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Bio- By Shripriya Singh, PhD Contributing Editor

Nature’s very own nanoparticles are the new delivery agents: The promising role of Exosomes in

Evolving of the field of therapeutics: therapeutics to cellular therapeutics and From organ therapeutics to molecular finally to molecular therapeutics. Thereby, we therapeutics are gradually heading towards an era of The word research spells curiosity, intrigue, precision medicine where the focus is on and uncertainty and the term medicine spells molecular targets such as specific . treatment, healing and cure. Whereas, when Gene therapy, genetic and the two come together in form of medical nucleic acid based therapeutics have shown research, the term spells change, hope and immense potential and hold a promise to good health. Research potentially comprises abrogate diseased states in their infancy. two main phases, first in which we develop an The manipulation of genetic understanding of the subject and delve at the machinery of an is one of the most grass root level of the existing problems and interesting aspects of and has the second phase of research deals with opened endless avenues for researchers. solutions and tries to provide answers for the However, the delivery of any foreign particle rediscovered problems. In the present times such as a drug, a xenobiotic, an antigen or a we have successfully transitioned to the fragment of nucleic acid is the trickiest part of second phase where researchers across the the job. Every living organism has an efficient globe are focusing on answers and solutions. natural defense system via which it restricts This aspect of research has brought a and resists the entry of any foreign tremendous change in the field of medicine substance. This efficient defense mechanism where diseases no longer intrigue doctors which is popularly known as the ‘immune and practitioners; instead they are tackled system’ is by far the biggest challenge in the with precision and accuracy. Medical field of medicine. In case of stem based advancements have led us systematically therapeutics, , organ from organ therapeutics to organellar transplantation and even gene therapy, this

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major hurdle has to be overcome and cells cultured in-vitro. Their nano scale size resolved. Thus the approach is to use the (40–150 nm in diameter) and abundant patients’ own tissues and cells for any further presence have compelled biologists to treatment. consider them as potent tools of Bio nanotechnology offers hope in gene biotechnological use. Gene therapy and therapy nucleic acid based therapeutics hold great promise for treating multiple human In one of our previous articles we had diseases. However, the inefficient delivery of discussed about nanochips and tissue nano- negatively charged and relatively large (TNT), an innovative molecules into cells and tissues of interest which allows body to regenerate any type of has been a major drawback of the process. cell through genetic reprogramming. As Several in vivo methods and mentioned earlier the nanochip is a simple techniques have been developed and yet unique miniscule device designed around improved over the past years to accomplish the novel concept of tissue nano-transfection. precision and efficiency. These techniques TNT is an based technique comprise synthetic nanocarriers such as that facilitates the direct delivery of polymeric and liposomal nanoparticles and reprogramming factors (DNA) into the cytosol viral vectors. However, these strategies have via the application of a focused and highly suffered on grounds of , intense electric field through arrayed toxicity and manufacturing costs. The quality nanochannels. The electric field benignly control of manufactured products and the nanoporates the cell membrane and the high cost incurred have added to the burden reprogramming factors are further. However, the biggest drawback of the electrophoretically driven in (1). Keeping in existing techniques has been their inability to pace with this advancing technology we take deliver the cargo across specific the story forward and our current article is physiological barriers such as the blood- focused on exosome mediated gene delivery barrier (3). via cellular nanoporation (2). Recently exosomes have emerged as What is exosome mediated gene delivery potential carriers for nucleic-acid-based - the concept and details of the therapeutics (4) and the method takes technology advantage of these fluid-filled sacs that cells Exosomes are membrane bound release as a way to communicate with other extracellular vesicles (EVs) secreted cells. The scientists used the technique of routinely by all cells of the body. They are cellular nanoporation in which they placed considered the smallest organelles possibly around one million donated cells (such as present inside a cell and are capable of mesenchymal cells collected from human fat) expelling metabolic byproducts and help in on a nano-engineered silicon chip and used clearance. Exosomes are also an electrical stimulus to inject synthetic DNA routinely present in biological fluids such as into the donor cells. As a consequence of this blood, plasma and even the secretome of DNA force-feeding the cells are compelled to

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eject the unwanted substance as part of DNA nucleic acid fragment of interest. The transcribed messenger RNA and induced to electrical stimulation had an additional effect repair the holes that have been induced in of a thousand-fold increase of therapeutic their membranes. This serves two main genes in a large number of exosomes which purposes, first the leakage to the cell were released by the cells. This is a clear sign membrane is fixed and secondly the garbage that the technology is scalable to produce is dumped outside the cell. In this case as enough nanoparticles required for human mentioned by the authors the garbage bag is use. Figure 1 depicts the proposed the exosome which carriers the drug or the mechanism of CNP.

Figure 1: Schematic representation of the proposed mechanism for Cellular Nanoporation (CNP) triggering of exosome release in CNP-transfected cells. [Source: Nature 2019.] The knowledge of the exact molecular target brain tumors of mice by delivering a cancer- and the exact gene responsible are crucial for suppressor gene called PTEN. in the success of gene therapies. To test the the PTEN gene interfere with the suppression efficacy of the above mentioned technique role and thus allow cancer cells to proliferate scientists chose to test the results on glioma and grow unchecked. Gliomas represent

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about eighty percent of malignant brain control. Further these gene carrying tumors in humans and thus were a target of exosomes restored the tumor suppressor interest. The results clearly showed that function, inhibited the growth and spread of labeled exosomes carrying the gene of malignant tissues and increased overall interest not only travelled to the brain tumors survival. Figure 2 depicts the overall accurately but slowed their growth as technique and its application in mice gliomas. compared to the test substances used as

Figure 2: Diagrammatic representation of CNP-generated exosomes for targeted gene delivery. Left: the CNP system comprises a nanochannel array (red rectangles). DNA added to the buffer enters attached cells via nanochannels under transient electrical pulses. Attached cells subsequently release large quantities of exosomes containing transcribed mRNA that can be collected for tumor-targeted delivery through the blood–brain barrier (BBB) or blood–brain tumor barrier (BBTB) (right). [Source: Nature Biomedical Engineering 2019.]

The current and future aspects of the encapsulated exosomal contents and helps exosome based nano-carriers in tissue-directed delivery of genes. The only Exosomes are superior to any artificially drawback was the large scale production and made nano-carriers as they are non toxic, purification of exosomes which have been biocompatible and do not evoke an immune overcome by the technique of cellular response in the patients. They express nanoporation (CNP) discussed in this article. transmembrane and membrane-anchored Thus CNP helps in injecting of the nucleic acid (gene of interest) into the cell and which prolong their circulation in blood and help them in transport. These simultaneously produces a large number of proteins further facilitate the cellular uptake of exosomes (50 fold more than normal)

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carrying the same genetic transcript. The body’s own natural process has been tapped biggest advantage of these exosome based and manipulated to serve as a drug delivery carrier is that they can efficiently cross the system. The breakthrough research has blood brain barrier and thus will provide transformed human cells into mass endless opportunities for treating producers of miniscule nanoparticles full of neurodegenerative diseases such as genetic material that could possibly cure or Alzheimer's and Parkinson's disease. Since even reverse disease conditions. the study was published in December the References authors have fondly termed their innovative method as a small Christmas for the 1. Gallego-Perez D, et al. (2017) Topical medical and research fraternity. The tissue nano-transfection mediates non- exosomes are like nature induced parcels viral stroma reprogramming and rescue. that will carry the healing gift (gene or drug) Nature nanotechnology 12(10):974. inside and will efficiently deliver to the 2. Yang Z, et al. (2019) Large-scale molecular target of interest. They are being generation of functional mRNA- referred to as “Mother Nature-induced encapsulating exosomes via cellular therapeutic nanoparticles”. A small nanoporation. Nature Biomedical modification in tissue nanotransfection (TNT) Engineering:1-15. has led us towards cellular nanoporation. 3. Singh A, et al. (2016) Multifunctional This small innovative technology has once nanoparticles for crossing the again proven how smoothly we are blood–brain barrier and effecting optically progressing towards achieving goals that trackable brain theranostics. Advanced seemed miraculous a while ago. This functional materials 26(39):7057-7066. technique holds immense potential as it is a simple, efficient and cost friendly method via 4. Andaloussi SE, Mäger I, Breakefield XO, which we can deliver drugs of interest and & Wood MJ (2013) Extracellular vesicles: genes required for treating and repairing and emerging therapeutic medical issues at a very nascent stage. This opportunities. Nature reviews Drug is a new gene therapy technique where the discovery 12(5):347-357.

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