Nanotechnology-based therapeutics

While Martina Vijver, a professor of toxicology at the Leiden University, states that the nanoparticles are the new asbestos, they are actually used as therapeutics!!!

https://www.plastichealthcoalition.org/latest-news/nano-should-be-the-new-asbestos/

https://link.springer.com/article/10.1007/s13346-020-00818-0 Nanotechnology-based antiviral therapeutics | SpringerLink Published: 03 August 2020

In recent years, many new technologies have been explored for diagnosis, prevention, and treatment of viral infections. Nanotechnology has emerged as one of the most promising technologies on account of its ability to deal with viral diseases in an effective manner, addressing the limitations of traditional antiviral medicines.

The initial part of the paper focuses on some important proteins of influenza, Ebola, HIV, herpes, Zika, dengue, and corona virus and those of the host cells important for their entry and replication into the host cells. This is followed by different types of nanomaterials which have served as delivery vehicles for the antiviral drugs. It includes various

lipid-based, polymer-based, lipid–polymer hybrid–based, carbon-based, inorganic metal–based, surface-modified, and stimuli-sensitive nanomaterials

and their application in antiviral therapeutics. The authors also highlight newer promising treatment approaches like

nanotraps, nanorobots, nanobubbles, nanofibers, nanodiamonds, nanovaccines,

and mathematical modeling for the future.

In recent times, there have been outbreaks of several viral infections caused by corona virus, Nipah virus, Ebola virus, Zika virus, dengue virus, chikungunya virus and different strains of influenza virus—H5N1 (avian flu) and H1N1 and H3N2 (swine flu).

Application of nanotechnology in antiviral therapeutics

With the advent of nanotechnology, it has been possible to comprehend the cellular mechanisms of the living cells and to develop relevant technologies which facilitate early diagnosis and treatment of various viral infections [38, 39]. Some of its applications consist of drug and gene delivery; use of fluorescent biological labels, detection of proteins, pathogens, and tumors; separation and purification of biological molecules and cells; tissue engineering; MRI contrast heightening; and pharmacokinetic studies [40,41,42]. Thus, it has opened up a vast field of research and application with its ability to deal with viral diseases in an efficient manner and address the problems posed by traditional antiviral medicines [43]. Nanoformulations have not only helped to overcome the problems related to drug solubility and bioavailability but also by themselves have acted as antiviral agents through various mechanisms (Fig. 2).

Lipid nanoparticles for siRNA delivery

Recently, gene silencing by RNA interference (RNAi) strategy has been exploited for antiviral treatment. Small interfering RNA (siRNA) is the most commonly used RNAi tool which can target particular genes to cause their short-term silencing and thus blocking the production of respective proteins. They are short (19–21 nucleotides), double-stranded designed and synthesized to target a particular mRNA. They are transfected into the cells with the help of cationic lipid (as liposomes or lipid nanoparticles) or polymers [65]. The structural features which make lipids efficient vehicles for the delivery of siRNA are their polyunsaturated chains significant in the destabilization of intracellular membranes and the ionizable tertiary amino group which helps in the initial fusion of lipid nanoparticles (LNPs) with the cell membrane. Along with cationic lipids, helper lipids like fusogenic phospholipids or polyethylene glycols are also added in the formulation. They help to enhance the transfection efficiency and reduce the immunogenic response by protecting them from the macrophages. In addition, cholesterol is added in such formulations to enhance the stability of LNPs and enhance the activity of cationic lipids [66].

Polymer-based nanoformulations

Different types of polymers are used in the preparation of nanoformulations like natural hydrophilic and synthetic hydrophobic polymers.

Though polymers can serve various functions, their use remains limited due to high cost

and safety and compatibility issues [47].

Researchers are working to solve these issues and are trying to provide polymers that are biodegradable and safe to use.

Polymer drug conjugates

Polymer drug conjugates are comprised of a polymer and a therapeutic agent covalently bound, the therapeutic agent being a small molecule or a large molecule like protein [116]. The purpose of conjugation is to achieve better efficacy through extended plasma stability and safety through targeted delivery [117, 118]. Besides cancer, such conjugates have found tremendous potential in antiviral therapy. Some polymers are known to possess their inherent antiviral activity and their conjugation with antiviral drugs may act in a synergistic manner.

AZT conjugates with naturally occurring polymers like chitosan, dextrin, and κ-carrageenan through succinic ester linker have resulted in longer plasma half-lives and high loading of AZT. Phosphoamide-based prodrugs have been explored in which the antiviral drug stavudine was conjugated with chitosan through phosphoamide linkage. This resulted in greater antiviral potency and reduced toxicity. Synthetic polymers like vinyl esters and methacrylates have been explored for conjugation with AZT and ribavirine showing beneficial results [123].

Dendrimers

In another study carried out by García-Gallego et al.,

metal complexes of carboxylated and sulfated PPI dendrimers

with ethylene diamino core demonstrated dual therapeutic–preventive activity against HIV-1 infection by inhibiting internalization of HIV-1 into the epithelial cells.

These dendrimers were found to exhibit dual activity against HIV and enterovirus 71 (EV71) responsible for hand-foot-and-mouth disease prevalent among children below 6 years of age.

Antimicrobial peptides with antiviral activities

Nanotechnology-based solutions have been explored for the delivery of antiviral peptides. Peptide–nanoparticle conjugate systems have been extensively studied.

Carbon-based nanoformulation

Carbon-based nanoformulations are comprise of carbon nanotubes, graphene oxide nanoparticles, and fullerenes.

Carbon nanotubes

Carbon nanotubes (CNTs) are cylindrical-shaped hollow nanomaterials, viewed as tubes made by rolling up of planar graphene sheets. They can be viewed as coming from the rolling up of a graphene sheet, named as single-walled carbon nanotubes (SWCNTs), or a series of concentric rolled-up graphene sheets termed as multi-walled carbon nanotubes (MWCNTs) [157]. The cylindrical structure is capped with fullerene sheets on one end or both ends. The sp²-hybridized carbon atoms in graphene sheets impart a unique strength to CNTs. In addition, they display other unique characteristics like high aspect ratio, high surface area, cell penetration capacity, and ultralight weight [158]. The chemical vapor deposition (CVD) technique, laser-ablation technique, and electric arc-discharge techniques are commonly employed for the preparation of carbon nanotubes [159]. Though CNTs are widely explored for delivering chemotherapeutic agents at the target site,

their overall application in the biomedical field is limited due to pulmonary toxicity

and high hydrophobicity [160]. The proposed mechanisms for toxicity are uptaken by macrophages with subsequent generation of ROS and inflammatory mediators. However, functionalized CNTs have shown decreased toxicity and increased biodegradability. CNTs can be decorated with peptides, carbohydrates, and polymers and can be used for targeted therapy, when needed [161]. In one study, Kumar et al. stated about protoporphyrin IX (PPIX)-conjugated multi-walled nanotubes (MWNTs) and its ability to treat influenza using photodynamic therapy. It was found that in the presence of visible light, PPIX-MWNT may indulge in mechanisms like RNA strand breakage, protein oxidation, or protein–RNA cross-linking caused by reactive oxygen species (singlet oxygen and superoxide anion) leading to inactivation of the influenza viral strain. Probing into the inactivation mechanism of carbon nanotubes, they concluded that PPIX-MWNTs can be used for treating any viral infection as it displays nonspecificity in treating viral diseases. Also, PPIX-MWNT can be easily recovered through filtration and reused. Due to its multitarget mechanisms of antiviral action, it was proposed that PPIX-MWNTs have less chances of development of drug resistance [162].

Nanostructures have shown antiviral effect in respiratory syncytial virus, a virus causing severe bronchitis and asthma. The treatment is generally done by combining nanoparticles and gene-silencing technologies. In a novel approach, MWCNTs were functionalized with recombinant dengue virus 3 envelop proteins. This induced significant immune responses in mice [163]. Similarly, conjugation of functionalized CNTs with B and T cell peptide epitopes could generate a multivalent system that was able to induce a strong immune response; thus,

CNTs were considered to be good candidates for vaccine delivery [164].

Further, functionalized CNTs were used for the transport of peptides (such as foot-and-mouth virus peptide) for vaccination [165].

Graphene

One of the most promising carbon-based nanomaterials with great potential for antiviral application is graphene.

Graphene (G) is a two-dimensional (2-D) planar sheet of hexagonally arranged sp²-hybridized carbon atoms obtained from its three-dimensional (3-D) material of graphite [166]. It is chemically oxidized to graphene oxide (GO) to acquire oxygen bearing functional groups like hydroxyl, epoxide, and carboxylic acids [167]. Graphene-based nanomaterials (GBNs) have high surface area, high loading capacity, and superior mechanical strength which make them attractive candidates for carrying antiviral agents [168]. The oxygen-containing functional groups allow surface functionalization and conjugation strategies and show biocompatibility, reduced toxicity, and good dispersibility [169]. The amphiphilicity of GO makes the incorporation of hydrophilic as well as hydrophobic moieties possible [170]. In addition, these functional groups also provide attachment sites for various biological molecules like proteins, DNA, and RNA [171].

Recently, Pokhrel et al. studied the interactions between graphene and VP40 (viral matrix protein) of Ebola virus using molecular dynamics simulations and graphene pelleting assay. Graphene was found to interact strongly with VP-40 at various interfaces crucial for the formation of the viral matrix.

They proposed the use of graphene-based nanoparticle solutions as disinfectant to prevent the Ebola epidemic [172].

In another study, 18 sulfonated magnetic nanoparticles were anchored onto reduced graphene oxide (SMRGO) sheets and used to trap and destroy HSV-1 photothermally, upon their irradiation with near-infrared light. It was found to be effective against 28 viral infections including HSV. It was found that SMRGO has higher entrapment efficiencies in comparison to magnetic nanoparticles due to increased entrapping efficiency, larger surface area, unique sheet-like structure, and outstanding photothermal characteristics shown by graphene [173].

Fullerenes

Fullerenes are among the first discoveries in symmetric carbon nanostructures and have received considerable attention in the case of antiviral research. Fullerenes are comprised fully of carbon atoms forming a nanosized caged hollow sphere. Buckminster fullerene (C60), also known as buckyball, is the most common form of fullerenes with 60 carbon atoms arranged in a spherical structure showing high symmetry [174].

Few studies aimed at screening fullerene derivatives for anti-influenza activity.

Inorganic-based nanoformulations

Quantum dots (QDs) (2–10 nm) are semiconductor nanocrystals having the shape of dots.

They are comprised of a semiconductor core, overcoated by a shell, and a cap leading to improved solubility in aqueous buffers [190]. Fundamental semiconducting character and unique optical and electronic properties are attributed to the presence of the inorganic core consisting of semiconducting materials like silicon, cadmium selenide, cadmium sulfide, or indium arsenide [191]. Quantum dots find interesting applications in biomedical imaging due to its limited light scattering, narrow emission bands, and low tissue penetration. Quantum dots have been widely explored as theranostic platforms for simultaneous sensing, imaging, and therapy [192]. The advantages of quantum dots as a drug carrier system include improved bioavailability and stability of drugs, increased circulation times, active targeting, and localized therapy. In addition to this, QDs can be surface modified with targeting ligands [193, 194].

In vitro studies demonstrated that higher concentrations of saquinavir were able to cross the BBB by this method [195].

Metal and metal oxide nanoparticles have been widely explored for their antiviral activity. Among the various metal nanoparticles showing high efficacy are silver and gold, and among the various metal oxides are CuO, SiO₂, TiO₂, and CeO₂. These nanoparticles have shown great efficacy against a broad spectrum of viruses like influenza (H3N2 and H1N1), HBV, HSV, HIV-1, HSV, dengue virus type-2, foot-and-mouth disease virus, and vesicular stomatitis virus [196].

Lipid–polymer hybrid nanoformulations

In order to deliver two or more chemotherapeutic drugs with different physicochemical properties in a single delivery vehicle, lipid–polymer hybrid nanoparticles are developed [227].

Nanovaccines

In the last decade, a wide variety of nanoparticulate systems have been designed to induce humoral and cellular immunity against various viral infections. Various phospholipidic, polymeric, inorganic, carbon-based, metallic [269,270,271], and protein-based systems [272] have been explored for the development of vaccines [273,274,275,276,277,278].

Advanced methods include the formation of self-assembling units to form nanoparticles surface decorated with antigens [242, 281].

Nanotechnological approaches currently under investigation against SARS-CoV2

According to the statistical analysis conducted by StatNano, there is a total of 9217 patents filed at different patent offices with respect to coronaviruses till February 13, 2020; 5.2% of these are based on nanotechnology.

The majority of these patents are on diagnostic technology followed by RNAi therapeutics, vaccines, and nanomaterial-based filters [292]. (!!!!!)

The Iranian government is supporting a manufacturing plant producing 4 million N95 masks per day based on nanofiber technology. They propose that nanofibers produced by electrospinning as perfect filter materials for manufacturing N95 masks. The Czech nanofiber technology firm Respilon Group has incorporated copper oxide into nanofibers to be used for producing mask that has the capability of trapping and destroying the virus. The Advanced Institute of Science and Technology (KAIST), Korea, developed nanofiber-based nanofilters using an insulation block electrospinning process which maintains its filtering efficiency even after 20 washes with ethanol. The orthogonal and unidirectional alignment of nanofibers is claimed to minimize the air pressure toward the filter and maximize filtration efficiency [301].

The World Nano Foundation (WNF) has created a second-generation rapid COVID-19 IgM/IgG antibody assay kit using gold nanoparticles into a testing strip. Also, an MIT spin out startup company has developed strips based on gold nanoparticles which could give the color reaction within 20 min of the start of the test. The strip is coated with antibodies that bind to the specific SARS-CoV2 viral protein and the second antibody is attached to gold nanoparticles. The patient’s sample is placed on the strip, and if it has viral antigen which binds to both these antibodies, a colored spot appears on the strip. Sona Nanotech Inc., a Canada-based company, is developing a nanorod-based lateral flow test to screen the patient for the nCoV virus, which is expected to produce results within 5–15 min [302].

PreLynx in their portals have used vapors of a nanopolymer-based sanitizer which gets sprayed on individuals entering through this portal along with scanning of body temperature [304].

Nanotoxicity as a major limitation

With the rapid advancement of nanoscience in the healthcare sector, its adverse effects and toxicities are parallelly assessed by many scientists. On the virtue of the same reasons that lead to its increased potency, i.e., small size, larger surface area, specific shapes, and surface charges, their interaction at nonspecific target sites also increases [307]. The major mechanism responsible for the toxicity of nanomaterials is considered to be enhanced generation of

oxidative stress and inflammatory mediators in various tissues which damage the biological molecules of the cell, namely proteins, lipids, and DNA [308]. Some of the most perfused organs in the body include the liver, lungs, spleen, kidney, and heart.

On account of this, they receive a maximum amount of any material that is absorbed or injected.

The liver is the major site where accumulation of free radicals takes place.

Hence, nanomaterials may cause hepatotoxicity, nephrotoxicity, cardiotoxicity, immunotoxicity, and genotoxicity [309].

Nanotraps

Nanorobots

Nanorobots are multifunctional controllable machines, made up of inorganic or polymeric nanomaterials, modified with biomimetic materials performing various functions like actuation, propulsion, sensing, signaling, self-replicating, and delivering various materials with high accuracy [332]. In general, nanorobotic systems consist of a power source, sensors, actuators, onboard computers, pumps, and structural support. Additionally, it has a payload compartment to load the drug and a miniature camera to navigate through the bloodstream [333].

This kind of nanorobots can be comprised of a nanobiosensor established by nanoelectronics engineer experts, a nanochip, a nanotube, and a nanocontainer.

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