Introduction

More than 36.9 million people around the world are infected with HIV [1]. Sexual transmission of HIV-1 is a main reason for the increasing number of infected people [2]. Effective protection options for women against the sexual transmission of HIV are clearly needed [3]. Because some microbicidal agents with antiviral activity can be administered via the vaginal route, these components may be considered as potential agents for the prevention of HIV infection through intercourse.

The important stages in the treatment-based inhibition of the life cycle of HIV are (1) binding to host cells, (2) reverse transcription, (3) integration, (4) protein processing. Any of these stages can be considered as the target for HIV inhibition, but most research attention is focused on the binding and reverse transcription steps [4].

Research has been conducted on nanotechnology as a potential approach for interfering with the HIV entry, and several nanoparticles have been examined in this regard due to their chemical properties [5]. Among the new generations of nanosystems, dendrimers are potential drug carriers. These highly symmetric and branched polymers have attracted much attention in recent years because of their physical and chemical properties due to their structure [6]. Dendrimers have been designed with specific functional groups that can prevent a virus from entering its host cell. Furthermore, dendrimers with reactive surface groups and their broad interior space are potential carriers for chemical drugs, peptides, and genes to inhibit the HIV virus. The compounds mentioned can be conjugated to the surface groups of dendrimers or encapsulated in interior spaces. Dendrimers can enhance the sustainability of chemical drugs and their cellular uptake and provide potential carriers for RNAi (RNA interference) delivery rather than viral vectors to HIV inhibition [4].

SPL7013 is a dendrimer with a polyanionic surface which attaches to viruses and blocks viral attachment to cells. Among all dendrimers, SPL7031 has successfully completed phase-I clinical trials [7]. Polyanionic carbosilane dendrimers with sulphated and naphthylsulphonated functional groups have shown effective microbicidal activity against HIV [8].

The current study evaluated G2 dendrimers (citrate-PEG-citrate) as biocompatible agents against HIV infection.

Materials and methods

Materials

PEG 600, dicyclohexylcarbodiimide (DCC), dimethyl sulfoxide (DMSO), citric acid, and calcium chloride were used in the synthesis of the dendrimer (Merck, Darmstadt, Germany). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), antibiotics, and trypsin were used in cell cultures (Gibco, Paisley, UK). The pmzNL4-3, pMD2.G, and psPAX2 plasmids were used to produce pseudotyped HIV virions. The pmzNL4-3 plasmid (single-cycle replicable HIV-1 virions) were obtained from Pasteur Institute of Iran [9]. The pMD2.G and psPAX2 were purchased from Addgene (Massachusetts, USA). The cell proliferation kit II (XTT) was obtained from Sigma Aldrich (Missouri, USA) and the HEK and HeLa cell lines were procured from the Pasteur Institute (Tehran, Iran). The HIV p24 ELISA kit was obtained from Biomeriux (Marcy-l’Etoile, France), and the PolyFect transfection reagent was procured from Qiagen (Hilden, Germany).

Synthesis of G1 and G2 dendrimers

The G1 dendrimer synthesis was begun by reacting 3.7 mM of PEG 600 (as the dendrimer core) and 2 mM of dicyclohexylcarbodiimide (DCC) in dry DMSO under dark and vacuum conditions. The reaction vessel was stirred for 15 min on a stirrer. Then, 2.7 mM of citric acid was added, and the solution was stirred with a stirrer for 1 h. The reaction was terminated by adding water. Then, the dendrimer was obtained by filtering the yellow and brown solution. The G1 product was purified through dialysis. The cut off of the dialysis bag was 100–500 Dalton. The G1 dendrimer structure was then characterized by FTIR and NMR spectroscopy.

To synthesize the second generation (G2), 15 ml of dry DMSO solvent was added to 0.52 mM of G1 dendrimer. Then, 6 × 0.52 mM of DCC was added, and the solution was stirred on a stirrer for 15 min. At this stage, 6 × 0.52 mM of citric acid was added to the reaction and re-stirred for 1 h by a stirrer. Then, about 20 ml of distilled water was added and the reaction was terminated (Fig. 1). After filtering and dialysis, the solution was lyophilized and FTIR and NMR tests were done to determine and confirm the structure. The zeta and size of the dendrimer were determined by dynamic light scattering (DLS).

Fig. 1
figure 1

Sample prepration. PEG-Citrate dendrimer was synthesized in the reaction of PEG (as dendrimer core) with citric acid in DMSO solvent

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Virus production

Virus stocks were produced with 4 µl of PolyFect transfection and 400 ng of plasmids (pmzNL4-3, pSPAX2, and PMD2G) based on a 2:1:1 ratio, respectively, in a 24-well plate. Transfected cells were incubated in 5% CO2 and 37 °C. Virion-containing supernatants were collected at 24, 48, and 72 h post-transfection. The supernatant was centrifuged at 60,000×g for 90 min. The viral sediment was dissolved in a 1:20 ratio of RPMI medium and stored at − 70 °C [9].

Cytotoxicity assays

XTT cell proliferation was performed to assess viability. Hela cells were seeded in a 96-well plate (9 × 103 cells/well) with 100 µl of complete DMEM medium and incubated in 5% CO2 at 37 °C. After treating the cells with different concentrations (0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, 1.2 mM, 2.4 mM) of G2 dendrimer for 48 h, XTT solution was added to each well and incubated for 8 h according to the manufacturer’s protocol. The absorbance of the wells was read at 450–500 nm with a reference wave length of 650 nm using an ELISA plate reader Biotek EL340 (Winooski, USA).

Inhibition of HIV infection

Pre-treatment assay

The cells were treated with G2 dendrimer at different concentrations (0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, 1.2 mM, 2.4 mM) for 6 h. Then they were washed with PBS and infected with 250 ng of HIV viral load as assessed through p24 antigen titration for 6 h. Ultimately, the cells were washed three times with PBS to remove any unbound viruses and incubated at 37 °C and 5% CO2 for 48 h.

Co-treatment assay

The cells were co-treated with G2 dendrimer at appropriate concentrations (0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, 1.2 mM, 2.4 mM) and 250 ng of HIV viral load as assessed through p24 antigen titration for 6 h. Then they were washed three times to remove all unbound viruses and incubated at 37 °C and 5% CO2 for 48 h.

Quantification of p24 antigen through ELISA

The supernatants were collected at 48 h post-treatment, and the production of p24 antigen was quantified by ELISA kit protocol, according to the manufacturer’s instructions.

Results

FTIR spectra

The FTIR spectrum of each dendrimer was taken to determine the structure of the synthesized products. Figure 2G1 shows the FTIR spectrum shape of the G1 dendrimer. The peak observed at 1279 cm−1 (Fig. 2G1-B) refers to CO groups in the ester bond that confirm the synthesis of the G1 dendrimer and the formation of an ester bond between PEG and citric acid. The wavenumber range of 1154–1279 cm−1 (Fig. 2G1-C) shows alcohol CO in PEG as well as citric acid. The wavenumber range of 3370–3500 cm−1 (Fig. 2G1-A) indicates the acidic OH in citric acid.

Fig. 2
figure 2

FTIR spectrum was prepared to confirm the synthesis of G2 dendrimer. G1 is related to the G1 dendrimer. The peak at 1279 cm−1 (G1-B) refers to the steric bond between PEG and citric acid. G2 is related to the G2 dendrimer. The peak at 1232 cm−1 (G2-F) refers to the steric bond between the end citric acid of the G1 dendrimer and new citric acid groups

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Figure 2 G2 shows the FTIR spectrum of the G2 dendrimer. The peak observed at 1232 cm−1 (Fig. 2G2-F) refers to CO groups in the ester bond, which confirm the synthesis of the G2 dendrimer and the formation of an ester bond between citric acid groups at the end of the G1 dendrimer and new citric acid groups. Also, the peak at 1726 cm−1 (Fig. 2G2-E) corresponds to the C=O groups of citric acid at the end of G2. The peaks within the range of 2500–3430 cm−1 (Fig. 2G2-D) are also related to the acidic OH groups of citric acid.

H-NMR spectra

As seen in Fig. 3, the hydrogen methylene groups of the G1 and G2 dendrimers corresponding to the –CH2 groups of the citrate were found at 2.5–2.7 ppm and 3.3 ppm, respectively. The two signals at 4 ppm and 5.7 ppm are related to the CH2–CH2–O and O–CH2–CO structures, respectively, that are assigned to PEG.

Fig. 3
figure 3

The H-NMR spectrum of the product was taken by Bruker 500 Htz in DMSO solvent. The peaks at 2.5–2.7 ppm and 3.3 ppm correspond to the hydrogen methylene groups of the G1 and G2 dendrimers. The signals at 4 ppm and 5.7 ppm are related to the PEG structure

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Dendrimer size and zeta measurements

The G2 dendrimer particle size was measured using dynamic light scattering (DLS). The size of the G2 dendrimer was 90 nm, and the negative zeta potential for the diluted (5 × 10−3 g/ml) G2 dendrimer was − 2.46 mv due to the presence of free carboxylic acid groups.

Cytotoxicity assay

The mitochondrial metabolism for HeLa treated with the G2 dendrimer was measured by XTT assay. HeLa cells were incubated at various concentrations of G2 dendrimer (0.05, 0.1, 0.2, 0.4, 0.6, 1.2, 2.4 mM) and their viability was evaluated at 48 h post-incubation. As shown in Fig. 4, the G2 dendrimer did not exhibit significant toxicity at 0.05–0.4 mM concentrations after 48 h. The 50% cytotoxic concentrations (CC50) on the HeLa cell line was 0.6 mM.

Fig. 4
figure 4

Cytotoxicity assay: the influence of the various concentrations of G2 dendrimer after 48 h on the viability of the HeLa cell line

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Inhibition of HIV-1 infection

The inhibitory behavior of the G2 dendrimer was evaluated both pre-treatment and co-treatment to measure its therapeutic and preventive effects against HIV infection. In this experiment, infected cells (negative control) and treated infected cells with nevirapine (positive control) were used as control samples. In the pre-treatment assay (Fig. 5), the CC50 (50% cytotoxic concentrations) and IC50 (50% inhibitory concentration) were 0.6 mM and 2.4 mM, respectively (Table 1). The inhibitory effect was observed to be greater in the co-treatment than in the pre-treatment assay. As shown in Fig. 6, the CC50 and IC50 in this test were 0.6 mM and 0.4 mM, respectively (Table 1).

Fig. 5
figure 5

Pre-treatment assay: As shown, by increasing the concentration of the polymer, the virus replication was reduced. In the pre-treatment assay, CC50 and IC50 were 0.6 mM and 2.4 mM, respectively. Infected cells (negative control) and treated infected cells with nevirapine (positive control) were used

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Table 1 CC50 and IC50 in pre-treatment and co-treatment assays
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Fig. 6
figure 6

Co-treatment assay. In the co-treatment assay, CC50 and IC50 were 0.6 mM and 0.4 mM respectively. Infected cells (negative control) and treated infected cells with nevirapine (positive control) were used

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Discussion

Antiretroviral therapy (ART) for HIV has been developed, and approximately 30 approved antiviral drugs with different mechanisms for HIV inhibition are now used [5]. Nanotechnology is of interest globally to the designers of novel antiretroviral agents and microbicides [5]. Dendrimer-based nanotechnology such as SPL7013 inhibits entry of the virus. More noteworthy, the BRI2923 dendrimer that is coated with polysulfonate groups not only blocked entry, but also inhibited the reverse transcription process in HIV replication [10]. Polyanionic compounds are known to be anti-HIV agents. Anionic polymers inhibit virus attachment to cells, because they interact specifically with the virus envelope rather than with the negatively-charged cell membranes [11]. In this study, different concentrations of G2 dendrimer against HIV infection were examined.

Alavidjeh reported a safe concentration range of up to 0.25 mM for both (G1, G2) dendrimers in vitro [12]. As shown in Fig. 4, a significant reduction (50%) in cell viability (CC50) was observed for the G2 dendrimer at the concentration of 0.6 mM. These results suggest that the G2 dendrimer is biocompatible with the HeLa cell, because G2 is made of two biocompatible components (PEG and citric acid). The use of PEG could increase solubility and decrease cytotoxicity [13].

Studies have shown that a series of quinolone-3-carboxylic acids have considerable anti-HIV activity [14]. Also, poly(propylenimine) decorated with carboxylate (PPI-C) and sulfonate (PPI-S) groups could inhibit HIV-1 infectivity [15]. The PPI-S and PPI-C dendrimers showed an HIV-1 inhibition rate of between 20 and 30% [15]. Therefore, the carboxylate groups, which are present in the PEG-citrate dendrimer, are effective functional groups which can provide anti-viral properties.

The XTT test results (Fig. 4) showed that the G2 dendrimer in high concentrations is biocompatible and suitable for biomedical applications. Based on this result, the high concentrations were selected for surveys of the antiviral efficiency of G2. The anti-HIV activity of G2 showed that all concentrations of this dendrimer could inhibit HIV. To measure HIV inhibition, both pre-treatment (therapeutic properties) and co-treatment (prophylactic behavior) approaches were used. As seen in Table 1, both the pre-treatment and the co-treatment had equal CC50s, but different IC50s. This difference in TI (therapeutic index) or SI (selectivity index) between the two treatments suggests a better inhibitory effect in the co-treatment assay than in the pre-treatment test (TI or SI = CC50/IC50). The association of anti-fusion and anti-replication effects in anionic polymers suggests that the antiviral properties of these polymers are linked to anti-fusion activity [6]. Different IC50s in both assays indicate that the viral attachment to cells can be blocked by the G2 dendrimer, because the IC50 concentration in the pre-treatment was greater than the CC50, indicating that HIV inhibition is due to the reduction in cell viability. As shown in Fig. 6, the 1.4 mM concentration had an inhibitory effect of only 50%, but its viability was 18%, indicating that HIV inhibition was due to cell death in the pre-treatment approach.

Garcícó emphasized that the antiviral property of a dendrimer depends on the structure of its compounds (generation, nucleus, peripheral groups) [15]. One reason for the low antiviral activity of the G2 dendrimer may be related to its low negative charge (− 2.46 mv). It is known that polyanionic compounds have antiviral activity, and polyanions interfere with the binding process in viral entry steps [6]. Many carboxylated polyanions have shown high anti-HIV activity, such as aurintricarboxylic acid, carboxylated dextrins, polymerized carboxylated surfactants, etc. [7].

The BRI2923 dendrimer at 500 times its EC50 (100 g/ml) has an inhibitory effect on the reverse transcription step in HIV replication. This dendrimer was made of sodium 3,6-disulfonapthylthiourea terminated PAMAM dendrimer (generation 4) [19]. PAMAM-G4 has an approximate diameter of 4.5 nm. Higher generations or uses of a long core in dendrimers produce larger molecular diameters. The size of the G2 dendrimer was 90 nm. This large dendrimer size was probably unable to inhibit the HIV replication stages in host cells (reverse transcription or integration process), and this denderimer is able to interfere with the attachment of the virus to cells. The detailed molecular mechanism of the G2 dendrimer interference with HIV replication is not well known and needs to be studied in the future.

Conclusion

In this study, a PEG-citrate dendrimer (G2) was synthesized and its anti-HIV activity was evaluated. For microbicide development, some characteristics are important, such as activity against virus, the ability to prevent infection of cells, and lack of toxicity at high concentrations [16]. The G2 dendrimer has several interesting properties, including direct conjugation with drugs and chemical components through its functional groups, high solubility in water, anti-HIV activity, and biocompatibility. The design of such dendrimers opens new paths for protection against HIV infection.