In situ stabilized AgNPs and (Cu-Cur)CD dispersed gel, a topical contraceptive antiretroviral (ARV) microbicide

Abstract

The increasing scenario of sexual HIV-1 transmission and unintended pregnancies demands modern approaches to concomitantly tackle these problems. Dually active strategies with “microbicidal” anti-human immunodeficiency virus (anti-HIV) as well as contraceptive properties constitute one such strategy. Nano-metal and metalo-herbal technology was explored to develop such an approach with minimal toxicity. Synthesis of in situ stabilised silver nanoparticles (AgNPs) of 2–24 nm and a (copper-curcumin)β-cyclodextrin (Cu-Cur)CD inclusion complex was carried out. Cell viability and activity analysis revealed an acceptable dose of 500 μg ml⁻¹ of silver nanoparticles and 10 mg ml⁻¹ of (Cu-Cur)CD respectively. AgNP dispersed (Cu-Cur)CD incorporated carbopol 974p gel was prepared and characterized for its pharmaceutical properties. AgNPs, (Cu-Cur)CD and the whole formulation was evaluated for in vitro anti-HIV, spermicidal and antifungal potential. Results revealed that HIV-1 propagation and sperm motility was completely inhibited at folds of dilution levels. In vivo mating studies proved the contraceptive potential of the formulation. A pre-clinical toxicology study assures the acceptability of our formulation as an intravaginal product. The novel nano-metalo-herbal strategy was found to possess a potential for topical contraceptive antiretroviral (ARV) microbicide action with an undeniably safe profile to be used as a vaginal product.

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1. Introduction

The incessant high rates of the acquired immune deficiency syndrome (AIDS) and the brutal increase of unintended pregnancies, specifically in less developed countries, provoke the development of novel strategies to help individuals avoid these risks. Unprotected sexual intercourse is the most common mode and accounts for more than 75% of infections worldwide.^1–3 Sexually transmitted HIV infection and sperm fertilization share the same anatomical and functional context, and therefore offer an opportunity for simultaneous intervention.⁴ Condoms are considered as the best way to tackle this problem. But their denial, inconsistent or incorrect use and failure favour this misfortune. Further, a large population is not comfortable with oral contraceptives; this creates a thought of having a women oriented approach which can be easily used to avoid this epidemic.⁵ Dually active compounds displaying “microbicidal” anti-human immunodeficiency virus (anti-HIV) as well as contraceptive properties are the best strategy to deal concomitantly with this problem. “Contraceptive Microbicides” is the suitable term for such an approach.^4,6,7

Surface active agents, pH buffering agents, receptor blockers and enzyme inhibitors acting on sperm and HIV during primary events, antimicrobial peptides etc. represent the brief categorization of the compounds already targeted for the purpose of dual efficacy.⁸ But clinically very few were tested and found to be unacceptable because of either lack of efficacy or safety.⁷ Applied intravaginal microbicide (viral and sperm membrane disrupting agent) nonoxynol-9 (N-9) has shown contraceptive efficacy but the product failed in clinical trials due to the lack of safety, which unfortunately resulted in increased HIV incidents.⁹ Exploring compounds having dual characteristics with the advent of nanotechnology and herbal considerations will be a new direction. Moreover using combinations, with one compound having anti-HIV and the other contraceptive potential, can also be a good approach. With this viewpoint a selection of silver nanoparticles (prominent anti-HIV agents) and a pre-synthesised metaloherbal complex (having spermicidal potential) were explored simultaneously.

In this work we explored the potential of nano-metal and metalo-herbal technology for the development of contraceptive microbicide compounds having a dual activity profile and minimal toxicity. In our previous research we successfully explored a contraceptive moiety i.e. the (copper-curcumin)β-cyclodextrin inclusion complex (Chauhan et al. 2014).¹⁰ The present work is based on a hypothesis to deliver this inclusion complex along with a nanometal i.e. silver nanoparticles (AgNPs). Here the cyclodextrin inclusion complex helped to resolve the major problem of aqueous solubility related with this metalo-herbal complex. In situ stabilized AgNPs were synthesised by regulating the reduction process of the tannic acid (a polyphenolic compound derived from plant extracts) mediated reduction of silver salt. Capping of AgNPs with tannic acid residues during this reduction process provides a surface stable silver dispersion to be used for combinational use with the dextrin complex.

The mode of action of the silver nanoparticles against HIV-1 is not fully elucidated. The mixed viewpoints from different researchers make us unable to agree with one mechanism.^11–13 Studies suggested that AgNPs act at the primary stage of viral replication, as a virucidal agent or as an inhibitor of viral entry. Their affinity for the gp120 viron receptor prevents CD-4 dependent binding, fusion, and infectivity.¹⁴ Some mechanistic explanations revealed that AgNPs might preferentially interact with the negative cavity of gp120 where the interaction with the two disulphide bonds situated in the carboxyl half of the glycoprotein modifies this viral protein by denaturing its disulphide-bonded domain.¹⁵

In this study, both the moieties were initially evaluated for vaginal epithelial cell cytotoxicity, anti HIV-1 activity and spermicidal activity. Dose selection of both the components was done by analysing the efficacy levels and the toxicity levels (on vaginal epithelial cell toxicity). Finally a hydrogel formulation containing an appropriate dose of both these moieties was prepared and characterised. The nano-herbal microbicidal gel (NHM gel) was then tested for in vitro pre as well as post exposure prophylactic studies using different tropisms of HIV-1 strains. The NHM gel was then analysed for its contraceptive efficacy using both in vitro tests and in vivo mating challenges in Wistar rats. Supplementary to the above activities, the NHM gel and its components were tested against a most common sexually transmitted candida infection, as these kinds of infections assist the dissemination of HIV-1 from the vaginal epithelium. Finally its vaginal safety was evaluated by pre-clinical toxicology studies on Wistar rats, Albino rabbits and vaginal lactobacillus bioflora.

2. Materials & methods

Silver nitrate, tannic acid, mucin, Dulbecco’s modified eagle medium (DMEM), RPMI-1640, fetal bovine serum, phytohaemagglutinin (PHA), ficoll-hypaque and 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium (MTT), curcumin, bovine serum albumin (BSA), β-cyclodextrin, tergitol, and triethanolamine were purchased from Sigma Aldrich (Bangalore, India). Carbopol 974p was procured as a gift sample from Lubrizol (Mumbai, India).

R5 HIV-1_Ba-L was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, US. HeLa cells were procured from NCCS, Pune while the cell culture exposed in the MTS assay (HeLa, HEL, VERO, CRFK, MDCK) was included in the study protocol performed at the Department of Virology, Leuven, Belgium. C. albicans, C. tropicalis, L. acidophilus and L. jensenii were procured from IMTECH Chandigarh.

2.1 Synthesis, optimization and characterization of in situ stabilized silver nanoparticles

AgNPs were synthesized by using the tannic acid (a polyphenolic compound derived from plant extracts) dependent reduction of silver nitrate.¹⁶ The molar concentration ratio of tannic acid to silver nitrate was controlled with a constant silver nitrate (3 mM) concentration, while varying the concentration of the tannic acid (0.0375, 0.075, and 0.15 mM) sequence in order to achieve the respective molar concentration ratio patterns of 0.125, 0.025, and 0.05. Furthermore, at each molar ratio, three different pH value exposures of the tannic acid solution were made (pH 7, pH 8 and pH 9, adjusted with K₂CO₃) for the synthesis of AgNPs.

At ambient temperature, the flow rate of the silver nitrate addition is kept constant (55–60)μl s⁻¹ under continuous magnetic stirring. Stirring is stopped immediately after the complete addition of the silver nitrate solution and the whole reaction is strictly done in the absence of light. The synthesized AgNP dispersions were optimized on the basis of ultraviolet-visible spectroscopy, particle size and polydispersity index. Optimized AgNPs were then purified and washed by centrifugation at 21 913 × g for 30 min at 4 °C using distilled water and characterized by ultraviolet-visible spectroscopy using a UV-VIS spectrophotometer (UV-1700, Shimadzu, Japan), and particle size, polydispersity index, and surface potential were obtained using a particle size analyser (Zeta sizer, DLS 4C Beckman Coulter, Japan). X-ray powder diffractometry was carried out using a Bruker diffractometer (AXS D8 Advance, Karlsruhe, Germany). Thermogravimetric analysis (TG) was carried out using a Perkin Elmer TG/DTA analyzer (STA 6000, Massachusetts, U.S.A) operating in the range of 40 °C to 740 °C at a temperature rise of 10.00 °C min⁻¹, and the morphology was characterized using a transmission electron microscope (TEM) (Hitachi H-7500, Georgia, U.S.A) operating at 100 kV. Surface characteristics were studied using atomic force microscopy in contact mode on a multimode scanning probe microscope equipped with a Nanoscope IV controller at a scan rate of 5.086 Hz (Veeco Instruments, New York, U.S.A). Stability studies were also performed as per the ICH guidelines (ESI†).

2.2 Synthesis, optimization and characterization of the copper-curcumin (Cu-Cur) and copper-curcumin-β-cyclodextrin (Cu-Cur)CD inclusion complex

The metal ligand (M–L) complex of copper (M) and curcumin (L) had already been synthesized by many researchers. The inclusion complex was prepared by the solvent evaporation encapsulation method.¹⁰ (Work has been published already by Chauhan et al., 2014, also provided in the ESI†.)

2.3 Cell viability assay (MTS assay), peripheral blood mononucleated cell (PBMC) toxicity assay and haemolytic assay

2.3.1 MTS assay. The (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) dye reduction assay, in the presence of phenazinemethosulfate (PMS), produces a formazan product that has an absorbance maximum at 490–500 nm in phosphate-buffered saline.¹⁷ The MTS assay was performed to determine the cytotoxicity of the optimized AgNPs and MC-40 (pre-optimized metal herbal complex) in different cell cultures. The cell viability assay was performed to study both the specific cell toxicity on HeLa cells and the non-specific toxicity on the HEL (human embryonic lung), VERO, CRFK (Crandell-Rees Feline Kidney cells) and MDCK (Madin Darby canine kidney cell) cultures. Moreover the assay was also performed on CEM–GFP cells (HIV-1 host cells) to assess the direct cytotoxic potential of AgNPs and MC-40 which may result in an erroneous conclusion of the anti-HIV data.

2.3.2 Haemolytic assay. The haemolytic assay was done on isolated RBCs from Wistar rat blood (as per the ethical guidelines) to assess the irritation potential of the challenged moieties. Varying concentrations of AgNPs and MC-40 in phosphate buffered saline (i.e. PBS 7.4) and 0.1% Triton X-100 are used and treated as the positive control and negative control, respectively, and incubated at 37 °C for 30 min with mild shaking to attain complete haemolysis. The reaction was quenched in ice, the samples were centrifuged at 1500 rpm for 2 min, and the supernatants were analysed by a UV-Vis spectrophotometer at 576 nm.¹⁸ The percentage haemolysis was then calculated by the eqn (1).

%H = 100% (Abs − Abs_PBS)/(Abs_tx − Abs_PBS) (1)

2.4 Anti-HIV activity

Anti-HIV assays were performed on two host cells viz. CEM–GFP cells (expressing CD4, CD8, CCR5 and CXCR4 receptors) and phytohaemagglutinin (PHA) stimulated peripheral blood mononucleated cells (PHA-PBMCs), challenging them against the HIV-1_NL4.3 and HIV-1_Ba-L viral strains respectively.

In order to mimic the prophylactic challenge, the study was designed to cover two different possible interactions. Pre-interaction (interaction-1 assay) was performed, where the test compounds interacted with the virus first and later that virus was allowed to infect the cells. Here we tried to elucidate the probable surface receptor blocking or direct virus denaturing mechanism of the test compounds. On the other hand in post-infection (interaction-2 assay), the virus was allowed to do the job, i.e. the cells were initially infected with the virus, followed by the addition of the test compounds. Here we tried to inspect the ability of our test compounds to halt the infection after the virus has established an infection. Both these interaction can be better understood from the cartoon presented in Fig. 1.

Fig. 1 Designing the anti-HIV study on the basis of possible HIV-1 interactions with either the formulation components or the host cell.

2.4.1 Anti-HIV assay using CEM–GFP cells. CEM–GFP cells in an RPMI-1640 medium were supplemented with 10% FBS, penicillin (100 units per ml), streptomycin (100 μg ml⁻¹) and amphotericin B (250 ng ml⁻¹). For the pre-infection interaction study (interaction-1), different concentrations of AgNPs and (Cu-Cur)CD i.e. MC-40 were allowed to interact separately with 50% tissue culture infectivity doses (TCID₅₀) of HIV-1_NL4.3 for 4 hours and later added to different wells in the 96-well plate seeded with CEM–GFP cells (2.0 × 10⁵ cells per ml) and incubated at 37 °C and 5% CO₂. While for the host-interaction study (interaction-2), different concentrations of AgNPs and MC-40 were incubated with the CEM–GFP cells (already plated in each well of the 96-well microculture plate) for a period of 4 h. After this incubation, these cells were exposed to a TCID₅₀ dose of HIV-41_NL4.3 and incubated at 37 °C and 5% CO₂.

After the 7^th day of incubation of both the incubated challenged cultures, cell free supernatants (100 μl each) were collected and investigated for HIV-1 p24 antigen levels using a microplate ELISA reader. Percent inhibition of the p24 antigen by the test compounds was compared to the positive control (infected cells without any test compound).^19–21

2.4.2 Anti-HIV assay using phytohaemagglutinin-stimulated peripheral blood mononuclear cells (PHA-PBMCs). Healthy uninfected PBMCs were isolated from the blood by ficoll-hypaque density gradient centrifugation and cultured in a RPMI-1640 culture medium supplemented with 20% fetal bovine serum (FBS), penicillin (50 U ml⁻¹), streptomycin (100 μg ml⁻¹), PHA (10 μg ml⁻¹) and interleukin-2 IL-2 (10 units per ml) for 3 days prior to the anti-HIV assay. For selective growth of the T cells and over-expression of the surface markers, like CD4, PBMCs were activated with PHA and IL-2 prior to infection. On day 3, PHA-PBMCs were resuspended in a minimal growth medium (RPMI-1640, 10% FBS and IL-2 (10 units ml⁻¹)) and plated in each well of the 96-well microculture plate at a density of 0.5 × 10⁶ cells per ml. In this study, the potential of the formulation components was evaluated against the R5 HIV-1_Ba-L virus.

For the pre-infection interaction study (interaction-1), different concentrations of AgNPs and MC-40 were allowed to interact separately with a TCID₅₀ dose of HIV-1_Ba-L for 4 hours and later added to different wells in the 96-well plate seeded with PBMCs and incubated at 37 °C and 5% CO₂. For the host-interaction study (interaction-2), different concentrations of AgNPs and MC-40 were incubated with PBMCs (already plated in each well of the 96-well microculture plate) for a period of 4 h. After incubation, these cells were exposed to a TCID₅₀ dose of HIV-1_Ba-L and incubated at 37 °C and 5% CO₂.

After the 7^th day of incubation of both the incubated challenged cultures, the cell free supernatants (100 μl each) were collected and investigated for HIV-1 p24 antigen levels using an ELISA reader. Percent inhibition of the p24 antigen by the test compounds was compared to the positive control (infected cells without any test compound).

2.5 Spermicidal activity

2.5.1 Modified Sander–Cramer assay (sperm motility inhibition assay). Human sperm were obtained from three consenting healthy donors after 72 h of abstinence. The samples were washed once in Ham’s F10 containing 0.1% human serum albumin (HSA) and centrifuged. The sperm were resuspended to give a concentration of 60 million motile sperm per millilitre. The spermicidal activity of AgNPs and MC-40 was evaluated. Sequential dilutions of AgNPs and MC-40 were prepared in Ham’s F10. Tergitol NP-9 was used as a positive control, while a medium containing no test compounds was employed as a negative control. Aliquots of sperm of 50 μl were incubated (37 °C in the presence of 5% CO₂) with various concentrations of the test ingredients in a final volume of 100 μL. After 120 s, the reaction was terminated with 1.5 ml Ham’s F10 and centrifuged for 10 min at 290 × g to obtain the sperm pellet, which was further resuspended in 100 μL of Ham’s F10. Sperm motility was assessed under an inverted phase contrast microscope (MKX-41, Olympus, Tokyo, Japan).²⁰

2.5.2 Hypo-osmotic swelling (HOS) test. The hypo-osmotic swelling test is based on the loss of semi-permeability of the intact cell membrane, after an exposure to a membrane attacking moiety. Effective concentrations of AgNPs and (Cu-Cur)CD treated spermatozoas were exposed to the HOS solution (75 mM fructose and 20 mM sodium citrate) for at least 30 min at 37 °C to detect changes in the sperm membrane integrity. The number of spermatozoa showing a characteristic tail curling or swelling was counted under an inverted phase contrast microscope.²²

2.5.3 Sperm viability test (fluorescent staining). Dead and alive sperms were differentiated by staining with two different dyes by using the fluorescent red propidium iodide to stain the dead spermatozoa only while the fluorescent green SYBR-14 dye stained the alive ones only. AgNPs (250 μg ml⁻¹) and MC-40 (5 mg ml⁻¹) treated human spermatozoa were dual stained with SYBR-14 and propidium iodide to distinguish the green-fluorescing live from the red-fluorescing dead spermatozoa, in contrast with the dual stained untreated spermatozoa. The sperm count was taken from 200 spermatozoa under a phase contrast microscope.²³

2.5.4 Apoptotic and necrotic changes in the plasma membrane of the human spermatozoa. The mechanism of the sperm cell death was further studied by dual fluorescent labelling with fluorescein isothiocyanate (FITC)-Annexin V (to study the expression of the phosphatidylserine on the apoptotic sperm cells) and propidium iodide (to study the enhanced membrane permeabilization of the necrotic cells). Highly motile human sperm (2 × 10⁶) was incubated with AgNPs (250 μg ml⁻¹) and MC-40 (5 mg ml⁻¹) in Ham’s F10 media. After incubation, the sperms were washed in 1% BSA in Tyrode’s buffer and labelled with fluorescent dyes. Apoptotic study was done using a detection kit and the percentages of the cells positive for FITC and PI were determined using a flow cytometer (BD Accuri C6, CA, U.S.A). During identification, the unstained cells were marked as viable, FITC stained were marked as apoptotic, both FITC and PI labelled were marked as late apoptotic cells and only the PI stained were marked as necrotic.²⁴

2.6 Anti-candida activity

Candida infection is the most commonly seen sexually transmitted infection which can prominently assist vaginal HIV-1 transmission. The minimum inhibitory concentration (MIC) was assessed for C. albicans and C. tropicalis using a serial dilution assay using a 96-well flat-bottomed microtiter plate.²⁵ (Detailed study protocol is given in the ESI.†)

2.7 Formulation and characterization of the nano-herbal microbicidal gel (NHM gel)

Dose selection for AgNPs and MC-40 was done on the basis of the selectivity index (IC₅₀/EC₅₀) which provides a safe and effective range to work with. Nano-herbal gel was formulated using an optimized concentration of AgNPs and MC-40 (i.e. 500 μg ml⁻¹ of AgNPs and 10 mg ml⁻¹ MC-40) in 1% w/v carbopol 974p base. In vessel #1 the carbopol 974p gel was hydrated in distilled water and triethanolamine was added dropwise to make it a clear gel and incubated for 12 h. MC-40 was dissolved in 5 ml distilled water and added dropwise to vessel #1 with constant stirring at 500 rpm. Finally the AgNP suspension was added dropwise to vessel #1 with a mild stirring at 100 rpm until the homogeneous gel was obtained. The developed nano-herbal gel formulation was characterized for pharmaceutical aspects including macroscopic properties, pH, thixotropic/rheological properties and texture analysis using a Rheometer (R/S CPS, Brookfield, Middleboro, U.S.A.) and Brookfield texture analyzer (CT3 10K, Brookfield, Middleboro, U.S.A.) respectively.^26,27

2.7.1 In vitro challenge study for the pharmaceutical parameters of the NHM gel. A Box–Behnken statistical screening design was used to statistically evaluate the vehicle’s behaviour in simulated vaginal conditions (during sexual intercourse) with respect to the pH changes, viscosity, mucoadhesion and spreadibility alterations. The 3-factor, 3-level design used is suitable for exploring the quadratic response surfaces and constructing the second order polynomial models with Design Expert®, Version 9.0.3.1 (Stat-Ease Inc., Minneapolis, U.S.A). A design matrix comprising of 15 experimental runs was constructed. The dependent and independent variables selected are shown in Table 1. Physiological ranges for the vaginal fluid secretions (using vaginal simulated fluid “VSF”), semen secretions (using semen simulation “SS”) and application quantity of the formulation (that can vary in population) were taken as independent variables to mimic the physiological challenges. Different challenges as per the design were evaluated for the NHM gel behaviour with respect to pH, viscosity, mucoadhesion and spreadibility variations. (The experiment was detailed in the ESI.†)²⁸

Table 1 Independent and dependent variables considered during the in vitro challenge study for the NHM gel using a Box–Behnken design

Factor	Levels used
Independent variables	Low (−1)	Medium (0)	High (+1)
X1 VSF (mg)	500	3000	5500
X2 SS (ml)	2	4	6
X3 NHM gel (g)	1	2	3

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Dependent variables (at 37 °C)	Observation basis
Viscosity	An exploratory study to analyse the behaviour of the vehicle gel in the simulated challenged conditions
pH
Mucoadhesion
Spreadibility

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2.8 Pre-infection interaction (interaction-1) anti-HIV study of the NHM gels

2.8.1 On CEM–GFP cells. Different dilutions of the NHM gel were allowed to interact separately with 50% tissue culture infectivity doses (TCID₅₀) of HIV-1_NL4.3 for 3–4 hours (pre-infection interaction), and later added into the different wells which were prior fed with CEM–GFP cells and incubated at 37 °C and 5% CO₂. On day 7 of the culture, the cell free supernatants were harvested and investigated for the HIV-1 p24 antigen (Ag) levels in the supernatant by ELISA. All the experiments were performed in duplicates and the percent inhibition of p24 Ag by test formulation was compared to the control/placebo group (infected cells only).

2.8.2 On PBMCs. A similar experiment as mentioned above was run, challenging the PBMCs with the HIV-1_Ba-L virus. The percent inhibition of p24 Ag by sequential formulation dilutions was compared to the negative control/placebo group (infected cells only).

2.9 Spermicidal activity of the NHM gel (modified Sander–Cramer assay)

The study was performed in a similar manner as described above for the individual formulation component. Different dilutions of the NHM gel in VSF were observed for the in vitro spermicidal activity. 5% tergitol NP-9 was used as a positive control while the medium containing no test compounds was employed as a negative control. The sperm motility was assessed manually under an inverted phase-contrast microscope.

2.10 Challenge mating study in rats

Eighteen female Wistar rats having regular and nearly similar four day estrous cycles were selected in this evaluation. The animals were divided into three groups, each consisting of six rats. The first group (G-1) was left untreated; the second group (G-2) was treated with the placebo, and the third group (G-3) received the NHM gel. In the second and third groups, a 100 μl dose was administered intra vaginally in each animal (twice a day) for seven days. The animals were allowed to cohabit with males of proven fertility at a ratio of 1 male : 2 females on the 8^th day. The animals were examined the following morning for evidence of successful copulation. Animals with spermatozoa in the vaginal smears or mucus plug were separated from the male partners and this was considered as day one of pregnancy.^29,30

2.11 Efficacy of NHM gel against candida infections

C. albicans and C. tropicalis fungal cell strains were used in the present study. Sterile molten potato dextrose agar (PDA) was poured into sterile Petri dishes with (a) different NHM gel dilutions (X, 2X and 4X with VSF) and (b) without treatment (as a control). These test plates containing X, 2X and 4X dilutions (250 μl) were inoculated with 50 μl cell suspensions (1 × 10⁵ cells per ml) of C. albicans (in one set of the triplicate) and C. tropicalis (in another set of the triplicate) using a sterile inoculating loop. Plates were incubated at 25–30 °C in an inverted position keeping the agar side up with increased humidity. Control plates were incubated in a similar manner for 24 h. The number of colonies and their size was measured at the end of the experiment.³¹

2.12 Pre-clinical toxicology study of the nanoherbal gel

Toxicology studies were conducted in both rodent (viz. Wistar rat) and non-rodent (viz. Albino rabbit) species. All the procedures were conducted in accordance with the guidelines approved by the institutional ethics committee of ISF-CP Moga with approval no. TAEC/M4/CPCSEA/P64/2012. The study was performed in 24 female Wistar rats and 12 female Albino rabbits according to the protocol recommended by the US Food and Drug Administration (US-FDA) for products meant for vaginal use. Animals were maintained under light and temperature control (complying with standard husbandry conditions) with food and water ad libitum.^32,33

2.12.1 Pre-clinical toxicology study on female Wistar rats. The animals were divided in three different groups, the untreated “control” (G-I), placebo (G-II) and NHM gel (G-III) groups. Each group consists of 8 rats with an average weight in the range of 160–220 g. 100 μl of intravaginal application was done twice a day for 21 days. Individual observation for overt clinical signs (vaginal swelling, redness, and discharge including bleeding) was done daily. The change in the regularity of the estrous cycle was observed by microscopical examination of the vaginal lavage.³³
2.12.1.1 Effect on local tissues, vaginal tissue proliferation, and in situ apoptosis (after the 21 day protocol). Animals from each group were sacrificed to obtain the vaginal tissues by opening the slit ventrally between the urethral orifice and fornix. Macroscopic examination of the excised vaginal tissue was done for surface defects, marks of inflammation and any kind of ulceration. After that, representative samples of the proximal, middle and distal portions were collected and fixed in a 10% neutral buffered formalin fixative. Fixed tissues were embedded in the paraffin, sectioned at a thickness of 5 μm, stained with hematoxylin and eosin, and examined under an inverted phase-contrast microscope. The vaginal tissue sections were observed for epithelial ulceration, edema, leukocyte infiltration, and vascular congestion.³⁴
2.12.1.2 Haematology study. A study was performed to predict the chances of systemic infection when compared with the placebo and control groups. After 7 days blood samples were collected from the rats and CBC (complete blood count) and DLC (differential leucocyte count) were carried out using a flow cytometer as well as a standard microscopical analysis.
2.12.1.3 Organ distribution study. Because AgNPs possess a lower therapeutic safety window as compared to MC-40 (as determined in in vitro results) systemic effects were observed by studying the AgNP’s organ accumulation. After 21 days, the rats were sacrificed and the visceral organs (kidney, lungs, liver, brain and heart) of the dissected rats were removed and washed to remove any adhered debris and dried using a tissue paper. Isolated organs were weighed separately and homogenized in PBS pH 7.4 using a tissue homogenizer. They were centrifuged at 6000 rpm for 30 minutes. The supernatant was separated, filtered and AgNPs quantification was done in the supernatant using AAS spectroscopy (Shimadzu AA-7000, Japan).

2.12.2 The standard rabbit vaginal irritation test. The animals were divided in three different groups, the untreated “control” (R-I), placebo (R-II) and NHM gel (R-III) groups, each consisting of 4 rabbits. Once a day intravaginal application of the placebo and NHM gel (250 μl each) was made on R-II and R-III groups respectively for 7 consecutive days. Vaginas were examined daily for the macroscopic signs of irritation, inflammation and ulceration.³⁵

2.12.3 Lactobacillus toxicity screening. This study is a part of the toxicity screening, for the acceptability of any intra-vaginal product. Using two lactobacilli strains i.e. L. acidophilus and L. jensenii, lactobacillus toxicity was determined for the different formulation dilutions.^35,36 Sterile, molten (45–50 °C) lactobacilli MRS agar was poured into sterile Petri dishes with (a) different NHM gel dilutions (X, 2X and 4X with VSF) and (b) without treatment (as control). These test plates containing X, 2X and 4X dilutions (250 μl) were inoculated with 50 μl cell suspensions (1 × 10⁶ cells per ml) of L. acidophilus and L. jensenii (in the respective set of the triplicates) using a sterile inoculating loop. The plates were incubated at 37 °C in an atmosphere containing 5% CO₂ and 95% air for a period of 72 h. The number of colonies and the size were determined at the end of the experiment.

2.13 Statistical analysis

All biochemical observations were based on 3 independent experiments. Data were expressed as mean ± SEM or percentage. The results were analyzed by a 1-way analysis of variance (ANOVA), and chi-square test, as applicable, using the Microsoft excel (Microsoft office 10, Washington, U.S.A) and Graph Pad Prism 3.0 software (GraphPad Software, Inc., CA, U.S.A).

3. Results and discussion

3.1 Synthesis, optimization and characterization of in situ stabilized silver nanoparticles

The nanoparticle dispersion was yellowish brown in colour. Table 2 represents the effect of the molar concentration ratio (TA/AgNO₃) and the pH of the tannic acid solution on the average particle size, particle size distribution (PDI), and λ_max. Investigation revealed that a molar ratio of 0.025 (TA/AgNO₃) and pH 9 (tannic acid solution) showed the least average particle size, around 17 nm, with optimal polydispersity. Particle size distribution data (Fig. 2) at these conditions (molar ratio of 0.025) revealed that, AgNPs ≤ 20 nm were present in a 14% higher concentration when compared with the distribution of nanoparticles at a molar ratio 0.05. This climb was 9% when the concentration of the particles ≤30 nm was compared.

Table 2 Concomitant optimization of the molar concentration ratio of tannic acid to silver and the pH of the tannic acid solution [n = 3]

Sr. no	Molar ratio (TA : AgNO3)mM	pH	P-size (nm)	PDI	λmax
1	0.0125 (0.0375 : 3)	pH 7	47 ± 3.1	0.320 ± 0.105	424 ± 2.7
		pH 8	51.2 ± 2.2	0.410 ± 0.127	419 ± 1.5
		pH 9	40.1 ± 2.6	0.391 ± 0.016	418 ± 2
		pH 10	78.5 ± 6.4	0.644 ± 0.27	427 ± 0.7
2	0.025 (0.075 : 3)	pH 7	32.8 ± 1.8	0.307 ± 0.12	418 ± 3.6
		pH 8	22 ± 1.3	0.321 ± 0.163	413 ± 1.8
		pH 9	17.3 ± 0.2	0.311 ± 0.146	413 ± 1.2
		pH 10	55.9 ± 1.7	0.487 ± 0.52	425 ± 0.4
3	0.05 (0.15 : 3)	pH 7	31.1 ± 2.4	0.330 ± 0.053	420 ± 2.9
		pH 8	25.4 ± 1.4	0.245 ± 0.119	418 ± 3.4
		pH 9	19.6 ± 0.2	0.203 ± 0.068	412 ± 2.9
		pH 10	63.2 ± 2.7	0.148 ± 0.083	426 ± 1.3

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Fig. 2 Tannic acid based synthesis and stabilization of silver nanoparticles. The figure also signifies the stabilization status of AgNPs and a graphical comparison of the effect of the molar ratio and pH variations on the particle size distributions.

The optimization study revealed that, the size of the AgNPs at a lower molar ratio (0.0125) for all pH values was found to be undesirably large (i.e. >40 nm). A considerable reduction in the size was observed when the molar concentration of tannic acid is doubled to 0.075 mM (i.e. at molar ratio 0.025). Among the various pH values, the smallest size was found to be 17.4 ± 0.2 nm at pH 9. Overall studies revealed that at each molar ratio, pH 9 was providing a relatively smaller size than pH 7 and 8. Increasing the pH of the tannic acid solution above 9 results in an abrupt increase in the particle size.

The beauty of this synthesis procedure is the in situ stabilization of the synthesized silver nanoparticles, see Fig. 2. The glucose moiety is one of the hydrolyzed products of tannic acid, and it acts as a weak reducing agent at room temperature but as a strong stabilizing agent at alkaline pH. There exists an ideal requirement for the partial hydrolysis of tannic acid to produce an optimum quantity of gallic acid (for the reduction of AgNO₃ to AgNPs) and glucose (for stabilization of AgNPs in their smallest possible size). The pK_a of tannic acid is around 10, which means that pH >7 can easily hydrolyse tannic acid to an appreciable extent.³⁷ But experimental results showed that pH 9 provided an optimum extent of hydrolysed tannic acid for the reduction and stabilization process.

The concept of molar ratio optimization can be easily understood on the basis of the effective reduction and stabilization requirements. At a molar ratio <0.025, the concentration of glucose (hydrolysis product of tannic acid) was not enough for the effective stabilization of small sized AgNPs. Understandably, the large surface area and thus high requirement of glucose for an effective surface stabilization of the small sized growing nanoparticles could be the possible reason behind the increased size. Moreover at a smaller molar ratio, the incorporation efficiency of the atoms into the nuclei per particle (growth) will be higher per collision, resulting in higher growth rates and thus a size increase. At molar ratios >0.025, increasing the reagent’s concentration i.e. (tannic acid) results in the increased reaction rates that further lead to a higher monomer (hydrolysed products) concentration. The increased monomer concentration may facilitate the effective nucleation (growth step) to such an extent, which resulted in an increased particle size. It could be understood that apart from the reducing and stabilizing role, tannic acid was also acting as an organizer for facilitating nucleation.³⁸ In contrast, at a molar ratio of 0.025, the incorporation efficiency of the atoms into the nuclei per particle (growth) as well as the in situ stabilization, both take place in such an effective and timely manner that results in the smallest sized and stabilized AgNPs, ranging from 2 to 24 nm.

3.2 Characterization of the synthesised AgNPs

The optimized AgNP suspension with the lowest size of 17.3 ± 0.2 nm and polydispersity index of 0.311 ± 0.146 possesses the UV-vis absorption maxima at 413 ± 1.2 nm. Since there was only one transverse peak, it indicates that the nanoparticles are isotropic in nature. This absorbance maximum corresponds to the observed particle size, justifying the Mie’s relationship between the surface plasmon resonance and the AgNPs size (<20 nm) (Mastro et al., 2008, Yang et al., 2003). The zeta potential value comes out to be −21.03 ± 0.1 mV, clearly relating to the colloidal stability of the nano-metal suspension. PXRD studies showed the sharp intense peaks of AgNPs (specifically at 2θ values of 38.15 and 49.3 degrees) confirming the crystalline nature of AgNPs. Thermogravimetric (TG) analysis of the synthesised AgNPs presented in Fig. S1 (in the ESI†) revealed a substantial weight loss only in the temperature region of 260–480 °C, signifying the loss of the associated water and degradation of the surface anchored stabilizing components. TEM analysis further confirmed the diametrical range and polydispersity of the synthesised AgNPs (between 2 to 32 nm) with a spherical shape and no definite signs of agglomeration. Further AFM studies provided a fine level of surface smoothness with appreciably regular circles with uniform edges. This can be attributed to the synchronized AgNO₃ reduction and stabilization process at the optimized conditions. This eventually provided an undeviating platform for the upstream nano-assembly course. Morphological symmetry was the result of the in situ stabilization control provided by the concomitantly available glucose flux during the nucleation process. Fig. 3 illustrates all the above discussed characterization parameters.

Fig. 3 Characterization of synthesised silver nanoparticles.

3.3 Synthesis, optimization and characterization of the copper-curcumin (Cu-Cur) and copper-curcumin-β-cyclodextrin (Cu-Cur)CD inclusion complex

A basic synthesis scheme is illustrated in Fig. 4. A reddish brown colored (Cu-Cur) complex was obtained and the (Cu-Cur)CD inclusion was prepared by incorporating (Cu-Cur) inside the β-CD hydrophobic cavity. An MC-40 batch was selected as optimized based on the (Cu-Cur) saturation inside the hydrophobic β-cyclodextrin cavity. (Synthesis details are detailed in the ESI.†)

Fig. 4 Synthesized (Cu-Cur) and (Cu-Cur)CD. Structure of (Cu-Cur) where metal is expected to bind with 1,3-diketone moiety of curcumin. The probable inclusion of (Cu-Cur) inside the hydrophobic β-CD cavities is shown.

3.3.1 Cell viability assay. Minimum cytotoxic concentration (MCC) and cell cytotoxicity 50% (CC₅₀) values revealed the uniform response among all the cell cultures with no sign of specific cell damage as mentioned in Table 3. Toxicity at the cellular level is a characteristic for metal nanoparticles, but the synthesised AgNPs presented a safer and uniform cell toxicity response. This signifies the comparatively bio-friendly nature of the tannic acid reduced and stabilized AgNPs. Unrelatedly MC-40 showed negligible toxicity, describing its highly safe nature at both specific and non-specific levels. This can be attributed to the highly biologically safe nature of the β-CD shell preventing the direct exposure of the Cu-Cur complex.

Table 3 Cytotoxic concentration, as determined by measuring the cell viability with the colorimetric formazan-based MTS assay

Sample	HeLa		HEL		Hep G2		CRFK		U-87 MG
	MCC μg ml^−1	CC50 μg ml^−1	MCC μg ml^−1	CC50 μg ml^−1	MCC μg ml^−1	CC50 μg ml^−1	MCC μg ml^−1	CC50 μg ml^−1	MCC μg ml^−1	CC50 μg ml^−1
AgNPs	6.3 ± 0.7	431.7 ± 4.1	4.9 ± 1.2	396.2 ± 5.7	5.5 ± 0.3	403.5 ± 2.5	6.8 ± 0.8	465.2 ± 7.5	7.1 ± 2.1	458.1 ± 4.6
MC-40	>100	>100	>100	>100	>100	>100	>100	>100	52.3 ± 3.9	>100

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3.3.2 Hemolytic study. The hemolytic activity of AgNPs and MC-40 was determined by quantifying the released haemoglobin spectrophotometricaly. At 500 μg ml⁻¹ of AgNPs, the haemolytic effect observed is ∼28% whereas for MC-40 only 11% haemolysis was observed at the highest concentration 10 mg ml⁻¹. This reveals a highly safe and non-irritant profile of both the challenged moieties.

3.4 Anti-HIV activity

Both AgNPs and (Cu-Cur)CD i.e. MC-40 showed a dose dependent inhibition of HIV-1 transmission in all the challenged cases as mentioned in Fig. 5. AgNPs with IC₅₀ values of 218.2 ± 5.3 μg ml⁻¹ and 295.6 ± 4.7 μg ml⁻¹ during interaction-1 and 2 respectively, in the CEM–GFP host cells, presented a potent prospective for blocking the pre-interaction infection. The IC₅₀ value for viral dissemination in PHA-PBMCs was 236.9 ± 4.1 μg ml⁻¹ and 362.3 ± 7.4 μg ml⁻¹ for interaction-1 and 2 respectively. Observation provided a strong conclusion that AgNPs possess a better prophylactic potential by blocking the initial viral spread. 500 μg ml⁻¹ concentration showed >93.5% of viral inhibition in both the host cells during interaction-1, while this value declines to 82% while interaction-2 was studied. On the other hand MC-40 showed an IC₅₀ of 6.31 ± 0.15 mg ml⁻¹ and 8.17 ± 0.27 mg ml⁻¹ for the interaction-1 and 2 challenges respectively in the CEM–GFP host cells, whereas the IC₅₀ value in PHA-PBMCs was observed at 6.64 ± 0.08 mg ml⁻¹ and 8.4 ± 0.19 mg ml⁻¹ for interaction-1 and 2 respectively.

Fig. 5 Reduction in the virus load by AgNPs and MC-40 using CEM–GFP cells and PHA-PBMCs while exposing them to interaction-1 and interaction-2 challenges. Interaction-1 refers to the pre-infection interaction of the virus and test sample while interaction-2 states the host interaction of the test sample before challenging the virus. Culture supernatant was collected for p24 estimation by ELISA. Levels of the p24 antigen by the test compounds were compared to the positive control for calculating % inhibition values. The p24 value for the positive controls while exposing CEM–GFP cells to the interaction-1 challenge ranges in 197.4 ± 2.9 pg ml⁻¹ while for interaction-2 it is 192.6 ± 0.4 pg ml⁻¹, while in the case of the PHA-PBMCs challenge p24 concentration ranges 164.8 ± 4.7 pg ml⁻¹ and 142.7 ± 6.1 pg ml⁻¹ for interaction-1 and 2 respectively. The results obtained were found significant after applying two-tailed Student’s t’-test with P value <0.05.

Both the moieties reduced the virus spread as estimated by the p24 levels in the culture supernatants collected from the infected cells. AgNPs definitely showed a potent prospective as compared to MC-40 at both pre and post infection levels. Lower p24 titers, specifically observed during pre-infection interactions, are a possible outcome of the direct contact with the viral functional molecules (viral envelop, receptors/co-receptors). Moreover interaction-2 (post infection) includes the challenge of both the cell free and cell associated virus, where the inhibiting cell associated virus presents some serious challenges, like targeting the intercellular viral proceedings. Previous studies predicted the interaction of AgNPs with two disulfide bonds situated in the carboxyl half of the HIV-1 gp120 glycoprotein and finally denaturing its disulfide-bonded domain. The mechanistic basis of the MC-40 action can be primarily explained as the direct inhibitory effect of the Cu-Cur complex released from the inclusion complex. The multiple mechanisms of HIV-1 inhibition related with both the curcumin and copper moieties need more specific studies to define the exact mechanism responsible. In addition to the improved Cu-Cur presentation by β-CD, it can also interfere with HIV-1 transmission by disruption of the lipid raft functionality or membrane cholesterol balance.³⁹

3.5 Spermicidal activity

3.5.1 Modified Sander–Cramer assay (sperm motility inhibition assay). Scoring of the motile and immotile sperm under the phase contrast microscope revealed that AgNPs and the MC-40 complex presented a concentration dependent inhibitory effect as shown in Fig. 6 (left and centre). Terminating the assay after 120 s revealed the irreversible immobilization of sperms by MC-40 at a minimum effective concentration (MEC) of 290 ± 15 μg ml⁻¹ and complete immobilization at 5.3 ± 0.1 mg ml⁻¹. On the other hand AgNPs showed inhibition with MEC of 20 ± 2.5 μg ml⁻¹ and 82% motility inhibition at 450 μg ml⁻¹.

Fig. 6 Percentage motility inhibition after the AgNPs and MC-40 challenge (left and centre), observed during Sander–Cramer assay. (Right) Percentage HOS positive sperms after the AgNPs and MC-40 challenge.

3.5.2 Hypo-osmotic swelling test. The percentage of tail curling observed in the control is quite high (83.8%) while after treatment with 250 μg ml⁻¹ AgNPs and 1 mg ml⁻¹ (MC-40) the tail curling of the spermatozoa was significantly reduced to 34.2% and 23.5% as shown in Fig. 6 (right). The loss of HOS responsiveness after these treatments clearly indicated the compromised sperm membrane integrity in both the challenges. The study revealed an overall loss of the sperm membrane physiology which is comparatively prominent in the case of MC-40.

3.5.3 Sperm viability test (fluorescent staining). Viability testing using two different fluorescent stains for the live and dead sperms revealed a significant sperm death in both the challenged cases. AgNPs and MC-40 treated sperms showed a significant uptake of the red (PI) dye whereas the control group showed only (Sybr-14) green dye uptake (Fig. 7a–c). PI uptake was quantitatively higher in the case of the MC-40 challenged sperms, reflecting a higher sperm damage. PI uptake by the AgNP and MC-40 treated cells is a result of the sperm death which can also be understood by the compromised sperm membrane.

Fig. 7 Sperm viability assessment by SYBR-14/PI staining. The control human sperm (a) appears green due to the uptake of SYBR14 only. AgNPs and MC-40 treated human spermatozoa (b and c respectively) displaying red fluorescence due to the preferential uptake of PI. Apoptotic/necrotic changes in the plasma membrane induction by control.

3.5.4 Apoptotic and necrotic changes in the plasma membrane of the human spermatozoa. The selective spermicidal action of AgNPs and MC-40 was observed after 3 h incubation at different concentrations. By labelling with FITC-Annexin V (for the detection of phosphatidyl serine on the cell surface) and PI (for cell membrane integrity), cells were identified in different quadrants by flow cytometry. The data from the dual fluorescent labelling with Annexin V-FITC and PI showed that the control sample contained 94.1 ± 0.7% viable, 2.7 ± 0.2% apoptotic, 0.8 ± 0.1% early apoptotic and 2.4 ± 0.2% necrotic sperm. After treatment with AgNPs the number of apoptotic, early apoptotic and necrotic cells rose considerably to 25.4 ± 1.2%, 16.1 ± 0.8% and 3.8 ± 0.1% respectively, while the populations of the viable sperm cells reduced to 54.9 ± 1.4%. Treatment with MC-40 revealed a slightly lower amount of apoptotic cells (21.5 ± 0.5%) whereas early apoptotic and necrotic cell populations increased to 26.3 ± 0.2% and 5.2 ± 0.1% respectively. The results provide a mechanistic understanding of the spermicidal activity of AgNPs and MC-40. Apoptotic cell death is prevalent in the case of the silver nanoparticles challenge whereas MC-40 displayed the prevalence of the apoptotic as well as necrotic events in the challenged sperm cells.

3.6 Anti-candida activity

The serial dilution assay revealed a significant anti-fungal potential of AgNPs whereas MC-40 displayed much higher MIC values. MIC of AgNPs was found to be approximately three times more than the standard clotrimazole drug (MIC of clotrimazole against C. albicans is 6.3 ± 0.2 μg ml⁻¹ and for C. tropicalis is 7.1 ± 0.4 μg ml⁻¹). The MIC value of AgNPs against C. albicans is 35.2 ± 2.3 μg ml⁻¹ and for C. tropicalis is 37.5 ± 1.8 μg ml⁻¹, while the IC₅₀ value for both the strains lies at around 380 ± 15 μg ml⁻¹. On the other hand, no appreciable inhibition was seen in case of MC-40 with the MIC value ranging >250 μg ml⁻¹. AgNPs may exert an antifungal activity by disrupting the structure of the cell membrane and inhibiting the normal budding process due to the destruction of the membrane integrity.

3.7 Formulation of the nano-herbal microbicidal gel (NHM gel)

The selectivity index for both AgNPs and MC-40 is estimated in Table 4. AgNPs provided a narrower window due to the deemed toxicity associated at the nanoscale. MC-40 on the other hand is effective at comparatively higher concentrations but a very high selectivity index allowed the use of higher concentrations. Finally nano-herbal gel was formulated using 500 μg ml⁻¹ of AgNPs and 10 mg ml⁻¹ MC-40 in 1% w/v carbopol 974p base.

Table 4 Selectivity index of AgNPs and MC-40 calculated on the basis of their respective anti-HIV-1 potentials (approximately calculated for both HIV-1 strains and for both pre and post infection challenges in different host cells), spermicidal potential and specific HeLa cell cytotoxicity.

Treatment	HIV-1 EC50	Spermicide EC50	Specific (HeLa) cytotoxicity IC50	Selectivity index IC50/EC50
AgNPs	214 ± 21.6 μg ml^−1	62.5 ± 12.2 μg ml^−1	431.7 ± 4.1 μg ml^−1	>2
MC-40	7.1 ± 0.39 mg ml^−1	0.411 ± 5.3 mg ml^−1	—	Extremely high

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The prepared gel was a non-greasy homogenous orange-yellow clear gel, with good lubrication when textured on the hand. With its pH value of 4.2 ± 0.1 the gel displayed its acidic nature, well-suited for vaginal use. Thixotropic analysis of the gel formulation revealed its pseudoplastic flow with a very narrow deformation at the end of the descending protocol. Viscosity values at a shear rate of 100–500 γ(1/s) ranged from 0.1 Pa s to 0.34 Pa s. The detachment force of the gel evaluated during mucoadhesion studies was found to be 19.2 ± 0.4 g cm⁻². The spreadibility parameter of the gel was found to be in the range of 25.1 ± 1.7 g cm s⁻¹. All these parameters are critical for a vaginal gel’s performance and specifically for a contraceptive microbicide, which needs a uniform spread (depending on the viscosity and spreadibility) and a mucoadhesive nature to prevent unwanted leakage. These parameters significantly govern the product acceptability and the ease of the product use. Viscosity and texture parameters of the NHM gel meet some other vaginal products such as KY Jelly® Lubricant and Lacta-Gynecogel® Acidifying gel.

3.7.1 In vitro challenge study for the pharmaceutical parameters of the NHM gel. Contour plots and the response surface analysis during the Box–Behnken statistical screening, revealed the exact behaviour of the NHM gel in the simulated in vitro challenged conditions. Analysis showed a nominal buffering capacity of the gel. Because maintenance of an acidic pH is critical for such prophylaxis, it is not a good choice to depend on the safeguard provided by the buffering capacity of the formulation. A gel is efficient to provide sufficient viscosity, spreadibility and mucoadhesion in challenged conditions to provide a comfortable application and to resist the chances of leakage and related discomfort (details of the study are mentioned in the ESI†). Analysis of the pH and viscosity variations in different simulated runs revealed that a quadratic model governs both these responses. On the other hand mucoadhesion and spreadibility variations are governed by linear models (Table 5).

Table 5 In vitro challenge study for the pharmaceutical parameters of the NHM gel using Box–Behnken statistical screening. The table explains the model fit and regression analysis of pH, viscosity, mucoadhesion and spreadibility (dependent variable)

Response (Y)	R^2	Adjusted R^2	Predicted R^2	SD	% CV	Model fit	Regression equations
pH	0.96	0.8831	0.3320	0.19	3.14	Quadratic	pH = 5.66 − 0.32A + 0.39B − 0.24C + 0.19AB − 0.055AC − 0.058BC + 0.65A^2 + 0.064B^2 + 0.17C^2
Viscosity	0.81	0.5593	2.0848	0.18	36.16	Quadratic	Viscosity = 0.36 + 0.077A + 0.18B + 0.046C + 0.058 AB - 0.014 AC - 0.22 BC - 0.11 A^2 + 0.17 B^2 + 0.21C^2
Mucoadhesion	0.93	0.9188	0.8849	2.57	9.51	Linear	Mucoadhesion = 27 + 11.50A − 3.12B − 3.13C
Spreadibility	0.89	0.8650	0.8047	3.09	16.64	Linear	Spreadibility = 18.59 + 10.63A – 2.88B − 2.25C

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3.8 Pre-infection interaction (interaction-1) anti-HIV study of the NHM gel

A significant reduction in the viral propagation was observed during the pre-infection interaction challenge study of the NHM gel at various dilutions as presented in Fig. 8. Placebo gel, placed as the control group, offered an inert nature by not interrupting with the viral proliferation. With the undiluted NHM gel, around 97.5% inhibition in p24 Ag was observed on both the challenged cells. The effect of the VSF dilution was insignificant up to 4X dilution level, where >90% inhibition in p24 Ag levels assures the prophylactic efficacy of the formulation in such physiological challenges. A significant drop in the p24 inhibition was observed after 8X dilution, explaining the protection failure during the possible conditions where the dose application is not adequate or there are excessive genital secretions.

Fig. 8 Inhibition in the viral propagation/prophylactic potential of the NHM gel at different dilutions with undiluted placebo as the control. The challenge in both the viral strains showed 211.6 ± 4.8 pg ml⁻¹ of the p24 titer in the placebo group, whereas sequential dilutions of the NHM gel showed titer values of 5.2 ± 0.7 pg ml⁻¹ (at 1X or undiluted), 14.6 ± 2.1 pg ml⁻¹ (at 2X), 20.3 ± 1.5 pg ml⁻¹ (at 4X) and 126 ± 3.8 pg ml⁻¹ (at 8X). The results obtained were found to be significant after applying a two-tailed Student’s t’-test with P value <0.05.

3.9 Spermicidal activity of the NHM gel (modified Sander–Cramer assay)

Different dilutions of the NHM gel were tested for the spermicidal activity at different time intervals. Table 6 represents the percentage of motility inhibition at different dilutions after different time intervals. The gel showed a potential reduction in sperm motility, signifying the contraceptive nature of the product. Similar to the anti-HIV response, dilutions presented an inverse proportionality to the spermicidal profile. Up to 4X dilutions, a complete motility inhibition was noted at 90 s observation, whereas this inhibition potential fell significantly at the next dilution.

Table 6 Sperm motility inhibition study of different dilutions of the NHM loaded gel [n = 3]

Dilution	% inhibition time (0 s)	% inhibition time (30 s)	% inhibition time (60 s)	% inhibition time (90 s)
(No dilution) X	68.4 (±3.7)%	100%	100%	100%
2X	54.1 (±1.3)%	78.3 (±1.7)%	96 (±0.4)%	100%
4X	41.8 (±0.6)%	67.2 (±2.3)%	88 (±1.5)%	100%
8X	37.2 (±1.6)%	48 (±2.7)%	64.5 (±0.9)%	91 (±2.7)%
5% tergitol NP-9	51.6 (±3.4)%	94.3(±2.4) %	100%	100%

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3.10 Challenge mating study in rats

The study revealed the potential antifertility activity of the NHM gel when compared to the placebo treated and untreated rats. All the females mated successfully and no deviation of mating efficiency was observed. A 100% absence of fertilization (no positive pregnancy outcome) was observed in the NHM gel treated group i.e. G-3. On the contrary, group G-1 and G-2 showed 100% and 66.6% positive pregnancy outcomes respectively. These observations suggest that the NHM gel could effectively block the sperm potential for fertilization. Mechanistic explanation can be given by using the reference of the in vitro results. The seized motility and compromised membrane nature by the gel components probably resulted in disturbing the normal fertilization mechanism. The placebo effect can be taken statistically, where the viscosity, pH and ionic strength of the semisolid gel could account to the possibility of interrupted fertilization.

3.11 Efficacy of the NHM gel against candida infections

As per the mentioned protocol, the antifungal activity of different NHM gel dilutions was estimated. Although the activity showed an inverse proportionality with dilution, still the results showed a significant reduction in the fungal colonies even at 4X dilution level. Results for the reduction in the number of colonies of C. albicans are: control: 37 ± 0.26 and NHM gel: X: 17.4 ± 1.3, 2X: 24.3 ± 0.73 and 4X: 29.6 ± 0.1. For C. tropicalis the strain activity pattern is as follows: control: 33.4 ± 1.71 and NHM gel: X: 21.6 ± 0.9, 2X: 25.8 ± 0.24 and 4X: 27.2 ± 0.4. In accordance with the in vitro results for the individual components, the NHM gel showed a prominent potential of controlling the candida infections, which eventually lessens the chances of viral escape through vaginal epithelium.

3.12 Pre-clinical toxicology study of the NHM gel

3.12.1 Pre-clinical toxicology study on female Wistar rats. Examining the structural integrity of the vaginal epithelium during the course of the 21 days study protocol, there was no macroscopic sign of redness, edema, abnormal discharge, bleeding and inflammation. Microscopic examination of the in-vaginal lavage revealed the regularity in the estrous cycle.
3.12.1.1 Effect on the local tissues, vaginal tissue proliferation, and in situ apoptosis (after the 21 day protocol). Macroscopic examination of the excised vaginal epithelium after the 21 days protocol revealed that there was no macroscopic sign of any kind of redness, ulceration, edema and inflammation as displayed in the biopsy images (Fig. 9). Histological examination of the excised vaginal tissue showed the presence of the columnar epithelium in all samples with the nucleus in the basal portion and the rest cell showing the cytoplasm. The sub mucosa shows an infiltrate of lymphocytes while the deeper zone shows the muscular layer with no sign of any type of damage. Macroscopic examination and histopathology confirmed no sign of alteration when the control, placebo and treated groups were compared. Fig. 9 shows the microscopy images of the histopathology samples.

Fig. 9 Macroscopic examination of the excised vaginal epithelium and histopathology images of the vaginal epithelium tissues of the “control” (G-I), placebo (G-II) and NHM gel (G-III) treated groups.

3.12.1.2 Haematology study. A systemic safety conformation was made from the haemotogram results from animals of each group. CBC and DLC data showed no significant variations in the control, placebo and treated groups as mentioned in Table 7.

Table 7 Haematogram of blood taken from the control (G-I), placebo (G-II) and (G-III) treated rats after 21 days of the in-vaginal application protocol

Investigation	Groups
	G-I	G-II	G-III
Complete blood count
Haemoglobin (g dl^−1)	11.2(±0.7)	11.3(±0.2)	11.6(±0.4)
Total leucocyte (count per mm^3)	11 600(±31)	12 600(±18)	10 200(±82)
RBC (millons per mm^3)	5.82(±0.1)	6.24(±1.4)	6.11(±0.5)
HCT	38.3(±3.6)	32.4(±1.7)	38.4(±6.8)
MCV	58.8(±3.1)	52(±7.2)	61.7(±4.5)
MCH	22.6(±2.7)	18.1(±2.1)	20.8(±3)
MCHC	38.6(±4.7)	34.8(±1.9)	35.8(±1.6)
Platelet (lakh per mm^3)	4.12(±0.2)	4.61(±0.6)	3.92(±0.1)
Differential leucocyte count
Neutrophil%	46.3(±1.6)	45.8(±2.7)	46.5(±1.4)
Lymphocyte%	51(±2.1)	51.5(±1.3)	51.9(±0.8)
Eosinophil%	02(±0.4)	02.5(±0.5)	2(±0.3)
Monocyte%	01	01	01
Basophil%	00	00	00
ESR (mm per 1^st h)	05(±0.1)	04(±0.1)	05(±0.1)

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3.12.1.3 Organ distribution study. Despite the long term vaginal application protocol, the kidneys showed a maximum AgNP accumulation of 1.6(±0.2)μg (the rest of the distribution data is given in Fig. 10). Results of the organ uptake studies confirmed a minor systemic absorption of AgNPs by the vaginal cavity. Results obtained from the in vitro cell cytotoxicity (MTS) assay served as a reference to eliminate the discussion of the organ toxicity on consistent use of the NHM gel.

Fig. 10 Organ distribution data of AgNPs after the 21 days toxicity studies in G-III (NHM gel treated) rats.

3.12.2 The standard rabbit vaginal irritation test (after the 7 day protocol). No macroscopic alteration in the vaginal morphology was observed. No sign of redness, inflammation and edema on comparison of the control, placebo and treated groups confirmed the safety of the NHM gel.

3.12.3 Lactobacillus toxicity screening. A study was conducted on the two lactobacillus strains i.e. L. acidophilus and L. jensenii to assure the safety margins of the NHM gel. The study revealed that minor signs of toxicity were observed in the undiluted formulation; with dilution the effect was insignificant. Results for the reduction in the number of colonies of L. acidophilus are: control: 41 ± 0.72 and NHM gel: X: 35.2 ± 3.5, 2X: 38.1 ± 1.5 and 4X: 39.1 ± 1.2. For L. jensenii the strain toxicity pattern is as follows,: control: 38.2 ± 2.2 and NHM gel: X: 32.5 ± 0.7, 2X: 36.2 ± 1.1 and 4X: 37.6 ± 0.7. Minor signs of toxicity were observed on both lactobacillus strains but this effect had an insignificant presence after dilution.

4. Conclusion

Strategic delivery of in situ stabilized AgNPs and MC-40 showed a promising approach for the development of contraceptive microbicides. In vitro anti HIV-1 studies conformed the potential of AgNPs to interact with the cell free (pre-host interaction level) as well as cell associated virus (post-host interaction level). Contraception assays proved the potential of MC-40 as well as AgNPs to hinder the conception pathway by disrupting the sperm membrane. The NHM gel formulated with a rationally selected dose, showed a response harmony with the in vitro results of its components. The anti-HIV-1, contraception as well as anti-candida studies revealed the potential of the gel at several dilution levels. The pre-clinical toxicology protocol strengthens the candidature of the NHM gel as a safe contraceptive microbicide.

Acknowledgements

Author acknowledges Department of Biotechnology (DBT) India, Punjab Technical University, Jalandhar and Punjab State Council of Science and Technology (PSCST) India for providing the supportive facility and financial base for this research. Author also acknowledges the technical support from Prof. R. Snoeck & Prof. G. Andrei (Laboratory of Virology and Chemotherapy, Department of Microbiology and Immunology, Rega Institute, Belgium).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra16353f

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