Prospective treatment procedure of COVID-19 with siRNA-LDH nanoconjugate

Rituparna Acharya

School of Bio-Science and Engineering, Jadavpur University, Kolkata 700 032, India

Abstract

Keywords:corona virus, COVID-19, LDH, siRNA, SARS-CoV-2

Introduction

Corona virus is a type of RNA viruses that gives rise to several respiratory syndromes such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS).The commonest signs of this disease condition are cough and cold, respiratory symptoms, fever and breathing difficulties [1].This disease may be transmitted from human to human when they are in close contact with the infected person.There are altogether six species of corona viruses and that makes seven strains.Among them severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)is the most dangerous one that have produces the pandemic corona virus disease 2019 (COVID-19) [2].In December, 2019, the first outbreak was reported in Wuhan, China and rapidly it spread all over the world.The virus was found to be a new strain with 70%genetic similarity with SARS-CoV and was named as SARS-CoV-2 [3].As the virus is new the treatment procedure is also unknown to the mankind.

This is the era of nanotechnology that may help in the exploration of the treatment procedure of COVID-19.In this era a new therapeutic application method is RNAi therapy.RNAi therapy includes several types of RNA that are used for therapeutic reasons.siRNA,shRNA and miRNAs are the main types of RNAs that may help in the treatment of several incurable deseases.In this research study we are intended to deliver a hypothesis for the treatment of this COVID-19 using siRNAs.siRNAs are nothing but small interfering RNAs that have double stranded non-coding RNA molecules with 20-25 bp.It inhibits the translation procedure by reducing the expression of specific genes and degrading its mRNA after transcription [4].siRNAs may degrade the important viral mRNAs by gene silencing.However, the use of bare siRNAs has several drawbacks.Bare siRNAs usually get degraded by the enzymes in out body.In order to overcome this limitation nanoparticles play a vital role.There are several types of nanoparticles like inorganic, organic and polymeric nanoparticles.Among them Layered Double Hydroxide (LDH) is a type of inorganic nanoparticle that have several advantages such as,they are highly biocompatible and may deliver high payloads [5].In this study, we will discuss about the method that may be implemented for the delivery of siRNA intercalated within LDH and may be delivered to the target site.

1 Hypothesis

As COVID-19 doesn't have any treatment modality it makes this disease condition incurable for most of the population of the affected countries.But in this era of nanotechnology we can make a hypothesis of the drug that maybe used to cure the disease.We may use an inorganic nanoparticle called LDH for the delivery of specific types of siRNA with targeted sequence to the respiratory tract through nasal spray.The siRNAs will target ORF1ab, ORF3a, S, M and N region of the virus.

1.1 Literature review in support of the hypothesis

A publication demonstrated the nanoconjugate of siRNA-LDH nanoparticle, with siRNA-Htt sequence was studied on NIH3T3 fibroblast cells and in C57Bl/6 mice embryos.The efficacy of the delivery of the siRNA on cortical neurons of telencephalic lobes were studied by electron microscopy, fluorescent-activated cell sorting (FACS) and confocal microscopy [6].Another publication was conducted on HEK293T cells (Human embryonic kidney cells).In this study LDH was loaded with double stranded oligo deoxynucleotides (ODNs) and the delivery of the same was confirmed by western blotting [7].One more publication showed the co-delivery of siRNA and drug achieved in breast cancer, bone osteosarcoma and human colon cancer cell lines [8, 9].

2 Possible methodology

2.1 Synthesis of Mg-Al LDH

LDH maybe synthesized by co-precipitation method as mentioned in the publications [10-12].An aqueous solution of 100 ml of 0.5N NaOH was added drop wise to a mixed metal solution comprising of 500 ml of 32 mmol Mg(NO3)2· 6H2O and 16 mmol of Al(NO3)3·9H2O with vigorous stirring at 1100 rpm under a stream of nitrogen gas until the pH of 11.5 is reached.The resulting solution was aged at room temperature of 25°C for 24 h, after that centrifuged at 8519 g rcf for 15 min at 4°C.The precipitate was washed several times with decarbonated water and freeze dried at 20 Pa pressure and 82°C to get fine free flowing nanocrystalline LDH powder.The method is quite easy with commonly used reagents.After the synthesis,LDH may be characterized by X-RayDiffraction (XRD),thermal and particle size analysis.

2.2 siRNA intercalation within the LDH layers

The following siRNA sequences may be used to target the virus [13](Table 1).The intercalation of the siRNA within LDH layers may be done according to the previously published protocol [10-12].The siRNA and LDH nanoparticles were mixed in several mass ratios ranging from 1:5 to 1:20 and RNAse free water was added until the final volume is reached to 10 µL,keeping the final siRNA concentration constant at 0.1µg/µL.The above samples were incubated at 37°C for 10 min, to obtain siRNA-LDH nanoconjugate.The method is very easy and may be performed within 10 minutes.The accuracy of the intercalation may be checked by Gel mobility assay, FACS etc.For making a homogeneous solution of the LDH, it may be mixed with RNAse free water and the solution may be sonicated before it is using for the mixture of siRNA and LDH.However, the same procedure may be repeated in larger scale for industrial use.

Table 1 The following siRNA sequences may be used to target the virus

Conclusion

Currently, the treatment of COVID-19 is still not invented although there are many of them in clinical trial.The treatments available today are mainly based on blocking the host cells against virus entry or stopping virus infection by inhibiting replication or by inhibiting protease activity.The potential application of this proposed drug will be suppressing viral RNA synthesis by targeting viral mRNA molecules and degrading them, leading to the inhibition of viral replication and infection.

The main route of administration of the currently available drugs is intravenous delivery and there are challenges in intranasal administration.The intravenous delivery may lead to systemic toxicity that was detected as an accumulation of LDH molecules in the lung of the mice after 1hr of intravenous administration and 0% survival rate after 6 repeated injections.However, in case of low doses such as 30mg/kg of repeated injection are found to be lethal [14].In this publication we are not intended to make intravenous administrations but a nasal spray that requires very low level of dosage and helps in the targeted delivery.Moreover, Layered double hydroxide nanoparticle has a property of protecting the siRNA in adverse conditions and makes them stabilize in the body.So it may be concluded that siRNA-LDH nanoconjugate via intranasal routemay prove to be highly effective to combat with the COVID-19 condition.