The Benefits of Going Small: Nanostructures for Mammalian Cell Transfection

Tay, Andy

doi:10.1021/acsnano.0c04624

Cited by 30 publications

(24 citation statements)

References 59 publications

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“…A variety of nanoscale structures have been developed in which either high mechanical pressures (e.g., nanotubes or nanospears) or localized electric shock (e.g., nanostraws, nanofountain probes, nanospikes, and nanochannels) is induced to the nano–bio interface. [ 292 ] Carbon nanotubes (CNTs), [ 293 ] mesoporous silica nanoparticles (MSNs), [ 294 ] gold nanoparticles (AuNPs), [ 295 ] quantum dots (QDs), [ 6 ] silicon nanoneedles, [ 296 ] and silicon nanowires [ 297 ] ( Figure A,B) are examples of nanostructures, which have been widely used for the intracellular cargo loading applications. The chemophysical properties of these nanostructures such as size, charge, and displayed ligands on their surface can be tuned for intracellular delivery purposes.…”

Section: Micro‐ and Nanoengineered Intracellular Deliverymentioning

confidence: 99%

A Comprehensive Review on Intracellular Delivery

Rad

Bazaz

et al. 2021

Advanced Materials

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Intracellular delivery is considered an indispensable process for various studies, ranging from medical applications (cell‐based therapy) to fundamental (genome‐editing) and industrial (biomanufacture) approaches. Conventional macroscale delivery systems critically suffer from such issues as low cell viability, cytotoxicity, and inconsistent material delivery, which have opened up an interest in the development of more efficient intracellular delivery systems. In line with the advances in microfluidics and nanotechnology, intracellular delivery based on micro‐ and nanoengineered platforms has progressed rapidly and held great promises owing to their unique features. These approaches have been advanced to introduce a smorgasbord of diverse cargoes into various cell types with the maximum efficiency and the highest precision. This review differentiates macro‐, micro‐, and nanoengineered approaches for intracellular delivery. The macroengineered delivery platforms are first summarized and then each method is categorized based on whether it employs a carrier‐ or membrane‐disruption‐mediated mechanism to load cargoes inside the cells. Second, particular emphasis is placed on the micro‐ and nanoengineered advances in the delivery of biomolecules inside the cells. Furthermore, the applications and challenges of the established and emerging delivery approaches are summarized. The topic is concluded by evaluating the future perspective of intracellular delivery toward the micro‐ and nanoengineered approaches.

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Section: Micro‐ and Nanoengineered Intracellular Deliverymentioning

confidence: 99%

A Comprehensive Review on Intracellular Delivery

Rad

Bazaz

et al. 2021

Advanced Materials

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“…The transfection process can be broken down into four main steps. [ 7 ] The first step is the preparation of cargo such as oligonucleotides and proteins for delivery. Cargo preparation differs based on the transfection method.…”

Section: Chimeric Antigen Receptor‐immune Cell Transfection and Manufacturingmentioning

confidence: 99%

“…FDA-approved gold standard viruses and bulk electroporation suffer from low transfection efficiency while also perturbing the critical biological attributes of cells such as proliferation, metabolism, and gene expression. [7] This increases the time and costs for cell expansion, and without an efficient and cost-friendly transfection technology, the price (between USD 0.4-0.5 million per patient) for FDA-approved CAR treatments (Kymriah and Yescarta) will remain unaffordable and untimely. [8] The limitations in conventional transfection techniques have motivated the development of micro-and nanoplatforms such as microfluidics, nanoparticles, and high-aspect-ratio nanostructures to improve immune cell viability and throughput during transfection.Herein, we first provide an overview of CAR-T cell manufacturing, with emphasis on the science of transfection and limitations of traditional technology using viruses and bulk electroporation.…”

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confidence: 99%

Materials for Improving Immune Cell Transfection

et al. 2021

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recognize and bind to tumor-associated antigens such as CD19 and epidermal growth factor receptor (EGFR) before releasing cytotoxic granules and effector cytokines to destroy cancer cells. [3] CAR-T therapy has shown remarkable success against CD19 expressing B cell hematological malignancies and, thus far, five products (Kymriah from Novartis; Yescarta and Tecartus from Kite Pharma/Gilead Sciences; and Breyanzi and Abecma from Bristol-Myers Squibb) have been approved by the US Food and Drug Administration (FDA) for clinical use. [4,5] Currently, there are at least 500 clinical trials registered on ClinicalTrials.gov database using engineered immune cells to treat various cancers including that of the brain, lungs, and skin.Despite the clinical success of CAR-T cells against B cell leukemia, CAR-T therapy still face tremendous challenges in manufacturing, safety, and affordability before it can become a viable clinical option. [6] One of the biggest technical difficulties is to transfect sensitive, primary T cells whereby biomolecules like oligonucleotides and proteins have to be delivered intracellularly and into the nuclei of cells. FDA-approved gold standard viruses and bulk electroporation suffer from low transfection efficiency while also perturbing the critical biological attributes of cells such as proliferation, metabolism, and gene expression. [7] This increases the time and costs for cell expansion, and without an efficient and cost-friendly transfection technology, the price (between USD 0.4-0.5 million per patient) for FDA-approved CAR treatments (Kymriah and Yescarta) will remain unaffordable and untimely. [8] The limitations in conventional transfection techniques have motivated the development of micro-and nanoplatforms such as microfluidics, nanoparticles, and high-aspect-ratio nanostructures to improve immune cell viability and throughput during transfection.Herein, we first provide an overview of CAR-T cell manufacturing, with emphasis on the science of transfection and limitations of traditional technology using viruses and bulk electroporation. There will also be discussion of other cell-based cancer immunotherapy using other types of promising immune cell types. Next, we describe emerging transfection platforms and companies that have been established to overcome gaps in CAR-T transfection. We then provide a list of assays constituting the polyfunctionalities of immune cells that we believe will help the field better assess the robustness and suitability of their transfection methods. Finally, we end off with existing challenges in CAR-T transfection and how overcoming these challenges can significantly enhance the clinical impact of CAR-T therapy.Chimeric antigen receptor T cell (CAR-T) therapy holds great promise for preventing and treating deadly diseases such as cancer. However, it remains challenging to transfect and engineer primary immune cells for clinical cell manufacturing. Conventional tools using viral vectors and bulk electroporation suffer from low efficiency while posing risks ...

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“…These nanoscale materials have distinct advantages over other transfection methods as they are minimally perturbative and only deliver the cargo of interest without viral transgene or remnant, toxic materials such as during nanoparticle synthesis. [29] In the past decade, our group has combined nano-scale straw-like structures with electro-injection to deliver a variety of biomolecules into various adherent cell lines and primary stem cells with great success. [30][31][32] Through these endeavors, we have identified DNA delivery and expression in non-adherent primary immune cells as one of the most significant challenges for the transfection field which motivated this study.…”

Section: Introductionmentioning

confidence: 99%

Mechanical Stimulation after Centrifuge‐Free Nano‐Electroporative Transfection Is Efficient and Maintains Long‐Term T Cell Functionalities

Tay

Melosh

2021

Small

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that produces transient gene expression which necessitates repeated, expensive doses for treatment to work. [1] However, transfection using viruses, biochemicals, and bulk electroporation warrant considerable concerns due to high costs, low efficiency, limited cell types, high cell death, and toxic immune reactions. For instance, the size of cargo that can be delivered by most viral vectors is typically restricted to 5-10 kbp, although the use of retroviruses [2] and viral engineering could overcome this limitation. This method also faces safety concerns as immunogenic viral genetic elements can be randomly integrated into host genome, necessitating precautionary and costly long-term patient follow-up. [3] During bulk electroporation, the most popular non-viral method, inhomogeneous electric fields can cause Joule heating and bubble formation that dramatically reduce transfection efficiency and cell viability. [4] The longer time needed for cell manufacturing means delayed and costlier treatments for patients. [5] Advances in flow electroporation has also made this process less biologically perturbative as the cells are only briefly treated with electric fields. Such flow-based electroporation systems can also be scaled up to process billion of cells unlike conventional cuvette-based bulk electroporation platforms which have lower throughput. [6][7][8] The limitations of viral and bulk electroporation have motivated the development of non-viral micro and nano cell transfection technologies. [9] Microfluidic platforms coupled with mechanisms including cell squeezing, [10] micro-injection, [11] shear stresses, [12] deterministic mechanoporation, [13] and vortex shedding [14] have been created for high throughput cell transfection. While excellent performance has been achieved with immune cell lines like Jurkat, few papers have reported success for engineering primary immune cells, except for a handful of publications. [13,[15][16][17] The use of highly concentrated, expensive cargo freely floating in microfluidic channels can also quickly increase operating cost. Clogging of microfluidic channels can too disrupt high throughput cell processing.Nanoparticles with their programmable properties are attractive tools for cargo delivery. Smith et al. made use of nanoparticles to deliver DNA [18] and mRNA [19] for reprogramming T cells. McKinlay et al. also created a library of amphiphilic Transfection is an essential step in genetic engineering and cell therapies. While a number of non-viral micro-and nano-technologies have been developed to deliver DNA plasmids into the cell cytoplasm, one of the most challenging and least efficient steps is DNA transport to and expression in the nucleus. Here, the magnetic nano-electro-injection (MagNEI) platform is described which makes use of oscillatory mechanical stimulation after cytoplasmic delivery with high aspect-ratio nano-structures to achieve stable (>2 weeks) net transfection efficiency (efficiency × viability) of 50% in primary human T cells. This is, to the best of the a...

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The Benefits of Going Small: Nanostructures for Mammalian Cell Transfection

Cited by 30 publications

References 59 publications

A Comprehensive Review on Intracellular Delivery

A Comprehensive Review on Intracellular Delivery

Materials for Improving Immune Cell Transfection

Mechanical Stimulation after Centrifuge‐Free Nano‐Electroporative Transfection Is Efficient and Maintains Long‐Term T Cell Functionalities

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