Vertical Droplet-Squeezing Platform for Highly Efficient Biomolecular Delivery

This study presents a novel optoporation technique using a titanium-coated TiO2 microstructure (TMS) device activated by an infrared diode laser for highly efficient intracellular delivery. The TMS device, fabricated with 120 nm titanium coating on a titanium dioxide (TiO2) microstructure containing microneedles (height ∼2 μm and width ∼4.5 μm), demonstrates enhanced biocompatibility and thermal conductivity compared to the conventional TiO2 microstructure (MS). Exposure to the TMS device with an IR diode laser (980 nm) generates heat, forming photothermal bubbles that disrupt the cell membrane and create transient pores for biomolecular delivery. Unlike traditional optoporation methods, which rely on large, vibration-sensitive lasers, the IR diode laser-assisted TMS device-based optoporation technique offers a compact, cost-effective, and portable alternative, making it suitable for clinical and research applications in resource-constrained environments. The performance of the TMS and MS devices was compared in various cancer cell lines (HeLa, L929, and N2a), with the TMS device showing superior delivery success rates for biomolecules of varying molecular sizes. Notably, the TMS device achieved a 99.30% delivery success rate for the smallest molecule, PI dye, and an 85.17% success rate for the largest studied molecule, β-galactosidase enzyme-Cy5. Furthermore, the TMS device consistently provided a higher delivery success rate at lower laser power, minimizing cellular stress and preserving cell survivability. Moreover, using Western Blot analysis, the TMS device demonstrated lower levels of apoptosis compared to the MS device, with statistically significant differences, highlighting its potential for efficient intracellular delivery while minimizing cellular stress and damage. These results highlight the potential of the TMS device as an advanced tool for large-size intracellular biomolecular delivery, offering significant improvements in stability, efficiency, and cell survivability.

This study investigates the effectiveness of optoporation, activated by a titanium nitride micro-array device, in transporting a wide range of biomolecules into cells. Titanium nitride is a propitious plasmonic material exhibiting localised surface plasmon resonances within the near-infrared biological transparency window. Thin films of titanium nitride were deposited on a glass substrate via sputtering, and a periodic TiN micro-array device was fabricated (1 cm × 1 cm) through photolithography and chemical etching. When the device is irradiated with a laser at 1064 nm with a laser fluence of 10.28 mJ cm-2 and a motorised scanning speed of 5 mm s-1 in the presence of biomolecules, photothermal bubbles form near the plasma membrane and temporarily create pores that facilitate the smooth entry of biomolecules into the cells. Our TiN micro-array device with a laser-scanning setup can transfect more than a million cells within 1 minute. Utilising this device, a variety of biomolecules, including propidium iodide (PI) dye (668.4 Da), EGFP-plasmid DNA (229.4 kDa), and β-galactosidase enzyme (465 kDa), were efficiently delivered into several mammalian cell lines (L929, SiHa, and NIH/3T3), achieving high delivery efficiency and excellent cell viability. The results highlight that the maximum delivery efficiency for the PI dye is 95%, and the cell viability reaches 98% in L929 cells. Similarly, for large molecules like the β-galactosidase enzyme, the delivery efficiency is as high as 94%, with 97% cell viability. The MTT assay confirmed that the device exhibits no cytotoxicity during cell transfection. Thus, it holds potential for applications in cell therapy and diagnostics.

High throughput intracellular delivery of biological macromolecules is crucial for cell engineering, gene expression, therapeutics, diagnostics, and clinical studies; however, most existing techniques are either contact-based or have throughput limitations. Herein, we report a light-activated, contactless, high throughput photoporation method for highly efficient and viable cell transfection of more than a million cells within a minute. We fabricated reduced graphene oxide (rGO) nanoflakes that was mixed with a polydimethylsiloxane (PDMS) nanocomposite thin sheet with an area of 3 cm2 and a thickness of ∼600 μm. Upon infrared (980 nm) nanosecond pulse laser exposure, the rGO nanoflakes induced heat and created photothermal bubbles, leading to cell membrane deformation and biomolecular delivery. Using this platform, we achieved delivery of small to large size molecules, such as propidium iodide (PI) dye (668 Da), dextran (3000 Da), siRNA (20-24 bp), EGFP (6159 bp) and enzymes (465 kDa), in L929, N2a, and HeLa cells as well as in hard-to-transfect NiH3T3 and HuH7 cells. The best results were achieved for enzymes with ∼97% transfection efficiency and 98% cell viability in Huh7 cells. This highly efficient cargo delivery tool is simple and easy to use, and its dimensions can be varied according to the user requirements. Moreover, this safe and successful method has applicability in diagnostics and cell therapy.

Efficient drug and biomolecular delivery into cells is an important area of biomedical research. Intracellular delivery relies on porating cell membranes to allow exterior molecules to enter the cell efficiently and viably. Various methods, including optoporation, electroporation, and viral techniques, can deliver molecules to cells, but come with significant drawbacks such as low efficiency, low throughput, and low viability. We present a new laser-based delivery method that uses laser pulses to excite plasmonic, Titanium Nitride (TiN) microstructures for cell poration and offers high efficiency, throughput, and viability. TiN is a promising plasmonic material for laser-based delivery methods due to its high levels of hardness and thermal stability. We fabricate these microstructures by sputtering thin films of TiN on patterned sapphire substrates. We then optimize plasmonic enhancement and stability by investigating different fabrication conditions. We deliver dye molecules, siRNA, and microspheres to cells to quantify poration efficiency and viability by using flow cytometry and by imaging the target cells at defined time intervals post laser irradiation. Additionally, we study temperature effects via simulations and experiments, as well as oxidation of the TiN films over time. We also use scanning electron microscopy (SEM) techniques to study microstructure damage and cell adhesion. Overall, TiN presents a promising opportunity for use as a reusable material in future biomedical devices for intracellular biomolecular delivery and regenerative medicine.

A major challenge with the use of quantum dots (QDs) for cellular imaging and biomolecular delivery is the attainment of QDs freely dispersed inside the cells. Conventional methods such as endocytosis, lipids based delivery and electroporation are associated with delivery of QDs in vesicles and/or as aggregates that are not monodispersed. In this study, we demonstrate a new technique for reversible permeabilization of cells to enable the introduction of freely dispersed QDs within the cytoplasm. Our approach combines osmosis driven fluid transport into cells achieved by creating a hypotonic environment and reversible permeabilization using low concentrations of cell permeabilization agents like Saponin. Our results confirm that highly efficient endocytosis-free intracellular delivery of QDs can be accomplished using this method. The best results were obtained when the cells were treated with 50 μg ml−1 Saponin in a hypotonic buffer at a 3:2 physiological buffer:DI water ratio for 5 min at 4 ° C.

Nanoparticle-mediated photoporation has arisen as a universal intracellular delivery tool; however, the direct interaction of nanoparticles and cells hampers its clinical translation. Here, we report a uniform contactless intracellular delivery that transfects a large number of cells within a minute and avoids direct contact of nanoparticles and cells, thereby improving the cell viability. Our platform consists of an array of polydimethylsiloxane (PDMS) mixed reduced graphene oxide (rGO) nanoflakes on pyramidal microtips, uniformly distributed at the apex of the tip. The extraordinary optoelectronic properties of rGO were combined with micro-pyramidal cavities to entrap light in micro-cavities and efficiently convert it into heat through multiple reflections and absorptions. As a result, ultralow infra-red laser pulse irradiation could create cavitation bubbles followed by cell membrane deformation and biomolecular delivery. Using this delivery platform, we have achieved the delivery of small to large cargo (668 Da to 465 kDa) in various mammalian cells, including hard-to-transfect H9C2 cardiomyocytes. The best results were achieved for enzyme (465 kDa) delivery with a transfection efficiency and cell viability of 95% and 98%, respectively, in SiHa cells. The highly efficient cargo delivery tool demonstrated a safe and effective approach for cell therapy and diagnostics.

Electroporation is a proven technique that can record action potential of cardiomyocytes and serve for biomolecular delivery. To ensure high cell viability, micro-nanodevices cooperating with low-voltage electroporation are frequently utilized in research, and the effectiveness of delivery for intracellular access is typically assessed using an optical imaging approach like flow cytometry. However, the efficiency of in situ biomedical studies is hampered by the intricacy of these analytical approaches. Here, we develop an integrated cardiomyocyte-based biosensing platform to effectively record action potential and evaluate the electroporation quality in terms of viability, delivery efficiency, and mortality. The ITO-MEA device of the platform possesses sensing/stimulating electrodes which combines with the self-developed system to achieve intracellular action potential recording and delivery by electroporation trigger. Moreover, the image acquisition processing system analyzes various parameters effectively to assess delivery performance. Therefore, this platform has the potential for drug delivery therapy and pathology research for cardiology.

With the growing demand for precise and minimally invasive intracellular delivery, photoporation has emerged as a powerful non-viral strategy. This review presents a comprehensive analysis of photoporation as a versatile intracellular delivery platform, with particular emphasis on the role of micro- and nanostructured materials in enabling efficient transport across a wide range of biomolecular sizes. A key novelty of this review is its size-centric organizational framework, which systematically classifies photoporation strategies based on biomolecular cargo size, from small molecules and nucleic acids to ultralarge assemblies and bacteria, rather than conventional material- or laser-based categorizations. The review examines laser-induced mechanisms responsible for transient membrane permeabilization and highlights critical material parameters, including composition, size, shape, surface charge, and optical properties, that govern light-matter interactions and delivery efficiency. Comparative evaluation of micro- and nanostructured materials across different size regimes provides a practical framework for rational material selection and platform design. In addition, key challenges related to delivery precision, biocompatibility, scalability, and clinical translation are critically discussed alongside emerging optimization strategies. By integrating mechanistic insights with translational considerations, this review provides a structured roadmap for developing safe, efficient, and size-adaptive photoporation platforms for biological research and therapeutic applications.

Autophagy inhibitors enhance biomolecular delivery efficiency of extracellular vesicles

MASSIVELY PARALLEL HIGH THROUGHPUT SINGLE-CELL PRINTING AND HIGHLY EFFICIENT LARGE BIOMOLECULAR DELIVERY INTO MAMMALIAN CELLS

The use of cell-penetrating peptides (CPPs) as biomolecular delivery vehicles holds great promise for therapeutic and other applications, but development has been stymied by poor delivery and lack of endosomal escape. We have developed a CPP-adaptor system capable of efficient intracellular delivery and endosomal escape of user-defined protein cargos. The cell-penetrating sequence of HIV transactivator of transcription was fused to calmodulin, which binds with subnanomolar affinity to proteins containing a calmodulin binding site. Our strategy has tremendous advantage over prior CPP technologies because it utilizes high-affinity non-covalent, but reversible coupling between CPP and cargo. Three different cargo proteins fused to a calmodulin binding sequence were delivered to the cytoplasm of eukaryotic cells and released, demonstrating the feasibility of numerous applications in living cells including alteration of signaling pathways and gene expression.

In this pioneering study, an infrared light‐activated highly efficient and uniform, small to large biomolecular delivery into various cell types is developed using a flower‐shaped microstructure device (FMD). Featuring a unique structural design, this FMD consists of 8 µm in length, with edges of ≈3 and 20 µm gaps between FMD microstructure. When subjected to IR laser exposure at 1050 nm, the FMD triggers the generation of photothermal cavitation bubbles, exerting jet fluid flow on the cell's plasma membrane surface, and facilitating biomolecule delivery into cells. The platform achieves efficient intracellular delivery spanning various biomolecules — from low‐molecular‐weight propidium iodide dye to higher molecular weight siRNA, plasmid, and enzymes — across human cervical (SiHa), mouse fibroblast (L929), and neural crest‐derived (N2a) cancer cells, ensuring consistently high efficiency without compromising cell viability. 95% delivery efficacy and 96% cell viability are achieved for smaller molecules like PI dye in L929 cells. For larger biomolecules such as enzymes, transfection efficiency reached 82%, and cell viability is nearly 90% in SiHa cells. This is confirmed via confocal microscopy and flow cytometry, the FMD‐based delivery system holds broad potential for cellular diagnostics and therapeutics, promising significant advancements in cellular research and biomedical treatments.

Cell patterning is a powerful technique for the precise control and arrangement of cells, enabling detailed single-cell analysis with broad applications in therapeutics, diagnostics, and regenerative medicine. This study presents a novel and efficient technique that enables massively parallel high throughput cell patterning and precise delivery of small to large biomolecules into patterned cells. The innovative cell patterning device proposed in this study is a standalone, ultrathin 3D SU-8 micro-stencil membrane, with a thickness of 10 μm. It features an array of micro-holes ranging from 40 μm to 80 μm, spaced apart by 50 μm to 150 μm. By culturing cells on top of this SU-8 membrane, the technique achieves highly efficient cell patterns varying from single-cell to cell clusters on a Petri dish. Utilizing this technique, we have achieved a remarkable reproducible patterning efficiency for mouse fibroblast L929 (80.5%), human cervical SiHa (81%), and human neuroblastoma IMR32 (89.6%) with less than 1% defects in undesired areas. Single-cell patterning efficiency was observed to be highest at 75.8% for L929 cells. Additionally, we have demonstrated massively parallel high throughput uniform transfection of large biomolecules into live patterned cells by employing an array of titanium micro-rings (10 μm outer diameter, 3 μm inner diameter) activated through infrared light pulses. Successful delivery of a wide range of small to very large biomolecules, including propidium iodide (PI) dye (668.4 Da), dextran (3 kDa), siRNA (13.3 kDa), and β-galactosidase enzyme (465 kDa), was accomplished in cell patterns for various cancer cells. Notably, our platform achieved exceptional delivery efficiencies of 97% for small molecules like PI dye and 84% for the enzyme, with corresponding high cell viability of 100% and 90%, respectively. Furthermore, the compact and reusable SU-8-based membrane device facilitates highly efficient cell patterning, transfection, and cell viability, making it a promising tool for diagnostics and therapeutic applications.

Cellular delivery of biomacromolecules is vital to medical research and therapeutic development. Cationic polymers are promising and affordable candidate vehicles for these precious payloads. However, the impact of polycation architecture and solution assembly on the biological mechanisms and efficacy of these vehicles has not been clearly defined. In this study, four polymers containing the same cationic poly(2-(dimethylamino)ethyl methacrylate) (D) block but placed in different architectures have been synthesized, characterized, and compared for cargo binding and biological performance. The D homopolymer and its diblock copolymer poly(ethylene glycol)-block-poly(2-(dimethylamino) ethyl methacrylate) (OD) readily encapsulate pDNA to form polyplexes. Two amphiphilic block polymer variants, poly(2-(dimethylamino)ethyl methacrylate)-block-poly(n-butyl methacrylate) (DB) and poly(ethylene glycol)-block-poly(2-(dimethylamino)ethyl methacrylate)-block-poly(n-butyl methacrylate) (ODB), self-assemble into micelles, which template pDNA winding around the cationic corona to form micelleplexes. Micelleplexes were found to have superior delivery efficiency compared to polyplexes and detailed physicochemical and biological characterizations were performed to pinpoint the mechanisms by testing hypotheses related to cellular internalization, intracellular trafficking, and pDNA unpackaging. For the first time, we find that the higher concentration of amines housed in micelleplexes stimulates both cellular internalization and potential endosomal escape, and the physical motif of pDNA winding into micelleplexes, reminiscent of DNA compaction by histones in chromatin, preserves the pDNA secondary structure in its native B form. This likely allows greater payload accessibility for protein expression with micelleplexes compared to polyplexes, which tightly condense pDNA and significantly distort its helicity. This work provides important guidance for the design of successful biomolecular delivery systems via optimizing the physicochemical properties.

The recent advancements of single-cell analysis have significantly enhanced the ability to understand cellular physiology when compared to bulk cellular analysis. Here a massively parallel single-cell patterning and very large biomolecular delivery is reported. Micro-pillar polydimethyl siloxane stamp with different diameters (40-100µm with 1cm × 1cm patterning area) is fabricated and then imprint distinct proteins and finally pattern single-cell to small clusters of cells depending on the micro-pillar diameters. The maximum patterning efficiency is achieved 99.7% for SiHa, 96.75% for L929, and 98.6% for MG63 cells, for the 100µm micro-pillar stamp. For intracellular delivery of biomolecules into the patterned cells, a titanium micro-dish device is aligned on top of the cells and exposed by infrared light pulses. The platform successfully delivers small to very large biomolecules such as PI dyes (668Da), dextran 3000Da, siRNA (20-24bp), and large size enzymes (464KDa) in SiHa, L929 and MG63 cells. The delivery efficiency for PI dye, Dextran 3000, siRNA, and enzyme for patterned cells are ≈95 ± 3%, 97 ± 1%, 96 ± 1% and 94 ± 3%, with cell viability of 98 ± 1%. Thus, the platform is compact, robust, easy for printing, and potentially applicable for single-cell therapy and diagnostics.