Nanomedicine Based Approaches for Combating Viral Infections

Abstract

Millions of people die each year from viral infections across the globe. There is an urgent need to overcome the existing gap and pitfalls of the current antiviral therapy which include increased dose and dosing frequency, bioavailability challenges, non-specificity, incidences of resistance and so on. These stumbling blocks could be effectively managed by the advent of nanomedicine. Current review emphasizes over an enhanced understanding of how different lipid, polymer and elemental based nanoformulations could be potentially and precisely used to bridle the said drawbacks in antiviral therapy. The dawn of nanotechnology meeting vaccine delivery, role of RNAi therapeutics in antiviral treatment regimen, various regulatory concerns towards clinical translation of nanomedicine along with current trends and implications including unexplored research avenues for advancing the current drug delivery have been discussed in detail.

1. Introduction

Viruses pose significant threats to the existence of human beings. In agreement with the McMichael’s theory, we have entered in the fourth translational era surpassing the agrarian, Eurasian and European interventionism [1]. Till date, the world has witnessed several viral outbreaks such as Rabies, influenza, Spanish flu, viral hepatitis, measles, HIV, swine flu, COVID-19, etc. with a death toll of millions of lives each year [2]. The WHO estimated that the number of patients exposed to hepatitis B to be about 2 billion comprising the 240 million chronic carrier patients [3].

In 2015, WHO assessed that globally 257 million people infected with chronic hepatitis B infection, out of which 887,000 deaths were mostly from cirrhosis and hepatocellular carcinoma. As per WHO findings, an estimated 4.5 million deaths could be circumvented in underdeveloped and developed nations by 2030 through vaccines, diagnosis, medicines and counseling [4]. Approximately, 35 percent of the patients suffering from Middle East Respiratory Syndrome (MERS) die each year [5]. COVID-19 cases have surged till 135 million cases, with a global death toll of 2.92 million spread across 220 countries [6]. Globally, greater than 90 million new influenza cases, every year have been testified in children aged less than five years [7]. During the last few years, dengue cases are on the rise. WHO reports suggest up to 100–400 million global cases each year [8]. To quantify the fatality caused by viral infections, case fatality ratio was estimated for the most prevalent viral diseases (Figure 1). Case fatality ratio is the percent of total number of patients died due to a disease to the total number of patients suffering from a disease [9].

Figure 1.

Case fatality ratio of various viral infections

With evolution in the viral strains, medicine and therapeutic regimen has also evolved tremendously with novel drug molecules and vaccines arriving in the clinic in the last few decades [10,11]. However, due to reduced incubation period and high reproduction number of the viral strains, the spread of the infection is rapid compared to the time it takes for the clinical translation of new drugs and vaccines [12]. Despite of surpassing numerous developmental stages, proving safety and efficacy in the pre-clinical and clinical stage, several investigational new drug moieties fail to demonstrate efficacy in the clinic. The failure in efficacy could be attributed to their poor solubility, permeability, bioavailability, increased dose and dosing frequency, poor selectivity, targeting insufficiency and adverse effects leading to severe toxicities, rampant resistance, etc. making their translation difficult [13]. Nanotechnology based medicines aim to bridge the gap created by these obstacles to render targeting, safety and efficacy attributes to new chemical moieties. This review highlights the significance of nanotherapeutics based strategies to surpass and combat the major hurdles of the conventional antiviral therapy and hasten their clinical translations.

2. Types of viruses

Viruses are sub-micron sized intracellular parasites that utilize the host replication machinery owing to the absence of genetic material required for macromolecular synthesis and energy production [14]. This demeanor leads to the development of a unique relationship between host and virus cells. In accordance with the Baltimore classification system, viruses have been categorized into seven types as shown in figure 2 based on their genetic make-up [15].

Figure 2.

Categorical distribution of viruses and their mechanism of cellular interaction and replication

3. Barriers to viral entry

Viruses act as intracellular parasites which are transmitted as inert elements. In order to invade its host, it is imperative for a virus to first attach to the host cells [16]. This is mediated via ligand receptor interaction wherein specialized glycoproteins present on the viral capsid act as ligand and have an affinity towards the host membrane surface protein which further behaves as a receptor [17]. The position of the host receptor decides the site of infection of the virus. For instance, SARS-CoV-2 intermingles with the ACE-2 receptor which is present abundantly in the lungs via the Spike protein [18]. Similarly, different viruses depending on their membrane proteins attach the host cells or tissues at different sites. This type of selectivity that a virus possess towards particular type of tissue is known as tissue tropism [19,20]. However, the human body possesses several defense barriers in order to restrict the systemic entry of any foreign material. The respiratory tract lumen is lined by a layer of viscous mucus secreted by goblet cells where virions entering through the pulmonary route get trapped and are carried through the pharynx, where they either get swallowed or get coughed out [21]. Additionally, the distance travelled by the inhaled particles throughout the respiratory tract is predominantly size dependent. Larger particles (> 10 μm) are confined in the mucous layer whereas smaller particles (< 5 μm) have the ability to penetrate deeper into the lungs but they are engulfed by alveolar macrophages [22]. Additionally, the respiratory system is secured by innate as well as adaptive immunity along with specialized lymph patches i.e. Nasal-associated lymphoid tissue (NALT) and Bronchi associated lymphoid tissue (BALT) [23]. Despite its rigorous protective ways, the respiratory tract is one of the most communal gateways for viral entry. Post entry, depending on the affinity of the viral capsid proteins, some reside in the respiratory tract while others enter the systemic circulation through the lymphatic system. In such cases, muco-adhesive nano-medicine through the pulmonary route for prolonged duration may help in reducing the viral bioburden in the entire respiratory tract. This may also help in arresting the transmission from one host to another which may prove to be beneficial in viral infections linked to the respiratory tract [24]. Enteric viruses like Rota virus, disseminate into the hosts by consumption of viral contaminated eatables or beverages [25]. These infections usually commence in the mucosal epithelium of the gastro-intestinal tract. The entire digestive system is secured by an arsenal of defense system which includes acidic pH of the stomach, thick layers of mucus over the entire gastro-intestinal tract, the proteolytic activity of digestive enzymes, detergent action of bile and pancreatic juices. Adjunct role of innate and adaptive immunity, role of defensins and Immunoglobulins (Ig) A secreted by B lymphocytes in the gastro-intestinal tract and Mucosa-associated lymphoid tissues (MALT) provides additional protection to diminish the transmission via oral route [26,27]. However, enteric viruses have an innate resistance to bile and acidic pH. The failure of the gastro-intestinal tract in limiting the enteric viruses lies in their ability to invade the specialized M cells present within the Peyer’s patches which is a major defense base of the tract [28]. In such cases, use of lectins as ligands specific for M cell targeting is desirable to deliver drugs by imparting site specificity to the nanocarriers. Skin is composed of a thick external stratum of keratin which imparts a mechanical barricade to viral entry. Slightly acidic pH and occurrence of fatty acids impart further protection, along with the lymphatics which provide additional immunological elements sensitive to viral invasion [29]. Several antiviral peptides are expressed in different layers of the skin which are highly selective to viral proteins and genetic material. For instance, oligo adenylate synthetase protein selectively targets ssRNA while MxA peptide targets nucleocapsids of both RNA and DNA viruses. Inquisitive readers may direct their attention to reviews by Chelsea Handfield and co-workers [30] and on the role of defensins by Klotman and Chang [31]. Compared to the other barriers, the conjunctiva is much less resistant to viral manifestation but it is continuously rinsed with the lacrimal secretions and mechanical action of the eyelids [32,33]. Another route of entry includes the genital tract or the venereal route opted by herpes and papilloma viruses during copulation [34]. VivaGel^® Condom by Starpharma, lubricated with SPL7013 astrodrimer, has the ability to inactivate HIV, herpes simplex virus (HSV) and human papilloma virus (HPV) which are responsible for viral mediated sexually transmitted diseases. The product was marketed in Japan (Okamoto’s 003), Australia and Canada (Lifestyles^® Dual Protect™) [35]. The blood brain barrier (BBB) consists of an endothelial cell layer near the microvasculature as well as pericytes and astrocyte projections and microglial cells. Endothelial cell components such as Junctional Adhesion Molecule (JAM-1), Claudins, Cingulin AF-6/afadin, Occludins, etc. cohabit to reduce the intercellular junctional spaces. This results in restriction of the paracellular transport leading to increased trans-endothelial electrical resistance (TEER) of 8,000 Ω cm² [36]. Viral transport pathways include viral dissemination across brain microvascular endothelial capillaries (BMEC) due to inflammation, transport of nascent virus across the barrier and the ‘trojan horse’ pathway where the infected monocytes carry the virus from the blood to the brain. Few viruses have the ability to traverse the BBB entirely by retrograde axonal transport of virions via peripheral nerves into the CNS, viral invasion of the olfactory epithelium followed by transport of virus into the CNS across the cribriform plate [37].

4. The viral life cycle

Viruses as intracellular parasites invade the host cell and use the host cell machinery for their own protein synthesis [38]. The preliminary step consists of viral host cell attachment. The binding of viral proteins to the host receptor is highly specific and may require certain lipids or glycoproteins for viral entry to take place. These dynamics alleviate the viral binding and allow its entry into the host cell [39,40]. Contact between viral surface proteins and host receptors constitute an increased affinity which reduce the conformational energies required during the signaling process [41–43]. This free energy reduction promotes the destabilization of the host cell membrane resulting in an endosome vesicle formation. The endosomal vesicle promotes the entry of viral genetic content within the host cell. Prominent protein motifs which mediate the entry process include the integrin-binding tripeptide RGD protein, clathrin and caveolin mediated endocytosis, etc. [44]. Depending on the replication site, viruses migrate to the site where the viral genome integrates with the host cell genome. This migration process post-internalization is known as intracellular trafficking. Microtubule assisted transportation along with receptor-mediated endocytosis is the major intracellular transport mechanism for viruses [45,46]. For viruses whose replication site is the nucleus, nucleocapsid is routed to the perinuclear space via microtubule assisted transport [47]. As the virus reaches the replication site, the viral genome initiates the uncoating process. For viruses with a smaller genome, the capsid enters the replication site with the genome, e.g. polyomavirus while for larger genome, there is partial destruction of the capsid prior to its transit into the replication site e.g. adenovirus [48]. The viral genome replication stages are a finger print which is used to identify the type of virus and the family it belongs to [49]. Retroviruses undergo an additional reverse transcription step to convert viral RNA to cDNA prior to integration with the host cell genome [50]. Once the integration process has been initiated, transcription and viral protein synthesis occurs. These viral proteins are further assembled into virions which exit the host cell via cell lysis, migrate and infect other healthy host cells [51]. In a nutshell, this whole process is known as viral life cycle. Several drugs have been designed to inhibit this cycle at various stages – attachment, entry, intracellular trafficking, uncoating, replication, transcription and release of virions for various diseases however, there are several challenges associated with antiviral therapy. Figure 3 implicates various drug targets throughout the virus life cycle. Table 1 describes significant viral and host proteins interaction in various viral infections.

Figure 3.

Drug targets throughout the different stages of viral life cycle

Table 1.

Viral and host proteins interaction

Virus	Host proteins interacting with the viral proteins	Viral protein	Reference
Adenovirus	Integrins αvβ3, αvβ5	Chimeric antigenic receptor, E3	[52]
Herpes simplex virus	Nectin-1/HVEM, PILRα	gD, gC and Gb	[53]
Epstein Barr virus	CD21	gp350	[54]
HIV-1	CD4 (CXCR4 or CCR5) expressed on macrophages and T lymphocytes	gp120	[55]
Hepatitis B virus	NTCP	Pre-S1	[56]
Hepatitis C virus	CD81, Claudin-1, Occludin	E2	[57]
SARS CoV-2 virus	ACE2, surface serine protease TMPRSS2, HSP90/HSP70, ER chaperonin GRP78 TIM/TAM Macrophages’ Fcγ receptors	Spike protein	[58]
Rhinovirus	ICAM-1 or LDL receptor	VP1, VP2, VP3	[59]
Poliovirus	PVR/CD155	VP1, VP2, VP3	[60]
Rabies	NCAM-1/CD56	G protein	[17]
Influenza virus	Sialic acid residues	Hemagglutinin Neuraminidase Nucleoprotein	[61]
Ebola virus	C-type lectins, Phosphatidylserine receptors, C domain of NPC1	Nucleoprotein (NP) Capsid proteins - VP30, VP35 Matrix proteins - VP40 and VP24 Membrane fusion proteins - GP1,2	[62]
Zika virus	AXL, TIM, TAM, DC-SIGN, and Tyro 3 gas6	Capsid, envelope, membrane protein precursor	[63]
Dengue virus	Glycosaminoglycans like heparan sulfate, lectin and mannose receptors on macrophages, adhesion molecule of dendritic cells (DC-SIGN), lipopolysaccharide (LPS) receptor CD14	Membrane protein M, envelope protein E, capsid protein C	[64]

5. Challenges associated with current antiviral therapy

Incessant efforts made by the research community have ameliorated the quality of life and reduced the mortality associated with life-threatening viral infections. Medicine has evolved to a great extent with respect to newer drug molecules and vaccines making their way to the clinic. Still, there are certain challenges associated with current antiviral therapy. This section of the article aims to highlight noteworthy obstacles and could potentially contribute towards clinical success of antiviral therapy. For HIV, anti-retroviral therapy (ART) constituting of a single drug was not effective in treatment and arresting the spread of the disease [65]. This was attributed to immediate emergence of viral mutant strains resistant to ART therapy. Therefore, multiple agents must be utilized in combination for treating viral infections. Such combinations are recognized as highly active anti-retroviral therapy (HAART) and aimed to prevent resistance development. HAART has been successful to diminish the mortality rate substantially and enhance the life expectancy from few months up to 10 years [66,67]. However, HAART presents formidable difficulties like development of multi-drug resistant strains, viral reservoir site formation in areas unapproachable for the conventional drug delivery, dormant cells having hybridized viral DNA (Lysogenic pathway) which are diagnosed at an advanced stage, severe untoward effects and organ toxicities attributed to high dose and dosing frequency [68]. Pediatric HAART is particularly problematic because only 12 out of 25 drugs are approved for use among children [69,70]. During monotherapy or combination therapy of same class of drugs, resistance develops quickly due to mutation in the target protein by under-expression or modification of the binding site sequence, efflux or intracellular metabolism or prevention of the generation of the active moiety from the drug molecule [13]. Nucleoside reverse transcriptase inhibitors (Tenofovir and Adefovir) fail to develop resistance rapidly for prolonged duration of therapy even greater than a year. Viral resistance cases have been observed with lamivudine against Hepatitis B infection after a therapy of greater than 6 months. It has been found that in immunosuppressed patients, HSV could develop resistance to Acyclovir, and Cytomegalovirus to Ganciclovir [71]. Another major pitfall is the specificity and limited number of viral replication and metabolism steps which renders the broad spectrum antiviral drugs ineffective [72,73]. In such cases, novel drug synthesis exclusive and selective for particular step inhibition of the viral cycle is a laborious and costly affair. Even after drug designing for a specific target in the viral life cycle, incidences of point mutations could lead to rapid emergence of resistance. This bypasses the need of virus for the target leading to reduced effectiveness of the drug. For instance, impaired production of viral thymidine kinase, modified thymidine sensitivity and altered phosphorylation of acyclovir contribute towards the emergence of resistance of Acyclovir [74]. Inquisitive readers may refer to some of the excellent publications relating to antiviral drug resistance and causes for drug failure cited here [75–79].

Biopharmaceutical classification of the drug moiety must be taken into consideration during product development. Low solubility and low permeability may tremendously reduce the clinical efficacy of the drug since its absorption across the gastro-intestinal tract is hindered. Reduced absorption or permeation further diminishes the amount of drug reaching the blood stream. This prevents the drug from achieving sufficient therapeutic amount at the bioactive site which increases the incidences of resistance due to survival of viruses. The conventional therapy utilizes an increased dose and dosing frequency to surpass the bioavailability challenges however, as described previously, adverse effects increase proportionately. Acyclovir and ganciclovir, display poor oral bioavailability while, Lamivudine, Didanosine and Zidovudine are BCS Class III drugs with greater aqueous solubility, poor permeability and display extensive variability in bioavailability [80]. Sharma and Garg summarized that most of the antiviral drugs suffer from the limited absorption [81]. Acyclovir suffers from poor oral bioavailability (15–20%) attributed to its sluggish and inadequate absorption [82]. Large doses (1200 mg per day) are required to achieve sufficient plasma concentration to be an effective antiviral agent. In order to overcome this obstacle, Valacyclovir, valine ester of Acyclovir and Famciclovir, showed enhanced oral bioavailability compared to Acyclovir [83]. Topical acyclovir therapy for Herpes simplex keratitis infection showed little efficacy owing to its reduced penetration, which could be attributed to its hydrophilic nature and high aqueous solubility as well as an increased dosing frequency i.e. five to six times daily [84]. Ganciclovir, another antiviral drug suffers from diminished oral bioavailability (6–9%), with greater than 1000 mg per day. Pro-drug approach with the help of valine ester of Ganciclovir has been utilized to improve its bioavailability on oral administration [85]. In order to enhance the antiviral activity, it is imperative to modify the traditional dosage forms. Certain formulations include the application of depot parenteral, extended release tablets and enhanced topical delivery systems. Such modifications of dosage forms can alter the residence time, dose and dosing frequency reduction, help to surpass non-compliance and reduce the adverse effects. For instance, long acting PEGylated interferons have been designed for dosing per week compared to three times per week [86,87]. Another major disadvantage of current antiviral therapy is the reduced half-life of drugs which results in a decrease in the amount of drug due to extensive metabolism in distant viral depot sites which include lymphatic system, central nervous system and the pulmonary tract [88]. Additionally, the delivery of drugs through the conventional approaches via oral route to such distal sites poses extreme difficulty. For example, Zika virus, Ebola and HIV manifest into the central nervous system, lymphatic fluid and synovial fluid which significantly reduces the efficacy of the conventional therapy due to lack of attainment of sufficient concentration at such bioactive sites [89]. Other flaws associated with antiviral therapy include severe drug-drug interactions and organ toxicities [90,91]. Clinical adverse reactions are fickle and dependent on single drug, dosage regime and genetic make-up of patients. A reduced half-life is usually intended to prevent the incidences of viral resistances due to reduced contact time of the drugs with the viral bio-burden. However, this increases the dose, dosing frequency and could potentially increase the propensity of adverse effects. Multiple dosing frequency further promotes prolonged exposure of the drug moiety to the viral burden. For instance, Indinavir exhibits a half-life of about 1 h. Clinicians, therefore find it extremely difficult to treat patients who miss doses, especially in populations in developing nations. A newer generation of antiviral, Rilpivirine, which is active at significantly lower doses (100 mg) is currently being investigated for once a month extended release through the intramuscular route [92]. However, target specificity, incidences of mutation, absorption and bioavailability pitfalls, delivery to viral reservoir sites, etc. still remain unanswered even by use of extended release dosage forms. Nano-medicine based approaches have the potential to address these drawbacks effectively. Figure 4 indicates various currently approved antiviral agents while figure 5 describes hurdles linked with current antiviral therapy.

Figure 4.

Classification of approved antiviral agents along with their generic name and marketed brands used for treating various viral infections or diseases

Figure 5.

Biopharmaceutical, pharmacokinetic and pharmacodynamic challenges associated with current antiviral therapy

6. Rationale for the use of nano-medicine based approaches

From above findings, it is evident from the challenges associated with the conventional antiviral therapy that there exists a gap between the current conventional therapy and successful clinical translation against viral infections. In a nutshell, the underlying reasons for this interstice include short half-life, poor solubility, absorption bioavailability, high dose and dosing frequency, multi-drug resistance occurrences, lack of specific viral targeting, severe adverse effects and drug-drug interactions, inability to reach viral reservoir sites in the lymphatic circulation, BBB, etc. (Figure 5). The characteristics which control the pharmacokinetic profile of antiviral drugs can be modified with the help of nanoparticulate carriers to bridge the gap and ameliorate the efficacy and side effects of antiviral therapy. Nano-medicine based approach could be used to protect the drugs from gastro-intestinal tract degradation, first pass metabolism, enhance the pharmacokinetic profile and promote stimuli responsive release and intracellular drug delivery. These characteristics are profoundly beneficial in delivery of oligonucleotides like siRNA, miRNA, vaccines, etc. Nanocarriers can harness the specificity, targeting efficiency and deliver high drug payloads towards the viral bio-burden at the reservoir sites reducing the emergence of resistance. Aforementioned advantages offered by nano-medicine based approaches, make this therapy as a boon for treating viral infections. Figure 6 indicates different nanoformulations used for antiviral drug delivery.

Figure 6.

Lipid, polymer, carbon, metallic and emulsion based nanoformulations for delivery of antiviral drug/s

6.1. Lipid based delivery systems

6.1.1. Vesicular systems

Liposomes are self-assembling, sub-micron sized vesicles which consist of phospholipid bilayers entrapping an aqueous phase discovered by Sir Alec Bangham in 1965 [93]. They possess the potential to incorporate both hydrophilic as well as hydrophobic drug/s. Once liposomes are injected into the body, plasma opsonins adhere onto the surface of liposomes. This adherence helps macrophages to identify the liposomes as foreign material and eliminate them from the systemic circulation [94]. Susceptibility of liposomes towards macrophage uptake have been widely explored to deliver drugs against viruses like HIV, which infiltrate the macrophages and form reservoirs inside them. During viral infections, macrophages migrate towards the infection sites and possess a discriminatory property between healthy and viral infected cells. Just as the viruses like HIV, which use macrophages as depot sites, liposomes and nanoparticles have the potential to use the endosomal environment in the macrophage as depot until the drug cargo is delivered at the active site with the assistance of macrophages. This type of delivery system is known as Trojan horses [95]. Macrophage uptake depends on certain factors like size, shape, surface charge, surface functionalization, etc. In order to increase the efficiency of macrophage uptake, an interplay of such factors is desirable. Particle size greater than 100 nm, reduced aspect ratio (less than 20), high surface charge with both positive as well as negative polarity are prone to macrophage recognition and uptake [36,96]. Several strategies have been reported to achieve high macrophage uptake for antiviral drug delivery.

Liposomal surfaces concocted by amalgamation of surface charges (anionic or cationic) and ligands like mannose, galactose and other carbohydrates to target lectin receptors on the macrophages and enhance liposomal uptake and lymphatic distribution to lymph nodes and spleen [97]. Positive charge over the liposomal surface is imparted by cationic lipids like DOPE, DOTAP, etc. Charge inducers like chitosan and stearyl amine can be utilized to impart cationic charge distribution and stability of the formulation. Florescent microscopy of zidovudine loaded liposomes performed by Kaur and co-workers implicated an enhanced uptake and localization where mannose conjugated > cationic > anionic > conventional liposomes (p < 0.05) [98]. Tahara and colleagues explored the influence of stearyl amine and dicetyl phosphate on baculo virus infected A549 cell lines. Cytotoxicity studies, plaque reduction assays, cell uptake and antiviral activity was determined to check the efficiency of charge inducers compared with the conventional liposomes. They found a 22% greater reduction in baculo virus infected A549 cell lines for stearyl amine coated liposomes compared to dicetyl phosphate coated liposomes (p < 0.05). Stearyl amine played an adjuvant role in antiviral activity in A549, B16 and conjunctival cell lines infected with baculo virus along with improving the stability of the liposomal formulation [99].

Cationic liposomes have been recognized to deliver labile oligonucleotides intracellularly owing to the increased uptake by ionic interactions of the positively charged liposomes with the anionid cell membrane. Proton-sponge effect is one of the most important phenomenon used for intracellular delivery of small molecules and oligonucleotides. This effect elicited by cationic lipids and polymers depends on the acidic pH 6.5–5.5 of the endosomes attributed to the proton pumps which transport the protons from cytosol to the endosomes. When cationic polymers are used in the formulation, they take up the protons via electrostatic interactions. This promotes acidification of the endosomal vesicle leading to an increased proton entry along with chloride ions which causes swelling attributed to osmotic pressure differential followed by endosomal rupture leading to the endosomal escape of delivery system. This leads to translocation towards the nucleus and transfection of oligonucleotides [100,101]. This could prove to be beneficial in viral infected cells in which intracellular delivery preventing oligonucleotide degradation and nuclear targeting is imperative. Fusogenic lipids like Dioleoyl phosphatidyl ethanolamine could be used to fuse the liposomal and endosomal membrane to prevent their degradation during the endo-lysosomal stage [102]. The primary disadvantage of cationic liposomes is the rapid systemic clearance which prevents a sustained action and increases the dosing frequency in case of drugs with short half-life. In such cases, a combination of PEGylation and proton sponge effect principles must be utilized to address this problem. Slepushkin and co-workers added polyethylene glycol-phosphatidyl ethanolamine PEG-PE and found that a longer duration was achievable without compromising the cytotoxicity of the cationic lipids [103]. Liposomes have been utilized for traversing the BBB. Magnetoliposomes containing 3′-azido-3′-deoxythymidine-5′-triphosphate loaded into macrophages ameliorated the in vitro BBB transmigration by approximately three folds compared to the plain drug without compromising the BBB integrity (p < 0.001) [104,105]. Pollock and co-workers encapsulated N-butyl deoxynojirimycin, an HIV gp120 folding inhibitor, into CD4+ surface functionalized pH sensitive liposomes to target PBMCs. The uptake of CD4+ surface functionalized pH sensitive liposomes improved by five folds in HIV infected cells compared to healthy cells. Incorporation of N-butyl deoxynojirimycin into 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine : cholesteryl hemisuccinate pH sensitive liposomes increased its IC50 by 10⁶ folds (p < 0.0001) [106].

Immunoliposomes are liposomal carriers whose surface has been functionalized with antibodies or antibody fragments which impart high selectivity towards target infected cells which express specific antigens [107]. Ramana and co-workers developed a stealth anti-CD4+ conjugated immunoliposomes to selectively target HIV infected cells incorporating drugs - nevirapine and saquinavir, with a view that nevirapine was effective in the early stage, while saquinavir was active in the advanced stage of the viral life cycle. Incorporation of drugs into anti-CD4+ conjugated liposomes reduced the p24 level by four folds than the free drugs in combination demonstrating the promising potential of immunoliposomes in antiviral drug delivery [108]. Gagneé and colleagues incorporated indinavir anti-HLA-DR immunoliposomes, to target HIV primary reservoirs in the lymph nodes in C3H mice. They found a 126 fold greater drug accumulation in the lymph nodes than the plain drug without compromising the antiviral activity of indinavir [109]. Ludewig and colleagues investigated the immunogenic potential of liposomes loaded with antigenic peptides obtained from the lymphocytic choriomeningitis virus. They observed strong intradermal immune response post encapsulation of antigenic peptides within liposomes. Liposomes were found to form antigen depots which mediated sustained stimulation of dendritic cells. Furthermore, the immunogenicity was improved by employing immunostimulatory oligonucleotides which successfully induced CTL responses which were similar to peptide-presenting DC indicating strong potential for antiviral and antitumor activity in the future [110].

Although liposomes are excellent carriers for antiviral agents and possess so many attributes in targeting and crossing the physiological barriers, they suffer from certain drawbacks like reduced physicochemical stability, lability towards oxidative reactions, cost, etc. which restricts their use [111]. In the purview of these drawbacks, there is a need to develop a vesicular system with the attributes of liposomes concurrently overcoming its limitations. This was achieved with the utilization of niosomes. Similar to liposomes, niosomes are sub-micron sized vesicular structures where the phospholipid is replaced with a non-ionic surfactant to overcome its stability challenges [112]. Niosomes have the ability to encapsulate a plethora of agents including hydrophilic and hydrophobic drugs. Akhter and colleagues loaded Ganciclovir into niosomes to improve its oral bioavailability. Ganciclovir, a BCS class III drug having good aqueous solubility and poor permeability characteristics. Encapsulation into niosomes increased its oral absorption and led to a 5-fold amelioration in bioavailability in comparison with the marketed tablet [113]. However, antiviral activity studies were not performed to judge its efficacy against free drug. Not much attention has been paid to niosomes for antiviral therapy. This could open new opportunities for the researchers to develop cationic, pH sensitive and immune responsive niosomes for advancing the current antiviral therapy.

6.1.2. Lipid based nanoparticle systems

Lipid nanoparticles are colloidal dispersion which comprise of a solid lipid matrix where the drug is incorporated into or adsorbed over the matrix crevices. The lipids used are physiological fatty acids and triglycerides which are solid at room temperature which ensures good biocompatibility. Since the lipids are physiologically derived, most of them possess generally regarded as safe status. They were invented independently by J.H. Lucks and R.H. Muller in the year 1993 [114]. Solid lipid nanoparticles (SLN) have been widely explored and have been successful in traversing the major physiological barriers and delivering the active ingredient at the bioactive site. SLN protect the active ingredient from pre-systemic metabolism and punitive conditions of the gastro-intestinal tract thereby preventing its physiologic and enzymatic degradation. They show a controlled biphasic release profile which includes an preliminary burst release along with a sustained zero order release. This could be beneficial in achieving an immediate onset proceeded by a sustained action duration which is desirable in antiviral agents with reduced half-life [115]. Xing-Guo et al. developed adefovir dipivoxil loaded SLN which demonstrated time and dose dependent reduction in hepatitis B surface antigen and DNA levels and demonstrated enhanced antiviral activity compared to plain drug [116]. Li et al. incorporated Yak alpha interferon into the solid lipid matrix of SLN and demonstrated an in vitro biphasic controlled release till 16 days. Yak interferon loaded SLN showed prominent antiviral activity in Madin Darby bovine kidney cells infested with vesicular stomatitis virus with reduced systemic toxicity [117]. Chattopadhyay and co-workers loaded Atazanavir, a protease inhibitor for enhancing its accumulation into the brain. They demonstrated a three-fold improvement in brain deposition of [³H]-Atazanavir loaded SLN compared to plain drug within 1 h which was further confirmed by fluorescence microscopy. Additionally, both Atazanavir and Rhodamine 123 are substrates of glycoprotein efflux transporters which prevent their accumulation into the BBB. Their incorporation into the lipidic matrix of SLN successfully circumvented the efflux transporters and increased accumulation of the substrates across the BBB (p < 0.05) [118]. Mishra and colleagues synthesized mannosylated cationic SLN to investigate their efficiency as a carrier for HBV vaccine through the subcutaneous route. Mannosylated SLN exhibited greater cellular uptake, reduced cytotoxicity and superior induction of TH1 helper T cells. They also demonstrated greater immunization potential by generating pronounced and sustained antibody titer as a prospective approach towards vaccine delivery [119]. Choi and colleagues investigated the formation of a complex between Indivavir lipid nanoparticles with Tenofovir and Calcein. They observed a pH responsive release behavior demonstrated by the two drugs. In a previous study, HIV infected macaques were administered these lipid drug complexes via subcutaneous route which resulted in localized accumulation of the drugs in the lymph nodes. Indivavir levels were found to be ranging between 2.5–22.7-fold higher than plain drug itself. Additionally, viral RNA titer levels diminished substantially as compared to the CD4 T-cell levels which increased consecutively (p < 0.05) [120]. Despite such advantages imparted by SLN, they suffer from certain severe drawbacks like drug expulsion due to polymorphic changes in the lipid matrix, gelling tendency, stability issues, limited drug loading capacity, etc. These drawbacks can severely diminish their efficacy. In order to overcome these obstacles, nanostructured lipid carriers (NLC), came into the limelight. NLC are second generation SLN which differ from them only by substituting a portion of solid lipid with a liquid lipid. The existence of liquid lipid imparts stability, enhanced drug loading, reduces drug leakage and polymorphic changes associated with SLN [121–123]. The presence of liquid state lipid reduces the crystallinity of solid matrix during storage and release the drug by diffusion and/or erosion mechanisms. Based on the method of preparation, drug moiety could be distributed within the core, outer layer or homogenously throughout the matrix similar to SLN. Kasongo et al. loaded Didanosine into NLC for the management of AIDS dementia complex. A reduction in the degree of crystallinity was recorded through differential scanning calorimetry studies when compared to the formulations in which liquid lipid was lacking. Additionally, wide angle X-ray scattering revealed a β-modified form which demonstrated that Didanosine was molecularly dispersed within the lipid matrix. Two dimensional poly acryl amide gel electrophoresis studies revealed potential surface adsorption interactions between NLC and Apolipoprotein E which indicated its foreseeable use in brain targeting for neuro AIDS and related disorders [124,125]. Beloqui and colleagues produced Saquinavir loaded NLC which enhanced the transport of Saquinavir by 3.5 folds (p < 0.001) across Caca-2 cell lines and helped to circumvent the glycoprotein efflux which was prominent in plain drug. Since antiviral activity was not performed their potential to inhibit the viral titer remains questionable and further studied are warranted [126]. Endsley and Ho conjugated two CD4+ binding peptides (CD4-BP2 and CD4-BP4) to lipid nanoparticles and investigated its targeting efficiency against HIV-2 infected host cells. They found substantial drug accumulation in the lymphoid tissue with enhanced antiviral activity for CD4-BP4 compared to CD4-BP2 (p < 0.05) [127].

6.1.3. Emulsifying delivery systems

Nanoemulsions are bi- or multi-phasic thermodynamically unstable systems comprising oil, surfactants, cosurfactants and water, with a droplet size ranging between 50–500 nm although they may appear as a single phase [128]. The oils used in nanoemulsions may be physiologically derived triglycerides, edible oils like soyabean oil, peanut oil, etc. hence they are generally regarded as safe [129]. Nanoemulsions were extensively employed to enhance the solubility and dissolution rate of hydrophobic agents thereby enhancing the absorption and bioavailability through lymphatic uptake [130,131]. Similar to other nanoformulations, nanoemulsions also undergo rapid reticulo-endothelial system uptake. In case of viral infections like HIV, which form a depot within the macrophages, the inherent selective targeting is achievable by nanoemulsion systems by RES uptake. In order to harness the uptake attributes, cationic lipids like stearylamine and cationic polymers like chitosan have been used to impart cationic surface charge with attracts the macrophages to the carrier surface. Donalisio and colleagues studied the role of cationic Acyclovir loaded chitosan nanospheres formulated using W/O nanoemulsion as a template on skin permeation and antiviral activity against topical Herpes virus infections. In vitro skin permeation studies revealed a permeation enhancement of six folds compared to plain drug commercial cream within 24 h. Acyclovir loaded chitosan nanospheres showed 11- and 16-folds greater activity to counter HSV-1 and HSV-2 respectively. Fluorescent microscopy studies confirmed improved internalization of the loaded chitosan nanospheres which could be responsible for its enhanced antiviral activity (Figure 7) [132].

Figure 7.

Acyclovir containing chitosan nanospheres from nanoemulsion template for treating Herpes infection A) TEM of Acyclovir-loaded chitosan nanoparticles, B) Anti-viral activity of plain drug, drug loaded nanospheres, and blank nanospheres against HSV-1, C) Anti-viral activity of plain drug, drug loaded nanospheres and blank nanospheres against HSV-2, D) Skin permeation from the acyclovir containing chitosan nanosphere gel and commercial cream, and E) Confocal laser scanning microscopy and phase contrast images for studying the cellular uptake and internalization of fluorescent nanospheres across Vero cells. Reprinted from Donalisio et al. [132] licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/legalcode) Copyright^© 2018. Manuela Donalisio, Federica Leone, Andrea Civra, Rita Spagnolo, Ozgen Ozer, David Lembo and Roberta Cavalli. Published by MDPI.

The nanometric droplet size causes a large increase in surface free energy, surface area and Brownian motion which prevents its sedimentation under the influence of gravity. Nanoemulsions have been recognized for topical delivery of moieties; owing to the extraction of lipids from the stratum corneum and inducing keratin denaturation for enhanced penetration across the skin layers [133]. Measso do Bonfim and co-workers used curcumin as a photosensitizer in a clinical study and explored its potential against different variants of HPV in vulvar intraepithelial neoplasia by incorporating Curcumin into nanoemulsion. Curcumin loaded nanoemulsions were found to have greater biocompatibility and caused epithelial cell fragmentation only after irradiation using photodynamic therapy (p < 0.01) [134]. Nabila and colleagues observed the antiviral activity of Curcumin against four serotypes of dengue virus. Reduced viral titer and better biocompatibility to A549 cells infected with dengue viral serotypes and uninfected A549 cells was achieved by incorporating Curcumin in nanoemulsion form [135]. Argenta and coworkers developed a topical nanoemulsion for the delivery of Coumestrol with unique muco-adhesive properties. In this approach, Coumestrol was incorporated into phospholipid nanoemulsions (DOPC and DSPC) which was further dispersed in a hydroxy ethyl cellulose based gel. They revealed that coumestrol was successfully incorporated into positively charged nanoemulsions which enhanced their cellular internalization and cell surface of Herpes infected Vero and GMK AH1 cell lines. Greater flux across the porcine esophageal mucosa was observed from DOPC based nanoemulsion in intact and injured mucosa. Further, decrease in IC₅₀ values suggested improved antiviral spectrum against HSV-1 and HSV-2 for Coumestrol loaded DOPC nanoemulsion (p < 0.05) [136]. Vyas and co-workers developed Saquinavir loaded nanoemulsion for ameliorating its bioavailability and brain distribution. The amount of Saquinavir in plasma was found to be three-fold greater post oral administration (p < 0.05). The concentration in the brain increased by five folds and area under the curve increased by three folds compared to plain drug suspension (p < 0.05). These results implied that the nanoemulsions prepared with polyunsaturated fatty acid oils could be effective for targeting viral sanctuary reservoirs at distal sites [137]. Donovan and co-workers optimized two nanoemulsion preparations (8N8 and 20N10) which consisted of soybean oil, tributyl phosphate and Triton X-100 respectively against murine influenza virus associated pneumonia. Animal subjects receiving two nanoemulsion mixtures (8N8 or 20N10) and a lethal dose of virus endured the challenge. The results demonstrated that greater than 80 percent of mock pre-treated animals (p < 0.005), whereas only 26 percent (8N8 at 1 %), 31.25 percent (20N10 at 1 %), and 37 percent (20N10 at 0.2%) of animals pre-treated with nanoemulsion were deceased from pneumonitis. These outcomes conclude strong in vivo prospective use of surfactant based nanoemulsions for the treating viral influenza infections [138]. Chepurnov et al. developed novel nanoemulsions of detergents and vegetable oil dispersed in water. The nanoemulsion termed ATB incapacitated the purified Ebola viral strains within 20 min, despite of its 100-fold dilution. This study emphasizes on the efficiency of nanoemulsions as disinfectants for Ebola virus associated infections [139]. Such surfactants could be used in synergism with antiviral drugs preventing the occurrence of resistance and rapidly reducing the viral titer.

Self-nanoemulsifying delivery systems (SNEDDS) have also gained tremendous importance in successful clinical translation of two HIV protease inhibitors - Ritonavir (Norvir^® by Abbott Laboratories) and Saquinavir (Fortovase^® by Roche Pharmaceuticals) [140,141]. SNEDDS are monophasic dispersions which form fine sub-micronized o/w emulsions when they come in contact with water. In terms of stability, SNEDDS are found to be superior compared to nanoemulsions since emulsification process takes place in situ post administration to the test subject [142]. Patel et al. prepared liquid and solid SNEDDS for Nelfinavir mesylate, a BCS Class IV drug for overcoming its reduced solubility and permeability challenges. Oral bioavailability of Nelfinavir in rabbits increased by approximately two folds. However, the antiviral activity still remains unanswered [143]. Obitte et al. evaluated the efficacy of SNEDDS to improve solubility, dissolution rate and bioavailability of CSIC (5-chloro-3-phenyl sulfonyl indole-2-carboxamide) to combat HIV infection. They reported that SNEDDS were successful in overcoming the solubility and bioavailability challenges of CSIC without compromising its antiviral efficacy [144]. However, their viral inhibitory potential has not been investigated. Additionally, use of cationic SNEDDS has not been yet explored by researchers in the antiviral field.

6.2. Polymer based systems

6.2.1. Polymeric nanoparticles

Polymeric nanoparticles are particles with a nanometric diameter range. They consist of a polymer matrix which is composed of synthetic/natural polymers. The therapeutic agent/s are encapsulated, adsorbed or covalently bound with the polymeric matrix [145]. Owing to their polymeric configuration, they possess better physiological stability compared to liposomes and other nanocarriers in systemic circulation as well as during long term storage [146]. The method of preparation impacts the distribution of therapeutic agent throughout the matrix, size, drug loading and release rates. Polymeric nanoparticles can entrap both hydrophilic and hydrophobic drugs. However, encapsulation of hydrophilic drugs is a challenge and is usually limited [147]. Although natural polymers like gelatin, dextran, chitosan, alginate, etc. are available, prolonged release and duration of action is hindered due to their susceptibility to various polysaccharidases present throughout the human body [148]. This throws light on the attributes of synthetic polymers such as poly lactic acid (PLA), poly lactide–poly glycolide copolymers, poly (lactic-co-glycolic acid) (PLGA), poly caprolactone (PCL), poly acrylate (PCA), etc. Destache and colleagues explored the release of antiviral drugs from PLGA nanoparticles in BALB/c mice. Intraperitoneal injection Ritonavir, Lopinavir and Efavirenz was compared with drugs incorporated into nanoparticles. Drug concentrations were found to be traceable till 72 h. In case of PLGA nanoparticles, mice possessed detectable Ritonavir, Lopinavir and Efavirenz amount till 28 days [149]. Yang et al. conjugated anti-CD4 antibody to the surface of saquinavir loaded PLGA nanoparticles for targeting CD4⁺ cells specifically to prevent HIV infection in vaginal tract. Significant differences were observed in the antibody conjugated group with two fold increase in targeting efficiency compared to its unconjugated counterpart (p < 0.05) [150]. Li and co-workers prepared a novel delivery strategy with the help of biocompatible Malemide-PEG-PLA to engineer the nanoparticles encapsulated with DAAN-14f, a reverse transcriptase inhibitor and surface functionalized with T1144 peptide, an HIV fusion inhibitor since both drugs suffered from pharmacokinetic inconsistencies. The hypothesis was that the T1144 peptide would attach to the exposed curvatures over pre-fusion intermediate thereby preventing viral fusion. The intracellular component, DAAN-14f, would internalize and inhibit viral reverse transcriptase non-competitively, thereby blocking viral DNA synthesis. In a nutshell, the combination of T1144 and DAAN-14f in a single nanoparticle at the surface and core respectively offers synergistic antiviral efficacy against a broad range of sensitive as well as resistant HIV-1 strains. Furthermore, the incorporation of the new cocktail-like delivery offers other advantages like superior intracellular uptake, prolonged release profile and prolonged systemic circulation in vivo (p < 0.001) [151]. Roberta Cavalli and co-workers evaluated the antiviral potential of Acyclovir by its incorporation in beta cyclodextrin-poly (4-acryloyl morpholine) conjugate nanoparticles to counter two clinical strains of HSV-1. They demonstrated that the conjugate nanoparticles to be far superior compared to plain drug and a soluble beta cyclodextrin-PACM complex. Antiviral potency against HSV-1 BGM increased by three folds while against HSV-1 MRC increased by four folds compared to plain drug. Fluorescent microscopy of nanoparticles demonstrated enhanced cellular internalization and localization in the perinuclear compartment. Quantitative uptake by Vero cells indicated a three-fold greater uptake compared to plain drug. However, without in vivo experimentation, the efficacy of the conjugate nanoparticles remains unanswered [152].

Garzón et al. used the proton sponge effect for preparing PEI-gp 120 complex to induce the interferon -γ secreting CD8+ T cells and neutralizing antibodies. The PEI-gp 120 complex elicited a robust immune reaction to counter the virus and proposed this strategy to be effective in development of vaccines in the future [153]. A study was conducted by Kuo and Su to investigate the traversion of Stavudine, Delaviridine and Saquinavir through the in vitro BBB model using polybutyl cyanoacrylate, methyl methacrylate sulfopropyl methacrylate and SLN. The results displayed about 12–16 fold increase in permeability with polybutyl cyanoacrylate, 3–7 fold increase with methyl methacrylate sulfopropyl methacrylate and 4–11 fold with SLN. Interestingly, the order of BBB permeability for Delaviridine and Saquinavir was PBCA > SLN > MMA-SPM while for Stavudine, it was PBCA > MMASPM > SLN. An inverse relationship was observed between particle size and permeability coefficient attributed to its reduced mass transfer across the monolayer [154]. Jack Hu and co-workers used PEG-PLGA diblock copolymers and independently loaded Diphyllin and Bafilomycin into nanoparticles with an entrapment efficiency of 42% and 100% individually. The prepared nanoparticles displayed a prolonged release beyond 72 h in vitro and mediated intra-cellular localization in MH-S and ARPE-19 cell lines. Compared with plain drugs, the nanoparticulate system demonstrated reduced cytotoxicity and superior viral inhibition. The efficacy of Diphyllin and Bafilomycin indicated 3- and 5-times greater escalation compared to plain drugs respectively. Diphyllin loaded PEG-PLGA diblock copolymeric nanoparticles exhibited broad range antiviral activity against two strains of influenza viral strains - H1N1 and H3N2. Diphyllin loaded nanoparticles were found to be safe in mice and decreased the loss in weight and lung viral titre post H1N1 viral infection. The nanoparticles additionally enhanced the survival rate in mice after a lethal challenge by 33 percent [155]. Recently, Pedroso-Santana and co-workers formulated the alpha interferon loaded chitosan nanoparticles stabilized by sodium tripolyphosphate. Cellular internalization was studied by bovine serum albumin fluorescein isothiocyanate system. Post 20 h of incubation, the nanoparticles were localized within the cytoplasm. Prolonged interferon release was recorded till 90 h in vitro. Cytotoxicity study demonstrated the safety of the formulation, Chitosan nanoparticles loaded with interferon alpha displayed greater antiviral activity in vitro and sustained antiviral response in pigs through overexpression of the OAS2 and PKR proteins in vivo. This study showed the retention of porcine alpha interferon activity with a lasting response [156]. Jack Hu and co-workers prepared an innovative nanoformulation containing ATPase blocker, Diphyllin for ameliorating the viral inhibition to counter type II feline infectious peritonitis virus (FIPV). They revealed that Diphyllin inhibited endosomal acidification in fcwf-4 cell lines in a concentration dependent manner thereby altering the cellular susceptibility to FIPV preventing its viral replication. The nanoparticles consisted of a PEG-PLGA matrix which displayed superior inhibitory activity against FIPV. In vitro antibody based model demonstrated predominant antiviral activity without compromising the safety profile on intravenous injection (Figure 8) [157].

Figure 8.

A) Schematic representation of mechanism of Diphyllin and Diphyllin loaded nanoparticles against FIPV infection, B) Particle size distribution of Diphyllin loaded nanoparticles, C) Transmission electron microscopic image of prepared nanoparticles, D) Time dependent assays of diphyllin treatment – 1 h before infection, during infection and 1h after infection, E) Cytopathic effect displayed by infected cells without Diphyllin or Bafilomycin, F) Plaque assays of viral infected cells, G) ) 0.2 μM of bafilomycin A1 or diphyllin (0.25, 0.5, 1, 2 μM) treatment to fcwf-4 cells 1 h prior to infection. Fluorescence images of expression of viral nucleocapsid and nucleus at 400X magnification, H) Dose-dependent cytotoxicity of plain drug and loaded nanoparticles I) IC50 of plain drug and loaded nanoparticles against FIPV (NTU 156) and J) IC50 of Diphyllin and loaded nanoparticles against FIPV (NTU 204). Reprinted from Hu et al. [157], licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/legalcode) Copyright ^© 2017. Che-Ming Jack Hu, Wei-Shan Chang, Zih-Syun Fang, You-Ting Chen, Wen-Lin Wang, Hsiao-Han Tsai, Ling-Ling Chueh, Tomomi Takano, Tsutomu Hohdatsu & Hui-Wen Chen. Published by Scientific Reports, Springer Nature.

6.2.2. Polymeric micelles

Polymeric micelles are sub-micron sized colloidal structures ranging between 10–100 nm. They consist of amphiphilic block copolymers, where individual monomer contains a hydrophobic and a hydrophilic sidechains [158]. The monomers self-assemble into a micelle once their concentration crosses the critical micelle concentration. The hydrophobic core incorporates the poorly aqueous soluble drug whereas the hydrophilic shell reduces the surface free energy, stabilizes the system and protects the drug from untoward interactions with enzymes, plasma proteins and the RES system [159]. Chemical conjugation of micelle polymers with the drug could lead to enhanced drug loading and alter the premature drug release characteristics. Tethering specific ligands on the micellar surface imparts high specificity and targeting efficiency. Sik Ahn and co-workers synthesized a poly (L-lactic acid)-b-poly (ethylene glycol) (PLLA-b-PEG) copolymer functionalized with methyl-b-neuraminic acid, a sialic acid derivative and loaded amantadine within the co-polymer micelles. The functionalized micelles were hypothesized to bind selectively to hemagglutinin of influenza viruses, behaving as a decoy to trick the virus and release the drug post attachment. Enhanced antiviral activity along with an extended release profile was observed from the functionalized micelles [160]. Use of smart polymers as copolymers in the micellar composition enables stimuli responsive drug release has been widely explored. For instance, Sosnik et al. prepared Indinavir loaded muco-adhesive, thermo-sensitive, chitosan-g-poly (N-isopropyl acrylamide) polymeric micelle and reported 24 times greater water solubility. However, pharmacokinetics and antiviral activity were not performed in order to decipher its true efficacy [161]. Qian Li and coworkers prepared Lamivudine stearate loaded stearic acid-g-chitosan oligosaccharide micelles to impart pH dependent release and enhance its antiviral efficacy. The developed micelles showed improved entrapment, drug loading and pH responsive drug release. Reduced cytotoxicity, viral antigen expression and diminished DNA replication was seen in comparison with plain drug implying the utilization of pH sensitivity as an encouraging strategy in micellar antiviral drug delivery [162]. Du et al. prepared Adefovir lipid derivative nanoassemblies with cytochrome P450-stimulated release and galactose mediated hepatocyte targeting to the asialoglycoprotein receptors for site specific delivery to the liver. The nanoassemblies displayed prolonged circulation time, pronounced liver targeting and remarkably high antiviral activity against HBV in mice model [163]. Jiménez-Pardo et al. prepared an amphiphilic dendritic–linear–dendritic block copolymer, obtained from Pluronic F127® and 2,2- bis (hydroxy methyl) propionic acid dendrons and loaded Camptothecin to enhance solubility and drug transport across biological barriers. The dendrons promote the formation of covalent crosslinked micelles, a suitable carrier system for loading hydrophobic drugs. Cell uptake study demonstrated that the nanocarrier promotes the uptake and localization of the encapsulated cargo within the cell. Enhanced antiviral activity against HCV and reduced toxicity supports the suitability of micellar system for antiviral delivery [164]. Chiappetta and co-workers deciphered the relationship between reduced size of micelles and increased bioavailability. They evaluated the poly (ethylene oxide) - poly (propylene oxide) (PEO-PPOs) block copolymer micelles for treating pediatric and adult patients and reported an 8400-fold increase in water solubility of Efavirenz from 4 μg/mL to 34 mg/mL. Increase in bioavailability was achieved with decrease in particle size (p < 0.05). However, in vitro and in vivo antiviral efficacy was not performed to prove its effectiveness in therapy [165,166].

6.2.3. Dendrimers

Dendrimers are extremely branched, 3-D structures discovered by Vogtle [167]. The name ‘dendrimers’ which translates to be ‘tree-like’. Dendrimers are made of smaller units known as ‘dendrons’ [168]. As the functional groups attaching to the core increases, its 3-D configuration changes. The dendrimer converts into a bulkier or globular structure, which leads to modifications in solubility and reactivity of the end groups. The empty spaces within the dendrimer network expedite the loading of guest molecules [169]. The surface functional groups promote covalent as well as non-covalent interactions via electrostatic forces, Van der Waals forces or chemical conjugation. The preparation method includes the addition of block molecules to the initiator via convergent or divergent synthesis with the help of click or lego chemistry [170]. Dendrimers possess several attributes like enhanced cell uptake, prolonged circulation, improved stability, ease of surface functionalization, etc. which could prove to be beneficial in antiviral therapy [170]. Wang and co-workers evaluated the impact of generation number of poly (amido amine) (PAMAM) dendrimer on Tat protein/TAR RNA binding inhibition, which is responsible for transcription of HIV-1 proteins. Langmuir equation was used to decipher the binding with a hypothesis that the binding was a monolayer adsorption and the combination coefficient (K_D⁻¹) was estimated. They found that the binding of dendrimer generation showed that PAMAM G3, G4 and G5 could play an adjuvant role as inhibitors of HIV-1 transcription [171]. Dendrimers could sever virus–host cell interactions at the time of attachment owing to their ability to form stable complexes with viral proteins. Dendrimers possess uniform size distribution and multi-valent end groups which enable their interactions with multiple receptors at the same time which increases the avidity of dendrimer protein interactions. The surface of dendrimers is mainly functionalized with three moieties – carbohydrates, peptides and anionic functional group ligands. Dendrimers with anionic functionalities bind to the V3 loop of gp 120 protein and restrict the early levels of viral replication. Polypropylene imine (PPI) dendrimers linked with sulfated galactose residues and Poly-l-lysine dendrimers associated with sulphated galactose and cellobiose residues have shown enhanced antiviral activity [172]. Poly anionic carbosilane dendrimers (PCD) have shown promising activity against HIV and HSV-2 as topical microbicides. Poly-anionic carbosilane dendrimers inhibit viral entry and prevent transmission of infections [173]. The dendrimers have been reported to exhibit superior in vitro synergistic activity with antiviral drugs to counter HSV-2. The amalgamation of PCD with Tenofovir and Maraviroc demonstrated efficacy, dose reduction and minimize the occurrence of multi-drug resistant HIV mutants [174]. The multiple mechanisms underlying the action of polyanionic carbosilane dendrimers includes its interaction with viral gp120, CD4 and CCR5/CXCR4 receptors on the host cell. Lasala et al. developed a glycodendritic assembly, (BH30sucMan), which inhibited the contact between intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) and Ebola virus (EBOV) envelope at nanomolar concentrations which further prevents viral entry [175]. Dutta et al. functionalized PPI dendrimers with tuftsin, a tetrapeptide natural macrophage activator (Thr–Lys–Pro–Arg), for delivering Efavirenz. Tuftsin is a peptide which is released in the systemic circulation upon enzyme mediated IgG cleavage. Attaching tuftsin improved the drug loading from 0.65 ± 0.39 to 0.87 ± 0.53 /g of Tuftsin-PPI and entrapment efficiency from 37.4 ± 0.3% to 49.3% ± 0.33%. Drug release studies was found to be sustained for 6 days compared to the non-functionalized dendrimer which released the drug completely within 24 h. Cell cytoxicity assays demonstrated a substantial reduction in the cytotoxicity of the tuftsin-PPI in comparison with pristine PPI dendrimers (p < 0.01). Cellular internalization studies showed substantial increase in Tuftsin–PPI uptake by 34.5 and 19-fold within a short duration of 1 and 4 h. Additionally, HIV-infected cells displayed tremendous increment Tuftsin-PPI uptake. Encapsulation of Efavirenz within tuftsin–PPI improved the antiviral activity 42 times greater in comparison with the plain drug in vitro (p < 0.001) [176]. A study performed by García-Gallego and colleagues showed that metal complexes of copper, zinc, cobalt and nickel linked with sulfonated and carboxylated poly (propylene imine) dendrimers were functionalizable antiviral agents which could be potentially used to counter HIV-1 infections. Inhibitory activity against HEC-1A and VK-2 cell lines was obtained which restricted viral entry and prevented interaction with both CCR5 and CXCR4 proteins. Both preventive as well as therapeutic behavior was seen in peripheral blood mononuclear cells (PBMC), which concluded that metallo-dendrimers could potentially open new horizons for antiviral treatment [177]. In vitro antiviral activity of dendrimers has been widely explored against various viral infections like the influenza virus [178,179], respiratory syncytial virus (RSV) [180], simian humanized immunodeficiency virus (SHIV) [181], etc. Another well-known example is the anionic sulfonated dendrimer, SPL7013 known as VivaGel™ was synthesized by Star Pharma. Naphthalene disulfonate groups were functionalized on dendrimer surface with an attempt to produce an HIV and HSV inhibitor. With a 3% w/w concentration, the gel was safe for vaginal/rectal administration in macaques and prevented macaques to combat vaginal SHIV infection [182]. Phase I clinical trial proved the clinical safety of VivaGel™ in humans. Following the success of the first trial, four more trials to evaluate various considerations like safety, tolerability and pharmacokinetics were performed on different subjects. Presently, clinical trials of VivaGel™ have been completed successfully and it has found its way in the market designated as Betafem® BV Gel (UK), Betadine BVTM (Europe), Betadine™ BV Gel (Asia) and Fleurstat BV gel (Australia & New Zealand). Recently, Kandeel and co-workers synthesized three varieties of different generations of polyanionic dendrimers differing in anionic group donors - carboxylate, hydroxyl and succinamic acid and polycationic dendrimers with a primary amine group to screen out the best possible dendrimer type against MERS-CoV. The highest inhibition of MERS-CoV was shown by sodium carboxylate (generation - 1.5) (40.5%). On the contrary, cationic dendrimers were found to be cytotoxic to Vero cells. They concluded the potential use of polyanionic dendrimers coupled with antiviral drugs could open new gateways for enhancing the current antiviral delivery against a broad spectrum of viruses [183].

6.3. Elemental delivery systems

Today, elemental based delivery systems have gained tremendous attention owing to their easy synthesis processes and various applications in the biomedical field. These systems require the application of metals, metal oxides or non-metals in the nanometric dimension [184]. These entities have comprehensive applications in therapeutic as well as diagnostic fields. They have multi-fold applications in the pharmaceutical field and possess unique properties like antiviral, antimicrobial, anti-inflammatory, wound healing, anticancer, etc. The general mechanism of action of inorganic nanoparticles as a part of effective antiviral therapy includes – attachment to viral cell coat and preventing viral entry, produce ROS and cause oxidative stress in the infected cells thereby ceasing viral transcription and replication processes [185]. Gold nanoparticles block the viral gp120 attachment with CD4+ cells to prohibit viral entry while silver nanoparticles hinder CD4+ dependant virion attachment and contact with gp120 in the free and infected host cell. The gold nanoparticles inhibit viral replication in dsRNA viruses [186,187]. Copper nanoparticles block attachment and viral entry, rescind the viral genome and destroy the viral capsid [188]. Zinc nanoparticles inhibit the viral DNA polymerase and arrest viral replication [189]. Iron nanoparticles favourably attach to the virus and prevent it from attaching to the healthy host cells and prevents transmission [190]. Selenium nanoparticles safeguard the host cells from apoptosis triggered by the viral infection during the lytic cycle [191,192]. Metals have a tendency to oxidise and lose electrons which provides a positive charge on their surface. This positive charge allows them to electrostatically interact with the anionic cell membrane. Once the nanoparticles are attached to the cell surface, their uptake occurs either by ion channels, phagocytosis or clathrin mediated endocytosis. Post internalization, the nanoparticles are let out by the endosomal membrane lysis [193]. This is beneficial for intracellular delivery of drugs where they can be conjugated with metals with the help of linkers. Lin et al. loaded Zanamivir with silver nanoparticles to impede H1N1 influenza viral infection. The viral inhibitory mechanism was studied and they found that the surface decoration of Zanamivir over Silver nanoparticles independently exerted antiviral activity by inhibiting neuraminidase enzyme along with the generation of excess ROS, DNA fragmentation, condensation of chromatin network and caspase-3 up-regulation. Additionally, they revealed that p38 and p53 signalling cascades were connected with the downregulated ROS and arbitrated cell death in MDCK cells infected with H1N1 viral infection (p < 0.01) [194]. Lee and colleagues synthesized a hyaluronic acid-gold nanoparticle complexed with PEGylated interferon-α for hepatitis C virus targeting. Compared to the conventional therapy of hepatitis C, PEGylated interferon-α hyaluronic acid functionalized gold nanoparticles exhibited enhanced antiviral activity and were traceable even after a week of injection in mice liver in contrast to the non-PEGylated interferon. This work describes the use of gold nanoparticle-based complex as an effective anti-Hepatitis C therapy alternative and superior to any marketed available therapy [195]. Halder and co-workers synthesized mono-dispersed spherical gold-nanoparticles against HSV. With the use of ultrasonic energy, gallic acid was attached onto the gold nanoparticles. The cytotoxicity assay demonstrated that gold nanoparticles were relatively inert and proved to be beneficial for therapeutic implications. Plaque assay demonstrated that the gallic acid decorated gold nanoparticles possessed good HSV inhibitory capacity. Compared to plain Acyclovir, the decorated gold nanoparticles failed to produce resistant strains and parade excellent virucidal characteristics by impeding virus binding to Vero cells and preventing its transmission [196]. Tavakoli and Hashemzadeh synthesized copper oxide nanoparticles and evaluated its antiviral activity against HSV-1. At its maximum safe concentration (100 ppm), copper nanoparticles led to a 2.8 log₁₀ TCID₅₀ decrease in infectious virus titre and caused oxidative damage to the viral genome [197]. Ghaffari and co-workers prepared zinc oxide nanoparticles against H1N1 influenza virus infection and revealed that PEGylated and non-PEGylated zinc oxide nanoparticles at their maximum safe concentrations led to 2.8 and 1.2 log₁₀ TCID₅₀ decline in viral titre compared to control (p < 0.0001) with an infection inhibition rate of 94.6% and 52.2%, respectively [198]. Titanium dioxide nanoparticles prepared by Mazurkova et al. destroyed the influenza virus H3N2 strain on direct contact between the nanoparticles and virus particles [199]. Yang and co-workers developed a beta-cyclodextrin decorated graphene oxide composite loaded with Curcumin to investigate the anti-viral efficacy of the prepared composite. A significant anti-viral activity (p < 0.05) was observed along with enhanced drug loading onto the composite with high biocompatibility. Graphene oxide nanoparticles loaded with Curcumin displayed tremendous inhibition of respiratory syncytial virus with substantial biocompatibility. Graphene oxide inactivates the virus by preventing its attachment to host cells [200]. Carbon nanotubes are well recognized for their unique applications in drug delivery, gene delivery, bio-sensing and bio-imaging fields. In the field of antiviral research, they are a rapidly emerging platform with a combination of the aforementioned applications. Carbon nanotubes have the potential to act as filters to prevent bacterial as well as viral entry. Vecitis and co-workers demonstrated the efficacy of carbon nanotubes as efficient filters against Escherichia Coli and bacteriophage MS2 virus. When the applied potential ranged within 2–3 V, the nanotube filter reduced the bioburden of bacteria and virus below the detection levels with an efficiency of greater than 75% and 99.6% for bacteria and viruses respectively [201]. Similar studies have been testified by Zoltán Németh et al. [202] and Dong and colleagues [203]. Zhao and co-workers observed the role of carbon nanotubes against infectious spleen and kidney necrosis virus which cause significant mortality of mandarin fish in China. Post seven days of infection, the mortality was 14.75% and the infection rate was 26.55% for plain Ganciclovir (40 mg/L), while Ganciclovir loaded single walled carbon nanotubes (40 mg/L) displayed mortality of 17.5% and infection rate of 5.49% compared to control, where mortality was 88.75% and infection rate was 100%. Additionally, sustained action of Ganciclovir was observed when it was incorporated in carbon nanotubes [204]. Carbon dots have demonstrated tremendous potential in the biomedical and biotechnology owing to their biological and environmental inertness with desired attributes of quantum dots. Du and co-workers explored the influence of carbon dots on viral replication by pseudorabies virus and porcine reproductive and respiratory syndrome virus portraying as DNA and RNA viral models. Substantial inhibition of viral multiplication via interferon-α (IFN-α) induction along with the IFN-stimulating gene expression was observed [205]. Lannazzo and co-workers reconnoitered the viral inhibitory probability of graphene quantum dots prepared from multi-walled carbon nanotubes against HIV. The sustained antiviral effect was observed compared to reverse transcriptase inhibitors. Reverse transcriptase inhibitors conjugated CHI499 graphene quantum dots depicted an IC₅₀ of 0.09 μg/mL and an EC₅₀ of 0.066 μg/mL supporting tremendous potential of graphene quantum dots as antiviral delivery systems [206]. Dong and co-workers assessed the antiviral activity of carbon dots against human norovirus like particles surface modified with 2,2′-(ethylenedioxy) bis (ethylamine) (EDA) and 3-ethoxypropylamine (EPA) respectively. At 5 μg/mL concentration, EDA carbon dots attained complete inhibition while EPA carbon dots attained 85–99% inhibition. At lower concentration level, positively charged dots demonstrated greater inhibitory response (~82%) compared with non-charged EPA dots implying the role of cationic surface charge against anionic virus like particles. The antiviral activity against noroviruses was mainly due to the inhibition of virus like particles binding to HBGA receptors preventing spread of viral cells to other healthy cells (Figure 9) [207]. Table 2 indicates the summary of recent advances in nanomedicine approaches for combating viral infections.

Figure 9.

1) Structural representation of carbon dot, 2) Inhibition rate of EDA- and EPA-carbon dots against GI.1 and GII.4 VLP (A) EDA-Carbon dots (2–32 μg/mL) and (B) EPA-Carbon dots (8–64 μg/mL), 3) Inhibitory potential of carbon dots on norovirus VLP binding to A, B, O, salivary HBGA receptors. (A) Type A HBGA; (B) Type B HBGA; (C) Type O HBGA, 4) Top - SDS-PAGE and Bottom - Western blot of GI.1 (A) and GII.4 VLP (B) Lane 1 - control; Lane 2, 3 – Treatment with 20 ppm; Lane 4, 5 - Treatment with 60 ppm of EDA- and EPA-carbon dots. TEM images of GI.1 and GII.4 VLP - (A, D) Untreated, (B, E) EDA- and (C, F) EPA- carbon dots. Reprinted from Dong et al. [207] licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/legalcode) Copyright^© 2017. Xiuli Dong, Marsha M. Moyer, Fan Yang, Ya-Ping Sun & Liju Yang. Published by Scientific Reports, Springer Nature.

Table 2.

Summary of recent advances in nanomedicine based approaches against viral infections

Nanomedicine based approaches	Anti-viral agent	Viral strain	Outcome	Reference
Immunoliposomes	Suberoylanilide hydroxamic acid and bryostatin	HIV-1	Immunoliposomes functionalized with CD4+ antibody was specifically taken up by latent HIV-1 infected Jurkat 10.6 cells. Synergistic effect in reactivation of latent HIV was observed.	[208]
Solid lipid nanoparticles	Acyclovir	Herpes Simplex virus	Acyclovir loaded SLN displayed significant enhancement in bioavailability and mean residence time compared to plain drug. Individual dose of SLN was equivalent to 400 mg of plain drug given for 5 days in mouse model.	[209]
Nanostructured lipid carriers	Cytosineguanine repetitions	HIV p24 antigen	p24 surface modified cationic NLC displayed substantial enhancement in immune response, T-cell activation and antibody production in mice and primates.	[210]
Nanoemulsions	Curcumin	HPV-16 E6 strain	Photodynamic therapy along with Curcumin nanoemulsion caused 90% cell death with Journal Pre-proof high caspase 3 and caspase 7 activity in A431 cells infected with HPV-16 E6 strain.	[134]
	Curcumin	DENV-1 JMB-034, DENV-2 SUB-011, DENV-3 SUB-006, and DENV-4 SUB-007 viral serotypes	Curcumin loaded nanoemulsion with mean particle size of 40.85 ± 0.919 nm inhibited growth of four dengue viral serotypes.	[135]
Polymeric nanoparticles	Cytosineguanine repetitions	Avian influenza virus H6N1 strain	Hollow PLGA nanoparticles induced dendritic cell maturation, caused expression of IL-1β, IL-6, IL-12 and IFN-γ and proved potent immuno-stimulation against viral challenge.	[211]
	Curcumin	Hepatitis C genotype 4a viral strain	In silico docking studies showed good binding of curcumin and curcumin chitosan nanocomposite with NS3 protease, NS5A polymerase and NS5B polymerase. Curcumin chitosan nanocomposite particles demonstrated 100% viral entry inhibition via caspase mediated mechanisms.	[212]
	Isoprinosine	Betanoda virus	Enhanced anti-viral activity in zebra fish model along with increased expression of IL-1β, IFN-γ and TNF-α was shown by Isoprinosine. Improved internalization inside the SSN-1 cells along with reduction in larvae mortality from 96.7% for control and 58% for plain drug to 39.3% was observed within a week of infection.	[213]
Dendrimers	-	MERS-CoV virus	Dendrimers functionalized with different groups were evaluated for their antiviral activity. Hydroxyl group containing dendrimers displayed 17.36% to 29.75% reduction in MERS-CoV plaque formation, while about 40.5% inhibition was shown by sodium carboxylate group which could be further used as antiviral drug carriers in a synergistic manner.	[183]
Metallic nanoparticles	-	Influenza viral strains	Iron oxide nanoparticles with particle size range of 10–15 nm showed plaque inhibition against pandemic influenza strain A/H1N1/66/PR8-H1N1 in a dose and time dependent manner. 50% viability was recorded at 4.25 pg. Plaque inhibition for PR8-H1N1 strain in MA104 cells was found to be 01 picogram after 72 h. An eight fold reduction in virus population revealed significant antiviral potential of iron oxide nanoparticles.	[214]
PEGylated cationic liposomes	Immunostimulating RNA	Influenza viral strains	PEGylated cationic liposomes prevented the emergence of viral encephalocarditis in L929 cells. Additionally, significant enhancement in interferon induction was observed along with substantial reduction in viral bio-burden in the lungs.	[215]
Silver nanoparticles	-	Respiratory Synctial virus	Silver nanoparticles demonstrated marked reduction in respiratory syncytial virus population in vitro along with reduction in pro-inflammatory cytokines.	[216]
Carbon dots	Glycyrrhizic acid	Psuedo rabies virus and porcine epidemic diarrhoea virus	Carbon dots inhibited porcine reproductive and respiratory syndrome virus, boosted immunity and minimized the reactive oxygen species production. Additionally, dots also suppressed pseudorabies virus and porcine epidemic diarrhoea virus indicating broad spectrum of activity.	[217]
Carbon nanotubes	Pristine and metal decorated carbon nanotubes	SARS-CoV-2 virus	Out of all the metal functionalized nanotubes studied, rhodium and ruthenium single walled carbon nanotubes showed excellent hydrogen peroxide adsorption while, platinum and copper decorated carbon	[218]

7. Leveraging the intrinsic immunogenicity of nanoformulations

In recent years, noteworthy attention is being paid towards enhancing the antiviral immune response by manipulating the intrinsic immunogenicity of nanoparticulate carriers as an adjuvant in generating strong immune response. Greater degree of surface charge imparted by lipids and/or polymers, stabilizers or charge inducers have been widely employed to improve the intrinsic immunogenicity of the nanocarriers [219–222]. Courant and co-workers investigated the immunogenic characteristics of NLC linked with ovalbumin to visualize the effect of size (80 nm against 120 nm) and surface charge (anionic against cationic). They showed that 80 nm anionic lipid based NLC were superior antigenic carriers for provoking greater humoral and cellular response with elevated levels of gamma interferon (IFN-γ) [223]. Andorko and colleagues employed degradable poly (β-amino esters) to reconnoiter the intrinsic immunogenicity with respect to polymer form and degradation. The prepared particles abridged by electrostatic contact strongly stimulated dendritic cells, followed by antigen presentation and improve T cell propagation. The particles also enhanced the number of immune cells in lymph nodes in mice indicating their potential as carriers in antiviral delivery in the foreseeable future [224]. Elemental delivery systems have been well recognized to possess strong intrinsic immunogenicity potential which could be leveraged to generate superior antiviral immune response. Zhou et al. explored the influence of particle size on spherical gold nanoparticles (15–80 nm) for co-delivery of CpG oligonucleotides with ovalbumin towards antigen presentation, dendritic cell stimulation and T helper cell levels. They found the nanococktail particles of 60 and 80 nm substantially enhanced dendritic cell stimulation and strong immunogenic response compared to other particle sizes owing to enhanced antigen cargo withholding capacity [225]. Borrego and co-wprkers established the antiviral activity of silver nanoparticles (Argovit™) against Rift Valley fever virus infected Vero cells along with type I interferon receptor deficient mice model. The ability of silver nanoparticles to inhibit the infection was limited, however a reduction in the antibody titer was observed in vitro and in vivo indicating its potential as an adjuvant with intrinsic antiviral activity [226]. Figure 10 depicts the stages of immune recognition and response generation against the immunogenicity of the nanocarrier.

Figure 10.

Stages of immune recognition to generation of immune response against immunogenicity of nanocarrier.

8. Delivery of Interference RNA therapeutics via nanomedicine

Interference RNA (RNAi) based therapeutics were initially discovered in Caenorabditis elegans by Mello and Fire in 1998 [227]. RNAi has been extensively used in gene silencing and has attracted tremendous attention of researchers today. The RNAi pathway act as a silencing technology to selectively subdue expression of a proposed genomic section. This is achieved by slicing dsRNA and converting it into siRNA which consist of 21–25 base pairs. An siRNA strand is then assimilated into an RNA induced silencing complex (RISC) which destroys the target mRNA thereby arresting genomic expression. For small interfering RNA (siRNA), as the RISC binds to the siRNA strands, the strands separate and the antisense siRNA dictates RISC towards mRNA degradation [228,229]. Short hairpin RNA (shRNA) differ from small interfering RNA in their expression which requires bacterial/viral vectors and possess a hair pin structure. They can be delivered either by plasmid DNA or viral vectors. They are acknowledged by an endogenous dicer enzyme which then converts shRNA to siRNA for mRNA binding and degradation [230]. Aptamer microRNA (miRNA) can also be used for gene silencing via mRNA downregulation. The transcription of chromosomal miRNA to primary miRNAs by RNase III endonuclease Drosha and dsRNA-binding protein Pasha, creating a 60–70 nucleotide loop precursor miRNA. The precursor miRNA is further sliced by RNase III endonuclease Dicer to a 22 nucleotide double stranded miRNA which finally causes gene silencing. For mRNA degradation, the mechanism is similar to siRNA-RISC complex [231]. Delivery of siRNA poses certain challenges to researchers with regard to its stability in body fluids, RNases present in the serum, cellular internalization and intracellular degradation limiting their use. These issues can be overcome with the help of nanomedicine based approaches which prevent its degradation and promote intracellular delivery. Li and co-workers used siRNA as a novel therapeutic alternative against enterovirus 71 which causes hand, foot, and mouth disease. The major drawback of siRNA, its inability to cross biological barriers was overcome with the help of silver nanoparticles, surface functionalized with polyethylenimine (PEI) and antiviral siRNA. They confirmed that PEI and siRNA-modified silver nanoparticles showed inhibition to combat enterovirus infection and reduced toxicity to Vero cells. The mechanistic insights conveyed that this strategy blocked EV71 from invading host cells and diminish host DNA breakup, chromatin condensation and activation of caspase-3. The accretion of reactive oxygen species (ROS) by the EV71 virus and activation of AKT and p53 was inhibited concluding that PEI coated siRNA modified silver nanoparticles as a promising strategy against enterovirus infection [232]. Jamali and colleagues incorporated siRNA into chitosan nanoparticles owing to the ionic interaction of cationic chitosan and anionic host cell membrane. Intracellular uptake of labelled siRNA into Vero cells was envisioned by fluorescence microscopy. Nanoparticle assisted knockdown of enriched green fluorescent protein was enumerated by flow cytometry. The chitosan/siRNA nanoparticles were easily internalized by Vero cells leading to inhibition of replication of influenza virus. Furthermore, nasal delivery of siRNA by chitosan nanoparticles safeguarded BALB/c mice from a lethal influenza challenge [233]. Intravaginal administration of siRNA by incorporation into poly (lactic-co-glycolic acid) (PLGA) nanoparticles developed against HSV-2 infection by nectin knockdown were reported by Steinbach and co-workers. Mice infected with a lethal dose of HSV-2 survived greater than 28 days on treatment with PLGA nanoparticles compared to control which survived only up to 9 days [234]. Dual antibody functionalized small interfering RNA loaded chitosan nanoparticles were prepared to traverse the BBB with a view to treat HIV infected brain astrocytes. Gu et al. used transferrin and bradykinin B2 antibody to explicitly link to the transferrin and bradykinin B2 receptor and target siRNA athwart the BBB. Chitosan nanoparticles were chemically linked to dual antibodies which showed enhanced cellular internalization and gene silencing efficiency compared to plain and individual antibody linked chitosan nanoparticles [235]. Khantasup and colleagues described the use of siRNA loaded immunoliposomes against H5N1 influenza viral infection. The high specificity of siRNA viral nucleoprotein mRNA was utilized as the chief antiviral agent. The liposomes encapsulating the siRNA were functionalized with humanized single chain Fv antibody against avian influenza specific hemagglutinin protein. The cationic PEGylated immunoliposomes displayed binding specificity towards hemagglutinin Sf9 cells and showed improved transfection efficiency. The siRNA transfection efficiency was tremendously attenuated after pre-incubation of the HA target cells with an excess amount of free antibody. The gene silencing effect was marked up to 6 to 12 h after H5N1 virus infecting the Madin Darby Canine Kidney cell lines compared to non-functionalized liposomes (p < 0.05). This study could demonstrate the future of siRNA delivery system for viral infections [236]. Some siRNA based products under clinical trials include ARB-001467 for Hepatitis B, Fluquit (STP 702) for H5N1 (avian flu), H1N1 (swine flu), H5N1 (influenza) and cervisil (STP909) for HPV16 and HPV18 [237,238]. Several reports regarding synergistic combinations of siRNA and small molecules delivered through nanocarriers have been published in recent years for various cancers and inflammatory diseases but not for viral diseases. Inquisitive researchers can focus their attention towards this avenue for advancing the current antiviral therapy. Table 3 describes RNAi used for various viral infections.

Table 3.

RNAi used for various viral diseases

Viral infection	Interference RNA	Outcome	Reference
DENV	NS5 siRNA	Inhibition of replication in cell lines	[239]
HCV	Internal ribosome entry site synthetic short hairpin RNA	Inhibition of viral HCV replication and infection	[240]
HIV	Anti-HIV Ribozyme	Safety in phase II clinical trials but failure in efficacy	[241]
HBV	HBV sh RNA	Reduced HBV titers, mRNA and DNA levels	[242]
HIV	Tat ribozyme	Safety in HIV patients in phase I trial.	[243]
Adeno virus	pTP primary miRNA	Decrease in viral burden.	[244]
Influenza	M950, NS570, NS595 and NS615 siRNA	Reduced nonstructural proteins type 1 and 2 expression and enhanced type I interferon expression by 50%. Combination siRNA treatment reduced 20.9% more viral burden compared to individual siRNA.	[245]
Chikungunya virus	E1 siRNA	Substantial decrease (99.6%) in viral titre in Vero cells.	[246]
HIV	Agosh mRNA	Protection against CCR5-tropic HIV-1 strains.	[247]

9. Nanotechnology meets vaccines

During the last decade, the research community has seen the potential of nanotechnology in various scientific fields. The inclusion of nanomedicine principles in vaccine development has attracted tremendous attention today. This includes the use of nanocarriers which act as an adjuvant to generate a strong cytotoxic T cell response against the antigen [248]. Nanocarriers linked with a viral component can be tuned with respect to their size, shape and surface charge to be phagocytosed by specific antigen presenting cells by recognition of the viral component and generate a strong immune response which can be remembered by the immune system for the next encounter [249]. The commercial development of two liposomal nanovaccines - Inflexal^® V and Epaxal^® against influenza and hepatitis infection have shined a bright light on the nanotechnology based vaccine development [250]. Even-Or and co-workers conveyed that polycationic sphingolipid N-palmitoyl D-erythro-sphingosyl-carbamoyl-spermine triacetate salt liposomes loaded with influenza hemagglutinin antigen, promoted strong humoral and cell immunity in various animal models and prevent influenza virus infection (p < 0.0001) [251]. Recently, in another study from the same group, the adaptor protein MyD88 was no longer required for immunization in TLR2- and TLR4 deficient mice concluding the efficacy of cationic liposomes as adjuvants in vaccine development [252]. Lin and colleagues reported an effective immunity enhancing capability by encapsulating avian toll like receptor-21 agonist CpG ODN 2007 into a poly (lactic-co-glycolic acid) (PLGA) hollow nanoparticle matrix. The results showed that CpG ODN 2007 loaded nanoparticles were phagocytosed efficiently and induce dendritic cell proliferation, maturation and upregulation of CD40, CD80 and CCR7. Inflammatory mediators like IL-1β, IL-6, IL-12 and IFN-γ, were also stimulated indicating the occurrence of both cellular and humoral immunity reactions [211]. Virus like particles is another domain where the particles constitute of one or few viral structural proteins to superficially mimic the virus [253]. These particles do not contain the entire viral genome but may contain structural proteins which represent the virus and could be sufficient in generating an immune response against the virus [254,255]. Recently, Lu et al. developed three virus like nanoparticles out of which only RQ3013-VLP formulated from a cocktail of mRNA from Spike, Membrane and Envelope proteins from SARS-CoV-2 elicited both humoral and cellular response with a high titre of neutralizing antibodies in mice [256]. Gregory and co-workers conjugated F1 antigen with 15 nm gold nanoparticles and investigated its immunogenicity against plain antigen. They found an enhanced Ig2a response (p < 0.01) in mice and improved immunity development [257]. Hence, the combination of nanotechnology and vaccines can prove to be a vital weapon against various newly emerging and old resistant viral infections. Table 4 indicates various clinical trials where nanotechnology has been used to deliver vaccines.

Table 4.

Clinical trials for Nanovaccine delivery

Formulation	Route of administration	Phase	Antigen	Virus	Clinical trial Identity number
Liposomes	Intramuscular	Phase 1	CN54gp140/MPLA	HIV	NCT03408262
	Intranasal	Phase 1/Phase 2	-	Influenza	NCT00197301
	Intramuscular	Phase 1	gp41 MPER-656	HIV	NCT03934541
	Intramuscular	Phase 2	VaxiSome™ Cholesterol CCS/C-adjuvant	Influenza	NCT00915187
	Intramuscular	Phase 1	Tetravalent Dengue Vaccine (TVDV)	Dengue fever	NCT01502358
	Intramuscular	Phase 1	FMP012 vaccine with AS01B adjuvant system	Malaria	NCT02174978
	Intradermal	Phase 2	Fluzone® vaccine with JVRS-100	Influenza	NCT00936468
Nanoparticles	Intramuscular	Phase 1	EBV gp350	Epstein Barr virus	NCT04645147
	Intravenous	Phase 2	RSV-F protein	Respiratory syncytial virus	NCT02247726
	Intramuscular	Phase 1	Ebola Virus Glycoprotein (GP) – Matrix M1™ adjuvant	Ebola	NCT02370589
	Intramuscular	Phase 3	SARS-CoV-2 Recombinant Spike Protein (SARS-CoV-2 rS) With Matrix-M1™ Adjuvant	COVID-19	NCT04611802
	Intramuscular	Phase 2	RSV-F protein	Respiratory syncytial virus	NCT01960686
	Intramuscular	Phase 1	RABV-G Protein	Rabies	NCT03713086
	Intramuscular	Phase 1	VAL-506440 mRNA	Influenza H10N8	NCT03076385
	Intramuscular	Phase 1	VAL-181388 mRNA	Chikungunya	NCT03325075
	Intramuscular	Phase 1 Phase 2	SARS-CoV-2 rS vaccine SARS-CoV-2 rS/Matrix-M Adjuvant	COVID-19	NCT04368988

10. Advancement in nanotechnology based vaccine delivery against COVID-19

U. S. Food and Drug Administration (FDA) has sanctioned the urgent use of three vaccines under Emergency Use Authorization (EUA) to combat COVID-19. These include Moderna vaccine, Pfizer-BioNTech and Jassen vaccine.

Moderna vaccine –

ModernaTX, Inc. mRNA vaccine given permission under Emergency Use Authorization (EUA) by FDA on December 18^th, 2020 for individuals who have completed 18 years of age or older. It is administered intramuscularly, twice within 2 months. Moderna vaccine is also based on mRNA technology, which encodes for the pre-fusion stabilized spike glycoprotein (S). Each dose accounts for 100 μg of nucleoside-modified mRNA, PEG 2000, lipids like dimyristoyl glycerol, cholesterol and 1,2-distearoyl-sn-glycero-3-phosphocholine, along with tromethamine, acetic acid, sodium acetate and sucrose [258].

Pfizer-BioNTech vaccine –

U. S. FDA has approved the urgent use of Pfizer-BioNTech vaccine under EUA on December 11^th, 2020 for COVID-19 for people who are 16 years and older. It is given as an intramuscular injection two times with 3 weeks interval. It is based on mRNA technology, where mRNA formulated in lipid particles which delivers it into host cells to express mRNA which encodes for viral Spike protein. Once produced, Spike protein triggers immune response which results in the production of antibodies. It encloses 30 μg nucleoside - altered messenger RNA, PEG 2000-N,N-ditetradecyl acetamide, lipids like 4-hydroxy butyl) azanediyl) bis (hexane-6,1-diyl) bis (2-hexyl decanoate), DSPC and cholesterol, sucrose and buffer salts and tonicity adjusters [259].

AstraZeneca, University of Oxford vaccine (Vaxzevria) and COVISHIELD –

Astra Zeneca and the University of Oxford developed Vaxzevria vaccine, which was given authorisation for emergency use by the UK Medicines and Healthcare products Regulatory Agency (MHRA) on December 30^th, 2020. Vaxzevria vaccine synthesized by Astra Zeneca and COVISHIELD prepared by Serum Institute of India and is sanctioned by the Drugs Controller General of India. Vaxzevria vaccine comprises of a recombinant chimpanzee adenoviral vector which encodes for Spike protein and it was produced using recombinant DNA technology in human embryonic kidney (HEK) 293 cells. It is given intramuscularly to adults aged 18 years and older two times with second dose incorporated between 4 and 12 weeks after the first dose. After administration, spike glycoprotein expresses locally stimulating neutralizing antibody and cellular immune response. It constitutes of 5 × 10¹⁰ virus particles along with buffer salts, hydroxypropyl β cyclodextrin, tween 80 and tonicity adjusters [260].

COVAXIN^® -

This vaccine manufactured by Bharath Biotech, in association with Indian Council of Medical Research (ICMR)-National Institute of Virology (NIV) is based on inactivated virus which is produced by using whole virion inactivated vero cell derived platform technology. COVAXIN^® is given authorization under emergency use by the Drugs Controller General of India on 3^rd January 2021. It is given intramuscularly, two doses with 4 weeks apart. When administered, immune cells distinguish the dead virus particles further prompting the immune system to generate antibodies [261].

Self amplifying RNA based vaccine –

Acuitas Therapeutics in alliance with Imperial College (London) synthesized a self-amplifying RNA vaccine and incorporated into lipidic nanoparticles condensed with the pre-fusion alleviated Spike protein. Humoral and cellular response was investigated against pseudo-type SARS-CoV-2 strain. Post 6 weeks, vigorous Spike protein antibodies were observed in animals in concentration-dependent pattern. At the lowermost dose, nanovaccine encouraged greater neutralizing antibody titers in mice in comparison with the recuperated patients as well as other vaccines. Superior neutralization capacity, cellular reactions and antibody titers suggested the use of lipid nanoformulation for vaccine delivery [262]. Interested readers can further find relevant information from an excellent review from Machhi and colleagues [263].

11. Hurdles to clinical translation of nanomedicine

Recent advances in nanomedicine have enabled the research community to deliver and target drugs to specific organs, tissues, cells and cell organelles overcoming various biological, pharmaceutical and pharmacokinetic barriers. The researchers have been successful in overcoming the limitations of the conventional therapy at the pre-clinical level, however majority of the publications fail to end up in a clinic. Although there are a large number of reports relating to the benefits offered by nanomedicine, the intricacy, differences and the lack of thorough protocol regarding experimentation and characterization have increased the gap for clinical translation of nanomedicine. Even though FDA has introduced guidelines like ‘Liposomal drug products’ and ‘Drug products, including biological products, that contain nanomaterials’, there still exists an unmet need in harmonizing the collaborative efforts required in clinical translation of nanotechnology based products [264,265].

Stability is a major concern for nanomedicine based products. In order to enhance the stability, attempts have been made to convert the product from liquid to solid powders by use of various methods like spray drying, freeze drying, precipitation, etc. [266]. However, vesicular formulations like liposomes, niosomes, etc. exhibit vesicular leakage and drug expulsion when the aqueous phase inside the bilayer membrane is removed by drying processes [267]. Rigorous studies must be performed to decipher the ideal storage conditions for nanotechnology based products to sustain them for an optimum duration. With rapid innovation in nanomedicine, the researchers are investigating the toxicities of various nanoformulations along with their potential efficacy. The size reduction to the nanometric dimension not only enhances the potency of the medication, but also enhances the interactions occurring due to increased surface area, surface charge and geometry with the normal cells which can precipitate toxicity. The foremost reason responsible for the toxicity of nanomaterials is the increased oxidative stress due ROS, free radical reactions and inflammatory mediators generation which cause damage the healthy cells. Since the highly perfused organs of the body like heart, liver, kidney, spleen, lungs and brain, they are the most prone to toxicity and adverse effects. For instance, gold nanoparticles exhibit cyanidation and oxidation predominantly in the kidney and hence are nephrotoxic [268]. Silver nanoparticles are known to exert pulmonary and dermal toxicity [269]. Copper oxide nanoparticles have the highest potential to oxidize, cause DNA damage and are hepatotoxic and nephrotoxic [270]. Biodegradable and biocompatible polymers like PLGA, PLA, PCL, etc. are declared safe by the FDA [271]. However, the safety of non-biodegradable polymers like polycyanoacrylates and other acrylates still remains questionable. Cationic lipids and polymers promote enhanced cellular internalization and are beneficial for gene delivery, however, they have the ability to cause hemolysis and are cytotoxic to healthy cells. Cationic dendrimers have showed hemolytic reactions owing to the untoward electrostatic attraction with the RBC surface. Free anime group in dendrimers imparts cationic charge which is associated with increased ROS, membrane integrity damage, membrane potential changes, acidification of intracellular environment, oxidative DNA damage by free radical generation, modification of intracellular calcium transport, etc. [272]. In these cases, a risk benefit ratio must be established to judge whether the necessity of therapy is worth the risk.

Scalability and technology transfer for continuous manufacturing and reproducibility for nanotechnology based medicines are difficult tasks. Surface functionalization with specific ligands which bind selectively to viral receptors impart target selectivity and reduces the dose and reduces the unwanted side effects of therapy. However, this is a major obstacle towards scalability and reproducibility along with quantification of the surface modification. The researchers in the field must take the scalability parameters into consideration working towards the end goal of clinical translation. Another aspect which must be considered is the cost of therapy. An increase in surface functionalization increases the manufacturing steps and subsequently increases the cost. This could prove to be a hurdle in developing and underdeveloped countries, hence the nanotechnology based products should be designed in such a way that the cost of therapy is reasonable. In many studies throughout the literature, we found that antiviral efficacy has not been performed in vitro as well as in vivo. Pharmaceutical scientists and virologists must work together to decipher which nanomedicine approach provides the highest efficacy possible against viral infections along with the highest potential for clinical translation. This could help in bringing new products in the market and make us all better prepared against the next viral pandemic yet to come.

After surpassing the above obstacles, the ultimate hurdle which needs to be overcome prior to clinical translation includes the compliance with the regulatory agencies for receiving approval of product for marketing. The US FDA approval of mRNA nanotechnology based vaccines against COVID-19 supports the benefit for treating or preventing viral diseases. The benefit to risk ratio, cost of nanomedicine and availability of innovator and generic pharmaceuticals for nanomedicine product development will dictate an appropriate market potential as well as translational potential. In such cases, nanomedicine based products could prove fruitful in availing market entry through NDA, ANDA and hybrid NDA pathways in accordance with the FDA. For seeking approval for new chemical entities whose pharmacokinetic-pharmacodynamic (PK-PD) profile has not yet been established, it is imperative to file NDA, while for an approval of generic product, abbreviated new drug application must be filed. For drugs whose PK-PD profile has been established, with a change in dose, dosage form, administration route, drug repurposing, increased efficacy compared to innovator, etc. hybrid new drug application, also known as 505(b)(2) pathway brings new and exciting opportunities for the clinical translation of various products. The major advantage of this pathway is the circumvention of clinical trials provided the drug is a reference listed drug whose safety and efficacy have been established. In our previous publication, we have enumerated various reasons which contribute towards this gap and the potential ways to address it [273]. Nanomedicine based approaches fit perfectly in this scenario where they are superior to the conventional marketed therapy in terms of dose, bioavailability, diminishing the adverse effects, targeting efficiency, etc. This opens up a whole new arena for nanotechnology based products with rapid approval potential and faster clinical translation.

12. Conclusion and future perspectives

In this review article, we discussed various barriers of viral entry and the importance of drug product development at various viral life cycle stages. The therapeutic efficacy of conventional antiviral therapy is circumscribed due to the diminished solubility, permeability and bioavailability of therapeutic agent/s, adverse effects and reactions, emergence of multi-drug resistance, drug-drug interactions, etc. It is expected that the advent of nanomedicine based approaches could surpass these shortcomings and advance the therapeutic efficacy as well as product development. We emphasized on how various lipid, polymer and metal based nanoformulations could be used to their fullest potential in achieving high antiviral efficacy with overcoming the limitations if conventional delivery systems and minimizing side effects. The emerging delivery systems like carbon nanotubes and carbon dots, nanovaccines and interference RNA therapeutics which could open new doors to advance the current antiviral therapy. However, there are primary obstacles such as manufacturing and cost towards the successful clinical translation of nanomedicine approaches for combating viral manifestations. Storage and in vivo stability, scalability aspects, targeting efficiency at the site of cellular or intracellular action, safety and undesired interaction with plasma proteins and modulation in the pharmacokinetic as well as pharmacodynamic profile especially in the metal and polymer based systems are the prospective challenges associated with nanomedicine based strategies. These challenges could be potentially overcome by uniting the efforts as well as expertise of the pharmaceutical scientists and virologists to provide a better understanding and bring about successful clinical translation of nanomedicine for combating viral infections. Exploring the potential and merging the principles of ‘smart’ nanosystems, immune-ligand anchored targeting approaches, co-delivery of siRNA along with antiviral drugs in overcoming multi-drug resistance are some of the avenues which must be explored by the scientific community in the field of antiviral research. The last obstacle towards the successful clinical translation includes compliance with the regulatory agencies. Nanomedicine based approaches of drugs whose PK-PD profile has been established could hasten the approval and could circumvent the clinical trials through hybrid NDA pathway. These formulations are proven to be superior in comparison with the conventional formulations and therefore could dictate their way into the market. Through this article, the significance of nanomedicine based strategy for product development to fight against viral infections and improving the quality of life was emphasized.

Highlights.

The challenges associated with the conventional antiviral therapy could be circumvented with the advent of nanomedicine based approaches.

Different types of nanoformulations along with their attributes and factors influencing their potential in antiviral therapy were discussed.

Role of RNAi therapeutics, clinical status, obstacles towards clinical translation and modes to overcome those challenges have been deliberated.

Dawn of nanotechnology meeting vaccine delivery offers exhilarating opportunities in the field of vaccine therapeutics.