraging activated photosensitizer to generate highly reactive singlet oxygen and hydroxyl radicals, and therefore, selectively cleave materials at specific 3D locations in scaffolds.

Fig. 2 provides a graph showing molecular weight reduction of poly(acrylate-co-acrylamide) as a function of light exposure time through gel permeation chromatography.

Fig. 3 provides fluorescent images of square patterns created by gradient laser powers. The left image is a control image and the right image shows results with the patterning conducted in the presence of ground oxygen and hydrogen peroxide. Ground oxygen and hydrogen peroxide improved the patterning efficiency of vacant structures compared to the control case without them. Fig. 4A-F provides fluorescent images of square patterns created by different photosensitizers with gradient laser powers. (Fig. 4A) Rhodamine 123, (Fig. 4B) Rhodamine 6G, (Fig. 4C) sulfo Cy3, (Fig. 4D) Rhodamine B, (Fig. 4E) Methyl Blue, (Fig. 4F) sulfo-Cy5 square pattern with increasing laser power activation.

Fig. 5A-B provides bright field and fluorescent images of different patterns created with (Fig. 5A rhodamine B and (Fig. 5B) sulfo-Cy5.

Fig. 6. shows fluorescent images of linear gradient patterns built with increasing laser power and different solution condition.

Fig. 7A-D shows fluorescent images and 3D reconstruction images of 3D free-form structure of (Fig. 7A) hollow cross array, (Fig. 7B) multi-layer networks, (Fig. 7C) dendritic, and (Fig. 7D) helices built in poly(acrylate-co-acrylamide) hydrogels.

Fig. 8A-D provides fluorescent images of vacant pattern built in different hydrogel materials of (Fig. 8A) agarose, (Fig. 8B) alginate, (Fig. 8C) gelatin, and (Fig. 8D) collagen.

Fig. 9A-B shows results demonstrating high precision and accuracy were testified by the comparison of the (Fig. 9 A) design and (Fig. 9B) fluorescent image of patterned structure.

Fig. 10 provides fluorescent images (left panel), refraction index tomogram image (right panel) and corresponded 3D reconstructed images (bottom) of crossed but unconnected 3D channel structures build in poly(acrylate-co-acrylamide) hydrogels.

Fig. 11 A-C shows the construct interconnected, layered mesh channel structures with hydrophobic side wall built with Octadecyl Rhodamine B Chloride (R18) in poly(acrylate-co- acrylamide) hydrogels. (Fig. 11 A) fluorescent images, (Fig. 1 IB) 3D reconstructed image, and (Fig. 11C) refraction index tomogram image. Fig. 12A-C shows diffractive lens with multi-level and multi-layer 3D structure. (Fig. 12A) bright field image, (Fig. 12B) fluorescent images, (Fig. 12C) 3D reconstructed image.

Detailed Description

The invention, in part, provides methods of 3D topological nanofabrication within hydrogel scaffolds and provides an innovative approach for creating highly intricate structures, unlocking new opportunities in fields such as tissue engineering, regenerative medicine, drug delivery, and more. Methods of the invention can be used for the construction of complex 3D micro and nanostructures, such as multi-layered, hierarchical, interlaced, suspended, and spiral configurations, directly within commonly used hydrogels. Methods set forth herein circumvent the need for photodegradable solid materials, which are not yet commercially viable. Additionally, the selection of hydrogels that can be used for building these micro and nanostructures is vast, broadening the applicability of methods of the invention across biomedical, optical, and microelectronic domains.

Methods of the invention facilitate the creation of complex free-form 3D micro and nano structures, such as multi-layered, hierarchical, interlaced, suspended, and spiral configurations, directly within commonly used hydrogels. Studies have been performed that included selection and use of highly active photosensitizers and catalysts that can be dispersed in a hydrogel and can be activated with light at a focus spot, causing the hydrogel's polymer chains to break down into fragments. This process creates micro and nano-cavities wherever the light scans. In some embodiments of methods of the invention, the light is a laser light. In certain embodiments of methods of the invention, the light is a UV light.

With this invention, it is possible to avoid the use of complex photodegradable materials, which are not yet commercially viable. Moreover, methods of the invention permit the creation of structures with the finesse and freedom of design at the nanoscale that additive manufacturing of hydrogels cannot achieve. Through this invention, micro and nano-devices can be engineered with enhanced performance characteristics, and methods of the invention can be used in advanced applications in the biomedical, optical, and microelectronic fields.

Initial studies were performed that focused on identifying highly active photosensitizers that could be dispersed in aqueous solutions and diffused into the hydrogel matrix. Utilizing direct laser writing techniques, these photosensitizers could be efficiently activated at the laser focus spot, leading to the targeted breaking of the polymer chains within the matrix, thus creating micro and nano-cavities where the laser scans. Studies have also been performed in which catalysts that enhanced the activation of photosensitizers were incorporated, allowing the hydrogel matrix to be ablated into nanoscale fragments at as low a laser power as possible. This subtractive manufacturing technique enabled the formation of complex, self-supporting 3D micro and nano topologies in a variety of hydrogels.

Furthermore, in some embodiments of methods of the invention the photosensitizers were functionalized with various groups (e.g., amino, carboxyl, thiol, lipid chains) which enabled the attachment of these functionalized photosensitizers to the exposed polymer chains within the hydrogel matrix. This dual-action process not only constructs intricate micro and nano topologies but also allows for the functionalization of the sidewalls. As a result, embodiments of methods of the invention can be used to engineer micro and nano-devices with enhanced performance characteristics, and methods of the invention can be used in advanced applications in numerous scientific and technological fields.

Advantages and improvements over existing methods, devices or materials.

In prior years, various nano/micro-fabrication techniques within hydrogel matrices have been developed or proposed, yet they often faced limitations in terms of material selection and structural resolution. For instance, 3D additive manufacturing requires the use of extrudable hydrogels, such as temperature-sensitive agarose, gelatin, or light-sensitive polymer precursors. These materials not only limit the choice of usable substances but also restrict the achievable structure resolution to above a hundred micrometers due to process constraints. Conversely, 3D subtractive manufacturing necessitates specially designed photosensitive polymers capable of laser ablation, which are not commercially available and require several days to modify existing polymer precursors. Such photosensitive hydrogels, though complex, restrict the choice of materials that can be 3D printed, thereby limiting application scope.

Previous literature and previous methods, one of which is called Implosion Fabrication (ImpFab, IP No.: US 2017/0081489) have demonstrated the construction of porous structures on polyacrylate-based hydrogels using direct laser writing. However, none have mentioned the use of more efficient photosensitizers and catalysts to achieve micro/nano vacancy structures, resulting in the necessity for higher laser power and often compromising the smoothness and precision of the patterned edges. Additionally, this method's applicability is limited to specific types of hydrogels, further narrowing its utility.

Certain methods of the invention, include use of efficient photosensitizers, optimized through concentration design and the introduction of catalysts, allowing various types of hydrogels (including PLGA, PEG, gelatin, hyaluronic acid, agarose, collagen, sodium alginate, etc.) to be ablated into nanoscale fragments under very low-power lasers (below 50mW). This obviates the need for photosensitive polymers, significantly enhancing the efficiency of 3D printing and broadening the substrate materials available, thus expanding the application range of 3D microfabrication.

Traditional 3D fabrication techniques are limited to constructing topology within a single hydrogel. Combining specific molecules on particular micro/nano 3D topologies becomes complex, such as requiring needle changes, secondary patterning, or modification with functional groups on special areas. Embodiments of methods of the instant invention allow for the direct integration of unique functional groups into the sidewalls while constructing micro/nano topologies. For example, creating vacant channels with hydrophobic surfaces within hydrophilic hydrogel matrices or channels that can selectively adsorb metal ions with thiol groups. These functional groups can be easily incorporated by designing selected photosensitizer to attach functional groups. Through methods of the invention comprising subtractive 3D printing, devices with micro/nano topological structures possessing special functional properties can be efficiently fabricated.

Certain Elements of Fabrication Process

Fig. 1 provides a schematic illustration of an example of a fabrication process of the invention. In some embodiments, the process starts with immersing a polymerized and expanded hydrogel scaffold [a non-limiting example of which is poly(acrylate-co-acrylamide) hydrogel] in a solution comprising photosensitizers. A non-limiting example shown in Fig. 1 includes photosensitizer Rhodamine B; (concentration: 150 pM) and isopropylamine (concentration: 500 pM) for maintaining expanded status. The photosensitizers are activated by light exposure, which in some instances is UV light exposure and in certain instances is laser light exposure. A non-limiting example of parameters of contacting a hydrogel in a method of the invention with a laser light comprises contacting the hydrogel with laser light generated with a multi-photon laser with a wavelength of 780 nm, a pulse width of 100 fs, and a frequency of 80 MHz) to selectively cleave materials and generate 3D void structures inside the hydrogel scaffold. At the patterning step, the structure resolution is the same as that in direct laser writing using the same laser system. More complex structures become possible because the structures maintain physical connections with their surroundings during the patterning process. The fabrication methods of the invention may include contacting an expanded hydrogel with one or more photosensitizers, catalysts, hydrogel stability agents, etc.

A feature of fabrication methods of the invention that has now been identified is that some photosensitizers are highly effective at cleaving polymer chain from a non-photocleavable solid. The mechanism shown in Fig. 1 illustrates that the photosensitizers contacted by light energy (such as but not limited to light energy from a multi-photon laser) are activated from ground state to excited state. The activated photosensitizers transfer the absorbed light energy to form singlet oxygen (from ground triplet oxygen) and hydroxyl radicals at their locations in the hydrogel. These singlet oxygen and hydroxyl radicals are extremely reactive to degrade and cleave the scaffold material in their locations. Contacting a hydrogel of the invention with light that activates photosensitizers in the hydrogel generates 3D hollow structures within the hydrogel. The process of generating the structures within a hydrogel is referred to herein as “patterning” the hydrogel.

The patterning and processing elements of embodiments of methods of the invention comprise three-dimensional exposure to a light, which may be a UV light or may be a laser light. In a non-limiting example, in some embodiments of methods of the invention, three-dimensional exposure is performed using a two-photon laser system (Mai Tai Ti: Sapphire laser; wavelength: 780 nm; pulse width: 100 fs; frequency: 80 MHz). The pixel dimension of photomasks may remain as 580 x 580 nm. The average power (5 - 200 mW), dwell time (2 - 20 ps), Z-step (0.1 - 2.5 pm), and the number of repetitions (1 - 10) is adjusted according to different application scenarios. As non-limiting examples, multiple water immersion objectives (CFI75 Apochromat LWD 20XC with a working distance (WD) of 2.80 mm and a numerical aperture (NA) of 1.00; CFI Apochromat Lambda S 40XC with a WD of 0.18 mm and a NA of 1.24) may be used in the patterning process.

Hydrogel Preparation and Expansion Diverse types of expandable hydrogels may be used in methods of the invention to fabricate a hydrogel comprising 3D hollow or vacant patterning within the hydrogel. A nonlimiting example of a type of expandable hydrogel that can be used in certain embodiments of methods of the invention is a poly(acrylate-co-acrylamide) hydrogel. In some embodiments of methods of the invention, an expandable hydrogel is a hydrogel comprising one or more of: poly(lactic-co-glycolic acid (PLGA), polyethylene glycol (PEG), gelatin, hyaluronic acid, agarose, collagen, and sodium alginate. Details for preparing expandable hydrogels are provided herein, and additional methods of preparing expandable hydrogels suitable for use in certain methods of the invention are known in the art.

Methods of the invention utilize expandable hydrogels and hydrogel expansion is performed prior to patterning the hydrogel. In some embodiments, an expandable hydrogel is contacted with (also referred to as “incubated in”) a solvent or liquid. The solvent or liquid is absorbed by the expandable hydrogel material, resulting in expansion of the hydrogel. Certain methods of hydrogel expansion are described herein and additional art-known methods may be used in conjunction with methods of the invention to expand a hydrogel.

In some embodiments of methods of the invention, prior to patterning, an expanded hydrogel is contacted with an agent that assists in maintaining the expanded state of the hydrogel. A non-limiting example of an agent that may be used to assist in maintaining the expanded state of a hydrogel of the invention is isopropylamine. An expanded hydrogel of the invention may be contacted with a solution comprising isopropylamine prior to patterning of the hydrogel. In some embodiments, the expanded hydrogel is placed in (which may also be referred to as incubated in) a solution comprising 500 pM concentration of isopropylamine. In certain embodiments, the expanded hydrogel is incubated in a solution comprising isopropylamine at a concentration between 200 pM and 600 pM, between 300 pM and 600 pM, 400 pM and 600 pM, 400 pM and 700 pM, or 400 pM and 800 pM in the solution.

In some embodiments of methods of the invention, an expanded hydrogel is incubated in a solution comprising one or more photosensitizers. Non-limiting examples of photosensitizers that may be used in certain embodiments of methods of the invention are octadecyl Rhodamine B chloride (R18), Methyl Blue (MB), Rhodamine B (Rhod B), and Rhodamine 123 (R123). In some embodiments of methods of the invention, an expanded hydrogel is incubated in solution comprising a photosensitizer such as xanthene, phenothiazine, and porphyrin. The photosensitizers xanthene, phenothiazine, and porphyrin have been determined to be highly effective at cleaving a polymer chain from a non-photocleavable solid. In some embodiments of methods of the invention, an expanded hydrogel is incubated in a solution comprising isopropyl amine and one or more photosensitizers.

Fig. 1 illustrates activation of photosensitizers by light energy introduced three dimensionally. Photosensitizers within the hydrogel are contacted with the light and as a result are activated from ground state to excited stated. The activated photosensitizers transfer the absorbed light energy to form singlet oxygen (from ground triplet oxygen) and hydroxyl radicals in their locations in the hydrogel. It has now been determined that these singlet oxygen and hydroxyl radicals are extremely reactive to degrade and cleave the scaffold material at their locations in a hydrogel, no matter whether the scaffold material is a photocleavable scaffold material or a non-photocleavable scaffold material.

It has been identified that one or more catalysts can be used in embodiments of methods of the invention to enhance the activation capabilities of a photosensitizer. Catalysts with oxidizing properties, non-limiting examples of which include hydrogen peroxide and azide increase the level of activation of photosensitizers in a hydrogel. Some embodiments of methods of the invention, include contacting an expanded hydrogel comprising a photosensitizer with one or more catalysts such as hydrogen peroxide and/or azide. Inclusion of such catalysts results in the ability to achieve fully vacant structures within an expanded hydrogel using levels of laser power that are significantly lower than needed in the absence of the catalyst(s). Methods of the invention in which the expanded hydrogel to be patterned comprises a catalyst with oxidizing properties, such as but not limited to hydrogen peroxide and azide can be used to generate fully vacant structures in the hydrogel using laser power that is less than 50 mW. In some embodiments, inclusion of catalysts hydrogen peroxide or azide permits generation of fully vacant structures in the hydrogel using levels of laser power of 30mW and 45mW, respectively.

It was determined that different photosensitizers presented different efficiency of vacancy generation. In a non-limiting example, contacting expanded poly(acrylate-co-acrylamide hydrogels that comprised different photosensitizers, with laser light, required contacting the hydrogels with different laser powers. It was determined that use of some fluorescent molecules (photosensitizers) in the absence of a catalyst was not efficient for photo-ablation of hydrogels. In a non-limiting example, when employing sufficient laser power and dwell time (30m W, Ips) to excite Cy5 and Rhol23 in the same scaffold material — poly(acrylate-co-acrylamide) hydrogels — resulted in distinctly different optical patterns. As shown in Fig. 5, the pattern created by Rhod B exhibited no fluorescence; the pattern appeared more transparent in bright- field images, indicating that the 3D structure formed by RhoB was purely hollow. In contrast, the pattern generated by Cy5 displayed strong fluorescence and significant polymer residue under bright-field conditions, demonstrating that Cy5 without catalyst was not an effective photosensitizer for ablation of the hydrogel matrix to create vacancies. Thus, certain methods of the invention comprise hydrogels comprising combinations of certain photosensitizers with catalysts in order to have effective vacancy generation when contacted with light.

Patterning

Methods of the invention include patterning steps in which the scaffold of an expanded hydrogel is cleaved, forming one or more 3D hollow structures within the hydrogel. It has been determined that scaffold cleavage in the hydrogel resulted from singlet oxygen and hydroxyl radicals generated by light-activated photosensitizers in the expanded hydrogel. In a nonlimiting example, in the presence of ground oxygen (also referred to as “triplet oxygen”) and hydrogen peroxide, an expanded hydrogel comprising photosensitizers that was contacted with laser light with a power of 50 mW resulted in full vacancy in the patterned region (see Fig. 2). These results demonstrate the role of singlet oxygen and hydroxyl radicals in the vacancy generation. In contrast, in the absence of ground oxygen and hydrogen peroxide contacting an expanded hydrogel comprising photosensitizers with laser light at a power ranging from 5 to 85 mW failed to generate void structures in the scaffold (Fig. 3). In the absence of ground oxygen and hydrogen peroxide, the photosensitizers degraded the scaffold partially and anchored to the patterned region, therefore leading to fluorescence in that region. It was also determined that for the vacant structures, there were photosensitizers depositing and anchoring on surfaces and edges due to the partial power generated on those locations, which a practitioner can use as an indicator to check and characterize the vacant structures using imaging, such as confocal imaging.

Patterning of an expanded hydrogel comprises a three-dimensional exposure of the hydrogel to a light. Exposure of the hydrogel to a light is also referred to herein as “contacting” the hydrogel with a light. In some embodiments, the hydrogel is contacted with a UV light. In certain embodiments of methods of the invention, the hydrogel is contacted with a laser light. In some embodiments of methods of the invention, the laser light is generated with a two-photon laser. When contacting a hydrogel with a laser light, elements considered include, but are not limited to, laser power and dwell time of the contact.

In some embodiments of methods of the invention, contacting with a laser light comprises contacting with a laser light having an average power of 5 - 200 mW. In some embodiments of methods of the invention, the power of the laser light contacting the hydrogel is between 5 mW and 200mW (inclusive), 5mM and lOmW (inclusive), 5 mW and 15mW (inclusive), 5mW and 20mW (inclusive), 5 mW and 25mW (inclusive), 5 mW and 30mW (inclusive), 5mW and 35mW (inclusive), 5mW and 40mW (inclusive), 5mW and 45mW (inclusive), 5mW and 50mW (inclusive), 5mW and lOOmW, lOmW and lOOmW (inclusive), or lOmW and 50mW (inclusive).

With respect to dwell times, in certain embodiments of methods of the invention, a hydrogel is contacted with a laser light at a dwell time of 2 - 20 ps per contact. In some embodiments of methods of the invention, the dwell time of the laser light contacting the hydrogel is between 1 and 10 ps per contact (inclusive), 2 and 10 ps per contact (inclusive), 5 and 10 ps per contact (inclusive); 5 and 20 ps per contact (inclusive); 5 and 30 ps per contact (inclusive), or 1 and 30 ps per contact (inclusive).

For patterning, a hydrogel may be contacted with the light once or a plurality of times. AS used herein the term “plurality” means more than one. In some embodiments of methods of the invention, a hydrogel is contacted with a light 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more times.

In some embodiments of methods of the invention, the patterning of the hydrogel is not performed with the hydrogel under deoxygenated conditions.

A hydrogel that has been patterned using a method of the invention may be washed with distilled water to remove the chemicals and polymer fragments. In some embodiments, after patterning the hydrogel is washed 2, 3, 4, 5, or more times with distilled water.

In some embodiments, following patterning of a hydrogel, the method of the invention includes shrinking the patterned hydrogel. Art-known methods may be used to shrink the patterned hydrogel.

Commercial applications (economic potential, etc.) 3D topological nanofabrication in hydrogel scaffolds such as can be done using certain embodiments of methods of the invention, represents a breakthrough technology with vast commercial potential, spanning multiple industries including healthcare, biotechnology, environmental engineering, cosmetics, and optics. This innovative approach allows for the creation of highly precise, functionalized materials that can mimic the natural structure and functionality of biological tissues, enhance the efficacy and specificity of drug delivery systems, and enable the development of advanced diagnostic tools. Its applications range from the development of customized biomedical implants and devices that seamlessly integrate with body tissues, to the creation of sophisticated drug delivery platforms capable of releasing therapeutic agents in a controlled manner, and even to the production of environmentally friendly agricultural and remediation solutions.

The versatility of methods of the invention extends to the other industries, such as but not limited to, the cosmetic industry for transdermal delivery systems and advanced skincare products, as well as to the development of soft robotics and actuators that could revolutionize wearable technology and medical devices. As 3D topological nanofabrication technology continues to evolve and become more cost-effective, it is poised to drive innovation across these sectors, offering solutions that were previously unattainable and opening new frontiers in both commercial and research domains.

Examples

Example 1

Methods and Materials

All chemicals were purchased from Sigma- Aldrich and used without further purification except when mentioned specifically. The starting materials for scaffolds included sodium acrylate (Gelest, Morrisville, PA), acrylamide, N,N'-methylenebisacrylamide (MBAA), ammonium persulfate (APS), tetramethylethylenediamine (TEMED), agarose, methacrylated alginate (Advanced Biomatrix, Carlsbad, CA), methacrylated gelatin (Advanced Biomatrix, Carlsbad, CA), 2-hydroxy-l-[4-(2-hydroxy ethoxy) phenyl]-2-methyl-l -propanone (Irgacure D- 2959). The photosensitizers included Rhodamine dyes (B, 6G, and 123), octadecyl Rhodamine B chloride (R18; Thermo Fisher Scientific., Waltham, MA), Methyl Blue (MB), sulfo-Cyanine dyes (3, 5, and 7) (Lumiprobe, Westminster, MD). Synthesis of hydrogel

The synthesis of poly(acrylate-co-acrylamide) hydrogels with a shrinkage factor of 12 began with dissolving sodium acrylate (26.0 wt%), acrylamide (7.5 wt%), and PBS (l x; 16.7 vol%) in ultrapure water to form solution A. Dissolving MBAA (2.0 wt%), APS (10.0 wt%), and TEMED (10.0 wt%) in ultrapure water formed solution B, C, and D, respectively. Mixing solution A (600 pL), B (13 pL), and ultrapure water (347 pL), followed by bubbling N2 for 3 min to remove most of the dissolved O2, resulted in a solution E. Further mixing solution E (192 pL), D (4 pL), and C (4 pL) in sequence and filling the solution in to a hydrophobic mold finished the hydrogel synthesis. The synthesis of poly(acrylate-co-acrylamide) hydrogels with a shrinkage factor of 5 was similar with only difference in solution A and solution E. The new solution A contained sodium acrylate (39.0 wt%), acrylamide (11.3 wt%), and PBS (lx; 16.7 vol%) in ultrapure water. Mixing solution A (600 pL), B (120 pL), and ultrapure water (240 pL), followed by bubbling N2 for 3 min to remove most of the dissolved O2, led to the new solution E.

The synthesis of agarose hydrogels included dissolving agarose (5.0 wt%) in ultrapure water, microwaving for 2 min, and cooling down gradually to room temperature. The synthesis of alginate or gelatin hydrogels included dissolving methacrylated alginate (5.0 wt%) or methacrylated gelatin (2.0 wt%) in ultrapure water, adding Irgacure D-2959 (0.25 wt%), and exposing to ultraviolet (UV; wavelength: 365 nm) for 60 min. Pre-immersing the glass mold into trichloro(octadecyl) silane (0.2 vol % in hexane) or Sigmacote (a siliconizing reagent) for 90 sec and rinsing with ultrapure water led to non-sticky glass surface. After hydrogel synthesis, soaking these gels in ultrapure water and replacing the water three times allowed the gels to reach the expanded status.

Patterning preparation

Patterning process began with soaking the hydrogel scaffolds in isopropylamine solution (concentration: 66.7 pM; pH: 9.5) for three times (each time for 30 min) for maximum expansion, and then soaking in photosensitizer solution (concentration: 150 pM; O2 gassing for 5 min) with isopropylamine (concentration: 500 pM) to remain expanded status and with catalyst of hydrogen peroxide (concentration: 10 mM) to increase the photosensitizer efficiency. Patterning and processing

Three-dimensional exposure utilized a two-photon laser system (Mai Tai Ti:Sapphire laser; wavelength: 780 nm; pulse width: 100 fs; frequency: 80 MHz). The pixel dimension of photomasks remained as 580 x 580 nm. The average power (5 - 200 mW), dwell time (2 - 20 ps), Z-step (0.1 - 2.5 pm), and the number of repetitions (1 - 10) were adjusted according to different application scenarios. Multiple water immersion objectives (CFI75 Apochromat LWD 20XC with a working distance (WD) of 2.80 mm and a numerical aperture (NA) of 1.00; CFI Apochromat Lambda S 40XC with a WD of 0.18 mm and a NA of 1.24) were used in the patterning process. After patterning, the hydrogel was washed with distilled water for 4 times to remove the chemicals and polymer fragments.

Results and discussion:

1. Mechanisms for 3D nanoscale topological engineering in scaffolds

The schematic illustrations of an embodiment of the entire fabrication process appear in Fig. 1. The process starts with immersing a polymerized and expanded hydrogel scaffold [for example, but not limited to a poly(acrylate-co-acrylamide) hydrogel scaffold] in a solution filled with photosensitizers (e.g., Rhodamine B; concentration: 150 pM) and isopropylamine (concentration: 500 pM) for maintaining expanded status. The photosensitizers were activated by light exposure (e.g., a multi-photon laser with a wavelength of 780 nm, a pulse width of 100 fs, and a frequency of 80 MHz) to selectively cleave materials and generate 3D void structures inside the hydrogel scaffold. At the patterning step, the structure resolution is the same as that in direct laser writing using the same laser system. More complex structures become possible because the structures maintain physical connections with their surroundings during the patterning process.

The core in the fabrication process included the discovery that some photosensitizers (e.g., xanthene, phenothiazine, and porphyrin) can be highly effective to cleave polymer chain from a non-photocleavable solid. The mechanism shown in Fig. 1 illustrates the photosensitizers were able to be activated, by light energy introduced three-dimensionally from multi-photon lasers, from ground state to excited state. These activated photosensitizers transferred the absorbed light energy to form singlet oxygen (from ground triplet oxygen) and hydroxyl radicals in their locations. These singlet oxygen and hydroxyl radicals were extremely reactive to degrade and cleave the scaffold material in their locations, no matter whether the scaffold material was photocleavable or not.

Gel permeation chromatography (GPC) and systematic confocal imaging were performed to prove the discovery. For GPC, the study began with dissolving poly(acrylate-co-acrylamide) chains (0.1 wt%; molecular weight: ~ 145 kg mol-1), Rhodamine B (150 pM), hydrogen peroxide (10 mM) in ultrapure water. Continuous ultraviolet (UV; wavelength: 365 nm; power intensity: 20 mW cm'²) exposure led to the reduction in the molecular weight, from 157 ± 5 kg mol-1 (initially), and 153 ± 7 kg mol-1 (4 hr), 150 ± 6 kg mol-1 (8 hr), 131 ± 6 kg mol-1 (20 hr), to 97 ± 8 kg mol-1 (40 hr), as shown in Fig. 2. The results showed the photosensitizers were able to cleave polymer chains and reduced the molecular weight under light exposure. And it is notable to mention that the UV used here had a much lower intensity compared to the multiphoton laser system, so the longer time was required for the cleavage.

Additionally, in order to prove the singlet oxygen and hydroxyl radicals generated by activated photosensitizers were responsible for scaffold cleavage, experiments without and with ground oxygen (also known in the art as “triplet oxygen”) and hydrogen peroxide were performed. In the situation without ground oxygen and hydrogen peroxide, the laser with power ranging from 5 to 85 mW was unable to generate void structures in the scaffold (Fig. 3). These photosensitizers degraded the scaffold partially and anchored to the patterned region, therefore leading to fluorescence in that region. By contrast, with ground oxygen and hydrogen peroxide, Fig. 2 shows that with only a power of 50 mW, full vacancy occurred in the patterned region, proving the critical role of singlet oxygen and hydroxyl radicals in the vacancy generation. It is notable to mention that for the vacant structures, there were photosensitizers depositing and anchoring on surfaces and edges due to the partial power generated on those locations, which can serve as an indicator to check and characterize the vacant structures by confocal imaging.

2. Photosensitizer and catalyst selection

Diverse kinds of photosensitizers were proven to be effective using methods of the invention, including but not limited to: octadecyl Rhodamine B chloride (R18), Methyl Blue (MB), Rhodamine B (Rhod B) and Rhodamine 123 (R123). Fig. 4 presents the fluorescent intensity of the patterned region (square patterns) by using these photosensitizers as a function of laser power. With increased laser power from the beginning, the normalized fluorescent intensity increased due to the anchored photosensitizers in the patterned region; continuing to increase power led to the decline of the fluorescein signals and the degradation of the scaffold materials. Further increasing the laser power resulted in complete vacant structures in the patterned region. Notably, different photosensitizers present different efficiency of vacancy generation, and therefore, required different laser powers for the same scaffold material (poly(acrylate-co- acrylamide) hydrogels).

Some fluorescent molecules without catalyst were not efficient for photo-ablation of hydrogels. Employing sufficient laser power and dwell time (30mW, Ips) to excite Cy5 and Rhol23 in the same scaffold material — poly(acrylate-co-acrylamide) hydrogels — resulted in distinctly different optical patterns. As shown in Fig. 5, the pattern created by Rhod B exhibited no fluorescence; the pattern appeared more transparent in bright-field images, indicating that the 3D structure formed by RhoB was purely hollow. In contrast, the pattern generated by Cy5 displayed strong fluorescence and significant polymer residue under bright-field conditions, demonstrating that Cy5 without catalyst was not an effective photosensitizer for ablation of the hydrogel matrix to create vacancies.

Additionally, the use of catalysts was found to enhance the activation capabilities of the photosensitizer. Catalysts with oxidizing properties, such as hydrogen peroxide and azide were primarily employed in certain studies. As shown in Fig. 6, the red fluorescent squares represent Rhod B patterns constructed on poly(acrylate-co-acrylamide) hydrogels with progressively increasing laser power from left to right. In the groups where azide and hydrogen peroxide were added, the activation of Rhod B improved with increasing laser power, achieving fully vacant structures at 30mW and 45mW, respectively. Without any catalyst, Rhod B required a laser power of around 60mW to form vacant structures. Even with sufficient laser power under deoxygenated conditions, Rhod B did not create non-fluorescent squares; it could only form fluorescent patterns or partially hollow structures.

3. Ability to build 3D free- form structure

The 3D topological engineering in solids can achieve some complex structures that are challenging in direct laser writing. Previously, constructing multi-layered and multi-tiered complex 3D hollow structures smaller than 10 microns in scale within hydrogels was highly challenging or not possible. Methods set forth herein did not require the use of photocleavable solid materials and did not need the photosensitive solvents' layer by layer self-supporting structure in 3D printing. By employing a subtractive printing approach within the solid material of the hydrogel, it was possible to construct various unsupported 3D structures, thus enabling the creation of true 3D structures with high degrees of freedom that are otherwise difficult to fabricate. For instance, as shown in Fig. 7, multi-layered suspended nano-hollow cross arrays, multi-layer networks, helices, and dendritic structures within the hydrogel, which demand high self-support, could be easily achieved using methods of the invention.

4. Versatility for diverse scaffold materials

One of the key features in the strategy used in methods of the invention is the versatility for diverse scaffold materials and photosensitizer chemicals because the strategy does not build on the photocleavable properties of scaffold materials. Besides the poly(acrylate-co-aciylamide) hydrogels, it was determined that the strategy is also applicable to wide range of hydrogels, including, but not limited to agarose, alginate, gelatin, and collagen. In some studies, Rhodamine B was utilized as an example of the photosensitizer as proof of effectiveness of the method. The detailed synthesis procedures for these hydrogel scaffolds are described in the Materials & Methods section, above herein. The patterning started with immersing these hydrogel scaffolds in a solution containing Rhodamine B (1 mM), hydrogen peroxide (50 mM), and with O2 gassing for 5 min. Corresponding patterns were successfully created in these hydrogel scaffolds, which are ‘Agarose’, ‘Alginate’, ‘Gelatin’, and cross array patterns inside agarose, alginate, gelatin, and collagen scaffolds, respectively, as shown in Fig. 8. Because these hydrogels are widely used in academic settings and in industry, the strategy disclosed herein can help users to structure these materials in 3D easily without further chemical modifications.

5. High precision and accuracy of 3D fabrication

The topological structures constructed using embodiments of methods of the invention exhibited high precision and accuracy. Based on the design shown in Fig. 9, columnar structures ranging from a few microns to tens of microns in scale were fabricated. As demonstrated by the fluorescence images, columnar structures larger than 2 microns in size were well-maintained after patterning. Furthermore, measurements of the patterned fluorescent structures confirmed that the fabrication deviation was less than 1% at the scale of hundreds of microns. 6. High Degree of Freedom Structural Applications and Demonstrations

Due to the high degree of freedom in the structural design of the micro and nanostructures, it was possible to create configurations and applications that were unachievable through other methods. For instance, as demonstrated in Fig. 10, it was possible to construct crossed but unconnected 3D channel units. Such structures significantly increased the contact area between fluids without enlarging the overall system size. This configuration induced more complex fluid flow patterns within the channels, thus accelerating mixing efficiency. It is particularly suitable for microfluidic reactions that require rapid and efficient high-resolution material exchange and mixing of multiple chemical substances within a compact space.

Fig. 11 illustrates the ability to use certain embodiments of methods of the invention to construct interconnected, layered mesh channel structures built with Octadecyl Rhodamine B Chloride (R18). These structures enable precise fluid distribution and merging across multiple layers, which is crucial for microfluidic systems that require multistep processing or multiple fluid inputs. This increases the reaction efficiency and yield, as well as the complexity and functionality of the microfluidic systems. Additionally, it was possible to modify microchannels with hydrophobic surfaces or other functional molecules and vary the porosity of the channels, facilitating the separation of multiple materials. Such configurations play a vital role in pharmaceutical development, environmental monitoring, and clinical diagnostics.

The ability to build complex 3D microchannel structures allows integration of separation, detection, and analysis functions on a single chip, enhancing the overall efficiency and functionality of the system. This provides efficient, precise fluid handling and management solutions for fields such as material science, energy technology, and environmental engineering. Furthermore, this fabrication method can also be applied in optical domains.

As shown in Fig. 12, multi-layered, multi-level metasurface structures have now been constructed using methods of the invention. These topological configurations are based on the technology's ability to create independent hollow feature units within a hydrogel matrix, resulting in regions with different refractive indices between the pattern area and the matrix. By constructing multi-layered, multistage metamaterials, it is now possible to apply them in the creation of optical elements related to diffraction and focused imaging. Equivalents

Although several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto; the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and/or methods, if such features, systems, articles, materials, and/or methods are not mutually inconsistent, is included within the scope of the present invention.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified, unless clearly indicated to the contrary.

All references, patents and patent applications and publications that are cited or referred to in this application are incorporated by reference in their entirety herein. What is claimed is:

Claims

1. A method for fabricating a hydrogel scaffold comprising 3D structures, the method comprising:

(a) preparing an expandable hydrogel;

(b) expanding the hydrogel, wherein, exposed polymer chains are present within the expanded hydrogel;

(c) contacting the expanded hydrogel with a photosensitizer; and

(d) patterning the contacted expanded hydrogel with a light, wherein the patterning results in the fabricated hydrogel scaffold comprising one or more 3D structures.

2. The method of claim 1, wherein the patterning of the contacted, expanded hydrogel comprises a three-dimensional exposure of the hydrogel to a light.

3. The method of claim 1, wherein the light is a UV light.

4. The method of claim 1, wherein the light is a laser light.

5. The method of claim 4, wherein the laser light is generated with a two-photon laser.

6. The method of claim 4, wherein the power of the laser light contacting the hydrogel is below 50mW.

7. The method of claim 4, wherein the power of the laser light contacting the hydrogel is between 5mW and 200mW (inclusive), 5mM and lOmW (inclusive), 5mW and 15mW (inclusive), 5mW and 20mW (inclusive), 5 mW and 25mW (inclusive), 5 mW and 30mW (inclusive), 5mW and 35mW (inclusive), 5 mW and 40mW (inclusive), 5mW and 45mW (inclusive), 5mW and 50mW (inclusive), 5mW and lOOmW, lOmW and lOOmW (inclusive), or lOmW and 50mW (inclusive).

8. The method of claims 4, wherein the patterning comprises a three-dimensional exposure of the contacted expanded hydrogel with the laser light.

9. The method of claim 1, further comprising contacting the expanded hydrogel in (b) with a catalyst that increases the photosensitizer effect in the hydrogel.

10. The method of claim 9 wherein the catalyst is an oxidizing catalyst.

11. The method of claim 9, wherein the catalyst is hydrogen peroxide or azide.

12. The method of claim 1, wherein the photosensitizer is xanthene, phenothiazine, and porphyrin.

13. The method of claim 1, wherein the photosensitizer is octadecyl Rhodamine B chloride (R18), Methyl Blue (MB), Rhodamine B (Rhod B), or Rhodamine 123 (R123).

14. The method of claim 1, wherein the one or more photosensitizer comprises a functionalized photosensitizer.

15. The method of claim 14, wherein the functionalized photosensitizer attaches to the exposed polymer chains within the hydrogel matrix.

16. The method of claim 1, wherein the hydrogel comprises one or more of: PLGA, PEG, gelatin, hyaluronic acid, agarose, collagen, and sodium alginate.

17. The method of claim 1, wherein the patterning comprises one or both of multi-layered and multi-tiered 3D hollow structures in the hydrogel.

18. The method of claim 1, wherein the one or more 3D hollow structures are each less than 10 microns in diameter.

19. The method of claim 1 , wherein the one or more 3D hollow structures are each between 5 and 10 microns in diameter, between 5 and 15 microns in diameter, between 1 and 15 microns in diameter, between 5 and 20 microns in diameter.

20. The method of claim 1, further comprising contacting the hydrogel with one or both of ground oxygen and a hydroxyl radical prior to the patterning.

21. The method of claim 1, wherein the step (d) is not performed with the gel under deoxygenated conditions.

22. The method of claim 1, further comprising contacting the expanded hydrogel with a solution comprising isopropylamine to maintain the hydrogel’s expanded state.

23. The method of claim 22, wherein the isopropylamine is at a 500 pM concentration in the solution.

24. The method of claim 22, wherein the isopropyl amine is at a concentration between 200 pM and 600 pM, between 300 pM and 600 pM, 400 pM and 600 pM, 400 pM and 700 pM, or 400 pM and 800 pM in the solution.

25. The method of claim 1, further comprising washing the hydrogel after patterning.

26. The method of claim 25, wherein the hydrogel is washed 1, 2, 3, 4, 5, 6, 7, 8, 9, or more times.

27 The method of claim 25, wherein the washing comprises washing with distilled water.

28. The method of any one of claim 1-27, further comprising shrinking the hydrogel after the patterning.