When it comes to negotiating safe sex, accessing to HIV prevention information and services, women and girls from developing countries are in the most disprivileged position, because of their economic and sociocultural status [1,2]. Therefore, effective strategies to stop spreading of HIV infection and HIV sexual transmission are urgently needed.
Up to now, HIV-preventive strategies such as behavioral and structural interventions (e.g. condom use) or preexposure prophylaxis [e.g., topical microbicides and the oral administration of antiretroviral (ARV) drugs] seem to be the only effective methods against the HIV infection [3–6]. The success of the first efficacious phase II clinical trial of an ARV-based microbicide (CAPRISA 004) using tenofovir-based gel to prevent man-to-woman HIV transmission showed that ARV can be effective being used as topical microbicides [3,7]. However, more recent failures in other antiretroviral microbicide clinical trials, such as the Vaginal and Oral Interventions to Control the Epidemic (VOICE) study , suggest that there is still more room for improvement and efforts to work out new effective microbicide strategies [9,10].
Nanotechnology offers suitable approaches in order to develop new antiviral agents. Therefore, different nanomolecules are being evaluated in clinical trials as antiviral agents to inhibit the spread of sexually transmitted diseases, for instance HIV [11,12]. Polyanions with anti-HIV activity have extensively been studied in vitro and in animal models (e.g. cellulose sulphate, carrageenan or PRO 2000) [13–15]. All of these polyanion-based microbicides examined up to now have a high level of efficacy in preclinical trials and safety in both preclinical and phase I clinical trials. However, none of them showed to have efficacy in further phases of clinical trials .
Dendrimers are a relatively new class of nanocompounds characterized by highly branched, well defined, three-dimensional structures . Dendrimers have shown antimicrobial properties [18,19] and capacity of preventing the HIV from sexual transmission in animal models, such as macaque . Moreover, different types of polyanionic dendrimers with anti-HIV properties, such as naphtalene sulphonated or carbosilane dendrimers, are being developed as a topical microbicide for human use [21,22]. The more successful results were achieved with the SPL7013 dendrimer (Vivagel) [23,24]. It has demonstrated great potency against both HIV-1 and HSV-2, although the phase I clinical trial evaluating their antiviral activity and microbiota tolerance revealed evidence of mild irritation after repeated vaginal use [25,26].
We have previously shown that the anionic carbosilane dendrimer 2G-S16 has antiviral activity in vitro and can be used as a potential microbicide candidate [22,27,28]. In this research, we have studied the microbicides properties of two new water-soluble and stable polyanionic carbosilane dendrimers (G3-S16 and G2-NF16) functionalized with sulphate and naphthylsulphonate ended groups, respectively. Our objective has been to evaluate both dendrimers as new potential microbicide candidates that could be used in well tolerated and effective therapeutic approaches to stop the HIV-sexual transmission.
Materials and methods
Polyanionic carbosilane dendrimers, third generation G3-S16 with 16 sulphated end groups (C256H508N48Na16O64S16Si29) and second generation G2-NF16 with naphthylsulphonated end groups (C184H244N24Na16O56S16Si13) were prepared according to reported methods by the Inorganic Chemistry group of University of Alcala de Henares (UAH) (article submitted). The dendrimers were dissolved in distillated water in a final volume of 1 mmol/l (6.978 mg/ml for G3-S16; 4.934 mg/ml for G2-S16). Dilutions to μmol/l range were generated in phosphate-buffered saline (PBS) (Lonza, Walkersville, Maryland, USA) from stocks.
Blood samples were obtained from buffy coats of healthy anonymous donors from the transfusion centers of Madrid and Albacete following national guidelines. Peripheral blood mononuclear cells (PBMC) were isolated with standard Ficoll gradient (Rafer, Spain) and cultured as already described [22,29]. HEC-1A, VK2/E6E7, HeLaP4.2.R5 MAGI that expresses β-galactosidase gene (β-gal) under the control of HIV promoter, TZM.bl that contains integrated copies of the luciferase and β-galactosidase genes under control of the HIV-1 promoter and U87.CD4.CCR5 cell line provenance and maintenance were already described .
Virus stock of R5 HIV-1NL(AD8), R5 HIV-1WT/BAL, X4 HIV-1NL4.3, dual HIV-189.6 –tropic laboratory strains were produced by transient transfection of pNL(AD8), pWT/BaL, pNL4.3 and p89.6, respectively [(all of them from NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID), in 293T cell lines (ATCC-LGC, Teddington, UK)]. The primary clinical R5-tropic HIV-1 isolates R23 (clade A1), X-1936 (clade C), X2160–2 (clade G), X3016 (clade F1), P2392–3 (CRF02_AG) and X-2457–2 (CRF47_BF) were kindly provided by Dr L Pérez (Instituto de Salud Carlos III, Spain). All viral stocks were titrated by p24gag ELISA kit (INNOTEST HIV Antigen mAb, Innogenetics, Belgium).
The reagents used as controls for inhibition of viral replication were: ZDV or zidovudine (Retrovir, GSK) nucleoside reverse transcriptase inhibitor; T-20 (Genentech, South San Francisco, California, USA) inhibitor of HIV fusion step; dextran (Sigma–Aldrich, St Louis, Missouri, USA) harmless molecule that was used as negative control of cellular toxicity and suramin (Sigma–Aldrich) was selected as a positive control of adhesion inhibition .
Cell viability assays
Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St Louis, Missouri, USA) or (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) (Promega, Madison, Wisconsin, USA) assay according of manufacturer's instructions. DMSO 10% (Sigma–Aldrich) was used as positive control of cellular death and nontreated cells were used as negative control. Each experiment was performed by triplicate.
HIV inhibition experiments
Inhibition of HIV internalization
HEC-1A or VK2/E6E7 were seeded in p96 well plates 24 h prior to the experiment (5 × 104/200 μl). After 1 h of pretreatment with a range of dendrimer concentrations, cells were infected 3 h with R5 HIV-1WT/BAL or X4 HIV-1NL4.3 (50 ng/106 cells). Then, cells were washed and lysed and HIV p24gag antigen was measured by p24gagELISA kit. Each experiment was performed by triplicate.
Inhibition of cell-free and cell-associated HIV transmission
To obtain a perfect monolayer of polarized epithelial cells, HEC-1A and VK2/E6E7 were cultured for 7 days on a 0.4 μm pore polycarbonate permeable support (Costar, Cambridge, Massachusetts, USA). Monolayer formation was monitored using an EVOM2STX2 device (World Precision Instrument, Sarasota, Florida, USA) as already described . Transepithelial Electrical Resistance (TEER, Ω × cm2) was measured and was followed for 1 h when monolayers were treated with dendrimers and then infected with 1 μg of HIV-1NL4.3. In cell-free HIV transmission, monolayer was treated for 1 h before addition of 100 ng of p24gag of HIV-1WT/BAL or HIV-1NL4.3 into the upper chamber. Supernatant was collected from the lower chamber at 24 h. On the contrary, PBMC-associated HIV-1NL(AD8), (1 × 105) were added on confluent monolayers of HEC-1A and VK2/E6E7 pretreated with dendrimers. Subsequently, the apical chamber was inserted in the basal chamber of a 24-well plate containing 2 × 105 phytohaemagglutinin (PHA)/IL-2 activated PBMC. After 24 h of incubation, the apical chamber was removed and the PBMC in the basal chamber were maintained for 7 days in IL-2 medium. The viral p24gag antigen concentration was quantified by p24gagELISA kit. Each experiment was performed by triplicate.
Inhibition of HIV replication
Activated PBMC were treated with different concentrations of dendrimers for 1 h and then infected with 50 ng of p24gag/106 cells of different HIV-1 strains for 3 h. After 72 h, supernatant of infected cells was collected and viral concentration was determined by p24gagELISA kit. U87.CD4.CCR5 were treated with serial dilutions of dendrimers. Cells were then infected for 3 h with 5 ng of p24gag of HIV-1NL(AD8), HIV-1WT/BAL or HIV-189.6 and after 72 h, supernatant was collected and HIV was measured using the p24gag ELISA Kit.
CD1(ICR) mice vaginal irritation study
This study was conducted in accordance with Hospital General Universitario Gregorio Marañón (HGUGM) Animal Care and Use Committee approval. Female CD1(ICR) mice, 6–8 weeks old were maintained under specific pathogen-free conditions in the Animal Facility of the HGUGM. Twenty estrous mice (selected by vaginal smears) were inoculated into vagina with 25 μl of G3-S16 or G2-NF16 in PBS at the concentration of 5 and 50 μmol/l or PBS alone (control mice). Two specimens were in each experimental group. Two hours and 24 h after the treatment, vaginas were excised, fixed, and embedded in paraffin. All tissue sections were examined in Anapath S.L (Animal Anatomopathologycal Laboratory, Granada, Spain).
Semen samples were stimulated by swim-up and aliquots of each sample were added to tubes containing 1 ml of Sperm Prep (MediCult, Denmark) to achieve a final concentration of progressive motile sperm of 5 × 106 per ml. Calculated amounts of each dendrimer and a mixture of them were added in test tubes. Nontreated sperm was used as control. All tubes were prepared in duplicate. The sperm progressive motility (%) and sperm survival index were examined at 24 h using the Sperm Class Analyzer automated v5.0 (Microptic, Barcelona, Spain).
Statistical analysis performed included the calculation of the mean, SD and P-values using Mann–Whitney U nonparametric test. The significance level was set as P = 0.05. It was performed with GraphPad Prism V5.0 (San Diego, California, USA).
G3-S16 and G2-NF16 dendrimers biosafety in HIV target cells
As microbicide compounds are in direct contact with female genital tract epithelium, nontoxic concentrations of both dendrimers were determined in human epithelial cell lines derived from uterus (HEC-1A) and vagina (VK2/E6E7). Dendrimer concentrations with a viability 80% or more compared with control cultures were considered as nontoxic. Cells were treated 24 h with a range concentration of G3-S16 and G2-NF16 (Supplemental Fig. 1, http://links.lww.com/QAD/A312). Both dendrimers were considered nontoxic at 5 μmol/l in HEC-1A and at 3 μmol/l in VK2/E6E7 by MTT assay (Fig. 1a–d). The biocompatibility of the dendrimers was also evaluated in PBMC and in U87.CD4.CCR5 cell line, which expresses high level of CCR5 co-receptor by MTT (Supplemental Fig. 2a, http://links.lww.com/QAD/A312). G3-S16 was nontoxic at 5 μmol/l in PBMC and at 10 μmol/l in U87.CD4.CCR5 and G2-NF16 was nontoxic at 10 μmol/l in PBMC and 5 μmol/l in U87.CD4.CCR5. To confirm these results, MTS assay was equally used to evaluate toxicity of dendrimers at 24 h in HEC-1A, HeLaP4.2.R5, TZM.bl and VK2/E6E7 cell lines derived from utero and vagina (Supplemental Fig. 2b-c, http://links.lww.com/QAD/A312). Neither of both dendrimers was toxic at 10 μmol/l in all cell lines tested, except in VK2/E6E7, with a nontoxic concentration of 3 μmol/l. Finally, an in-vitro working concentration of 5 μmol/l was chosen as nontoxic for all types of cells, except for VK2/E6E7 (3 μmol/l).
G3-S16 and G2-NF16 dendrimers activity in vaginal cell lines
Urogenital epithelial cells have the capacity to transfer infectious virus after the uptake of infectious particles. To establish the microbicidal potential of G3-S16 and G2-NF16 dendrimers, these compounds were tested to prevent HIV-1 particle internalization in pretreated HEC-1A and VK2/E6E7. At the higher concentration of G3-S16, 51 and 61% reduction of HIV internalization was achieved for X4 HIV-1NL4.3 and R5 HIV-1BALviral strains in HEC-1A in comparison to HIV-infected nontreated cells (Fig. 1b). In the case of VK2/E6E7 cells, 53 and 64% inhibition was achieved for X4 HIV-1NL4.3 and R5 HIV-1BAL (Fig. 1c). The same inhibitory effect was showed by G2-NF16. Thus, G2-NF16 was able to reduce the HIV internalization in HEC-1A 49% for both HIV strains and in VK2/E6E7 70% for X4 HIV-1NL4.3 and 68% for R5 HIV-1BAL (Fig. 1e–f).
We also studied if the dendrimers were able to prevent the transmission of cell-free and cell-associated virus through the epithelial barrier. For this purpose, we used transwell assays to test the ability of viral particles or PBMC-associated R5 HIV-1 to cross monolayers formed by epithelial cells pretreated with dendrimers. Day 7 was setting down for each transwell assay regarding our previous results . In case of cell-free HIV transmission, HEC-1A or VK2/E6E7 monolayers in transwell system were treated with G3-S16 or G2-NF16 for 1 h and then infected with X4 HIV-1NL4.3 or R5 HIV-1BAL. Transwell supernatants were taken off from the lower chamber at 24 h and viral antigen p24gag was quantified by ELISA (Fig. 2a–b). Non significant changes in HIV transmission though HEC-1A monolayer was observed for both G3-S16 and G2-NF16 when infected with X4 HIV-1NL4.3 (5 and 13% of reduction, respectively) (Fig. 2a) or R5 HIV-1BAL. Otherwise G3-S16 and G2-NF16 decreased the X4 HIV-1NL4.3 transmission (28% and 26%, respectively) through VK2/E6E7 monolayer (Fig. 2b). However, nonsignificant reduction was showed by G3-S16 and G2-NF16 in the transmission of R5 HIV-1BAL (12 and 17%, respectively). We also evaluated the passage of free X4 HIV-1NL4.3 particles across a HEC-1A monolayer during the first hours of infection (Fig. 2c). A delay in viral transmission was observed when HEC-1A monolayer was treated with G3-S16 or G2-NF16. Seventy percent and 86% of reduction in X4 HIV-1NL4.3 transmission was achieved after 1 h for G3-S16 or G2-NF16 treatment, respectively. Furthermore, the transmission of cell-associated R5 HIV-1NL(AD8) was evaluated. A reduction of PBMC infection in basal chamber was achieved in HEC-1A with G3-S16 and G2-NF16 (31 and 15% of reduction, respectively) (Fig. 2e). The same effect was observed in case of VK2/E6E7 (Fig. 2f) (24 and 22% of inhibition in PBMC-associated virus transmission for G3-S16 and G2-NF16, respectively).
Several clinical studies have shown that HIV-1 infection is associated with increased permeability of the intestinal tract and HIV-1 could directly breach the integrity of mucosal epithelial barrier, allowing translocation of virus . Therefore, we followed TEER value for 1 h when a monolayer of HEC-1A cells was treated or not with 5 μmol/l concentration of G3-S16 or G2-NF16 and then infected with X4 HIV-1NL4.3 (Fig. 2d). Sixty minutes later, TEER was reduced in 10% when the monolayer was treated with X4 HIV-1NL4.3 but TEER was maintained overtime when the monolayer was previously pretreated with dendrimers. These data suggest that G3-S16 and G2-NF16 could protect urogenital epithelial cells from the tight junction disruption induced by HIV-1.
G3-S16 and G2-NF16 dendrimers anti-HIV activity in primary blood human cells
Once viral particles have crossed the first genital epithelial barrier, activated PBMC are the main target of HIV. Activated PBMC were pretreated for 1 h with different concentrations of G3-S16 or G2-NF16 and infected with X4 HIV-1NL4.3 and R5 HIV-1NL(AD8) (Fig. 3a) and after 72 h antigen p24gag was quantified in the supernatant of the culture. 5 μmol/l nontoxic concentration of G3-SF16 or G2-NF16 inhibited in a significant and strong way the infection of activated PBMC by X4 and R5 HIV-1 (95 and 35% of inhibition for G3-S16; 90 and 52% for G2-NF16, respectively). The antiviral activity of both dendrimers in PBMC against several primary clinical R5 tropic HIV-1 isolates from different clades was also evaluated (Fig. 3b). The inhibitory effect of dendrimers was different in function of the viral subtype. The best results for G3-S16 were observed for clade C and G (80 and 88% of inhibition, respectively) whereas G2-NF16 was more effective against F1 and AG subtypes (84 and 85% of reduction). It is interesting to note that both dendrimers showed high inhibitory effect against R5 CRF02_AG subtype, one of the most prevalent in West Africa  (73 and 85% of inhibition).
Since CCR5-tropic HIV-1 is the predominant during initial HIV transmission , we evaluated the effect of dendrimers in R5 HIV-1 infection using U87.CD4.CCR5 cell line. The cells were pretreated with G3-S16 or G2-NF16 and then were infected with R5 HIV-1NL(AD8), R5 HIV-1BAL or R5X4 HIV-189.6 (Fig. 3c). At 5 μmol/l concentration both dendrimers were able to inhibit significantly the HIV infection of different R5 HIV-1 isolates in U87.CD4.CCR5 cells (Fig. 3c).
Moreover, in order to determine dendrimers efficacy, EC50 (50% reduction in virus production) were calculated in TZM.bl cells . Both dendrimers were more efficacious against X4 (G3-S16 EC50 = 0.0493 μmol/l; G2-NF16 EC50 = 0.0276 μmol/l) than against R5 tropic strains (G3-S16 EC50 = 1.22984 μmol/l; G2-NF16 EC50 = 1.09449 μmol/l; Supplemental Fig. 3, http://links.lww.com/QAD/A312).
Effect of G3-S16 and G2-NF16 dendrimers on human primary and epithelial cell lines proliferation and inflammation
We studied the dysregulation capability of these dendrimers in human primary cells or epithelial cell lines. First of all, we examined if pretreatment of PBMC with G3-S16 or G2-NF16 had any effect on proliferation activity. CFSE-labeled PBMC treated with PHA were used as proliferative positive control and proliferation was determined as the loss of CFSE at day 5 posttreatment. None of the tested concentrations stimulated the proliferation of PBMC in comparison to untreated cells (Supplemental Fig. 4A, Supplemental methods, http://links.lww.com/QAD/A312). Afterwards, we researched if pretreatment of PBMC with dendrimers had any effect on their subsequent proliferation at the onset of a proliferative stimulus. For that purpose, pretreatment with G3-S16 or G2-NF16 at different times before activation were used (Supplemental Fig. 4B, http://links.lww.com/QAD/A312). No proliferation differences were found when PBMC were pretreated with dendrimers for 1, 4 or 24 h previously to 2 μg/ml PHA stimulus, except for G2-NF16 at 5 μmol/l, when significant but a slight reduction in proliferation was found (Supplemental Fig. 4B, http://links.lww.com/QAD/A312).
The main application of topical microbicides is the protection of infection at level of urogenital and rectal mucosas. Thereby, we considered whether dendrimers could have a potential inflammatory or deregulate effect on epithelial cells. To determine the inflammatory process, changes on profile of cytokines expression were studied using Diaplex Human TH1/TH2/inflammation kit after 30 min, 1, 3, or 24 h of treatment. Treatment of HEC-1A and VK2/E6E7 monolayers with both dendrimers did not increase expression levels of cytokines tested at different times, compared with untreated cells (Supplemental table 1&2, http://links.lww.com/QAD/A312).
Summing up, our results suggest that G3-S16 and G2-NF16 did not induce cell proliferation and cellular inflammatory process. These properties are essential to make available a secure topical microbicide.
In-vivo assay of G3-S16 and G2-NF 16 in mice model
To assess whether the topical administration of dendrimers could cause vaginal irritation, experiment with female mice from CD1(ICR) strain was performed [35,36]. Mice were treated with different concentrations of G3-S16 and G2-NF16. At 2 and 24 h after vaginal application, irritation and other epithelial lesion parameters were studied. No mortality or sign of vaginal discharge, erythema or edema in mice studied at the lowest dose (5 μmol/l) or higher dose (50 μmol/l) of G3-S16 and G2-NF 16 was detected (Table 1). No characteristic abnormality was observed during histopathological analysis of vaginal tissues compared with the negative control (vehicle). The vaginal irritation index was calculated from the score of the microscopic observations and values of 0.5 and 1 for the higher dose of G3-S16 and G2-NF 16 at 24 h, respectively, confirmed the biocompatibility of both dendrimers (Table 1). We concluded that both dendrimers did not induce vaginal irritation or inflammation and they seemed to be well tolerated for topical application.
Antimicrobial and spermicidal activity of G3-S16 and G2-NF 16
In the primary screening assays to identify topical microbicides, compounds toxicity in sperm and normal vaginal flora have to be analyzed to assure the biocompatibility and safety of candidates . First, toxicity of the potential microbicidal dendrimers G3-S16 and G2-NF16 against Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, Lactobacillus plantarum, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans, observed in normal vaginal microbiota was measured. No antimicrobial activity against the listed microorganism was observed at the concentrations tested (data not shown).
Moreover, the human sperm survival assay was also performed. Sperm were cultured in the presence or absence of G3-S16 and G2-NF16, and progressive motility of the sperm was measured. Nonreduction in sperm survival index, calculated from changes in progressive motility compared with control condition, after 24 h of treatment was found (Table 2). Summing up, polyanionic carbosilane dendrimers tested are safe to use as topical vaginal microbicide since sperm and vaginal female flora were not affected.
For the last 20 years, the idea of alternative prevention strategies based on the use of topical vaginally dosed products to inhibit HIV infection in women has been established [9,15,38]. Dendrimers are being researched as nonspecific microbicides  and successful first results have been achieved in human clinical trials with SPL7013, a polyanionic dendrimer with 32 naphthalene sulphonate ended groups .
Carbosilane dendrimers are stable compounds and their microbicidal capacity has been previously reported . Two new anionic carbosilane dendrimers with sulphated and naphthylsulphonated peripheral groups, G3-S16 and G2-NF16, respectively, have been studied. We evaluated their biosafety in different types of cells and their effectiveness inhibiting HIV transmission and viral infection in order to characterize their microbicidal potency.
It has been reported that in heterosexual transmission of HIV-1 only virus utilizing CCR5 but not CXCR4 was apparently transmitted and dominant in the early stages of HIV-1 disease . However, X4-tropic HIV-1 variants were present in semen too , although their role in sexual infection is still unknown. G3-S16 and G2-NF16 were able to inhibit cell-associated R5 HIV transmission through transepithelial monolayer in vitro at the first moments of the infection process and could block HIV internalization inside epithelial cells showing ability to protect the integrity of the monolayer. As it has been reported, the direct exposure of genital epithelial cells to HIV-1 lead to breaching of the mucosal barrier and increased leakage of HIV-1 across the epithelium by direct interaction between epithelial cells and HIV-1 gp120 . Furthermore, both dendrimers were highly active against X4 HIV-1 infection in PBMC and showed great inhibitory effect against different R5 HIV primary subtypes in PBMC. Polyanionic HIV inhibitors are traditionally considered as entry inhibitors and their mechanism is associated with electrostatic interactions between HIV gp120 and different functional groups of these agents that ultimately prevent binding of the virus to the target cells [22,42]. However, the inhibitory mechanism of tested dendrimers has still to be elucidated and we cannot rule out that dendrimers could interact with others surface markers like CD4, CXCR4 or CCR5 or triggering a series of events that inhibit some steps of viral replication . Further experiments and molecular modeling assays should be performed in this way. Moreover, development of resistance to these entry inhibitors could be one of the main problems when testing as microbicides in human trials. Therefore, it will be interesting to test them in combination with other antiretroviral drugs as combined drug microbicides aimed to effectively block HIV transmission and avoid resistance profiles .
In the last years, from all ongoing clinical trials of topical microbicide candidates, only one had shown positive results in humans [7,9]. Moreover, some microbicides have been described as increasing HIV infection associated with damage and induction of local inflammation or irritation in the vaginal epithelium, such as cellulose sulphate [45,46]. We showed that treatment of epithelial cells with the G3-S16 or G2-NF16 did not produce changes in proinflammatory cytokines profile, proliferation of PBMC, microbiota or sperm survival. It is interesting to note that even if higher toxicity was detected with the specific vaginal cell line VK2/E6E7 when treated with the both compounds, no irritation, inflammation or vaginal lesions were detected in female CD1(ICR) mice after dendrimers vaginal administration. All these findings suggest the in-vivo safety of these candidates.
Despite the success of some microbicide candidates in preclinical evaluations, finally the majority of them failed in last phases of human clinical trials. Therefore, starting to evaluate topical microbicide candidates’ activity in more physiological condition as closer as possible of conditions at early stages during HIV-1 infection is essential. These include the study of the impact of microbicides on vaginal and rectal fluid, mucosal immune-cross-talk, physical trauma of sex and possible epithelial injury and inflammatory consequences in microbicide local application . The role of all of these parameters in carbosilane dendrimers G3-S16 or G2-NF16 activity should be evaluated in the future.
We acknowledge the Centers of Transfusion of Madrid and Albacete for the buffy coats and Spanish HIV BioBank for the process of these buffy coats and Laura Díaz from cytometry unit of HGUGM for FACS samples analysis.
This work was supported by grants from Fondos de Investigación Sanitaria (INTRASALUD PI09/02029, P509102669), Red Temática de Investigación Cooperativa Sanitaria ISCIII (RETIC RD06/0006/0035), INDISNET S-2011-BMD2332, FIPSE, Cost action TD0802, CTQ2011-23245 (MEyC), NANODENDMED S2011/BMD-2351 and the Spanish MICINN through the Ramon y Cajal (RYC-2009–05486) to M.P.
Authors’ contribution: M.M.F. conceived the study. M.M.F. and M.P. have participated in the design. E.V.C., carried out the majority of experimental work and MP collaborated in them. R.G. and F.J.D.M. participated in the design of dendrimers and R.G., F.J.D.M. and E.A. performed the synthesis and chemical and physical characterization of dendrimers. M.R. collaborated in the in-vivo experiments with CD1(ICR) mice. C.S.T. performed the assays of spermicidal activity with dendrimers. F.D. carried out the experiments related with the microbicidal activity of dendrimers. L.P.A. provided the primary human HIV-1 isolated and collaborated in the inhibition experiments with them. M.P., E.V.C. and M.M.F. have participated in writing, revision and discussion of the article. All the authors read and approved the final article.
Conflicts of interest
The authors confirm that there are no known conflicts of interest associated with this publication.
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