COVID-19 therapeutics: nanotechnology in antiviral treatments and vaccines

Summary

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is the causal agent of COVID-19. The infection with SARS-CoV-2 usually produces mild-to-moderate respiratory symptoms but some patients may need hospitalization and intensive care. The mainstay of conservative therapy for COVID-19 is a combination of corticosteroids, anticoagulants or anti-aggregants and, in cases of bacterial pneumonia, antibiotics. Several specialised treatments for COVID-19 (antivirals, monoclonal antibodies, and others) have been developed that may be used as pre-exposure prophylaxis or as treatment in the early stages of the infection in patients at high risk for complications. Modern nanotechnology offers a variety of high-tech solutions for the purposes of prevention of infection, diagnostics, post-exposure prophylaxis, as well as effective treatments in cases when SARS-CoV-2-related disease has already developed. It may be reasonably expected that nanomaterial-based sensors, drugs and vaccines will play a critical role in the management of the pandemic in the near future.

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

1. Vaccines and treatments for COVID-19 – an overview

1.1. SARS-CoV-2 and COVID-19 – an overview of causality, pathogenesis and potential approaches to decrease the burden of COVID-related complications

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.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is the causal agent of an infectious disease named COVID-19 (abbreviated from Coronavirus Disease of 2019). In most cases, the infection with SARS-CoV-2 will produce only mild-to-moderate symptoms. These may include any or all of the following: asthenoadynamia, respiratory symptoms, anosmia/dysosmia, headaches, joint and muscle pains, diarrhoea and/or abdominal pain, and others. In most cases the recovery is spontaneous, within several days up to two weeks. Some patients, however, may develop acute respiratory distress syndrome (ARDS) and/or progressive respiratory failure. Compared to its relatives SARS and MERS (Middle East respiratory syndrome), COVID-19 has milder clinical symptoms and lower fatality rates. Nevertheless, anyone can contract and may die from SARS-CoV-2, regardless of sex and age (although the risk for death is higher in males and children, and adults with underlying disease).

1.2 Pathogenesis

 

SARS-CoV-2 enters the cell by endocytosis via the ACE2 receptor on the cell surface, similarly to most coronaviruses. The process is mediated by binding of the receptor binding domain (RSD) of the S (spike) protein to the ACE2 receptor of the target cell [67]. When the virus enters the cell, it uncoats its single-stranded, positive-sense genome. The genomic RNA is translated into two replicase polyproteins that are subsequently cleaved into 16 non-structural proteins that form the RNA replicase-transcriptase complex that produces the viral genome and the subgenomic copies carrying open reading frames for protein synthesis. The viral RNA and the structural proteins are then assembled into new viral particles that are released by exocytosis to infect other cells [66, 38]. Since the ACE2 receptor gene is expressed in many tissues besides the lung (namely, the gastrointestinal tract, the kidneys and others [74, 15], the patients may develop lung injury and/or injury to multiple tissues and organs.

1.3. Pathomorphology and laboratory findings

The main pathomorphological finding in patients with COVID-19 is diffuse alveolar damage combined with pathological changes in the pulmonary vascular bed and alveolar hemorrhagic syndrome [Pathology of COVID-19: Atlas, 2020]. Systemic inflammation with inflammatory lymphocytic and monocytic infiltration also accounts for the impact on the target tissues, in particular the lung and the heart [22, 38]. Typical laboratory findings of severe COVID-19 are lymphopenia (although some patients may exhibit lymphocytosis, thrombocytopenia, elevated fibrinogen, D-dimer, LDH, ferritin, IL‑2, IL‑7, IL‑6, monocyte chemoattractant protein 1 (MCP1), macrophage inflammatory protein 1‑alpha (MIP‑1‑alpha), and tumour necrosis factor (TNF‑α) levels.

1.4. Transmission and prevention of infection

SARS-CoV-2 is unstable outside the human body and is transmitted by close contact with the infected person. It is spread mainly via airborne mechanisms. The most effective ways to prevent or, at least, to slow down its transmission are self-isolation, maintaining safe distance from others, washing hands often (and/or using 70 % ethanol-based hand disinfectants), cleaning regularly the surfaces and objects that are frequently touched, and wearing properly fitted facemasks when indoors. The World Health Organization (WHO) release regularly updated guidelines on prevention of the infection with SARS-CoV-2: https://www.covid19treatmentguidelines.nih.gov/about-the-guidelines/whats-new/

1.5. Pathogenetic targets for treatment of the SARS-CoV-2 infection

The SARS-CoV-2 genome encodes 4 major structural proteins, namely: spike (S), envelope (E), membrane (M), and nucleocapsid (N), along with non-structural and accessory proteins. Both the viral RNA and the structural proteins of the virus have been viewed as targets for treatment. The most commonly used antiviral drugs usually target the RNA-dependent RNA polymerase of the virus, while the monoclonal antibodies are more commonly targeted at the structural proteins of the virus.

1.6. Risk factors for complications of SARS-CoV-2 infection

Major risk factors for adverse outcomes are male gender and underlying medical conditions such as cardiovascular disease, renal disease, diabetes, chronic respiratory disease and cancer [51]. Obstructive sleep apnea is a specific risk factor that increases the risk for hospitalization and respiratory failure almost 8-fold [36]. Advanced age was initially believed to be a major risk factor for adverse outcomes but later it was recognised that the presence of underlying disease had significantly more weight than age alone [https://www.cdc.gov/aging/covid19/covid19-older-adults.html; https://gis.cdc.gov/grasp/COVIDNet/COVID19_5.html].

1.7. Treatments of SARS-CoV-2-infection and its complications

1.7.1. Conservative management

Uncomplicated cases of COVID-19 are best managed with home self-isolation from others, rest and symptomatic treatment. About 10-20 % of the infected are at risk of becoming seriously ill and may need specialised medical care [https://www.uptodate.com/contents/covid-19-clinical-features]. Complicated cases are managed according to designated WHO and Centre for Control of Disease (CDC) protocols, and local modifications of these protocols. As COVID-19 is associated with hyperinflammation and prothrombotic states, anti-inflammatory and anti-coagulation and/or anti-aggregation therapy are vital parts of virtually any protocol [https://covidprotocols.org/en/chapters/inpatient-management/]. The mainstay of anti-inflammatory therapy for COVID-19 are corticosteroids, mainly long-acting such as dexamethasone. In patients with severe respiratory failure, methylprednisolone may be added. Low-molecular weight heparin is most commonly used for anticoagulation in the protocols for treatment of COVID-19, but other options are available such as unfractionated heparin, indirect-acting anticoagulants, novel oral anticoagulants, aspirin, clopidogrel, dihydropyridamole, and others. The potent anti-inflammatory properties of corticosteroids come, among others adverse effects, at the price of gastric irritation; therefore, use of gastric protection by H2 antagonists or proton pump inhibitors is usually the norm. Antibiotics come into use in COVID-19 patients when there is evidence of secondary bacterial pneumonia. The latter is a common complication of severe COVID-19, as the virus produces, besides its other effects, a severe suppression of the innate immunity and a profound dysregulation of the immune signalling in the host [76]. Thus, treatment of severe COVID-19 often includes wide-spectrum antibiotics. Bactericidal antibiotics (e.g. penicillins, 4-quinolones and aminoglycosides) are typically being preferred over bacteriostatics (e.g. macrolides and tetracyclines). About 2 % of the infected patients may develop progressive respiratory failure and may need intensive care [https://www.uptodate.com/contents/covid-19-clinical-features].

1.7.2. Specific treatments against SARS-CoV-2 related disease

A variety of antivirals and monoclonal antibodies have been tried and tested for activity against SARS-CoV-2. Mostly, these are pre-existing drugs that have been previously tried for other disease caused by RNA-based viruses, such as favipiravir, remdesivir, lopinavir/ritonavir, tocilizumab, and others. Other, such as nirmatrelvir (a compound of Pfizer’s recently approved drug Paxlovid) and sotrovimab were specifically developed for the purposes of treating COVID-19, albeit on the base of previously known drugs or antibodies.

The advent of vaccines against SARS-CoV-2 brought new possibilities for management of infections and the prognosis for those who have developed COVID-19. The virus was isolated by the end of 2019 [70] and the first genetic sequence was published less than 2 weeks later [71]. Thus, the scientific and medical community along with the pharmaceutical industry had an early warning and could integrate and target their efforts toward developing effective treatments for COVID-19 and vaccines against infections with SARS-CoV-2 before the first wave of the pandemic hit the globe in March and April 2020. The first experimental anti-SARS-CoV-2 vaccine (Convidecia, developed by CanSino Biologics) was approved in China in late June 2020. Within the next few months about a dozen vaccines of the vector, peptide and mRNA type were fast-tracked and approved for use in adults, and, later, in children as well. By December 21, at least 14 vaccine products against SARS-CoV-2 have had their assessment finalized and about 10 more were currently under development (e.g. EpiVac Corona by Vector State Research Center of Virology and Biotechnology, and others) or under final assessment (e.g. vaccinal products by BioCubaPharma, CanSinoBio, Sanofi, Clover Pharmaceuticals, and others). More data may be viewed at Status_COVID_VAX_23Dec2021.pdf (who.int).

Applications for several products have been withdrawn. One of these was, unfortunately, the first entirely nanoparticle-based vaccine against infection with SARS-Cov-2 (zorecimeran, by CureVac). It was issued an expression of interest (EOI) by the WHO, but the application was later withdrawn by the manufacturer, as the demonstrated efficacy of protection of the vaccine against symptomatic disease was below 50 % (namely, 48%) in all age groups. The vaccine showed efficacy of 100% against hospitalization and death in the study group, but as it was comprised of 134 objects only, the final analysis pointed out that at least 80 additional cases were needed for proper assessment [https://www.reuters.com/business/healthcare-pharmaceuticals/curevacs-covid-19-vaccine-misses-efficacy-goal-mass-trial-2021-06-16/]. Thus, CureVac abandoned the zorecimeran project and started cooperation with Sanofi-GlaxoSmithCline in the work on their own brand of vaccine [https://www.reuters.com/business/healthcare-pharmaceuticals/curevac-withdraw-first-generation-covid-19-vaccine-candidate-2021-10-12/]. By January 2021, WHO has listed a total of 63 candidate vaccines in clinical development and further 173 in preclinical development.

2. Nano insights into treatment and prevention of COVID-19

Nanotechnology, as defined by the Centre for Disease Control in Atlanta, USA 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” 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. Nanomaterials usually have good solubility in biological liquids and a high surface-to-volume ratio that permits them to interact well with biological membranes and allows for better control of drug release than classic pharmaceuticals.

SARS-CoV-2 is a medium-sized virus (its particle is within the range of 60-140 nm), which fits very well into the nanoscale range. Modern nanotechnology offers a variety of high-tech solutions for the purposes of prevention of infection (development of efficient personal protection equipment (PPE); development of vaccines; pre-exposure prophylaxis, etc.); diagnostics (detection of the virus in biological and environmental samples), and management of the early stages of the infection (post-exposure prophylaxis), as well as effective treatments in cases when SARS-CoV-2-related disease has already developed. It may be reasonably expected that nanomaterial-based sensors, drugs and vaccines will play a critical role in the management of the pandemic in the near future [52, 41].

To date, a wide variety of nanomaterials have been employed for use in the detection of SARS-CoV2, prevention of transmission, drug and vaccine delivery. One of the best and most comprehensive reviews of the use of nanomaterials may be viewed here: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8308319/table/nanomaterials-11-01788-t001/?report=objectonly

2.1. Nanomaterials for prevention of transmission of SARS-CoV-2

The highest load of SARS-CoV-2 viral particles is found in fine respiratory droplets that are released when the patient (or the presymptomatic carrier) talks, coughs and sneezes; but large droplets containing viral particles also have high infective potential, when present on surfaces and objects that are frequently touched [https://www.cdc.gov/coronavirus/2019-ncov/science/science-briefs/sars-cov-2-transmission.html]. The significant infective potential of SARS-CoV-2 was acknowledged at an early stage of the pandemic, although it was significantly lower than the levels of infectivity of the later variants Delta and Omicron [https://www.cdc.gov/coronavirus/2019-ncov/variants/omicron-variant.html].

The size of the SARS-CoV-2 viral particles allows them to penetrate easily through usual protective equipment that has proven effective when used against other common respiratory diseases (influenza, chickenpox, and others). The development of modified types of PPEs called for the use of nanotechnology. Novel nanofibres and nanofibre webs were developed for use in protective facemasks worn by the medical personnel and first responders. High-performance facemasks (FFP2, FFP3, and N95 standard) use a combination of nanofiber webs and electrostatic charge that could protect from large respiratory droplets dispersed by the patient [9]. FFP-standard masks are 3D-printed to fit tightly the face of the wearer and may include a molded nosepiece to create an airtight seal. The filtration efficiency for high-performance masks for the highly virus-laden larger droplets (>0.3 μm) is expected to be at least 78% for FFP1, 92% for FFP2, and 98% for FFP3 masks [https://www.protectivemasksdirect.co.uk/ffp3-masks-hse-guidance].

Additional protection may be obtained by impregnation of the layers of the mask with compounds with antimicrobial and antiviral activities. Nanomaterials such as silver and copper oxide nanoparticles, alone or carrying other bacteriocidal and/or viricidal agents (e.g. iodine), have been used with success [6, 1].

Special nanocoatings (silicon-based, graphene based and others) have been developed to ensure that the masks are reusable after cleaning [14, 75].

Additionally, the size of the pores in the filter of the mask may be decreased by impregnation with polymers such as ß-cyclodextrin. It must be noted, however, that while the safety for the wearer of the mask is crucial, a perfectly fitting facemask may actually result in decreased compliance of the wearer. Typical surgical masks and respirators from the pre-nanotechnology era were known for their low air permeability, causing the wearer to fidget and try to touch and adjust the mask. Modern nanofibers produced by electrospinning create an exquisite web with excellent air permeability, improving the functionality of masks and increasing the antibacterial effects.

Similarly to facemasks, nanotechnology has also contributed to manufacturing medical gloves and other PPEs. Gloves with silver nanoparticles have shown bactericidal and virucidal effects.

A very interesting proposal about nanotechnology-based capture of viral particles by ligand-receptor interaction was made in 2021. It was based on the fact that the SARS-CoV-2 binds and enters the cell via the ACE2 receptor. Thus, it was proposed that nose filters, masks, gloves and other PPEs could be impregnated with nanotechnology-engineered ACE2-receptor that could bind the offensive agent and minimise the risk for the wearer [2]. The results, however, are yet to come.

Since SARS-CoV-2 (as well as influenza virus, HCV, HBV and HIV) is an enveloped virus, most currently used hand and surface disinfectants are ethanol or 2-isopropanol-based. The critical concentration of ethanol that completely inactivates the virus is believed to be over 35 % v/v [44]. Ethanol and isopropanol are, nevertheless, volatile, flammable, may contain compounds that are harmful. They are also relatively expensive and may have adverse effects such as headaches, nausea and dizziness when used as sprays, liquids and rubbing gels. Several nanoparticle-based sanitizers have been developed using titanium oxide and silver nanoparticles as well as engineered water nanostructures [49, 7]. Other types of nanotechnology-based hand sanitizers utilize hydrogen peroxide to ensure bactericidal and virucidal effect [65].

2.2. Nanomaterials in the diagnostics of SARS-CoV-2 viral particles

Almost all aspects of SARS-CoV-2 infection present a challenge. The virus is highly contagious, but not all people exposed to the virus will become infected. The infected person, even when symptomatic, does not produce a distinct clinical and laboratory phenotype that would allow COVID-19 to be reliably diagnosed on clinical appearance or results of laboratory tests. The incubation period may vary between 2 and 14 days (although it is, at present, believed that remaining asymptomatic beyond 7 days means that it is unlikely that the person is infected). The differential diagnosis of mild and moderate cases may include virtually any other respiratory infection and, in severe cases, may mimic other causes for rapid deterioration of the clinical status such as inflammatory disease of any origin, sepsis, vascular accidents, and others. A significant proportion of cases may present with radiological evidence only, repeatedly producing negative tests for viral RNA. Thus, development of a rapid, highly sensitive and highly specific COVID-19 diagnostic test is yet to come, especially in the view of the enormous potential of the virus to mutate further, evading routine diagnostics.

As COVID-19 is a modern pandemic, nanotechnology has been employed in order to improve the reliability of diagnosis of infection with SARS-CoV-2 since the first months after the initial outbreak. Most of the technologies and devices used in detection of SARS-CoV-2 are based on those previously developed for the detection of SARS and MERS.

Various nanomaterials have been tested and approved for sensitive and specific detection of viral agents prior to the outbreak of COVID-19. Among these are silver and gold nanoparticles loaded with gold-binding polypeptides and proteins [32, 47]; DNA probes immobilized onto a carbon electrode overlaid with gold nanoparticles; antisense oligonucleotides-covered gold nanoparticles coupled with colour substrate in order to ensure rapid visual detection [39, 30, 42], and others.

The typical SARS-CoV-2 test uses material from a nasopharyngeal or throat swab. This method of sampling, although scoring low in the invasiveness scale, may be viewed by the patients as an intrusion on their privacy. This may result in poor swab taking technique in order to avoid complaints and, respectively, in increased rate of falsely negative results. Less invasive approaches have been proposed for detection and rapid screening for SARS-CoV-2 in exhaled air using a biosensor-based gold nanoparticles [57, 19].

Magnetic nanoparticles have been used as a tool in molecular diagnostics of coronaviruses for extraction of nucleic acids from biological and environmental samples [20]. This was the basis for development of a specialised extraction protocol for SARS-CoV-2 using zinc ferrite nanoparticles [58; 76].

Quantum dots are nanoscale-sized crystals of artificial origin that contain excitable electrons with excitation and emission wavelengths that are subject to variance by the user. They possess semiconductor and fluorescent properties. The optical properties of quantum dots and, specifically, the correlation between size and optical characteristics allows them to be used for as imaging probes in biosensors. After the first SARS outbreak quantum dots have been used in a biosensor for detection of SARS nucleocapsid protein antigen [56]. In 2020, Liu et al., proposed that the same technology may potentially be adapted for detection for SARS-CoV-2 [33].

Carbon dots, carbon nanotubes and nanodiamonds have been used for enrichment and concentration of samples for the purposes of improved sensitivity of low-copy viral nucleic acid [72].

Nanozymes are nanoscale-sized molecules of artificial origin that possess catalytic activity. Recently, a rapid and sensitive paper strip-based assay for SARS-CoV-2 using Co-Fe hemin peroxidase nanozyme instead of natural peroxidase was developed [33]. The test provided, according to the authors, a lower detection limit as low as 10 pg/mL of the S protein antigen, with the test complete in about 15 minutes.

Serological detection of SARS-CoV-2 has also benefited from the development of nanotechnology. In 2020, Huang et al. developed an assay for rapid detection of IgM against SARS-CoV-2 using viral nucleoprotein conjugated to gold nanoparticles, obtaining results in less than 15 min [23].

3. Nanotechnology to combat COVID-19: therapeutics research

Nanotechnologies have the potential to become an integral component of development of treatments for COVID-19, providing novel platforms for drug delivery as well as regulation and coordination of temporal and spatial release of bioactive substances such as antivirals and monoclonal antibodies. The field is rapidly developing, although results lag behind the development of pharmaceuticals with antiviral and immunomodulatory properties. Still, research, technology and clinical medicine have presented a front as united as never before when humanity became face to face with SARS-CoV. It could be reasonably expected that development of novel nano-based drugs and vaccines against the virus would be significantly accelerated in the near future.

3.1. Antivirals for COVID-19

3.1.1. Favipiravir

Favipiravir is a guanosine analogue inhibitor of RNA-dependent RNA polymerase. It has been marketed in Japan as a treatment for influenza under the brand name Avigan (Fujifilm Toyama Chemical Co, Ltd.) Later, after the outbreak of SARS-CoV-2, favipiravir was entered into a trial drug for COVID-19 and in Feb 2020 was granted the status of a repurposed anti-COVID-19 drug in China (Favilavir, Zhejiang Hisun Pharmaceutical) https://www.pharmaceutical-technology.com/news/china-favilavir-testing-approval/. Later in 2020, favipiravir was approved in Italy for experimental use against COVID-19 [13]. Favipiravir is administered orally and its use is prescription-only. There have been reports that favipiravir may provide advantage in mild cases of COVID-19, especially in patients with fever [27, 62], but the drug is still not considered efficacious enough. The official statement of Fujifilm Toyama Chemical Co. is: ”… at this stage, clinical application of Avigan Tablet to treat Coronavirus disease (Covid-19) is under study and preparation in order to obtain clear evidence of the drug’s efficacy and safety” [https://www.fujifilm.com/fftc/en/avigan].

In 2020, a novel way of delivery of favipiravir directly to the pulmonary epithelium by means of a nanoemulsion was proposed [55]. In 2021, solid lipid nanoparticles have been developed for the purposes of local delivery of favipiravir by nebulization [64]. These trials are exclusively in in vitro settings. In vivo studies in the field have yet to come.

3.1.2. Molnupiravir (Lagevrio by Merck)

Molnupiravir is a pyrimidine ribonucleoside analogue that inhibits RNA-dependent RNA polymerase of SARS-CoV-2 [46]. Similarly to favipiravir, molnupiravir was initially developed for treatment of influenza. The Emergency Use authorization (EUA) for molnupiravir was granted by the Federal Drug Agency of US at Dec 23 2021, on the next day after the EUA of Paxlovid (see below) was granted [https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-additional-oral-antiviral-treatment-covid-19-certain]. Molnupiravir is administered orally. Its use is prescription-only and is indicated in non-hospitalised patients with “…mild-to-moderate coronavirus disease in adults with positive results of direct SARS-CoV-2 viral testing, and who are at high risk for progression to severe COVID-19, including hospitalization or death, and for whom alternative COVID-19 treatment options authorized by the FDA are not accessible or clinically appropriate”. At present, the EUA for Molnupiravir does not extend its use in children. Treatment with Molnupiravir must be initiated within five days of symptom onset in eligible patients. Current studies report that molnupiravir may reduce the risk of hospitalization or death by 50% in patients at risk [37, 25, 69]. The incidence of drug-related adverse events is comparable to the incidence occurring with other treatments [69]. Molnupiravir is one of the anti-COVID drugs that are considered for development in the direction of use of nanotechnologies to ensure better delivery and higher bioavailability [25], although trials of novel nano-formulations are yet to come.

3.1.3. Remdesivir

Remdesivir (Veklury, Gilead Sciences) was among the first specific treatments that exhibited significant effects in patients with COVID-19. Remdesivir was initially developed for treatment of hepatitis C and was found to be effective in the suppression of replication of virus RNA in animals infected with the Ebola virus [68] but inferior to other therapeutics for Ebola in humans [43]. It is an adenosine analogue that binds to the viral RNA-dependent RNA polymerase and inhibits viral replication by premature termination [21]. Unlike most other antivirals for COVID-19, Remdesivir is only used parenterally, as a slow IV infusion. Also, unlike other antivirals used against COVID, Remdesivir is indicated for use in hospital settings and in patients with severe disease only. It was initially approved for use in hospitalised adult and paediatric patients (provided they were aged ≥12 years and weighed  ≥40 kg) with COVID-19 that had severe disease and respiratory failure (evidenced by need to use supplemental oxygen) and were already on dexamethasone [https://www.covid19treatmentguidelines.nih.gov/management/clinical-management/hospitalized-adults–therapeutic-management/].

Those eligible for treatment with Remdesivir must have documented COVID-19 evidenced by positive results of direct SARS-CoV-2 testing (although, in cases where there is no positive test, evidence from radiographic or CT scan image such as ground-glass lung opacities may be enough for justification of the use of Remdesivir). Later, Remdesivir was approved for use in smaller children and in certain groups of non-hospitalized patients [https://www.fda.gov/news-events/press-announcements/fda-takes-actions-expand-use-treatment-outpatients-mild-moderate-covid-19]. Remdesivir use is subjected to review in each individual case but is generally contraindicated in patients with liver and kidney failure and is only administered after a negative cutaneous skin test for hypersensitivity.

Few research papers on use of nanotechnology to improve the delivery of remdesivir have been published so far. One, based on use of remdesivir-loaded nanovesicles made of poly(lactic-co-glycolic) apparently may serve as a base for creation of platform for improved delivery of remdesivir but results are yet not far away from the computer design [71].

3.1.4. Nirmatrelvir/Ritonavir (Paxlovid, Pfizer)

In late December 2002, the FDA issued an EUA for Pfizer’s combined anti-COVID drug Paxlovid (nirmatrelvir and ritonavir). Nirmatrelvir is a 3C-like protease inhibitor that inhibits the main viral protease 3CLpro that cleaves the polyprotein generated by translation of the viral mRNA in order to generate separate protein products [45]. It was developed onto the template of another covalent protease inhibitor, lufotrelvir. The latter was also previously a candidate drug in preclinical trials for treatment of COVID-19 [5]. Ritonavir, a well-known protease inhibitor commonly used in co-formulations in highly active antiretroviral therapy (HAART) for treatment of HIV infection works by inhibiting CYP3A. This results in decreased breakdown of nirmatrelvir by proteases and thereby increasing the its serum levels [40].According to a recent press release by Pfizer, Paxlovid reduced the risk of hospitalization or death by 89% [https://www.pfizer.com/news/press-release/press-release-detail/pfizers-novel-covid-19-oral-antiviral-treatment-candidate]. Paxlovid is administered orally and is indicated for the treatment of mild-to-moderate coronavirus disease (COVID-19) in adults and paediatric patients (12 years of age and older weighing at least 40 kilograms or about 88 pounds) that are at high risk for progression to severe COVID-19, including hospitalization or death [https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-first-oral-antiviral-treatment-covid-19]. For best results, treatment with Paxlovid must be initiated within five days of symptom onset. It is available by prescription only.

The studies of the effect of Paxlovid on the course of COVID-19 in high-risk patients are still scarce, calling for further studies to elucidate whether it really decreases the risk for hospitalization and death. One meta-analysis showed that patients on Paxlovid did experience lower incidence of adverse outcomes and that the rate of adverse events related to therapy with Paxlovid was close to the rate in the placebo group [69], but the amount of data is still deemed insufficient to draw conclusions on the efficacy and safety of the drug. Yet, nanotechnologies have not played a significant role in development of novel formulations of Paxlovid or its components.

3.1.5. Lopinavir/Ritonavir

Lopinavir/ritonavir (Kaletra or Aluvia by AbbVie) is another combo of protease inhibitors that has been tried in the treatment of COVID-19. It is a well-known and safe drug for treatment of HIV-infection. One of its known disadvantages is its poor water solubility, resulting in erratic bioavailability. In 2016, development of lopinavir granules that spontaneously produced drug-loaded self-assembling nanoparticles upon contact with water, greatly improving the delivery of the drug, was reported [48]. This could serve as a basis for further development of nanoparticle-based antivirals with improved safety profile and more effective delivery. Kaletra was, at one time, repurposed as a COVID-19 drug in hospitalized patients. Studies (including the RECOVERY study) show that the use of Kaletra in COVID-19 patients does not significantly decrease the risk for complications and death [53]. Later, Patel et al. demonstrated that the rate of adverse events was higher in the patients treated with lopinavir/ritonavir compared to patients receiving other treatments [60]. Therefore, its use is mostly discouraged at present.

3.2. Monoclonal antibodies (mAbs) for the treatment of SARS-CoV-2 infection and COVID-19

Monoclonal antibodies have been successfully used in the treatment of immune and auto-immune disease such as rheumatoid arthritis, psoriatic arthritis, ulcerative colitis and Krohn’s disease, some types of cancer, cytokine storms occurring in the course of infections and in transplanted patients, etc. The most common adverse effects of monoclonal antibodies are infusion-related reactions, including allergic reactions and anaphylaxis.

All monoclonal antibodies currently used in the therapy for COVID-19 are authorized for patients with mild and moderate disease that are at risk of developing severe complications. Most mAbs used for the treatment of SARS-CoV-2 infection are targeted towards structural proteins of the virus or may be directed against key players of the pro-inflammatory pathways that are inherent to the pathogenesis of systemic inflammation in COVID-19 (as is tocilizumab, see below). At present, only monoclonal antibodies that target the spike protein have demonstrated to have clinical benefit in infected patients [28]. Other monoclonal antibodies may be used as pre-exposure prophylaxis (for details, see below).

Several neutralising monoclonal antibodies have received EUAs from FDA for use in outpatient settings in patients with confirmed SARS-CoV-2. Four of these are used in combination (bamlanivimab and etesevimab; casirivimab and imdevimab), while sotrovimab, bebtelovimab and redganvimab may be used alone. Two antibodies are authorized for use as pre-exposure prophylaxis only.

3.2.1. Bamlanivimab and etesevimab (by Ely Lilly)

Bamlanivimab and etesevimab (generic only) bind to different, although overlapping epitopes in the spike protein receptor-binding domain of SARS-CoV-2. They are administered in an intravenous infusion. The BLAZE-1 trial showed a clinical benefit of the combination in patients with mild-to-moderate disease aged 2 years or older [12]. However, since the two combinations have shown reduced activity against the Omicron variant of concern, their use has been limited by the FDA [https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-limits-use-certain-monoclonal-antibodies-treat-covid-19-due-omicron].

3.2.2. Casirivimab and imdevimab ((REGEN-COV by Regeneron, Ronapreve by Roche)

Casirivimab and imdevimab bind to non-overlapping epitopes of the receptor-binding domain of the spike protein.  The Human Medicines Committee of the European Medicine Agency (CHMP) stated that the combined preparation of Ronapreve significantly reduces hospitalisation and deaths in COVID-19 patients at risk of severe COVID-19 [COVID-19: EMA recommends authorisation of two monoclonal antibody medicines | European Medicines Agency (europa.eu)].

3.2.3. Sotrovimab (Xevudy by GlaxoSmithKline)

Sotrovimab targets an epitope in the receptor-binding domain of the spike protein that is highly conserved between the related SARS-CoV and SARS-CoV-2. So far, Sotrovimab has shown efficacy against the Omicron variant of concern (VOC) [https://www.fda.gov/media/149534/download]. It is administered within 10 days of symptom onset as a single IV infusion or subcutaneously in non-hospitalized patients with mild-to-moderate COVID-19 that are at high risk of clinical progression. Sotrovimab is only administered to adult patients or children aged ≥12 years and weighing ≥40 kg.

3.2.4. Bebtelovimab (generic, by Ely Lilly)

Bebtelovimab is another spike-protein targeting mAb that reportedly retains its activity against the Omicron variant. It was granted an EUA by the FDA in February 2022 for use in adult and paediatric patients wirh mild-to-moderate disease [https://investor.lilly.com/news-releases/news-release-details/lillys-bebtelovimab-receives-emergency-use-authorization]. Bebtelovimab is administered to as a single IV injection.

3.2.5. Tixagevimab and cilgavimab (Evusheld by Astra Zeneca)

Tixagevimab and cilgavimab bind to non-overlapping epitopes of the receptor-binding domain of the spike protein [10]. FDA has granted an EUA for the combination tixagevimab and cilgavimab for pre-exposure prophylaxis. Eligible for treatment are individuals that have not been infected with SARS-CoV-2 and have not been in recent contact with an infected person, but may be at high risk for an inadequate immune response to COVID-19 vaccination, or have a documented history of severe adverse reaction to an available COVID-19 vaccine or any of its components [https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-new-long-acting-monoclonal-antibodies-pre-exposure].

3.2.6. Regdanvimab (Regkirona, by Celltrion)

Regdanvimab is a human monoclonal antibody directed against the spike protein of SARS-CoV-2. It is administered parenterally in a single infusion. Regdanvimab is indicated for the treatment of adults with mild-to-moderate COVID-19 who do not require supplemental oxygen and who are at increased risk of progressing to severe COVID-19 [https://www.ema.europa.eu/en/documents/product-information/regkirona-epar-product-information_en.pdf]. The CHMP stated that  the use of Regkirona significantly reduces hospitalisation and deaths in COVID-19 patients at risk of severe COVID-19 [COVID-19: EMA recommends authorisation of two monoclonal antibody medicines | European Medicines Agency (europa.eu)].

3.2.7. Tocilizumab (Actemra, RoActemra by Roche)

Tocilizumab has been successfully used as a biological treatment for a variety of autoimmune diseases, including rheumatoid arthritis and systemic juvenile idiopathic arthritis. It is a humanized monoclonal antibody against the interleukin-6 receptor. Since IL-6 plays a major role in inflammation and is among the early inflammatory markers in COVID-19, it was initially proposed that targeting IL-6 could reduce the rate of deaths among hospitalised patients. At the time, it was believed that the main pathogenetic mechanism of COVID-19-related complications was the cytokine storm resulting from dysregulation of the immunity signalling pathways. Tocilizumab was granted an EUA by the FDA for the treatment of COVID-19 in the in June 2021 [https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-drug-treatment-covid-19]. Several large trials (RECOVERY, REMAP-CAP) showed that the reduction of deaths produced by use of tocilizumab was added to those produced by use of dexamethasone [73]. The initial enthusiasm, however, was soon tempered down, as significant transaminase elevation (a known complication of treatment with tocilizumab) was consistently occurring in most hospitalized patients with COVID‐19 treated with tocilizumab. Indeed, many patients experience transaminase elevation as a result of treatment for COVID-19, but the use of tocilizumab added to the risk of serious liver injury. In July 2020, Hoffmann-LaRoche announced that tocilizumab treatment did not improve clinical status for patients with COVID-19-associated pneumonia and did not reduce patient mortality: [Roche provides an update on the phase III COVACTA trial of Actemra/RoActemra in hospitalized patients with severe COVID-19 associated pneumonia”]. Thus, tocilizumab is not recommended for use in patients with COVID-19. In the spring of 2021, a trial of a combination preparation of favipiravir-tocilizumab encapsulated in mucoadhesive vesicles and administered via nebulizer was started [61]. So far, no encouraging results have come through.

3.3. Other drugs with potential for use in patients with COVID-19

3.3.1. SQAd/VitE nanoparticles

A study published in 2020 described the development of anti-inflammatory drug based on nanoparticles [11]. These nanoparticles were made by conjugating the natural compounds squalene (a compound with anti-inflammatory activities) and adenosine (a natural immunomodulator), encapsulating them together with another biological compound, α-tocopherol (an antioxidant). The authors proposed that their nano-based drug serve as a novel therapeutic approach for safe treatment of inflammation. The practical applications are yet to come.

3.3.2. Baricitinib

In November 2020, FDA issued an authorization for the use of the combination remdesivir-baricitinib in hospitalized adults and paediatric patients two years of age or older with COVID-19 and severe respiratory failure [https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-drug-combination-treatment-covid-19]. Baricitinib (Olumiant, by Ely Lilly) is a pre-existing drug based on inhibition of the janus kinase (JAK)-mediated signalling pathways. It is usually used as a second-line treatment of rheumatoid arthritis. Baricitinib was not initially authorized as a stand-alone treatment for COVID-19. Baricitinib plus remdesivir was shown in 2021 to be superior to remdesivir alone in patients with severe COVID-19, especially those notably among those receiving non-invasive ventilation and had a satisfactory safety profile [26]. In July 2021 the EUA for the combination was revised. Presently, baricitinib alone may be used for the treatment of COVID-19 [https://www.thepharmaletter.com/article/fda-authorizes-baricitinib-alone-as-treatment-for-covid-19, https://www.fda.gov/media/143822/download]

3.3.3. Fluvoxamine (Fevarin by Mylan and Luvox by Solvay Pharmaceuticals, Inc.)

Fluvoxamine is a selective serotonine selective reuptake inhibitor that has been in use in clinical psychiatry for decades. It is usually prescribed for major depressive disorder and obsessive-compulsive disorder (OCD) but may also have its use in anxiety disorders [17]. It has a good safety profile, with lower incidence of the typical adverse effects associated with SSRI use – headaches, anxiety, irritation, sexual and sleep problems and cardiovascular complications. Psychiatric drugs usually have multiple targets and many potential adverse effects. Thus, the potential for delivery of drugs specifically to the target site has always been a major issue in clinical psychiatry. Design of fluvoxamine maleate-loaded solid lipid nanoparticles have been reported in 2019, demonstrating high entrapment efficiency and effective release of the drug [29].

At least three randomized trials have been conducted in order to study the effects of fluvoxamine in the treatment of outpatients with COVID-19. The first (STOP COVID) – showed reduction of the rate of clinical deterioration in the patients treated with fluvoxamine [69]. The second was stopped for futility by the data safety monitoring board as the treatments effects were significantly lower than expected [69]. The third was the TOGETHER study, which showed in 2022 that fluvoxamine may result in reduction of the severity of COVID-19-associated symptoms [54]. Namely, when used orally by non-hospitalized patients with early diagnosed COVID-19 and high risk for complications, fluvoxamine in standard therapeutic doses apparently reduced the risk for hospitalisation and death [54, 69]. The effects of fluvoxamine in COVID-19 patients remain to be seen in further trials. An application for an EUA for fluvoxamine use in patients with COVID-19 was submitted to the FDA [https://www.medpagetoday.com/special-reports/exclusives/96431] but, by present day, there is no information about an EUA being granted.

4. Nanoparticles’ vaccines

4.1. History and basics of vaccination as a method of prevention of transmissible disease

Vaccines have become a main weapon in the arsenal of medicine against infectious disease since the late XVIII century, when Edward Jenner applied his practical observations on the apparent immunity to smallpox of those that have previously had cowpox. This was not, strictly speaking, Jenner’s own invention, as he must have been familiar with the Eastern practice of ‘variolisation’, which was introduced in Britain earlier in the century by Mary Wortley Montagu and the studies of Dr. John Fewster that prevented contraction of smallpox by previously infecting his test subjects with cowpox [63]. In any case, the glowing reputation of Jenner and high social status undoubtedly contributed to the positive reception of his vaccinal practices. In the XX century, vaccines were rapidly developed for a variety of common infectious diseases and became an obligatory part of the vaccination calendar throughout the world. Vaccines against endemic infectious diseases such as yellow fever, tick-borne encephalitis, Japanese encephalitis, typhoid fever, and others are also available and some are obligatory for travelers in areas with vaccine preventable endemic disease.

Vaccines work by presenting an antigen typical of the infectious agent to the immune system of the host without occurrence of infection. Thus, the host may build a potent mechanism of defense against the offending agent prior to encounter or at very early stages of infection. Vaccination and immunization are different terms that are, nevertheless, very commonly confused. Immunity against a certain agent may be innate or acquired and may be developed either after vaccination or after the host has met with the infectious agent and has run the natural course of the infectious disease. Vaccination involves presentation only of the antigen (or more than one antigen, as is the case of inactivated vaccines) to the host’s immune system without the risk of (or, as with live vaccines, at a very low risk) of contracting the associated disease.

4.2. Types of vaccines

Multiple types of vaccines may exist for a single disease. Historically, there have been four major types of vaccines:

• Inactivated (e.g. Salk vaccine against poliomyelitis, rabies vaccine, Hepatitis A vaccine, and others);
• Live attenuated (Sabin vaccine against poliomyelitis, vaccines against measles, mumps, infectious parotitis, and others);
• Toxoid vaccines (e.g. tetanus and diphtheria toxoids);
• Subunit vaccines (e.g. some of the vaccines against influenza (e.g FluMist Quadrivalent by MedImmune), vaccines against hepatitis B, and others). These include peptide polysaccharide and conjugate vaccines.

Recently, two more types of vaccines have been added to this list: namely, vector vaccines (e.g. Zabdeno (against Ebola) and several vaccines against SARS-CoV-2) and mRNA vaccines (against SARS-CoV-2).

For some of oldest vaccines (such as the polio vaccine), several types may have been tried (in the case of polio – a live attenuated or inactivated vaccine, which are administered via different routes). Different countries may have a different policy regarding the type of vaccine used on the general population and in special cases.  Up to the COVID-19 era, however, there have not been such a large choice of vaccines against the same infectious agent.

mRNA vaccines have shown a comparable or superior safety profile to most other vaccines and, when administered in at least two doses, provided highest levels of protection (94-95 %) against development of COVID-19 and associated complications [50, 3]. The humoral immune response induced by most currently used mRNA vaccines against SARS-CoV-2 tends to decrease beyond 6 months of the second dose which was temporally overcome by the use of booster dose [31]. Also, booster dose was reported to increase vaccine effectiveness in terms of about 75 % less COVID-19 related emergency care department visits and 80 % less hospitalizations [16].

4.3. Vaccines against SARS-CoV-2

Presently, authorized SARS-CoV-2 vaccines belong to three of the four major ‘classic’ types listed above or may be of the newer mRNA or vector types.

4.3.1. Vaccines developed using ‘classic’ techniques

Inactivated vaccines against SARS-CoV-2 are both vaccines developed by Sinopharm (Sinopharm BIBP and Sinopharm WIBP); Turkovac (by Health Institutes of Turkey), CoronaVac (SinoVac Biotech), Covaxin (by Bharat Biotech), QazVac (by Kazakh Biosafety Research Institute).

Subunit vaccines are usually based on presentation of the S (spike) protein to the immune system of the vaccinated host. Such are: EpiVacCorona (by VECTOR center of biotechnology, Corbevax (by Biological E. Ltd.), Novavax, Soberana and the yet unnamed Sanofi-GSK vaccine;

Live attenuated vaccines against SARS-CoV-2 are, notably, among the least popular choices among the vaccines against SARS-CoV-2. At present, the only live attenuated vaccine is COVI-VAC by Codagenix Inc., in a phase 1 trial.

4.3.2. Nanotechnology for development of vaccines against SARS-CoV-2 infection

The newer generation of vector vaccines is strongly represented in the list of anti-SARS-CoV-2 vaccines. Such are Oxford/Astra Zeneca’s Vaxzevria, the vaccine developed by Janssen-Cilag, Sputnik V, Convidecia (CanSino Biologics), and others.

mRNA-based vaccines have been in development for at least 20 years but until the pandemic spread of SARS-CoV-2, the effort was targeted mainly at development of anticancer vaccines and novel vaccines against influenza. Representative members of the mRNA vaccines are Comirnaty (by Pfizer-BioNTech) and Spikevax (by Moderna). Basically, they both create and boost the host’s immunity against SARS-CoV-2 via presentation of artificially created and modified mRNA encoding parts of the protein sequence of the S protein of SARS-CoV-2. Development of the mRNA vaccines is, par excellence, a nanotechnology, as the mRNA encoding the S protein is packed into nanoparticles (e.g. liposomes). The particles are taken up by dendritic cells and the mRNA is readily translated by the host ribosomes. The foreign protein is exported on the surface of the antigen-presenting cells and presented to the T cells in order to mount an immune response.

Liposomes are closed vesicles composed of one (or more) phospholipid bilayers. Their structure resembles the structure of cell membranes. The phospholipids may be natural or artificial origin. Various compounds may be encapsulated within the vesicles and may be delivered to a specific target tissue or in a time-dependent manner, allowing liposomes to be used for controlled drug delivery. The phospholipids in the bilayers may be modified in order to achieve the properties desired by the user – increased lipo- or hydrophilicity (depending on the target), slow or rapid disintegration of the vesicle upon reaching their target (in order to control drug release), etc. Polyethylene glycol (PEG)-conjugation (PEGylation) is commonly used in drug design. It increases their hydrophobic properties and prolongs the circulation time of the liposomes in living tissues [24] (although repeated administration may result in the exact opposite – see below). PEG3350 (polyethylene glycol [PEG])-2000]-N,N-ditetradecylacetamide) is a common ingredient in many routinely used preparations – e.g. tablets, injectable depot preparations, oral laxatives, and others. PEG3350-conjugated liposomes are currently used in both Comirnaty (Pfizer BioNTech) and Spikevax (Moderna) vaccines. When injected into muscle, the active substance forms a depot that is slowly released over several hours up to several days, allowing for migration of dendritic cells to the place of deposition and uptake, processing and presentation of the antigen in order to mount an immune response.

In general, liposomes are biocompatible and non-toxic carriers for drug delivery. They have been successfully used in vivo for drug delivery to specific targets, mainly in clinical oncology [34, 35]. However, modifications (including PEGylation) may result in increased immunogenicity of the resulting preparation. Anaphylactic and other immune reactions have been described for a variety of PEG-containing preparations (e.g. laxatives, depot steroids) [59]. mRNA vaccines usually are described as having excellent safety profile that is readily demonstrated by an overall 0.2 % incidence of adverse events after vaccination [https://www.cdc.gov/mmwr/volumes/70/wr/mm7002e1.htm]. Anaphylactoid reactions, however, have been reported both for Comirnaty and Spikevax [4, 8].  Presently, almost all cases of anaphylaxis after administration of the Pfizer-BioNTech vaccine are believed to be associated with the presence of PEG3350 in the preparation [18]. Since Moderna does not list PEG in the safety sheet for their Spikevax, it is difficult to know whether the reported cases of anaphylaxis after vaccination are linked to the PEG content or some other compound. At present, the official recommendation of FDA is: “If you are allergic to PEG, you should not get an mRNA COVID-19 vaccine…If you are allergic to polysorbate, you should not get the J&J/Janssen COVID-19 vaccine.” https://www.cdc.gov/coronavirus/2019-ncov/vaccines/recommendations/specific-groups/allergies.html

Liposomes and the related encapsulation technology are only early precursors to the much wider variety of technologies based on the use of lipid nanoparticles. Potential next steps are: improving target specificity, better control of drug release, development of stimuli-responsive liposomes (sensitive to triggers such as changes in temperature, pH, binding of specific ligand, etc.) and many others.  Use of nanotechnology undoubtedly improves the defense against the new pandemics at all levels and allows for creation of reliable, more robust and easily accessible diagnostic tests, personal protection equipment, drugs and vaccines.

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Test LO 4.1

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