Introduction

Recently, we focused on the ability of various metal-containing polymers to inhibit different cancers including those responsible for human breast, prostate, lung, pancreatic, and colon. These efforts have been reviewed for organotin polymers [1,2,3,4]. During this time we have also begun to focus on the ability of these polymers to inhibit various viruses (Table 1) [5]. In this section the inhibition of various viruses by organotin polymers is described.

Table 1 Viruses used or discussed in this report

Antiviral Polymers Derived from Acyclovir

Acyclovir, a nucleoside analog derived from guanosine, is frequently used in the treatment against many viral infections, particularly infections with the herpes viruses HSV-1 and HSV-2 [6]. Structurally, acyclovir contains two Lewis acid–base functional groups-an alcohol group and an amine group (Fig. 1).

Fig. 1
figure 1

Structure of acyclovir

A variety of organotin poly(amine ethers) containing acyclovir were created (Fig. 2) and these polymers showed outstanding inhibition of herpes simplex virus-1 and varicella zoster virus [6]. These polymers also showed promising results against HSV-1 and VZV, moderate inhibition of vaccinia virus, and little inhibition of reovirus ST3, a double-stranded RNA virus. The dibutyltin, dimethyltin, and diphenyltin polymers showed better antiviral activity than acyclovir itself.

Fig. 2
figure 2

Structure of polymer repeat unit for the product of acyclovir and diorganotin dihalides where R represents the particular substituent on the tin and R1 simple chain extension

Organotin Polyethers

Increased cellular DNA replication is seen in cancerous cells, and it is also seen in most DNA and some RNA viral infections [7]. A variety of simple organotin polyethers were synthesized by reaction of organotin dihalides with Lewis bases containing two hydroxide groups, diols. Initially they were tested against HSV-1, a dsDNA virus which replicates in the nucleus of the host cell, and vaccinia virus, a dsDNA virus that replicates in the cytoplasm of the host cell. Results from the initial series of organotin polyethers showed that the polymer derived from dibutyltin dichloride and ethylene glycol (Fig. 3) had high activity against vaccinia virus only, while the water soluble dibutyltin dichloride and poly(ethylene glycol) (PEG) polymer (Fig. 4) showed good inhibition of both viruses. It is believed that the promising activity of the PEG polymer towards both viruses is due to its water solubility.

Fig. 3
figure 3

Synthesis of organotin polyethers derived from dibutyltin dichloride and ethylene glycol

Fig. 4
figure 4

Repeat unit for the product of dibutyltin dichloride and poly(ethylene glycol), PEG, where R represents simple chain extension

Organotin polyethers derived from a variety of hydroquinones were also studied (Fig. 5) [7]. These compounds showed the highest antiviral activity against HSV-1 for the organotin polyethers thus far studied. Three of the hydroquinone-derived polymers blocked HSV-1 infection in 35 to 80% of cells, while only the dibutyltin/tert-butylhydroquinone demonstrated activity against vaccinia virus. Structurally, hydroquinone possesses an aromatic ring with available pi bonds, and these pi bonds may be able to interact both extracellularly (at the point of viral attachment) and intracellularly (binding to DNA), which would explain the high antiviral activity for this series of polymers.

Fig. 5
figure 5

Repeat unit for the organotin polyethers derived from hydroquinone where R1 represents substituents on the tin and R simple chain extension

Organotin Polyamines

Organotin polyamines derived from the reaction of dibutyltin dichloride with 4,6-diaminopyrimidines were also studied (Fig. 6) [7]. Results showed that the polyamine derived from 4,6-diaminopyrimidine blocked the replication of HSV-1 in in about 30% of cells, while the dibutyltin derivative of 4,6-diamino-2-mercaptopyrimidine blocks infection in 20% of treated vaccinia cells. It has been surmised that the antiviral activity of these classes of polymers is due to the compounds being nucleoside analogs, and the virus may incorporate the polymer into its DNA during viral replication, thereby terminating the viral DNA synthesis.

Fig. 6
figure 6

Repeat unit for the polymer from dibutyltin dichloride and 4,6-diaminopyridine where R1 represents simple chain extension

Known Antibiotics

Ampicillin and norfloxacin are commonly used antibiotics. Ampicillin belongs to the penicillin family of antibiotics, while norfloxacin is a fluoroquinolone antibiotic used to treat a variety of gram-positive and gram-negative bacteria that often cause urinary tract infections. Multiple previous studies have demonstrated that polymers containing the dibutyltin moiety demonstrate the most antimicrobial activity, and thus, dibutyltin-containing polymers derived from ampicillin (Fig. 7) and norfloxacin (Fig. 8) were tested against a variety of viruses, including reovirus ST3, vaccinia virus, herpes simplex virus (HSV-1), and varicella zoster virus (VZV) [8]. Reovirus is a double-stranded RNA virus, while the other viruses are DNA viruses. Using a standard plaque reduction assay technique for each virus, the dibutyltin-ampicillin and dibutyltin-norfloxacin polymers were tested. Both the ampicillin and norfloxacin polymers showed good inhibition of both the RNA virus (reovirus) and the DNA viruses tested, and thus, may hold promise as future antiviral agents.

Fig. 7
figure 7

Repeat unit for the product of dibutyltin dichloride and ampicillin where R1 represents simple chain extension

Fig. 8
figure 8

Repeat unit for product of dibutyltin dichloride and norfloxacin where R represents simple chain extension

Materials and Methods

Cell Culture Maintenance

The cell lines used in this study were Vero cells and 143 cells. All the cell lines that were used are transformed cell lines with the ability to replicate indefinitely in cell culture. Vero cells are African green monkey kidney cells and support the growth of Zika virus. 143 cells, which allow for the growth of vaccinia virus, are derived from a human osteosarcoma cancer. All cells lines were maintained in 75 cm2 flasks in Dulbecco’s Modified Eagle’s Media (1× MEM) with 5% fetal bovine serum (or 10% bovine growth serum) and the antibiotics streptomycin/penicillin at a final concentration of 100 U/mL (100 units/mL based on biological activity rather than concentration. Sold as stocks at 100,000 U/mL). All cells were kept in a 5% CO2 incubator. The cells were subcultured as often as needed to seed new flasks or plates for experiments. Briefly, confluent cells were washed in the culture flask with 1 × sodium saline citrate (SSC), or MEM without serum. Trypsin (0.05%) was then added to the cells to detach them from their flask. The trypsinized cells were placed in the 5% CO2 incubator for 5 min. Following detachment, 5 mL of fresh media was added to the cells to wash them from the side of the flask. New media (up to 5 mL total for a 25 cm2 flask and 10 mL total for a 75 cm2 flask) was added to the flasks, usually in a 1:4 dilution (2 mL of cell suspension in 8 mL of fresh media). The cells were seeded at varying dilutions, depending on when they were next needed.

Virus Stock Preparation

Virus stocks were grown in their appropriate cell lines. Initially, cells were seeded in a 25 cm2 culture flask to 90–100% confluency. The following day, the cells were infected with virus. For the first passage, a multiplicity of infection (MOI) of 1 was used, which means that there was one virus particle per cell. The viral inoculum was made using diluent which consisted of 1× MEM with 100 U/mL of penicillin/streptomycin (100 units/mL: based on biological activity rather than a concentration. Sold as stocks at 100,000 U/mL) and no growth serum. After 1 h of virus adsorption with rocking every 15 min, 5 mL of 1× MEM with 5% BCS and 1% P/S was added. When the virus had killed 50% of the infected cells, which was seen microscopically as a cytopathic effect (CPE), the cells were sonicated to release all of the virus particles from the cells. Upon second passage, 1 mL of the passage 1 viral lysate was used to infect a confluent 75 cm2 flask. The third and final passage involved infecting cells in a 150 cm2 flask with 2 mL of the second viral passage. The third passage is the viral passage that was titered.

Viral Titration

To determine the titer of each virus stock in PFU/mL, standard plaque assays were used. Initially, the virus was serially passaged three times in a tissue culture of the appropriate cell line (vaccinia: 143; Zika: Vero), first in a 25 cm2 flask, followed by a 75 cm2 and then a 150 cm2 flask. The cells in the 150 flask cm2 were then sonicated to lyse the cells and release the virus. The third passage lysate was then used to infect confluent monolayers of cells for a plaque assay. Vero cells were seeded into 6-well plates for Zika virus and 143 cells were seeded into 12-well plates for vaccinia virus in 1× MEM containing 5% fetal bovine serum (FBS) and penicillin/streptomycin at the concentration seen above. When the cells reached around 80% confluency ~ 24 h later, the media was aspirated off, and the cells were infected with serial dilutions (10−1 to 10−7) of virus at an inoculum size of 250 μL (6-well plate) or 125 μL (12-well plate). To obtain serial dilutions, 100 μL of the third passage viral lysate was suspended in 900 μL of diluent, which is 1× MEM with no FBS, and 1% penicillin/streptomycin. This yields a 10−1 dilution. This dilution was then vortexed, and 100 μL of this dilution was then placed into another tube containing 900 μL of diluent. The process was repeated until all desired dilutions had been created. The control for each plate contained cells and 250 μL (or 125 uL) of diluent without added virus. Following 1 h of incubation with agitation every 15 min, the cells were washed, and fresh media was added to each well. After the assays were incubated (2 days for vaccinia, 5 days for Zika), both were stained with 1% crystal violet in 50% ethanol.

Compound Preparation

Compounds began as solid powers. Initially, 0.01 g (10 mg) of compound was dissolved in 1 mL of DMSO, giving a stock concentration of 10 mg/mL. One microliter of the stock solution was then placed into 1 mL of AquaPure sterile water, giving a concentration of 10 µg/mL. Finally, 1 mL of this solution was placed into 10 mL of AquaPure water, giving a beginning concentration of 1 µg/mL for compound testing.

Cytotoxicity Assays

To determine the highest concentration of compound that the cell lines could tolerate without showing cytopathic effects, cytotoxicity assays were performed for each compound. For each of the three cell lines, 125,000 cells were seeded in 500 μL of media in each well of a 24 well plate. When the cells reached confluency around 24 h later, compounds were added to the wells. The 1 µg/mL starting concentration for each compound was ten-fold serially diluted in 1× MEM, yielding 10−1 to 10−4 compound dilutions. The media from each well of the 24 well plates were removed, and 500 μL of the compound dilutions were added to each well. After 96 h, 50 μL of trypan blue were added to each well and allowed to stain the cells for 5 min. After 5 min, the cells were observed microscopically to determine the highest concentration of drug for each cell line that did not cause cell death.

Vaccinia Plaque Reduction Assay

For the vaccinia plaque reduction assays, 12 well plates were used. When the cells had reached confluency, the media was removed, and vaccinia virus at a 10−4 dilution was added to each test well. For control wells, only 1× MEM was added. Control wells of DMSO dilutions were also completed in which the DMSO was diluted in MEM as if it were a drug. All wells, whether control or test wells, received a 125-μL inoculum. After 1 h of virus adsorption with agitation every 15 min, the cells were washed with 1× MEM without serum to remove any remaining virus. The compounds were then added to the wells, 1 mL per well, at the concentrations previously determined by the cytotoxicity assays. After 48 h, the old media was removed, and 1% crystal violet was used to stain the cells.

Zika Plaque Reduction Assay

For the zika virus assay, 96-well plates were used. After the cells reached confluency in their wells ~ 24 h later, the old media was removed from each well and 100 μL of fresh media was added. For each compound, 200 μL of drug were added to the first well of each column. After mixing the solution, 100 μL were taken from the first well and placed into the second well. The compounds were serially twofold diluted across the 96-well plates. Following the addition of the compounds, 10 μL of Zika virus was added to the test wells. The assays were placed in the incubator for 5 days and then viewed microscopically. The lowest concentration of compound that prevented the Zika-infected cells from showing cytopathic effects (CPE) was recorded.

Polymer Synthesis and Characterization

Synthesis is rapid (generally < 30 s) occurring under mild conditions at room temperature employing the interfacial polymerization process.

Following is a general sequence for the synthesis of the polymers reported in this paper. As noted, reactions were carried out using the interfacial polycondensation technique. An aqueous solution containing the Lewis base and sodium hydroxide was transferred to a one-quart Kimax emulsifying jar fitted on top of a Waring Blender (model 1120; no load speed of about 18,000 rpm; reactions were carried out at about 25 °C). Stirring was begun and a hexane (or other suitable solvent for the organotin-containing reactant) solution containing the organotin dihalide was rapidly added through a hole in the jar lid using a powder funnel and the resulting solution blended for 15 s. The precipitate was recovered using vacuum filtration and washed several times with deionized water and heptane to remove unreacted materials and unwanted by-products. The solid was washed onto a glass petri dish using acetone and dried at room temperature.

Structural characterization includes product yield, infrared spectroscopy, mass spectrometry, molecular weight determination, nuclear magnetic resonance spectroscopy and preliminary ability to inhibit important solid tumor cancer cell lines. The results are given in the references associated with the results [9,10,11,12,13,14].

Results and Discussion

Due to the success of a variety of organometallic polymers as anticancer agents a number of these compounds were tested for their efficacy as antiviral agents. There are a few compounds which show promising activity against vaccinia virus (dsDNA) and none against zika virus (+RNA).

Vaccinia Virus

Vaccinia virus is an Orthopoxvirus within the Poxviridae family of viruses [15]. It is a double-stranded DNA virus that was previously used in the vaccination program against smallpox, which was eradicated through the World Health Organization (WHO) in 1979. Although smallpox has been eradicated, research into the disease remains invaluable, as smallpox remains a potential agent for bioterrorism. The worldwide vaccination program against smallpox was stopped in 1980, and thus, an outbreak of smallpox today would decimate the current population who lack smallpox immunity [16]. Smallpox is caused by two viruses: variola major and variola minor, with variola major producing most of the fatal disease burden, with a case-fatality rate of 20% or more. Vaccinia virus is the vaccine strain of smallpox, and although its origins are still unclear after over 100 years of research, it is surmised that vaccinia could be the product of genetic recombination.

Vaccinia virus is a unique virus because, although it is a DNA virus, it is replicated exclusively in the cytoplasm of infected cells through a viral DNA-dependent RNA polymerase [16]. It is also unique in that vaccinia produces two types of virions: intracellular mature virus (IMV) and extracellular enveloped virus (EEV), both of which are infectious [17]. The IMV virions are surrounded by a single membrane, while the EEV virions possess two membranes and are critical in the dissemination of the virus. The external membrane of the EEV virions is derived from the host cell, and thus, these virions can evade both host antibody and host complement immune responses.

Upon contact with a competent host cell, vaccinia virions enter the cell through phagocytic vacuoles where they are partially stripped of their outer core, releasing the vaccinia virus dsDNA genome into the cytoplasm [18]. Once inside the cytoplasm, the DNA-dependent RNA-polymerase of the virus transcribes the vaccinia viral genome into messenger RNAs (mRNAs) [16]. The transcribed genes can be classified as early, intermediate, and late genes. The early vaccinia genes are needed for viral DNA replication and for assisting the virus in escaping the host’s innate immune response. The intermediate genes encode for transcriptional regulators of the late genes, and the late genes encode the structural proteins and enzymes necessary to construct the new virions. Inside cytoplasmic inclusions known as virus factories, the vaccinia virus dsDNA is packaged into an immature virion (IV), with proteolytic cleavage leading to the formation of the intracellular mature virus (IMV), which are released through cell lysis. Some virions, however, will become wrapped in Golgi-derived membranes and are known as intracellular enveloped virus (IEV). These intracellular enveloped virus particles are transported to the surface of the cell, where they can either remain at the cell surface as CEV (cell- associated enveloped virus), or they are released as extracellular enveloped virus (EEV).

Vaccinia virus is a dsDNA virus with a unique intracytoplasmic replication mechanism. Although smallpox has been eradicated worldwide, vaccinia remains relevant because of its potential use in biowarfare. Using standard plaque reduction assays, several organometallic polymers were tested against vaccinia virus for their ability to inhibit viral replication in 143 cells. Most polymers were not active; however, the derivatives of 3-AT (Table 2) showed some inhibition, protecting between 15 and 35% of cells from infection.

Table 2 Inhibition of vaccinia virus by organotin polymers derived from reaction with 3-amino-1,2,4-triazole, 3-AT [19]

In Table 3, for example, all the organometallic/dicumarol polymers showed over 100% virus production. In this case, and in the case of other groups of polymers, the compounds made the cells more susceptible to lysis, resulting in an enhanced production of visible plaques compared to the number of visible plaques for the virus-infected control wells. Thus, in these instances, there was not an increase in virus production, but an increase in susceptible cells.

Table 3 Vaccinia plaque reduction assay results for organotin polymers derived from reaction with dicumarol [20]

The organotin polymers derived from camphoric acid and lamivudine also showed the ability to inhibit vaccine replication (Table 4). The inhibition was mild at best.

Table 4 Inhibition of vaccinia virus by polymers derived from camphoric acid, acyclovir and polymers derived from organotin dichlorides and camphoric acid, CA [21], and lamivudine, LAM [22]

Table 5 contains vaccine plaque reduction assay results for various organotin polymers. All polymers showed decent inhibition of vaccinia viruses.

Table 5 Vaccinia plaque reduction assay results for various organotin polymers [19, 23, 24]

In summation, a number of organotin polymers exhibit some inhibition of the vaccinia virus to merit further testing of newly synthesized polymers.

Zika Virus

Zika virus, an enveloped, plus-stranded RNA virus belonging to the Flavivirus genus, was first discovered in Uganda in 1947 [25]. Usually, Zika virus causes asymptomatic infection; if symptoms do occur, they include a low-grade fever, itchy rash, and arthralgia [25]. The concern for Zika virus, however, emanates from its potential to cause severe neurological disease, such as microcephaly in newborns, as well as a handful of cases which involved development of Guillain–Barre syndrome, which causes the immune system to attack the peripheral nervous system [25]. It gained much publicity during the recent Olympics in Brazil because of the fear by the participants, particularly women, and attenders of contracting the zika virus.

Zika virus particles contain an inner nucleocapsid surrounding the genomic RNA, and the nucleocapsid is wrapped in an envelope that contains the viral membrane protein (M) and the viral envelope protein (E) [25]. The RNA of Zika virus encodes 3,423 amino acids which are translated as a large polyprotein, which is subsequently cleaved into 10 + individual viral proteins. The viral nonstructural protein NS3 has helicase and nucleoside triphosphatase activities, while NS5 is the viral RNA-dependent RNA polymerase which is required for viral genome replication. Zika virus infects human neural progenitor cells (hNPCs) through clathrin-mediated endocytosis. The acidic environment of the endosome induces conformational changes in the viral envelope (E) glycoprotein leading to fusion between the viral and endosomal membrane and subsequent release of the zika virus RNA into the cellular cytoplasm. The viral RNA-dependent RNA polymerase is responsible for replication and translation of the viral genome, along with currently unidentified cellular factors. Immature virions bud into the endoplasmic reticulum, where they receive their cellular-derived envelope with embedded viral prM (membrane) and envelope proteins. Immature virions complete the process of maturation as they proceed through the trans-Golgi network, and the virions are eventually released from the cell through exocytosis (Figs. 9, 10, 11, 12, 13, 14).

Fig. 9
figure 9

Repeat unit for the organotin polymers derived from 3-amino1,2,4-triazole, AT, where R1 represents simple chain extension and R organic substitutions on tin

Fig. 10
figure 10

Repeat unit for the polymer formed from reaction between various diorganictin dichlorides and dicumarol where R1 represents simple chain extension

Fig. 11
figure 11

Repeat unit for the polymer formed from the reaction of camphoric acid and various organotin dichlorides where R1 represents simple chain extension and R organic substitutions on the tin

Fig. 12
figure 12

Repeat unit for the product of dimethyltin dichloride and lamivudine where R represents simple chain extension

Fig. 13
figure 13

Repeat unit for the products from diphenyltin dichloride and various poly(ethylene glycols), PEGs, where R represents simple chain extension

Fig. 14
figure 14

Repeat unit for the polymer formed from various organotin dichlorides and alpha-cyano-4-hydroxycinnamic acid where R1 represents simple chain extension

Zika virus is a single-stranded, plus-sense RNA virus that has garnered worldwide attention recently due to its connection to neurological birth defects. Table 6 shows the results for the compound assays for zika virus. Unlike vaccinia virus, in which a plaque assay technique was used to assess the efficacy of the compounds, we could not get zika virus to produce defined plaques, so we used a cytopathic effect assay. For the organotin polymers, results indicate that two groups of compounds show promise as antiviral agents against zika virus. These compounds are derived from camphoric acid and lamivudine.

Table 6 Inhibition of zika virus strain 502

Lamivudine (Fig. 15; also called 3TC) is a potent reverse transcriptase prodrug antivirial molecule employed in the treatment of AIDS [26,27,28,29,30]. Structurally, lamivudine is a nucleoside analogue. It is administered several times daily because of its short half-life of 5–7 h. It has additional problems including negative effects from the accumulation of the drug, high cost, and lack of patient compliance. Further, there is an increased incidence of co-infection of HIV with such diseases as tuberculosis. Co-treatments are being investigated. For instance, co-loaded polymer microspheres containing lamivudine and an anti-tuberculosis drug such as isoniazid have been described that allow the treatment of both the HIV and tuberculosis [31].

Fig. 15
figure 15

Structure of d-Camphoric Acid

All the organotin polymers derived from lamivudine and camphoric acid exhibit total inhibition of the zika virus, strain 502. They all inhibited infection of Vero cells at 0.025 µg/mL. Against Zika strain 502, the products from diethyltin dichloride and camphoric acid and diphenyltin dichloride and camphoric acid inhibited infection of Vero cells at concentrations of 0.000391 and 0.000781 μg/mL, respectively. Thus, it appears that the organotin and camphoric acid-derived compounds merit further testing against zika virus as potential novel antiviral agents. These concentrations are in the nano or near nanogram region (Fig. 16).

Fig. 16
figure 16

Structure of lamivudine

In hopes of determining additional products that exhibit good inhibition of the zika virus several polymers that had structural characteristics like camphoric acid were tested for their ability to inhibit the zika virus. We identified possible structural characteristics that we have begun to explore. Camphoric acid has two acid groups and further a ring system from which the acid groups are attached. We have synthesized many polymers that exhibit good anticancer activity. Table 6 contains results for some of these. Figure 17 contains the structures for some of these. In no case was any inhibition of the zika virus found. Additional structures were studied but thus far with no success. We continue to seek further structures that will inhibit the zika virus.

Fig. 17
figure 17

Structures of diacid-containing compounds whose dibutyltin polymer derivatives were tested for their ability to inhibit the zika virus. From left to right the structures are derived from chelidonic acid, 3,5-pyridinedicarboxylic acid, dipicolinic acid and 2-ketoglutaric acid

The influence of the polymeric nature was briefly studied. A dimer of the dibutyltin camphoric acid was synthesized through simply reacting the camphoric acid with tributyltin chloride. Because the organotin reactant has only one reactive group, it can only react at the ends of the camphoric acid without chain extension (Fig. 18).

Fig. 18
figure 18

Formation of the camphoric acid dimer

The dimer showed the ability to inhibit the zika virus, Table 6, but inhibition was significantly less compared with the dibutyltin/camphoric acid polymer. Thus, the polymeric nature is positive in this case.

Summary

In summary, a number of organotin polymers inhibit the vaccinia virus to some extent. Organotin polymer derived from camphoric acid and lamivudine totally inhibit the zika virus using drug concentrations in the nanogram/mL range.