New platforms to control viral infections: nano-scale carriers and drug delivery systems

Summary

The novel generation of nano-carriers holds a great promise for effective delivery of drugs and vaccines to their targets with significantly lower risk for adverse effects, modulation of immune reactions to self and non-self antigens, and development of sensitive imaging technologies for early detection of human diseases. COVID-19 was (and presently is) a challenge to medicine as well as to science. Novel nanotechnology-based biosensors, diagnostic devices, antiviral drugs, and vaccines have been developed or repurposed in order to combat the modern pandemic. The outbreak of SARS-CoV-2 triggered the research and development sectors to work together to create novel, versatile, and efficient ways to prevent and treat human diseases that are likely to continue their rapid development beyond the present pandemic situation.

Key words/phrases: COVID-19, antivirals, monoclonal antibodies, vaccines, nanotechnology

1. SARS-CoV-2 and COVID-19 – an overview of origins of a modern pandemic and its effect on thinking in research and clinical medicine

Human infection with SARS-CoV-2 occurred a zoonotic spill-over in the weeks and months preceding the first reported case in early December 2019 [69]. On 31 Dec 2019, the World Health Organization (WHO) was informed about a rapidly growing number of cases of pneumonia of yet unknown origin in Wuhan City, China. The causal agent was identified by the local authorities on 7 Jan 2020 as a hitherto unknown member of the beta-coronavirus family. In the end of January 2020, the WHO Director General D-r Tedros Adhanom Ghebreyesus declared that the novel coronavirus outbreak is a public health emergency of international concern [https://www.who.int/news/item/30-01-2020-statement-on-the-second-meeting-of-the-international-health-regulations-(2005)-emergency-committee-regarding-the-outbreak-of-novel-coronavirus-(2019-ncov)]. Remarkably, at this time there were a total of about 100 cases and no deaths yet, but the virus has managed to spread in almost 20 countries outside China. Research on the potential of development of etiologic treatment and prevention started immediately, uniting the efforts of clinicians, scholars and biotechnological and pharmaceutical companies.

COVID-19 was (and presently is) a challenge to medicine as well as to science. Nevertheless, it is also an opportunity to learn (yet again) from Nature about the unlimited variety of mechanisms of evolution, the causal relationship between changes occurring on nano-and micro-scale and the magnitude of the effects they may have on the macroworld. This modern pandemic triggered the research and development sectors to work together to create novel, versatile and efficient ways to prevent and treat human disease that are likely to continue their rapid development beyond the present epidemic. A prime example is the development of vector and mRNA vaccines. By the end of 2019 there was, at best, one vector vaccine approved for use in humans (rVSV-ZEBOV, or Ervebo, by Merck) and no approved mRNA vaccines, although the first clinical trials for a rabies mRNA vaccine started in 2013.

The timeline of development of vaccines against SARS-CoV-2 is, indeed, striking, but not because they were developed ‘too fast’, rather because apparently all the knowledge was already there and ‘a big push’ was all that was needed to invent novel types of vaccines that could be safely and efficiently used to prevent severe disease in millions of people. Same applies to the development of safe and efficient anti-COVID-19 drugs. The antivirals that have shown significant effects in the treatment of patients with COVID-19 in present day (remdesivir, favipiravir, and others) are, in their majority, repurposed drugs that were previously tried in the treatment of other viral diseases. Thus, the novelty of the anti-COVID treatments of present day is not exactly in their principle of action on their targets, but rather in the manner in which these targets are addressed.

2. Development of versatile nano-scale carrier platforms

The well-tried conservative therapies work, very generally speaking, by saturating the environment of living cells with a drug so that the chance for its binding to the target (e.g. a cellular receptor) is greatly increased. The majority of drugs enter the living cells via a receptor-ligand interaction. As receptors of a certain type are expressed on more than one type of cell and the same drug ligand may bind to more than one type of receptor, there is a (sometimes significant) risk for adverse effects related to the effects of the drug on tissues different from the target tissue. This is especially true for cytostatic and psychiatric drugs. Thus, safe and effective drug treatments (and vaccines, for that matter) do not depend solely on the active substance in the drug but also on its carrier and the manner this carrier interacts with the target tissue.

Nanotechnology is… “the manipulation of matter on a near-atomic scale to produce new structures, materials and devices… using materials with a length scale between 1 and 100 nanometres…at which size materials begin to exhibit unique properties that affect [their] physical, chemical, and biological behaviour”, according to the definition provided by the Centre for Disease Control (CDC) in Atlanta, USA: https://www.cdc.gov/niosh/topics/nanotech/default.html#:~:text=Nanotechnology%20is%20the%20manipulation%20of,new%20structures%2C%20materials%20and%20devices.&text=Nanotechnology%20refers%20to%20engineered%20structures,between%201%20and%20100%20nanometers.

The size (comparable to the size of biological macromolecules) and the significant surface-to-volume ratio of the nano-scale particles increases their solubility and may be used to modulate the segregation of nano-carriers loaded with bioactive substances into different cell and tissue compartments (depending on the desired effect). The novel generation of nano-carriers for pharmaceuticals for human use ensure effective delivery of the active substances to the target with significantly lower risk for adverse effects. Nanotechnology may provide ways to overcome the nature-provided barriers in order to improve penetration of drugs in certain target sites (e.g. the blood-brain barrier), to modulate the immune reactions to self and non-self antigens and may assist in the development of sensitive imaging technologies for early detection of human disease. There are also many other applications of nanotechnology in modern research and development, some of which may be viewed in Fig. 1\

Figure 1. Some of the applications of nanotechnology in modern research and development.

The size of most viruses may vary between 20 and 250 nm, which makes nanotechnology-based technology an excellent option for the purposes of antiviral research.

SARS-CoV-2 possesses high infectivity potential (comparable to the infectivity of the influenza and measles virus). Roentgen imaging, computerized tomography and laboratory (molecular biology) tests are routinely used to assist screening for infections and in clinical diagnosis of COVID-19. X-ray and CT findings that are considered ‘typical’ for COVID-19 (disseminated interstitial changes, ground-glass opacities) lag significantly behind the onset of symptoms and may persist after the clinical status has improved. Somewhat paradoxically, the first laboratory test developed for COVID-19 was the highly sensitive but elaborated, expensive and time-consuming RT-PCR test. PCR-based tests for detection of disease are, in essence, nanotechnology-based test devices (although the technology dates back to the 90-ties of the XX century). Antigen tests for SARS-CoV-2 (which are another example of nanotechnology-based tests) were developed later in order to speed up the diagnostics and make it easily available for patients and carers, but besides the common limitations of using nasopharyngeal and throat swabs, they have additional issues such as varying brand-to-brand sensitivity and specificity. Serological tests (for the presence of IgG, IgM and, sometimes, IgA) may reveal present and past SARS-CoV-2 infections but are less applicable for new infections, as they must be performed with significant delay from the onset of symptoms and may be unreliable in the case of previously vaccinated symptomatic patients. Biosensors (another nanotechnology-based type of devices) have been developed for the purposes of diagnosis relatively early (first similar report by Seo et al. was published in June 2020) [76] but are still mainly for research use due to their high cost.

The size of SARS-CoV-2 virion is about 100 nm (medium range). Thus, the potential of COVID-19 as a target for nanoparticles-based treatments has been considered from the very beginning of the pandemic. So far, nanotechnology-based approaches have been developed and tried for the purposes of prevention of infection, diagnostics and treatment of COVID-19.

Several nanotechnology-based devices have been developed for the sensitive and specific detection of COVID-19. At present, they augment the routinely used approaches based on RT-PCR and antigen tests. A novel biosensor based on gold nanoparticles has been proposed for detection of SARS-CoV-2 in exhaled air [75, 78, 27]. Magnetic nanoparticles were used in a specialised extraction protocol for SARS-CoV-2 [81, 97]. Nanoscale-sized crystals with semiconductor properties (commonly known as quantum dots) have been successfully used in a biosensor for detection of SARS-CoV-1 nucleocapsid protein antigen [70] and may well be used for detection for SARS-CoV-2 [40]. It could be expected that nanomaterial-based detection devices, sensors, drugs and vaccines will be the mainstay of the management of the pandemic in the foreseeable future [65, 50].

3. Nano-based delivery systems for antiviral drugs

3.1. Nanotechnology formulations – types and potential applications in the treatment of viral infections

A nanopharmaceutical, is, by definition, a material with particle size in the nanoscale range that has therapeutic potential. Nanoparticles may have different shapes and chemical compositions. The active compound in the nanoparticle may be dissolved, entrapped, encapsulated, adsorbed, conjugated or chemically attached, thereby the vehicle of the active compound is referred to as a nanocarrier. A schematic representation of the main types of nanocarriers is shown on Fig. 2.

Figure 2. Types of nanocarriers.

The most common types of nanocarriers are briefly listed below. Special attention is paid to liposomes, as they were the first and are, at present, the most commonly used type of drug nanocarrier.

3.1.1. Lipid-based nanoformulations

Lipids are, by far, the safest type of carriers. They are easier and cheaper to produce, are biodegradable, biocompatible, non-toxic, and, generally, non-immunogenic [61]. The lipids in the formulation are used together with other agents such as surfactants and solvents. Commonly, used lipid-based nanoformulations include liposomes, solid lipid nanoparticles (SLN), nanoemulsions, and nanosuspensions.

Liposomes

Liposomes are vesicles in which an aqueous core is entirely enclosed by a membranous lipid bilayer composed of natural or synthetic phospholipids. Depending on the method of preparation, lipid vesicles can be multi-, oligo- or unilamellar, containing many, a few, or one bilayer shell(s), respectively. The diameter of the vesicle may vary widely – between 10 and 1000 nm.

The aqueous core of the first-generation (conventional) therapeutic liposomes contained the hydrophilic bioactive compound. Later-generation liposomes may entrap hydrophobic compounds as well and may carry additional modifications to increase the time of circulation in vivo and to ensure targeted and timed delivery of the medicinal agent [37]. A schematic of the different types of liposomes may be seen in Fig. 3.

Figure 3. A representation of the different types of liposomal drug delivery systems.

Liposomes are prepared from an aqueous solution of phospholipids by a variety of methods: ultrasound sonication, dehydration-rehydration, reverse phase evaporation, freeze-thaw cycles, vesicle extrusion, etc. [31]. The resulting entrapment efficiency may be very different. Optimal control in the size and lamellarity of the resulting liposomes is usually achieved by extrusion using filters with pores of defined size. Targeting therapeutic liposomes to the desired site is achieved by adding ligands to the surface of the vesicle [37].

Liposomes have their drawbacks as nanocarriers. Among these are their relatively low drug-loading capacity and instability, as well as potential risk of immunogenicity due to various adjuncts (e.g. PEG3350) [11].

At present, several liposomal formulations are in clinical use. Among the applications are targeted delivery of cytotoxic agents, intraocular applications, post-irradiation treatments for patients with extreme UV sensitivity with preparations of T4 endonuclease V [95, 13, 49].

Polyethylene glycol (PEG)-conjugated liposomes are currently used in both Comirnaty (Pfizer BioNTech) and Spikevax (Moderna) vaccines.

Solid lipid nanoparticles (SLNs)

SLNs are composed of a solid lipid matrix (triglycerides, steroids, fatty acids, waxes, etc.). Unlike liposomes, SLNs are industrially scalable. SLNs have a history of use as carriers for a variety of antiviral drugs (ritonavir, maraviroc, darunavir, efavirenz, zidovudine, lopinavir, dolutegravir and others- for details see below).

A later generation of SLNs are the nanostructured lipid carriers (NSLCs). Unlike SLNs, the lipids in the NSLCs are in liquid state, conferring increased stability and improved controlled release pattern.

Nanoemulsions

Nanoemulsions (NE) are globular single-phase systems consisting of emulsified oils, water and surfactants. NEs have a high loading capacity, increased hydrophilicity and enhanced bioavailability. Nanoemulsions have been used to develop formulations of anti-HIV drigs such as protease inhibitors (saquinavir, indinavir) [90].

Self-nanoemulsifying drug delivery systems (SNEDDS) are another type of formulation of single-phase oil-water-surfactant systems that are used for carriers of hydrophobic drugs, such as the NNRTI drug nevirapine (see below) [Selvam and Kulkarni, 2014]. To date, a nanoemulsion [68] and a suspension of solid lipid nanoparticles [87] containing the antiviral drug favipiravir have been developed for targeted delivery to the pulmonary epithelium. So far, the results obtained in vitro studies are encouraging.

3.1.2. Polymer-based nanoformulations

A variety of natural and synthetic polymers may be used in the preparation of nanoformulations. Hydrophilic polymers may include gelatin, albumin, alginate, dextran, chitosan, agarose and others. Hydrophobic polymers used in nanoformulations may include polylactic acid (PLA), polylactide-co-glycolic acid (PLGA), polystyrene, polycaprolactone (PECL), polymethacrylate (PMMA) and others. Surface-modifications may be introduced in order to improve the pharmacokinetics, reduce potential immunogenicity and introduce stimulus-triggered release of the drug in response to changes in pH, temperature and other stimuli. Special polymers inhibiting P-gp and other multidrug resistance (MDR) proteins have been developed specifically for the purposes of oncology.

Polymer-based nanoformulations includes polymeric micelles, polymeric solid nanoparticles, nanocapsules, and nanospheres.

Polymeric micelles

Polymeric micelles are nano-sized structures made of amphiphilic copolymers, each structure (unimer) consisting of a hydrophobic and a hydrophilic subunit. Unimers associate to form structures with a hydrophobic core and a hydrophilic shell called micelles. The hydrophobic core contains drugs with poor aqueous solubility while the hydrophilic shell drug cargo is responsible for the transport and targeted release of the drug.

The hydrophobic core-forming polymers may be polyesters, polycaprolactone, poly(l-amino acids) and others. Polyethylene glycol is usually the major component of the hydrophilic shell. Various modifications such as adding sialic acid residues to the hydrophilic shell have been tried. In one study, sialylated outer shell was used to bind the haemagglutinin of the influenza virus in order to prevent viral entry [1].

Polymeric nanoparticles

Polymeric nanoparticles may be manufactured with a variety of natural or synthetic polymers. Nanocapsules are polymeric nanoparticles that entrap the drug into their core. When the drug is adsorbed onto the surface or embedded in the matrix of the nanoparticle, the structure is termed as a nanosphere. Polymeric nanoparticles have been used for developing newer formulations of anti-HIV drugs, such as efavirenz, lopinavir, ritonavir, and others.

Polymer drug conjugates

Polymer drug conjugates are comprised of a polymer and a covalently bound drug. The drug is typically a small molecule but may be a larger molecule, e.g. a protein. Most applications of polymer drug conjugates are in clinical oncology (e.g. daunorubicin, doxorubicin, methotrexate, melphalan) and ophtalmology (e.g. daunorubicin) [62, 20] but also in antiviral therapy. PEGylated interferons were, for a long time, the only highly effective treatment of HCV [17]. Zidovudine (AZT) conjugates typically exhibit longer plasma half-lives than conventional AZT [38]. In 2018, Andersen et al. used N-(2-hydroxypropyl) methacrylamide (PHPMA)-albumin copolymer for delivery of a combination of anti-HIV drugs of ART to primary human T cells with very promising results.

Nanocapsules

A nanocapsule is comprised of a core (where the drug is entrapped) and a shell. Nanocapsules may be loaded with significantly larger amounts of drug than liposomes and may be used for controlled and targeted drug delivery. Nanocapsules of poly(iso-butylcyanoacrylate) core, entrapping AZT triphosphate and polyethyleneimine shell have been designed.

Nanospheres

Nanospheres are spherical structures where the drug is not enclosed within but, is, rather, dispersed in the matrix. Nanospheres are smaller in size than nanocapsules and may be subjected to rapid drug clearance. Topical formulations of acyclovir in nanospheres has been tried for treatment of infections caused by herpes simplex virus.

Lipid-polymer hybrid nanoformulations

More often than not, a single drug is not enough for treatment of a viral infection.  On the other hand, patient compliance decreases when the patient is asked to take more than two drugs. Thus, more than one drug with different physicochemical properties may need to be delivered by single delivery vehicle. This may be achieved using lipid-polymer hybrid nanoparticles. Several hybrid nanocarrier systems have been developed, including polymer core-lipid shell nanoparticles, hollow core/shell lipid–polymer-lipid hybrid nanoparticles, lipid bilayer-coated polymeric nanoparticles, and polymer-caged nanoparticles. Basically, hybrid lipid-polymer nanoformulations consist of an inner polymeric core enclosed in one or more outer layers of lipid or lipid-polymer layers. Polymer-caged nanoparticles are somewhat different, as they are based on liposome technology with the liposome surface being modified by cross-linkage with polymers. Lipid-polymer hybrid nanoformulations exhibit very high capacity for drug loading and high encapsulation efficiencies.

A variant of lipid-polymer hybrid nanoformulations are biomimetic lipid-polymer hybrid nanoformulations, in which the surfaces of the nanoparticles are modified so that they mimic cell surface proteins. This category comprises virus-like particles (VLPs) and virosomes (see below).

Stimuli-based lipid-polymer hybrid nanoparticles are yet another variety of lipid-polymer hybrid nanoformulations that are capable of releasing the encapsulated drug in response to various stimuli such as pH, temperature, and magnetic field. Thus, timed delivery to the desired site is feasible. In 2012, methylcellulose stearate was proposed as a thermosensitive nanocarrier for slow intravaginal delivery of tenofovir [38]. Similarly, a hybrid thermosensitive hydrogel was developed for the co-delivery of theaflavin (an antioxidant, which is hydrophilic) and nifeviroc (an antiviral, which is hydrophobic) for intravaginal application as a pre-exposure prophylactic of transmission of HIV. Thermosensitive hydrogels have also been tried for intranasal delivery of antiviral drugs.

Nanoparticles may have different sizes and shapes. Shape-based lipid-polymer hybrid nanoformulations are based on the observation that shape of a nano-scaled particle may have significant impact on cell-cell interaction, drug uptake, and biodistribution [8]. For example, non-spherical nanoparticles have been found to provide higher therapeutic efficacy in comparison with spherical particles in the treatment of cancer [74]. Thus, different shapes and builds have been explored in order to improve the pharmacokinetic properties of drugs.

3.1.3. Dendrimers

Dendrimers are highly branched three-dimensional synthetic nano-architectures, 2–10 nm in diameter consisting of a central core, an inner shell, and an outer shell carrying various modifications [83]. A schematic of a very simple dendrimer may be seen in Fig. 4.

The dendrimer core allows for entrapment of various molecules and the functional groups on the surface allow interaction with the desired targets. Dendrimers exhibit longer circulation times, and enhanced solubility and stability and targeted delivery. Dendrimers are synthesized by sequential addition of building blocks molecules to an initiator molecule. Commercially available, at present, are dendrimers made of polyamidoamine, polypropylene imine, and poly-l-lysine dendrimers. The outer shell may be covalently modified (e.g. esterified, glycosylated, etc.) or conjugated to proteins, peptides, etc. Polyanionic carbosilane dendrimers have been used as topical microbicides for barrier protection against HIV and herpes simplex viruses. García-Gallego et al. in 2015 demonstrated that carbosilane dendrimers may inhibit the internalization of HIV-1 into the epithelial cells and the entry into peripheral blood mononuclear cells. Thiolated dendrimers exhibited sustained release of acyclovir.

Figure 4. Polyethylenimine dendrimer generation.

3.1.4. Carbon-based nanoformulations

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

Graphene

Graphene is a two-dimensional planar derivative of graphite. For biomedical purposes, graphene-based materials are modified with various functional groups to improve biocompatibility and to reduce toxicity. Both hydrophilic as well as hydrophobic drugs may be encapsulated. The functional groups may be designed to provide sites for attachment of diverse biological molecules.

Carbon nanotubes (CNTs) are hollow cylindrical nanotubes with walls made of one or more graphene sheets. The cylinder may be capped with fullerene on one or both ends. CNTs have excellent properties in terms of are drug loading and potential for controlled release but their medical applications are quite limited due to pulmonary toxicity and high hydrophobicity. Newer types of CNTs exhibit decreased toxicity and increased biodegradability.

Fullerenes

Fullerenes are carbon structures forming a nano-sized hollow cage. Fullerenes have been found to inhibit HIV, HCV and influenza virus replication by steric blocking  and inhibited the expression of viral nucleoprotein.

3.1.5. Inorganic nanoformulations

Quantum dots (QDs) are nanocrystals with semi-conductor properties. They are comprised of an inorganic core (made of silicon, cadmium selenide, cadmium sulphide, or indium arsenide, responsible for the semi-conductor properties), a hydrophilic shell, and a cap. Quantum dots find their applications predominantly in biomedical imaging, as they can absorb and emit light at preset wavelengths. As drug-carrier systems, modified QDs have been used in the treatment of HIV [42].

Metal and metal oxide nanoparticles

Metal nanoparticles may have microbicidal and antiviral activity of their own. Such are silver, gold, copper, titanium, cerium oxides and others. The surface of metal nanoparticles may be functionalized with various groups in order to improve molecular interaction, bioavailability and release of the conjugated drug. Small interfering RNAs have been conjugated to AuNPs and the resulting formulation inhibited dengue virus replication and the release of the virion. Silver nanoparticles were found to inhibit haemagglutination and H1N1 influenza A virus-induced apoptosis in cell cultures. Silver nanoparticles conjugated with tannic acid were shown to reduce HSV-2 infection vitro and in vivo. Zinc oxide nanoparticles have been studied in mouse models of vaginal infections with HSV-2 with promising results with regard to suppression of viral activity.

The use of inorganic nanoparticles is largely limited due to concerns for toxicity [43]. Newer formulations are being developed, but clinical trials in humans are still severely limited.

3.1.6. Nucleic acid-based nanotechnologies

Aptamers

Aptamers are, basically, short nucleic acid (oligonucleotides) or protein sequences (peptides) that can specifically bind to other molecules such as other nucleic acids and proteins [34]. In a specific third category fall X-aptamers that are a combination of natural and chemically-modified DNA or RNA oligonucleotides. Aptamers may have thiophosphate backbone to enhance nuclease stability and binding affinity [45].

Aptamers have been tried for therapeutic potential for at least 20 years. For example, PEGylated aptamers have been used to target vascular endothelial growth and the resulting preparation (pegaptanib, Macugen by Bausch & Lomb) has been approved as an intravitreal injection factor in macular degeneration [85]. Aptamers targeting key coagulation cascade members such as factor IXa, thrombin, and von Willebrand factor have been tested as potential anticoagulants [57].

Oncology is a very wide field for development of therapeutic aptamers.  Aptamers against nucleolin, prostate-specific membrane antigen (PSMA), carcinoembryonic antigen (CEA), MUC1, ErbB-2 and other proteins with significant roles in pro-carcinogenesis have also been investigated as cancer treatment strategies [72, 43, 22, 86]. Nevertheless, no aptamer has been approved for treatment of cancer yet.

In the field of infectious disease, aptamers have been found to inhibit various stages of HIV infection by binding to reverse transcriptase and integrase [19, 71]. The hemagglutinin of the influenza virus has been successfully targeted by aptamers [53]. Aptamer-based biosensors for SARS-CoV and SARS-CoV-2 been designed [16, 70; 63]. In 2021, aptamers were used by Sun et al. to prevent the binding of the spike protein to the ACE receptor [82].

Antisense nucleotides (ASOs)

Antisense nucleotides are short, single-stranded, artificially created nucleic acids that have been designed specifically to bind and inhibit mRNAs. ASOs with potential for applications to combat viral disease target mRNAs coding for proteins critically important for the deployment of the infective potential of the virus. The first approved member of this family was fomivirsen (Vitravene, by Novartis), a 21 bp oligodeoxyribonucleotide complementary to a sequence encoding the major early proteins responsible for cytomegalovirus (CMV). Vitravene was withdrawn ‘for commercial reasons’ from the market in 2002 [Public Statement on Vitravene (fomivirsen) (europa.eu)].

There are three generations of ASOs, differing in their chemical characteristics with regard to potency, pharmacokinetics, toxicity. First generation ASOs feature a phosphorothioate backbone in order to increase resistance to hydrolysis. Fomivirsen was a first-generation ASO. Second-generation ASOs are modified at the position 2 of the sugar moiety allowing them to act as steric blockers. One of the better known second-generation ASOs, Mipomirsen (Kynamro, by Genzyme) was rejected by the European Medicines Agency shortly after it was released due to severe adverse effects [http://www.fiercebiotech.com/regulatory/ema-committee-shoots-down-sanofi-s-cholesterol-drug-mipomersen]. Third-generation ASOs (e.g. eterlipsen, marketed as ExonDys by AVI BioPharma International for treatment of Duchenne’s muscular dystrophy) have superior nuclease resistance, increased target affinity and pharmacokinetics compared to previous generations.

In 2004, shortly before the first human beta-coronavirus outbreak, several second-generation ASOs against the causal agent SARS-CoV have been reported [56]. They were designed to target the open reading frame of the main polyprotein of the virus (ORF1) and its 5′-UTR regulatory sequence. In vitro, they demonstrated significant antiviral activity. Afterwards, ASOs targeting the translation initiation site of ORF1 and the site where frameshift occurs in order to transcribe more than one polyprotein (commonly termed as ‘pseudoknot’) were designed [1]. Later, AVI Biopharma developed ASOs targeting the 3-UTR of the viral RNA [AVI BioPharma’s NeuGene antisense drugs inhibit SARS coronavirus (pharmabiz.com)]. In 2020, a rapid and sensitive detection system for the presence of SARS-CoV-2 RNA based on ASOs bound to gold nanoparticles (AuNPs) was reported [51]. The ASOs belonged to the first generation and were specific for the nucleocapsid phosphoprotein gene of SARS-CoV-2. The authors claimed that their colorimetric naked-eye assay could detect several copies of the SARS-CoV-2 viral genome without need for special equipment.

Small interfering RNAs (siRNAs)

siRNAs are small double-stranded RNA molecules that show a promise in the treatment of COVID-19. Key members of the siRNA family are patisiran and givosiran that are presently used for treatment of genetic disease. Both patisiran and givosiran were first-in-class medications [9]. According to the definition by FDA, a ‘first in class’ is a drug that works by “new and unique mechanism of action” to treat a particular medical condition [FDA approves first-of-its kind targeted RNA-based therapy to treat a rare disease | FDA].

Patisiran (Onpattro, by Alnylam) is approved for threatment of polyneuropathy in patients with hereditary transthyretin (hTTR) amyloidosis [Onpattro | European Medicines Agency (europa.eu)]. Patisiran targets the mRNA of a mutant form of the TTR gene that produces a misfolded protein whose degradation products are deposited in many tissues and organs. A newer siRNA drug manufactured by Alnylam for treatment of hTTR amyloidosis is revusiran. Givosiran (Givlaari by Alnylam) is another siRNA drug used in the treatment of acute hepatic porphyria, a genetic disease caused by mutations in the ALAS1 gene. Givosiran is believed to decrease the incidence of attacks of acute porphyria in the affected patients and to decrease chronic pain, which is the cardinal symptom of AHP [Balwani et al., 2020]. One of the Ebola vaccines developed earlier, namely, TKM-Ebola (by Arbutus Biopharma Corporation) was comprised of siRNA encapsulated in LNPs. TKM-Ebola, however, did not reach later phases of clinical trials due to increased risk for allergic reactions.

Nanotechnology has already demonstrated its power for improvement of drug release with all three Alnylam siRNA formulations. Patisiran is delivered to the liver (where the mutant gene is normally expressed) in a lipid-nanoparticle formulation that increases nuclease resistance degradation and facilitates the delivery to the target cells. The siRNA in givosiran and revusiran is covalently linked to a ligand containing syalic acid residues to ensure better binding to the target and is packed into lipid nanoparticles for efficient hepatic delivery. siRNAs are transfected into cells using polymer carriers or cationic lipids (liposomes or lipid nanoparticles).

siRNAs packed in solid lipid nanoparticles have been highly effective against SARS-CoV-2 when used in animal models [33, 4]. The siRNA targets specifically the conserved regions of the SARS-CoV-2 genome. The formulation allows that the pharmaceutical is delivered preferentially to the lungs as a prime site for SARS-CoV-2 replication but by variation of parameters the authors have shown that the loaded nanoparticles may target the liver and the spleen and, possibly, other tissues. This versatility may be very valuable in COVID-19, as it is a multisystem disease and may extend its damage well beyond the lungs. Presently (as of Jan 2022), a database of siRNAs targeting SARS-CoV-2 is being established, containing more than 100 000 siRNAs ranging from 18 to 21 nucleotides in length [47]. This may be expected to speed up the process of design of novel siRNA-based drugs against COVID-19.

3.2. Nanoformulations developed specifically for the delivery of antiviral drugs

Modern antiviral treatment is very effective but has its limitations. Many patients will need a multidrug regimen, but antiviral drugs may interact with each other as well as with other prescription medicines. Some patients will require long-time treatment (COVID-19 makes no exception, as the incidence of long COVID syndrome reaches 40-50 % in some studies [54]). The antiviral drugs may have poor bioavailability and short half-lives, resulting in a need of multiple dosing and, respectively, poor patient compliance. Administration of higher doses in attempt to compensate for the low bioavailability may result in toxic effects. Development of drug resistance is common in patients who require treatment beyond several days or weeks. Many viruses may spread into sites that are typically inaccessible for most drugs (usually termed as virus sanctuaries, such as the CNS, lymphatic system, and synovial fluid. Lastly (but not in importance) is the fact that as the virus uses the host’s cellular apparatus to synthesize its proteins and nucleic acids, the differential selectivity of antiviral agents toward the virus and the target cells may be crucially important.

Nanotechnology-based novel platforms for drug delivery may assist in finding solutions to many of these issues. The largest amount of data we have about drug treatment of diseases caused by RNA-containing viruses are HCV and HIV-associated states and influenza.  Many drugs used successfully for treatment of HIV and HCV infections have been tried for activity against SARS-CoV-2 and several have been repurposed in COVID-19.

Antivirals approved for the treatment of HCV and HIV (and, some of them, for COVID-19) may, very broadly, be divided in several classes depending on their site of action and their principal mechanism:

1. Interferons (IFNs);
2. Entry blockers;
3. Nucleoside/nucleotide analogues;
4. Non-nucleoside analogues;
5. Protease inhibitors;
6. Integrase inhibitors;
7. Antisense nucleotides;
8. Small interfering RNAs (siRNAs);
9. Others.

The ASOs and siRNAs presently tested for activity against COVID-19 were already mentioned in the previous section.

The latter group, “Others”, is comprised of a variety of drugs that are normally used in the clinic for other purposes, such as antimalarials (e.g. hydroxychloroquine), antihelminthics (levamisole, ivermectin), antidepressants (fluvoxamine), etc. They may exhibit activities typical of the former groups (inhibitors of replication, protease inhibitors, etc.). Some of these, such as ivermectin, have antiviral activity in vitro, but the results in vivo have not come through yet or are not encouraging [39]. Others (such as bromhexine hydrochloride, with potential protease inhibitor activity) have shown some activity in vivo in terms of preventing symptomatic disease but the effects were not significant enough to justify more research in the field or the early-phase clinical trials have not been finished yet [48]. Chloroquine was shown to interfere with virus-endosome fusion of SARS-CoV, to inhibit the glycosylation of ACE2 receptors [93, 94] and to possess anti-inflammatory effects via inhibition of phospholipase A2 activity and blocking cytokine production and release [2], but significant antiviral effects have not been observed for SARS-CoV-2. Fluvoxamine, an antidepressant of the selective serotonine selective reuptake inhibitor (SSRI) group, initially showed more promise as a treatment for patients with COVID-19, but ongoing trials do not show anything except that larger studies may be needed to clarify whether there is an effect of fluvoxamine of disease severity [91]. A report about a fluvoxamine formulation containing solid lipid nanoparticles that reportedly ensured effective targeted release of the drug was published in 2019 [35]. No commercial preparation based on these studies has come out yet.

The COVID-19 pandemic caused significant psychological distress to those affected by the virus as well to those who have witnessed loved ones’ struggle with the disease. As is typical of such times, ‘wonder treatments’ emerged, widely proclaimed by the media. Most of these did not show, in the long term, any significant benefits to those taking them. Instead, many affected patients got worse after they used their ‘wonder cures’ and, respectively, missed the right time to seek proper treatment. Hydroxychloroquine, ivermectine, bromhexine and others all enjoyed their short periods of (yet) undeserved fame. None of them has shown significant clinical benefits and their use by patients with COVID-19 ought to be discouraged until reliable further studies have proven conclusively that they lower the rates of hospitalization and mortality.

3.2.1. Interferons

Interferons are signaling proteins normally produced by the immune system of the infected host and interfere (hence their name) with the replication of the virus within the cell. Previously used in the therapy of viral hepatitis, the capacity of IFNs to interfere with the synthesis of proteins needed for replication is, at present, utilized predominantly in the treatment of multiple sclerosis and various cancers. PEGylation of interferons as a basic nano-scale technology has been used to increase their clearance time in order to ensure that the drug binds effectively with its target [89]. PegIntron (by Merck, discontinued) is a commercial preparations of interferon alpha-2b that was used in the treatment of hepatitis B and C until 2016. Pegasys (Genentech) remains, to this day, the only PEGylated interferon approved for use by the FDA and EMA for treatment of hepatitis B in adults [https://www.natap.org/2005/HBV/051605_02.htm; https://www.ema.europa.eu/en/documents/product-information/pegasys-epar-product-information_en.pdf]. PEG-interferon alpha-2a (Roferone by Roche, used in the treatment of leukemia, malignant melanoma, polycythemia vera, essential thrombocythemia and others) is another interferon drug that was recently discontinued due to severe adverse reactions and the availability of safer drugs [https://pharmac.govt.nz/medicine-funding-and-supply/medicine-notices/interferon-alfa-2a/]. Interferon beta-1a (Rebif by Merck and Pfizer) is presently approved in the treatment of relapsing multiple sclerosis [https://www.pfizer.com/news/press-release/press-release-detail/emd_serono_and_pfizer_announce_fda_approval_of_rebif_rebidose_interferon_beta_1a#:~:text=announced%20today%20that%20the%20U.S.,of%20multiple%20sclerosis%20(MS).].

Gold nanoparticles have been studied as a potential carrier of interferon alpha for the clinical treatment of hepatitis C virus (HCV) infection [36]. A sustained drug delivery IFN-β-1a formulation based on nanoparticles composed of poly(lactic-co-glycolic acid and polymers was developed and tested in vitro in 2020 [24]. The authors report that the formulation increased the efficiency of interferon delivery and had the potential to decrease the associated adverse effects of IFN-β-1a. There have been numerous reports about trials of IFNs in the treatment of patients with COVID-19 [32, 64, 52]. IFN-alpha was shown to reduce viral shedding and suppress the inflammation associated with COVID-19, while IFN-beta was associated with faster viral clearance [46]. Sodeifian et al. point out that data from animal studies show that the administration of IFNs administration at the early stages of COVID-19 or in mild-to-moderate disease may have protective effect, but during the later stages and in severe disease may cause significant harm to the patients [80]. The effect of IFNs on mortality from COVID-19 may be reported as drastically different in different studies. One randomized controlled trial of the use of IFN-beta in patients with severe COVID-19 reported a lower mortality rate in the group receiving IFN, but the patients received other drugs as well, including standard antivirals (lopinavir/ritonavir, or atazanavir/ritonavir [44]. Another trial reported that the use of IFN beta showed no significant difference in the mortality [21]. Studies of the use of PEGylated IFN-alpha 2b as an addition to standard therapy showed that it resulted in rapid viral clearance and improved clinical status [12]. It is possible that the nanotechnology-based systems developed previously may be used for efficient delivery of antivirals in patients with COVID-19, but the controversial results from the use of interferons have prevented deployment of large clinical trials yet.

3.2.2.Nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs)r

NRTIs work by interfering with the replication of viral nucleic acid, usually presenting a nucleoside analogue that, upon being added to the growing polynucleotide chain, would terminate the elongation of the chain inhibit the replication of the nucleic acid of the virus. Reverse transcriptase inhibitors have been extensively studied in relation to their use in the treatment of HIV. Current clinical practice of antiretroviral therapy (ART) proposes first-line anti-HIV therapy being constituted by a combination of two (or more) NRTIs and a NNRTI or a protease or integrase inhibitor. Depending on the characteristics of the patient, an entry inhibitor may be added, although they are expensive, cause compliance issues (e.g. subcutaneous application two times a day, which may be problematic) and may have severe adverse effects. Lamivudine, stavudine, zidovudine, emtricitabine, zalcitabine, tenofovir, and others are some of the commonly used NRTIs.

NRTIs and NNRTIs are very efficient drugs. Nevertheless, they have their shortcomings, such as short half-life, high first-pass effect (resulting in low bioavailability), and variation in serum levels due to individual variation on the activity of the CYP system. A wide variety of technologies have been tried for improving the delivery and the stability of nucleoside analogues.

Nanotechnology has provided a number of highly efficient vehicles for delivery of NRTs used in the treatment of HIV infection. Liposomes, dendrimers, micelles, solid lipid nanoparticles, nanosuspensions, and polymeric nanoparticles have all been used to improve drug delivery in HIV [73, 29].

The data about the use of nanotechnology to improve the delivery of anti-COVID-19 antivirals is still sparse but the field is rapidly expanding.  Most of antivirals used in COVID-19 are NRTIs. Such are: favipiravir (a guanosine analogue); molnupiravir (a pyrimidine ribonucleoside analogue) and remdesivir (an adenosine analogue). Most of them are approved for patients with mild-to moderate disease at high risk of complications and must be started early in the course of the disease.  Only remdesivir is approved for treatment of hospitalized patients with severe disease and respiratory failure [https://www.covid19treatmentguidelines.nih.gov/management/clinical-management/hospitalized-adults–therapeutic-management/] although later it was approved for use in selected groups of outpatients also [https://www.fda.gov/news-events/press-announcements/fda-takes-actions-expand-use-treatment-outpatients-mild-moderate-covid-19]. In a study from 2020, HIV-positive patients receiving tenofovir/emricitabine exhibited a lower risk for COVID-19 and related hospitalization than those receiving other therapies [5]. Remdesivir-loaded polymer nanovesicles, a favipiravir nanoemulsion and favipiravir-loaded SLNPs intended to deliver the drug directly to the pulmonary epithelium have been developed and tried in vitro [68, 92, 87]. In vivo studies in the field have yet to come.

3.2.3. Non-nucleoside reverse transcriptase inhibitors (NNRTIs)

The non-nucleoside reverse transcriptase inhibitors (NNRTIs) directly inhibit the HIV-1 reverse transcriptase by binding in a reversible and non-competitive manner to the enzyme. The currently available NNRTIs are nevirapine (Viramune, by Boehringer Ingelheim), delavirdine (Rescriptor, ViiV Healthcare, and efavirenz (generic, by Mylan and Systiva, by Bristol Myers Squibb). Nano-based formulations have been tried to improve the bioavailability and the controlled release of NNRTIs. Manyarara et al. in 2018 developed a nevirapine nanoemulsion for pediatric uses. A self nanoemulsifying drug delivery system (SNEDDS) has been successfully developed for nevirapine in order to increase its bioavailability [60]. NNRTIs have been evaluated for anti-COVID-19 activity through molecular docking studies, but their clinical efficiency in COVID-19 remains to be proven [15, 25].

3.2.4. Entry inhibitors (fusion blockers)

Entry inhibitors, basically, prevent a virus from entering a cell by blocking the viral entry point, e.g. a preferred receptor. Presently, entry blockers such as maraviroc (Celzentry, by GlaxoSmithKline), enfivurtide (Fuzeon, by Genentech), bulevirtide (Hepcludex, by Gilead Sciences), and others) are used in the treatment of HIV and hepatitis D infections.  In 2015, a iron oxide nanoparticles coated with amphiphilic polymer were used for delivery of enfivurtide to the HIV sanctuaries in mice [23]. In 2020, polymer-lipid hybrid nanoparticles (PLN) loaded with a combination of a non-nucleoside reverse transcriptase infibitor (efavirenz) and enfivurtide were used to study the potential for delivery of the drugs to T-cells and macrophages sequestered in virus sanctuaries.

3.2.5. Protease inhibitors (PIs)

Protease inhibitors inhibit the protease that cleaves the viral polyprotein in order to form separate proteins. PIs are, similar to the above group of nucleoside analogues, are very well studied because of their common use in the treatment of HIV. Again, similarly to nucleoside analogues, PIs are subject to extensive first-pass metabolism and are substrates of CYP, hence the wide variance of the levels of the same drug in the same dosage taken by a different person. This is partly overcome by co-administering them with serum level enhancers (essentially, CYP3A blockers) such as ritonavir or cobicistat. Nanotechnology-based approaches have been developed in order to enhance the bioavailability of the most common PIs, such as lopinavir (solid lipid nanoparticles, polymer-based nanoparticles), atazanavir (nanoemulsion, nanosuspension), darunavir (lipid nanoparticles), nelfinavir (polymer-based nanoparticles) and others [55, 66, 88, 28, 79, 7, 18]. Saquinavir-conjugated quantum dots have been tried to improve bioavailability of the drug in virus sanctuaries [42].

Protease inhibitors are one of the exchangeable components of the standard antiretroviral therapy for HIV, as either protease or integrase inhibitor may be added to the reverse transcriptase blockers in ART. The PIs, however, seem to have risen to the heights of a novel drug for SARS-CoV-2 infection. In 2020, Beck et al. used a deep learning-based drug-target interaction prediction model to predict the potential anti-COVID-19 activity of a group of drugs of different classes that are used for treatment of HIV infections (remdesivir, efavirenz, ritonavir, and dolutegravir) [10]. They found that atazanavir had the highest affinity for the viral protease of SARS-CoV-2. A new oral drug (Paxlovid, by Merck) was only approved for use in patients with COVID-19 in December 2020. It consists of nirmatrelvir, a protease inhibitor and ritonavir, a well-known augmenting agent used in ART [59]. Undoubtedly, after enough data has been collected about the efficiency of the oral preparation, better and more efficient nanotechnology-based formulations will be developed shortly.

3.2.6. Integrase inhibitors

Integrase inhibitors work by preventing the insertion of a DNA copy of the RNA genome into the genome of the host cell. Typically, an integrase inhibitor such as dolutegravir (Tivicay, by ViiV Healthcare), elvitegravir (part of the fixed-dose combination Stribild, by Gilead Sciences) or raltegravir (Isentress, by Merck) is a key component in ART for HIV infections. Nanoformulations of integrase inhibitors have been developed in order to increase their solubility and for controlled delivery. Myristoylated dolutegravir was proposed as a long-acting formulation against HIV infection with slow-release potential [90]. In 2015, it was demonstrated that gold nanoparticles are readily internalized by lymphocytes and macrophages and an AuNP-conjugate of raltegravir was proposed that could inhibit the replication of HIV in peripheral mononuclear cells [26]. In 2020, dolutegravir was identified to have a potential inhibiting activity for the viral protease of SARS-CoV-2 [10]. The studies in the field are ongoing.

4. Virus capsids used for drug delivery

Virus-like particles (VLPs) are self-assembling particles that are formed by incorporating viral proteins of the capsid or the viral envelope into various naturally occurring proteins like ferritin or bacterial encapsulin. The proteins are typically produced in heterologous systems. VPLs may potentially be used in a wide range of applications, including development of drugs, immunotherapies, vaccines, gene therapies, imaging and others. Virosomes are VLPs that feature an additional phospholipid bilayer that incorporates the viral envelope glycoproteins (e.g. haemagglutinin). A schematic may be viewed in Fig. 5.

The proteins may be modified or conjugated with peptides, toxins, chemotherapeutics, siRNA, quantum dots, etc. in order to achieve targeting to a specific site and/or timing of release of the encapsulated agent.

Natural viral particles may, in response to triggers such as pH or temperature change, release units or parts of their capsid or envelope lacking the genome of the virus. Similarly, viral particles may be disassembled and reassembled into VLPs under specific conditions [58]. VLPs resemble the morphology of the wild-type viral particles, exhibit the same tropism and intracellular distribution, but do not include the nucleic acid of the virus that makes them, in fact, genome-free equivalents of viruses. Thus, the cargo of the vesicle (drugs, antibodies, fluorescent dyes, contrast agents, etc.) may be delivered to a specific type of the target cell using cell-cell interactions similar to the virus-cell interaction. VNPs may be used for development of novel materials such as catalysts, biomimetics or selectively targeted imaging agents [14, 58].

Figure 5. Schematic representation of a virus-like particle (the phospholipid envelope is not shown).

VLPs have been used to design vaccines against human disease since 2017, when Kanekiyo et al. created a haemagglutinin-ferritin fusion protein that would spontaneously self-assemble to create nanoparticles presenting trimers of haemagglutinin on its surface. Similarly, an anti-RSV vaccine has been developed later that presented the F-antigen of RSV [30].

Virus-like particles hold a great promise among the modern nano-based approaches to improve the safety and the efficiency of drugs. VLPs are, generally, safer to use than viral vectors in the process of vaccine development. A potential drawback of VLPs is their immunogenicity due to the presence of viral proteins. Since the proteins are produced in bacteria, however, their expression does not typically include post-translational processing that may actually decrease the risk for immune response following translation [58, 3].

Drugs with high toxicity may safely and efficiently be delivered to the target using VLPs. Ashley et al. used VLPs to deliver encapsulated doxorubicine to human hepatocellular carcinoma cells (Hep3B) that were known to use the Pgp efflux mechanism to expel chemotherapeutic drugs [6]. The results showed that the doxorubicine, cisplatine and 5-fluorouracil loaded viral particles could inhibit the growth of cancer cells at significantly lower levels of the drug.

Modification (functionalization, decoration) of the proteins in VLPs may increase their affinity to the target and improve the release of the drug. Decoration of VLPs with biotin moieties in order to ensure targeting via avidin-biotin interaction was reported in 2007 [67].

In 2020, three types of mRNA vaccine candidates based on VLPs were tested in mouse models. The VLPs presented the spike, membrane and the envelope protein of SARS-CoV-2. The expressed proteins were encapsulated in lipid nanoparticles and transfected into cultured cells, then VLPs were secreted by the cells in the culture medium. The results were encouraging, although the antibody levels of the vaccinated mice did drop rapidly shortly after the vaccination [41]. In 2022, Yilmaz et al. reported the first results of development and preclinical evaluation of VLP-based vaccine against SARS-CoV-2 tried in cultured cells ad in animal models. The VLPs expressed all 4 structural proteins of the SARS-CoV-2. The results showed that the vaccine triggered a potent T-cell response and significantly reduced viral load in the vaccinated infected animals [96].

Covifenz (by Medicago and GSK) is a vaccine based on VLPs. Its Phase III trials were finished in early February 2022. According to the manufacturers, Covifenz is already approved for use by Health Canada [Medicago’s Canada-made COVID-19 vaccine approved by Health Canada | Globalnews.ca].

As of February 2022, no VLP-based drug formulations for the treatment of COVID-19 are reported or approved. Future research about the possible applications of VLPs in the treatment of SARS-CoV-2 and the associated conditions may be needed before any products may be approved.

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Test LO 4.2

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