Month: June 2021

magnetofection, also known as magnetic transfection, is a procedure in which nucleic acid is bound to magnetic nanoparticles (MNPs) followed by their delivery into eukaryotic cells through applying magnetic force. The MNPs are usually made up of iron oxide and degraded within living cells, thus posing no hazard to cells.  This technique has broader applications in molecular, cellular biology research and potential therapeutic applications (1).

How Magnetofection Works

In Magnetofection, nucleic acids are first bound to MNPs, followed by their delivery into eukaryotic cells with the help of a magnetic field. The binding of nucleic acids [deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)] to MNPs is facilitated through electrostatic interactions. Both the DNA and RNA have negative surface charges and are attracted towards positive (cationic) materials on the surface of MNPs. The in vitro and in vivo experiments proved that nucleic acid molecules bound to MNPs are transferred inside the cell through magnetofection in a highly efficient manner (2).

Magnetofection Advantages Over Other Transfection Methods

Transfection is a procedure for delivering nucleic acids inside the eukaryotic cells utilizing either physical or chemical methods. Viral vectors mediated nucleic acid transfer into the cells is classified as transduction; however, sometimes their delivery is mediated through transfection methodologies. Magnetofection, including several other methods like microinjection, sonoporation, electroporation, gene gun, nucleofection, microneedles, and magnetoporation, fall in the category of physical methods. Chemical transfection methods are mainly lipid or polymers-based. Several modifications of chemical methodologies like nanoparticles, quantum dots, and graphene-mediated nucleic acid delivery with silica nanoparticles or carbon nanotubes are also used in some protocols. However, among all these, magnetofection is relatively more straightforward in use, efficient in delivering nucleic acid, and economical compared with all other methodologies that involve sophisticated equipment and complex formulations.

Delivery of free nucleic acids or therapeutic gene containing vectors into the eukaryotic cells/tissues is quite challenging. In intracellular environments, degradative enzymes impede delivering therapeutic genetic materials inside the cells/tissues. Magnetofection has been recognized as one of the most powerful tools for delivering intact/functional nucleic acids and vectors containing specific genetic material being used, thus having the potentials for correcting dysfunctional gene (3).

Suppose magnetofection is being utilized for the delivery of RNA. In that case, it should get transferred into the cellular cytoplasm for expression. In contrast, DNA first goes into the nucleus, followed by forming messenger RNA that goes out to the cytoplasm for expressing the desired product.

Several scientific reports have provided proof of concept about the superiority of magnetofection compared with other transfection procedures. According to a study in vitro gene transfer into the microglial cells that protect our brain, magnetofection efficiently transferred genetic material compared with conventional gene transfer methodologies (4).

Recent Advancements in Magnetofection:

Magnetofection has great application in gene therapy protocols in which defective genes are replaced with healthy ones into the human cell/tissues. Compared with other protocols, magnetofection mediated delivery of healthy genes into human cells is considered most promising. However, there is ongoing research to develop a proper formulation and appropriate magnetic field skills. Gene therapy through magnetofection for therapeutic purposes besides targeted delivery offers the unique advantage of being least invasive (5).

Magnetic nanoparticles mediated nucleic acid transfer into stem cells (6), and the flexibility of methodology (7) add to its versatility for its extensive utilization in regenerative medicine and genetic diseases.

Magnetofection in Clinical Practice

It has become a fact that magnetofection if properly used, could efficiently transfer nucleic acids as well as therapeutic genes containing viral vectors into the human cells/tissues. However, its development for clinical usage is hampered due to the chemical nature of MNPs fate in vivo. Research is ongoing on the bioevaluation of MNPs for their potential clinical use in gene therapy protocols involving magnetofection (8). As far as clinical applications of magnetofection are concerned, targeted delivery into cancerous tissues for controlling several cancer types holds a tremendous future promise in clinics (9).

Sources:

1. Plank C, Anton M, Rudolph C, Rosenecker J, Krotz F. Enhancing and targeting nucleic acid delivery by magnetic force. Expert Opin Biol Ther. 2003;3(5):745-58.
2. Bi Q, Song X, Hu A, Luo T, Jin R, Ai H, et al. Magnetofection: Magic magnetic nanoparticles for efficient gene delivery. Chinese Chemical Letters – doi: https://doiorg/101016/jcclet202007030. 2020.
3. Scherer F, Anton M, Schillinger U, Henke J, Bergemann C, Kruger A, et al. Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo. Gene Ther. 2002;9(2):102-9.
4. Smolders S, Kessels S, Smolders SM, Poulhes F, Zelphati O, Sapet C, et al. Magnetofection is superior to other chemical transfection methods in a microglial cell line. J Neurosci Methods. 2018;293:169-73.
5. Qunjie B, Song X, Hu A, Luo T, Jin R, Ai H, et al. Magnetofection: Magic magnetic nanoparticles for efficient gene delivery[J]. . Chinese Chemical Letters. 2020;31(12):3041-6.
6. Yamoah MA, Thai PN, Zhang X. Transgene Delivery to Human Induced Pluripotent Stem Cells Using Nanoparticles. Pharmaceuticals. 2021;14(4):334.
7. Blokpoel Ferreras LA, Chan SY, Vazquez Reina S, Dixon JE. Rapidly Transducing and Spatially Localized Magnetofection Using Peptide-Mediated Non-Viral Gene Delivery Based on Iron Oxide Nanoparticles. ACS Appl Nano Mater. 2021;4(1):167-81.
8. Dragar C, Kralj S, Kocbek P. Bioevaluation methods for iron-oxide-based magnetic nanoparticles. Int J Pharm. 2021;597:120348.
9. Belete TM. The Current Status of Gene Therapy for the Treatment of Cancer. Biologics. 2021;15:67-77.

Biological complexity and the challenges associated with its elucidation

Biological organisms are complex systems composed of a variety of cell and tissue types.¹ To elucidate the physiological and pathophysiological processes that occur in biological systems, research studies should take into consideration their complexity. However, many traditional experimental paradigms either do not allow the proper evaluation of this biological complexity or do not enable a fast and scalable assessment. Molecularly barcoded (magnetic) microparticles (beads) are one of the avenues to advance research on biological complexity.

Molecularly barcoded beads and their applications

Molecularly barcoded beads are tagged with unique barcodes (molecules) that enable single-cell evaluation. The number of codes varies depending on the used assay and may include fluorescent, chemical, electronic, or graphic tagging. Molecularly barcoded beads facilitate the identification of reads originating from individual cells. In the context of microfluidic systems, they enable flexibility, sensitivity, and high-throughput analysis. Therefore, barcoded beads have found applications in multiplex biological assays in the fields of molecular diagnostics, biomarker development, analysis of tumor heterogeneity, pharmacogenomics, understanding of tissue complexity, and gene mutational analysis.

Oligonucleotide-barcoded beads

Oligonucleotides are one of the types of molecular bead barcodes. The design of oligonucleotide-barcoded beads used with the Drop-Seq method has been described by Macosco et al. (2015).² The authors synthesized oligonucleotide primers on the beads in the 5′ to 3′ direction, which yields free 3′ ends available for enzymatic priming. The oligonucleotide tags consist of four parts: a constant sequence, a “cell barcode”, a unique molecular identifier, and a capturing and priming oligo-dT sequence. The constant sequence serves as a priming site for downstream PCR and sequencing. The “cell barcode” is identical on the surface of each bead across all primers but differs from the cell barcodes present on other beads. The unique molecular identifier differs among primers and identifies PCR duplicates.³ Finally, the oligo-dT sequence is responsible for capturing polyadenylated mRNAs and priming reverse transcription.²

Oligonucleotide-barcoded beads in the context of the Drop-Seq technology

When molecularly barcoded beads are applied in the context of sequencing, they enable tracing the sequence back to the individual cells. The Drop-Seq technology is a microfluidic-based experimental system, in which beads tagged with oligonucleotides facilitate the retrieval of single-cell transcriptomes.⁴ Using a microfluidic device, single cells are encapsulated together with barcoded beads and lysis buffer into aqueous droplets. Following encapsulation, the cells are lysed, and mRNA is released and hybridized to the oligonucleotide tags of the beads. Subsequently, the droplets are pooled and broken, and the beads are released. The single-cell mRNAs captured on the isolated beads are then subjected to reverse transcription with template switching. cDNAs are generated and amplified, and sequencing adapters are added. Finally, the generated barcoded mRNA samples can be sequenced.^2,4 More recently, methods for the identification and error-correction of molecular bead barcodes have been identified, including circularization of the RNA sequence.⁵

Overall, the Drop-Seq method, which utilizes oligonucleotide barcoded beads, not only analyzes mRNA transcripts from thousands of individual cells but also registers their cells of origin. Thus, the method enables high-throughput, single-cell transcriptomic analysis, which facilitates the elucidation of biological complexity.

Sources:

1. Lobo, I. Biological complexity and integrative levels of organization. Nature Educ. 2008;1(1):141.
2. Macosko EZ, Basu A, Satija R, Nemesh J, Shekhar K, Goldman M, Tirosh I, Bialas AR, Kamitaki N, Martersteck EM, Trombetta JJ, Weitz DA, Sanes JR, Shalek AK, Regev A, McCarroll SA. Highly parallel genome-wide expression profiling of individual cells using nanoliter d Cell. 2015;161(5):1202-1214.
3. Kivioja T, Vähärautio A, Karlsson K, Bonke M, Enge M, Linnarsson S, Taipale J. Counting absolute numbers of molecules using unique molecular identifiers. Nat Methods. 2011 Nov 20;9(1):72-74.
4. https://www.dolomite-bio.com/how-it-works/drop-seq/
5. Tambe A, Pachter L. Barcode identification for single cell genomics. BMC Bioinformatics. 2019;20(1):32.