DESC:Technical Field of the Invention

[0001] The present invention relates to a composition to treat sexually transmitted infections, and mediated inflammations in vagina, as well as contraception. More particularly, it relates to a method for preparing a composition for providing the formulation of efavirenz and curcumin loaded lactoferrin nano-particles to treat sexually transmitted infections and spermicidal activity for birth control in vagina.
Background of the Invention

[0002] Sexually Transmitted Infections (STI’s) or Sexually Transmitted Diseases (STD’s) have been rampant in recent years. The sexually transmitted infections in females are transmitted through vaginal and/or oral sexual conduct; most sexually transmitted infections do not show significant symptoms. Some sexually transmitted infections can spread through the use of unsterilized IV drug needles, from mother to baby during child birth or breast feeding and blood transfusions. Recent studies have shown that due to STI’s the females in low and middle income countries like sub-Saharan region experience about 41% of unintended pregnancy that leads to approximately 70,000 deaths.

[0003] The most common female symptoms of sexually transmitted infections may vary for each STI may include sores or blisters on or around the genital area or in the mouth, vaginal itching, rashes, unusual discharge, swelling, pain in or around the vagina and pain in the pelvic area or abdomen. Many sexually transmitted infections display no symptoms at all. The STI’s in females have higher risk of serious health problems as compared to male. The STI’s if left untreated may lead to fertility problems, increased of cervical cancer, chronic pelvic pain, increases risk of ectopic pregnancy and increase fatal mortality in pregnant women.

[0004] Several research groups have already reported various combinations of microbicides. Some of the formulations have undergone several stages of clinical trials and a large number of formulations have failed due to safety, toxicity and efficacy issues. The existing microbicides have failed to release optimum concentration of active drug in vagina along with higher bioavailability for longer period without causing discomfort and adverse effects to the biological barriers.

[0005] Currently there are multiple prevention technologies for both male and female from transferring sexual transmitted infections from one person to other person. Multiple prevention technologies include male and female condoms, female diaphragms, spermicides and microbicides. While the aforementioned MPTs are considered a viable solution to lower the risk of unwanted pregnancy and are not capable of reducing the risk of contracting STI’s.

[0006] There are several references for treating sexually transmitted infections using nano-particles. The prior art W.O. Patent Application No. 2009015214 discloses about treating chlamydial infection by using nano-particles. A method of treating a chlamydial bacterial infection comprising a therapeutically effective amount of folic acid-conjugated nano-particles comprising at least one antibiotic agent. Another prior art U.S. Patent No. 6432935 discloses about pharmaceutical compositions mainly vaginal suppository containing many different active ingredients. The composition comprises an antibiotics, sulphonamide, at least one an antiquagul agent, e.g., clotrimazol (bis-phenyl-(2-chlorophenyl)-1-imidazolyl-methane), natamycin, or nystatin,and 5-nitro-metronidazol. The compositions are useful mainly as vaginal suppositories. Hence there is need to develop efficient Nano-formulation to treat sexually transmitted infections.

[0007] As a result of the above, there is still a need for an effective composition for providing the formulation by using nano-particles to treat sexually transmitted infections and mediated inflammations by preventing unwanted side effects and toxicities.

Brief Summary of the Invention

[0008] In accordance with the teachings of the present invention, a method and composition for providing the formulation of Efavirenz or tenofovir or dapivirine and Curcumin loaded Lactoferrin Nano-particles (ECNP, TCNP, DPNP) to treat Sexually Transmitted Infections (STI’s), and STI-mediated inflammation in vagina and birth control is provided.

[0009] Lactoferrin protein has several pleiotropic functions like immune modulation, anti-viral and anti-cancer activities thus it can act as first line of defence to inhibit inflammation and infection. Curcumin an active principle of unique herbal compound turmeric has been used as the main spice ingredient in most parts of Asian subcontinent since centuries. It exhibits pleiotropic effect like anti-HIV, anti-inflammatory, anti-oxidant, vaginal contraceptive and many more. It can potentially provide an ideal non-steroidal contraceptive having both spermicidal and microbicidal properties against vaginal infections. Efavirenz and dapivirine is non-nucleoside reverse transcriptase inhibitors (NNRTI). Tenofovir is nucleoside reverse transcriptase inhibitor (NRTI). They are used as part of highly active antiretroviral therapy (HAART) and as a Microbicide for the treatment of HIV-1.

[0010] According to embodiments illustrated herein, there is provided a method for providing the formulation of Efavirenz (EFV) or tenofovir (TFV) or dapivirine (DPV) and Curcumin (Cur) loaded Lactoferrin Nano-particles (ECNP, TCNP, DPNP) to treat Sexually Transmitted Infections (STI’s), and STI-mediated inflammation in vagina. The method includes the step of formulating nanoparticle of Lactoferrin, 94 percent pure Curcumin and 98 percent pure Efavirenz or tenofovir or dapivirine. The method for formulation includes the step of dissolving 40 milligram of Lactoferrin 500 microliter ice cold Phosphate Buffered Saline (PBS) having a pH of 7.4. The method further includes the step of dissolving 20 milligram of Curcumin in 100microliter of Dimethyl Sulfoxide (DMSO) and dissolving 10 milligram of Efavirenz or tenofovir or dapivirine in 100microliter of Dimethyl Sulfoxide (DMSO). The method further includes the step of adding the Dissolved Curcumin and Efavirenz or tenofovir or dapivirine to the dissolved Lactoferrin and incubating on ice for 1 hour or until completion of binding to form the protein-Drug Mixture. The method further includes the step of adding the protein-drug mixture to 25 millilitre of ice cold olive oil while stirring continuously and sonicating on ice for 15 minutes using ultrasonic homogenizer with a pulse period of 30 seconds and an amplitude of 5 micrometre at an interval of 1 minute between sequential pulses, and the frozen in liquid nitrogen for 15 minutes and further incubation on ice for 4hours. Further, the method includes the step of centrifuging the protein-Drug Mixture at 6000g for 15 minutes to discard the containing olive oil and to form pellets. The method further includes the step of washing the pellets with ice-cold diethyl ether to remove traces of oil and resuspending in PBS to obtain Efavirenz-Cucumin loaded Lactoferrin Nano-particles (ECNP) or Tenofovir-Cucumin loaded Lactoferrin Nano-particles (TCNP) or Dapivirine-Cucumin loaded Lactoferrin Nano-particles (DCNP).

[0011] According to embodiments illustrated herein, there is provided a composition of Efavirenz and Curcumin loaded Lactoferrin Nano-particles (ECNP) or Tenofovir-Cucumin loaded Lactoferrin Nano-particles (TCNP) or Dapivirine-Cucumin loaded Lactoferrin Nano-particles (DCNP) to treat Sexually Transmitted Infections (STI’s), and STI-mediated inflammation, contraception in vagina. The composition comprises of nano-particles of 40milligram of Lactoferrin loaded with 20milligram of 94percent pure Curcumin and 10 milligram of 98percent pure Efavirenz or tenofovir or dapivirine.

[0012] These and other features and advantages of the present disclosure may be appreciated from a review of the following detailed description of the present disclosure, along with the accompanying figures in which like reference numerals refer to like parts throughout.

Brief Description of the Drawings

[0013] Other objects and advantages of the present invention will become apparent to those skilled in the art upon reading the following detailed description of the preferred embodiments, in conjunction with the accompanying drawings, wherein like reference numerals have been used to designate like elements, and wherein

FIG. 1 illustrates a process flowchart for preparing the formulation of efavirenz and curcumin loaded lactoferrin nano-particles according to the present invention.

FIG. 2 shows microscopic and DLS analysis of nano-particles according to the present invention.

FIG. 3 shows Fourier transform infrared spectroscopy (FTIR) spectral analysis of nano-particles according to the present invention.

FIG. 4A-4C shows pH and simulated fluid dependent drug release from nano-particles according to the present invention.

FIG. 5A-5C shows cellular localisation assay of curcumin in soluble and nano-forms according to the present invention.

FIG. 6A-6D shows sperms viability assay of nano-particles with varying concentrations of soluble and nano-forms according to the present invention.

FIG. 7A-7C shows anti-HIV-1 activity of combination of nano-particles according to the present invention.

FIG. 8A-8C shows lactobacillus viability test of both soluble and nano-forms in single and combination forms of nano-particles according to the present invention.

FIG. 9A-9I shows bioavailability studies upon topical application in vagina for combination of nano-particles according to the present invention.

FIG. 10A-10C shows safety analysis for combination of nano-particles according to the present invention.

FIG. 11A-11C shows histopathology analysis according to the present invention.

FIG. 12A-12C shows pharmacokinetic analysis for combination of nano-particles according to the present invention.

FIG. 13A-13C shows time course analysis for combination of nano-particles according to the present invention.
Detailed Description of the Drawings

[0014] The present disclosure is best understood with reference to the detailed description set forth herein. Various embodiments are discussed below. However, those skilled in the art will readily appreciate that the detailed descriptions given herein are simply for explanatory purposes as the methods and systems may extend beyond the described embodiments.

[0015] The present invention discloses a triple-combination topical formulation that can simultaneously act on HIV, HIV-mediated inflammation, other viral and bacterial infections with contraceptive action, based on the principle of multipurpose prevention technologies (MPT). This is a triple combination of broad spectrum lactoferrin and curcumin as preventive and protective agent and efavirenz as therapeutic agent against HIV and serves as a highly effective and efficient multi-protective and microbicide formulation.

[0016] The main benefit of protein nano-particles is that, these nano-particles can be prepared in relatively mild condition without use of any toxic chemicals. Protein could be an ideal vehicle for drug transportation in nano-form because its amphiphilic nature helps its cooperation with drugs as well as solvent. Curcumin loaded-apotransferrin nano-particles are shown to inhibit HIV-1 through inhibition of virus infection and down-regulation of host inflammatory responses.

[0017] Natural protein such as lactoferrin is water soluble, metabolizable, biodegradable, surface modification of such natural protein could be done very easily to facilitate required interaction of drugs and ligands. Lactoferrin protein has several pleiotropic functions like immune modulation, anti-viral, and anti-cancer and thus can act as first line of defence to inhibit inflammation and infection.

[0018] According to present invention, the composition to treat sexually transmitted infections and mediated inflammations in vagina comprises of a lactoferrin, curcumin of 20mg and efavirenz (EFV) of 10mg, curcumin of 20mg and tenofovir (TFV) of 10mg, curcumin of 20mg and dapivirine (DPV) of 10mg or the like. The lactoferrin nano-particles is prepared with the combination of curcumin and efavirenz through sol-oil chemistry method and used as a controlled delivery system through vaginal route as a microbicide and the composition required is of lactoferrin loaded with 94% pure curcumin and 98% pure efavirenz or tenofovir or dapivirine.

[0019] The nano-formulations of the present invention as shown in (Fig. 1) is prepared by the following method. At the beginning 102 the drugs such as curcumin (20mg), efavirenz (10mg), tenofovir (10mg) and dapivirine (10mg) are selected to prepare nano-particles. The lactoferrin nano-particles were prepared with the combination of curcumin and efavirenz through sol oil chemistry method. Lactoferrin (40mg) 104 was dissolved in 500µl ice cold PBS pH 7.4. In a separate tube, the curcumin (20mg) 106 was dissolved in 100µl of methanol/DMSO and fixed at 10 mg or varying concentration (5, 10, 15 and 20mg) of efavirenz or tenofovir or dapivirine is also dissolved 108 in 100µl methanol/DMSO. The above drug mixture is added to lactoferrin (40mg) 110 dissolved 1x PBS (ph-7.4) at 4ºC and kept it for one hour incubation on ice.

[0020] Then the whole mixture was slowly added to a 60ml plastic container 112 containing 25ml of ice cold olive oil followed by gentle manual vortexing. The container was kept on ice (4°C) and sonicated 114 using the narrow titanium probe for 15minutes at an interval of 5min. After sonication the whole mixture was transferred to 50ml Oak Ridge centrifuge tube 116 and immediately frozen into liquid nitrogen at-least for 15minutes118. After 15minutes the tube was thawed 120 on ice for four hours. The NP tube containing tube was centrifuged 122 at 6000rpm for 10min at 4°C. Supernatant (oil) was discarded 124 and the pellet was saved. The pellet was washed 126 thrice with ice cold di ethyl ether to remove any oil traces. Finally the NP pellet was resuspended in PBS 128 and used for further study or stored at 4°C for long term storage (up to 2months) 130.

[0021] The same protocol was followed for the preparation of individual nano-particles of curcumin or efavirenz in different ratios mentioned in Table.1. Four different types of nano-particles were prepared (Table. 1) for the study. In the table 1. Lacto nano (without any drug), 2. Lacto-nano-EFV + Cur (ECNPs), 3. Lacto-cur-nano (CNP) and 4. Lacto-EFV-nano (ENP).

Formulations Lactoferrin concentration (mg) Curcumin concentration (mg) Efavirenz concentration (mg) Loading Efficiency of curcumin Loading Efficiency of efavirenz
I.E of ECNPs
I 40 20 5 47% ±2.5 49% ±2.4
II 40 20 10 63% ±1.9 61.5% ±1.6
III 40 20 15 57.4% ±3.2 51.7% ±2.7
IV 40 20 20 48% ±2.8 53% ±3.7
I.E of Lacto-Cur-Nano
IA 40 5 0 38% ±1.45 NA
IIA 40 10 0 59% ±1.34 NA
IIIA 40 15 0 52% ±2.2 NA
IVA 40 20 0 49.5% ±2.5 NA
I.E of Lacto-EFV-Nano
IB 40 0 5 NA 41% ±1.73
IIB 40 0 10 NA 47.6% ±2.8
IIIB 40 0 15 NA 58.4% ±1.79
IVB 40 0 20 NA 57.83% ±2

Table 1. Loading efficiency (LE) of drug(s) loaded Lactoferrin nanoparticle.

[0022] Characterization Of Nano-particles
Nano-particles assembled were morphologically characterized using Field Emission Scanning electron microscope (FE-SEM), Atomic Force Microscopy (AFM), Transmission electron microscopy (TEM). For SEM, particles were coated with gold. For TEM, samples were fixed on 200 mesh type-B copper grid coated with carbon and staining was done using 2% Uranyl acetate. Particles characterized through AFM were spin coated on a cover slip.

[0023] Fourier Transform Infrared Spectroscopy (FTIR) Of Nano-particles
FT-IR studies have been performed using ALPHA’s Platinum ATR single reflection diamond ATR module (Bruker Corporation). Briefly one to two milligrams of lyophilized samples were directly kept on the sample holder and scanned from 500 cm-1 to 4000 cm-1. The spectra were visualized using OPUS software.

[0024] Calculation Of Loading Efficiency
Nano-particles were incubated with 1 ml of 1X PBS (pH5) and kept under gentle rocking for 30 minutes at room temperature. 100 µ l of 30% silver nitrate was added to resulting mixture to precipitate the protein. Then 1 ml of HPLC grade methanol as shown in the below HPLC condition was added to the mixture and this was centrifuged at 12000 rpm for 15 min and concentration of drugs in supernatant were estimated. Supernatant was analyzed in triplicate. Standard curve was developed using the different concentrations of curcumin and efavirenz through HPLC respectively. Loading efficiency was calculated as per equation (1).
Loading Efficiency (%) = X loaded/X total * 100 (1)
Xtotal - Xlost = Xloaded (2)
where Xloaded = amount of drug loaded, Xtotal = amount of total drug used and Xlost = amount of drug lost during preparation.

HPLC Condition:
Drugs Mobile Phase Wavelength Flow rate
Curcumin Acetonitrile : 5% glacial Acetic acid (75:25) 420nm 1ml/min
EFV Acetonitrile : 0.1% formic acid (75:25) 247nm 1ml/min
TFV Acetonitrile-water (75:25) 259nm 1ml/min
DPV Acetonitrile:0.1%glacial Acetic acid (80:20) 245nm 1ml/min

[0025] In Vitro Experiments
Analysis of Sensitivity of Nano-particles under Different Conditions of pH and simulated fluid
Nanoparticle pellet were re-suspended in 1 ml of 1X PBS (of different pH values in the range 19) and simulated vaginal fluid. The nano-particles are added with the PBS (Phosphate Buffer Saline) and these were then kept for incubation on rocker at room temperature for 2h. Then 300ml of 30% silver nitrate was added and drugs were extracted by adding 1 ml of methanol. These were centrifuged at 12000 rpm for 15 min and the supernatant was filtered using 0.2 micron syringe filter and analysed by using HPLC. Every experiment was conducted in triplicate.
The pH dependent drug release is analysed for more than include but not limited combination of nano-particles including curcumin, Efavirenz, Tenofovir, dapivirine, combination of curcumin and efavirenz, combination of curcumin and tenofovir andcombination of curcumin and dapivirine.

Cellular Localization Assay
The cellular localisation assay of curcumin is done for understanding the protein function of nano-particles. The experiment is conducted by selecting 0.2 million cells and seeding the cells in twelve well plates with SFM (Serum Free Medium). After seeding the supplements are placed for four hours at 370 and 5% Co2 and treated with 10µm concentration of soluble and nano-formulation for one hour, two hour, four hour and eight hour. Then wash and replace the cells with fresh media after each time point and observe the protein function of each cell under confocal microscopy.

Sperm Viability Assay
The sperm viability activity of nano-particles is examined and/or analysed for both soluble and nano-forms. The studies are conducted for many types of nano-formulations, such as Sol Cur, Nano Cur, Sol EFV, Nano EFV, Sol (Cur+ EFV), ECNPs, Sol (Cur + TNF) and Sol (Cur + DPV) nano-particles. The study is conducted by selecting 0.2 million sperms and seeding sperms in 96 well plates with M2 media. After seeding the plates are placed at 370 and 5% Co2 and treated at varying concentrations of soluble and nano-formulation for two hours. Then wash and replace the cells with fresh media for every two hours and add 20ml of 5mg/ml MTT. The cells are then incubated for two hours, removed from the media and dissolve the precipitate in 200µl.

In Vitro Stability Profile of Nano-particles
The stability profile of nano-particles was performed for ECNPs in suspension form and represented in Supplementary Table 1. The stability was measured in terms of the loading efficiency, size distribution, zeta (?) potential and poly dispersity index (PDI). Suspension ECNPs were incubated for indicated time points such as 0, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 days at two different temperature (4 °C and 25 °C). Loading efficiency was calculated according to equation (1). Size distribution, PDI and zeta potential were measured using dynamic light scattering methods.

Anti-HIV assay
Anti-HIV-1 activity of curcumin + efavirenz and ECNPs, curcumin + tenofovir and TCNPs and curcumin + dapivirine and DCNPs along with their soluble and nano-forms were analysed using HIV-1 for twelve hours. The concentrations of curcumin, efavirenz, tenofovir and dapivirine were taken as shown in the below tabular form in (Fig. 7). But the concentrations of all the drugs and Lactoferrin were reduced to half in the preparation of ECNPs. The experiment is conducted by selecting 0.5m Supt1 cells and seeding the cells with RPM1 1640 + 0.1% FBS. The seeded cells are challenged with HIV-1 of 20 ng/ml for twelve hours and incubated with the drug of soluble and nano-forms. After incubation the cells were washed and replaced with 10% FBS after twelve hours and incubated for 4 days i.e. 96 hours and after 96 hours the virus titer was measured using p24 ELISA assay.

[0026] In Vivo Experiments
Animal experimental design
Animals (0.160–0.240 kg, 6months) were housed for seven days prior to experiment at the animal housing facility, University of Hyderabad for 12 h in a light/dark condition. Animals were euthanized using sodium pentobarbital (50 mg/kg, IP) and final sacrifice was done by cervical dislocation. Blood was collected through heart puncture during terminal anesthesia. All the animal experiments were done in triplicate. All the experiments were performed on healthy female rats. 54 rats were used for pharmacokinetics (PK) study (study1) and these were reused for study 3 after a wash period of 48 h. 6 rats were used for time course bioavailability studies (study 2), and among these, 3 rats were randomly selected and reused for study 3. Study3 (dose dependent toxicity and bioavailability studies) were performed on pooled rats from study 1 and 2.

Dosage Schedule
For studies 1 to 3, drugs used were in the combination of 20 mg of curcumin (soluble curcumin) plus 10 mg of efavirenz (soluble efavirenz) and an equivalent amount of combination nano-formulation per rat. For single drug, curcumin (20 mg) or EFV (10 mg) was used either in soluble or nano-form. The best formulation ratio such as II, IIA and IIIB as given in Table 1 were used for this study. Nano-formulation of drugs used in this experiment was equivalent to its soluble form. All the drugs were topically applied in vagina. Saline treated rats were considered as control.

Pharmacokinetic Assessment in Vaginal Lavage
For study 1, Single dose of curcumin plus efavirenz either in soluble combination or nano-form combination as mentioned in the dosage schedule were topically applied in vagina to different groups of animals. After completion of various time points such as 0.5, 1, 1.5, 2, 2.5, 4, 6, 12 and 24 h, vaginal lavage was collected using sterile cotton swabs. Further, the swabs were mixed with methanol and the drug was extracted. Drugs were quantified using HPLC at the above mentioned time points and pharmacokinetics parameters were calculated using Kinetica v.5 software.

Time Course Experiment in Vivo
Sustained release of drugs in vaginal lavage: For study 2, all the animals were administered with single dose of Sol (EFV+ Cur) and nano (EFV+ Cur) or ECNPs. On the basis of Tmax obtained from PK study, the time course study was performed with a lag period of one hour (for soluble drugs) and two hour (nano-form drugs). The vaginal lavages were manually collected at fixed time points viz. 30 min, 1hr, 1.5hr, 2hr, 2.5hr, 4hr, 8hr, 12hr, 16hr and 24hr from injection. Drugs present in injection were extracted in methanol and estimated for efavirenz and curcumin separately using HPLC.

Lactobacillus Viability Assay
Lactobacillus is the most colonizing bacteria in the vagina and is generally considered as a gatekeeper of the vaginal ecosystem. Any microbicide could be considered as safe until it is non-toxic to the growth of Lactobacillus. To evaluate the effect of ECNPs on the growth of Lactobacillus crispatus. Lactobacillus viability test was performed according to standard protocol54. The lacto spores are cultured in nutrient broth at 370C for 24hr and inoculates of the particular bacteria were streaked on the Muller Hinton Agar (MHA) plates using a sterile inoculation loop. The drugs curcumin, EFV, TNF and DPV of 1mg, 2mg, 5mg of both soluble and nano-forms in single and combination forms were placed on sterile disc on MHA plates and inoculated plates were incubated at 370C for 24hr.

Dose Dependent Toxicity and Bioavailability Studies
For study 3, a dose dependent study was performed to examine the novelty of nano-particles as a microbicide. The study was carried out to compare the effect of soluble and nano-form of drug either in single or combination form. The studies are conducted for many types of formulations, such as Sol Cur, Nano Cur, Sol EFV, Nano EFV, Sol (Cur+ EFV), ECNPs, Sol (Cur + TNF) and Sol (Cur + DPV) were used and the study was carried out up to three doses. Doses were applied according to the schedule described below at an interval of 2 h between each dose. After the completion of each dose, rats were sacrificed under proper anesthetic condition. Vaginal lavage, blood and organs and vaginal tissue were collected. Blood was collected through cardiac puncture. Plasma was isolated from blood and used for the estimation of drugs. Vaginal tissues were collected and used for the estimation of inflammatory markers. Cytokines (IL-6 and TNF-alpha) levels were estimated in plasma and in the vaginal tissue homogenate. A small part of vaginal tissue were saved in 10% neutral buffered formalin and further processed for histopathology through Hematoxylin and Eosin tissue staining. Major part of vaginal tissues were homogenized in PBS using homogenizer. After final processing of vaginal tissues, blood plasma, and vaginal lavage concentrations of curcumin and efavirenz accumulation were determined using HPLC.

Drug Extraction from Vaginal Tissue
The Vaginal tissue collected was homogenized in PBS using rotor stator homogenizer for 15 minutes. 300 µ l of 30% silver nitrate per ml was added to the tissue extract to precipitate the protein. Drugs were extracted using 3 ml of HPLC grade methanol. Then curcumin and efavirenz were estimated through HPLC at 425 nm and 247 nm respectively.

[0027] Mobile Phase Chromatographic Condition
A reverse phase C18 column (25 cm × 4.60 mm, particle size 5 µ m) (Purospher® STAR RP-18 end capped (5 µ m) Hibar® RT 250-4.6, column No. 148837, Merck Millipore) was used for HPLC analysis. The mobile phase for efavirenz and curcumin were as follows. For efavirenz, mobile phase consists of 25% of 0.1% formic acid (Milli-Q water, pH 3.2) and 75% of acetonitrile, and the flow rate was set to 0.3 mL/min at ambient temperature55. For curcumin the mobile phase is composed of acetonitrile: 5% acetic acid in the ratio of (75:25, v/v) 56. All solvents were filtered using 0.4 µ m nylon syringe filter and degassed prior to use 10 µl of sample were analysed for detection of EFV and curcumin.

[0028] Safety Analysis
The safety of formulation is analysed to examine the safety of using nano-particles as a microbicide. The study was carried out to compare the effect of soluble and nano-forms of drug in combination form. The studies are conducted for many types of formulations such as Sol Cur, Nano Cur, Sol EFV, Nano EFV, Sol (Cur+ EFV), ECNPs, Sol (Cur + TNF) and Sol (Cur + DPV) were used and the study was carried out up to three doses.

[0029] Histopathology Study
The Histopathology study was done only for combination form of drugs either in soluble form or nano-formulation (ECNPs). Doses of soluble drugs in combination and ECNPs were given as described in dose schedule (n= 3). Negative and positive controls animals were treated with saline and 10 mg/kg nonoxynol-9 (N-9) respectively. Multiple doses were repeated up to three doses at time gap of 2 h. After the completion of measurements at the time points, animals were sacrificed under standard protocol. Vaginal tissue was fixed in 10% Neutral Buffered Formalin, followed by sectioning using cryomicrotome and Hematoxylin& Eosin staining. All pictures included in the results section were taken at 100× zoom using Olympus BX51P polarizing microscope.

[0030] Statistical Analysis
All studies were performed in triplicate. Data were presented as mean and standard deviation. The significance difference were calculated using one way ANOVA. The level of significance was used as ***P < 0.0005, **P < 0.005, *P < 0.05.
[0031] Results
Preparation and Characterization of Efavirenz and Curcumin Loaded Lactoferrin Nano-particle
Curcumin loaded Lactoferrin NPs (Lacto-Cur-nano), Efavirenz loaded Lactoferrin NPs (Lacto-EFV-nano) and Efavirenz plus curcumin loaded lactoferrin nano-particles (ECNPs) were prepared using sol-oil chemistry as described in materials and methods section. The nano-particles prepared were characterized through FE-SEM (Field Emission Scanning Electron Microscope), AFM (Atomic-force microscopy), TEM (Transmission electron microscopy) and DLS (Dynamic light scattering). Results presented in Fig. 2 shows microscopic and DLS analysis of nano-particles according to the present invention. Fig. 2 shows that nano-particles were uniformly dispersed spherical particles with size in the range of 20–80 nm. AFM images provide a three-dimensional surface profile reveal a particular type of projection which may help in binding with the receptor. DLS analysis of blank or drug/s loaded NPs has showed the hydrodynamic size in a range of 40 nm and 91–125 nm respectively as shown in below table. Increased apparent size in DLS is due to the surface water shell that contribute in DLS measurements. The zeta potential of freshly prepared blank or drug/s loaded NP were found to be in a range of - 21 to - 25 mv respectively (Supplementary Table 1) indicating their stability.

Drug Size ZP Value PDI
1 Lacto NPs 40nm -23 0.317
2 Cur NPs 109nm -25 0.619
3 EFV NPs 107nm -21 0.419
4 ECNPs 113nm -25 0.484

Supplementary Table 1

[0032] FT-IR spectral analysis
FT-IR spectral data showed the stability of lactoferrin, efavirenz, curcumin and efavirenz plus curcumin combination which remained conserved in their nanoformulation (Fig. 3). All relevant
FT-IR peaks related to the soluble and nanoformulation were indicated with black arrows and found to be almost identical with minor deviations in their intensities (Fig. 3 Table). All samples were lyophilized prior to scanning and data were collected using OPUS software.

[0033] Assessment of Loading Efficiency
Loading efficiency of Lacto-Cur-nano, Lacto-EFV-nano and ECNPs were assessed. ECNPs were prepared at four different concentrations of efavirenz by keeping the concentration of lactoferrin and curcumin constant (Table 1). Maximum loading was observed in formulation ratio II for ECNPs (63% ± 1.9 of Cur. and 61.5 ± 1.6 of EFV), IIA for Lacto-Cur-nano (59% ± 1.34) and IIIB for Lacto-EFV-nano (58.4% ± 1.79). This suggests that maximum amount of drugs has been entrapped in protein. It has also been observed that combination of EFV and Cur are synergistic in loading of one drug to the other.
Encapsulation Efficiency (%) = Mtotal – M lost
M total * 100
S. No. Nano-particles E.E (%)
1 CNPs Cur 59
2 ENPs EFV 58
3 ECNPs Cur/EFV 63/61

S. No Drug Name Size Z.P PDI E.E (%)
1 TNPs 108nm -19 0.374 TNF = 53
2 TCNPs 120nm -27 0.413 CUR = 61, TNF = 64

S. No Drug Name Size Z.P PDI E.E (%)
1 DNPs 102nm -26 0.304 DPV = 61
2 DCNPs 116nm -24 0.452 CUR = 60, DPV = 64

[0034] pH Dependent Release of Drugs from NPs
pH and simulated vaginal fluid dependent release profile of (Fig. 4A-4C) curcumin and efavirenz from ECNPs, curcumin from CNP (Lacto-nano-curcumin), efavirenz from ENP (Lacto-nano-efavirenz). The combination of nano-particles is studied for Curcumin + Efavirenz (Fig. 4a), Curcumin + tenofovir (Fig. 4b) and Curcumin + dapivirine (Fig. 4c). Each data points were repeated in triplicate (n = 3) and presented as Mean ± standard deviation (S.D). ECNPs were incubated at different pH conditions to mimic the in vivo environment of rat vagina; 300 µ g of drug loaded nano-particles were incubated with different pH values (1–9) of PBS and simulated vaginal fluid (SVF). Results showed that ECNPs are more sensitive at pH 5 and 6 with maximum drug release observed at pH 5 (Fig. 4). All three types of nano-particles either in single or combination form showed more than 80% of drug release at pH 5. At pH below 4 and above 6, only 10% of drug release was found. Thus suggests that nano-particles slowly release drugs in the vaginal lavage in the pH range of 4 to 4.5. Furthermore, higher concentrations of Cur and EFV will be released at = pH 4.5, a condition where higher virus infectivity was detected in vaginal lavage. In addition, bacterial vaginosis related-pH increase was seen which allows virus shedding41 and under these pH conditions ECNPs release higher concentrations of cur and EFV thus promoting higher viral neutralizing environment.

[0035] Stability studies of nano-particles-in vitro.
The stability of ECNPs in PBS (phosphate buffer saline pH7.4) suspension form was analyzed for at least 20 days at 4 °C and 25 °C. Data presented in Supplementary Table 1 shows that all the four parameters were found to be quite steady at both temperatures. An average negative charge of - 25 mV and a PDI of 0.4 indicates the high stability and homogenous colloidal solution property of
Nano particles. The loading efficiency and size distribution of ECNPs were found to be reasonably constant.

[0036] Cellular Localization assay
Fig .5a-5c shows cellular localisation assay of curcumin in soluble and nano-forms according to the present invention. The cellular localization is done to understand protein function of nano particles in both soluble and nano-forms. The study is done for SuPT1 cells (Fig. 5a), HI2/3 cells (Fig. 5b) and VK2 cells (Fig. 5c) was observed under confocal microscopy as shown in (Fig. 5) with 10µm concentration of soluble and nano-formulation for 1hr, 2hr, 4hr and 8hr.

[0037] Sperm Viability assay
Fig. 6a-6d shows sperms viability assay of nano-particles with varying concentrations of soluble and nano-forms according to the present invention. The sperm viability activity of nano-particles is examined and/or analysed for both soluble and nano-forms. The studies are conducted for many types of nano-formulations, such as Sol Cur, Nano Cur, Sol EFV, Nano EFV, Sol (Cur+ EFV), ECNPs, Sol (Cur + TNF) and Sol (Cur + DPV) nano-particles. The spermicidal activity is observed at 570nm, curcumin showed spermicidal activity at 200µm in both soluble and non-soluble forms as shown in (Fig. 6a-6d).

[0038] Anti-HIV Activity of Nano-particles
Fig.7a-7c shows anti-HIV-1 activity of combination of nano-particles according to the present invention. Virus inhibition in the presence of nano-forms either single or in combination remains sustained for a period up to 24 h of prior-exposure to cells while soluble forms show a slight decrease in activity upon this pre-exposure. Anti-HIV-1 activity of curcumin + efavirenz, curcumin + tenofovir and curcumin + dapivirine studies are as shown in (Fig. 7a-7c). The Anti-HIV activity study for combination of nano-particles Cur + EFV (Fig. 7a), Cur + TNF (Fig. 7b) and Cur + TNF (Fig. 7c) are found to be improved or intact even after 50 reduction in dose.

[0039] Viability assay Profile of Lactobacillus
Fig. 8a-8c shows lactobacillus viability test of both soluble and nano-forms in single and combination forms of nano-particles according to the present invention. Lactobacillus is the most colonizing bacteria in the vagina, and isgenerally considered as a gatekeeper of the vaginal ecosystem. Any microbicide could be considered as safe until it is non-toxic to the growth of Lactobacillus. Lactobacillus naturally produces hydrogen peroxide which provide a natural barrier for HIV transmission. In (Fig. 8a-8c) it is clear that there is no difference in the viability of bacteria between the media control and ECNPs, which confirms that ECNPs could be a safe microbicide and no bacterial growth inhibition was observed in both soluble and nonsoluble forms of drugs. Viability percentage of Lactobacillus cripatus when treated with ECNP, at 4 h and 48 h, media without ECNPs and 1% triton X served as negative and positive control respectively.

[0040] Dose Dependent Toxicity and Bioavailability Studies
Fig. 9a-9i shows bioavailability studies upon topical application in vagina for combination of nano-particles according to the present invention. Assessment of efavirenz and curcumin in vaginal lavage. The fixed doses mentioned in dose schedule (study 1) were administered intra vaginally and the drugs estimated in lavage are plotted in (Fig. 9a-9i). Results suggest that the nano-formulation of drugs curcumin & EFV either in single form or combination showed 1.8 (1st dose) to 2.2 (3rd dose) fold more availability of drugs at the topical site in a dose-dependent manner in vaginal lavage (fig. 9a). Efavirenz and curcumin concentration in vaginal tissue (Fig. 9b). Absorption of drugs in local cervical-vaginal tissue has been estimated and represented in (Fig. 9b). Efavirenz and curcumin were found in very low concentrations in the range of micrograms, 1000-fold less than vaginal lavage. Cur NPs and EFV NPs didn’t show appreciable difference in exposure of drugs as compared to sol counterpart. But in the case of ECNPs, we found there was 1.2 (1st dose) to 1.8 (3rd dose) fold lower drug concentrations as compared to sol drugs in combination. Systemic bioavailability of efavirenz and curcumin. (Fig. 9c) represents the nature of accumulation of drugs in the systemic circulation. We found that the sol form of drugs either in single or combination showed 1.5 (1st dose) to 1.7 (3rd dose) fold more accumulation in plasma as compared to its nano-form; leading to higher organ-associated toxicity in the case of sol formulation. In the comparison study between the ECNPs and its soluble combination form at the third dose, curcumin and efavirenz were found to be approx. 30 ng/ml & 20 ng/ml and 44 ng/ml & 35 ng/ml respectively, suggest that at higher doses also the NPs could me marked as safe.

Results for curcumin and tenofovir (Fig. 9d-9f) suggest that the nano-formulation of drugs either in single form or combination showed 2nd fold (1st dose) to 2.2 (3rd dose) fold more availability of drugs at the topical site in a dose-dependent manner in vaginal lavage (Fig. 9d). Tenofovir and curcumin concentration in vaginal tissue (Fig. 9e). Absorption of drugs in local cervical-vaginal tissue has been estimated and represented in (Fig. 9e). Tenofovir and curcumin were found in very low concentrations in the range of micrograms, 1000-fold less than vaginal lavage. Cur NPs and TNF NPs didn’t show appreciable difference in exposure of drugs as compared to sol counterpart. But in the case of ECNPs, we found there was 0.8 fold (1st dose) to 1.8 (3rd dose) fold lower drug concentrations as compared to sol drugs in combination. Systemic bioavailability of tenofovir and curcumin. (Fig. 9f) represents the nature of accumulation of drugs in the systemic circulation. We found that the sol form of drugs either in single or combination showed 1.4 fold (1st dose) to 1.8 fold (3rd dose) fold more accumulation in plasma as compared to its nano-form.

Results for curcumin and dapivirine (Fig. 9g-9i) suggest that the nano-formulation of drugs either in single form or combination showed 1.8 fold (1st dose) to 2.2 (3rd dose) fold more availability of drugs at the topical site in a dose-dependent manner in vaginal lavage (Fig. 9g). Dapivirine and curcumin concentration in vaginal tissue (Fig. 9h). Absorption of drugs in local cervical-vaginal tissue has been estimated and represented in (Fig. 9h). Dapivirine and curcumin were found in very low concentrations in the range of micrograms, 1000< fold vaginal lavage. Cur NPs and DPV NPs didn’t show appreciable difference in exposure of drugs as compared to sol counterpart. But in the case of ECNPs, we found there was 1.2 fold (1st dose) to 1.8 fold (3rd dose) fold when compared to soluble form. Systemic bioavailability (Fig. 9i) of dapivirine and curcumin represents the nature of accumulation of drugs in the systemic circulation. We found that the sol form of drugs either in single or combination showed 1.5 fold (1st dose) to 1.7 fold (3rd dose) accumulation in plasma as compared to its nano-form.

[0041] Safety Analysis
Fig. 10a-10c shows safety analysis for combination of nano-particles according to the present invention. The studies are conducted for many types of formulations such as Sol Cur, Nano Cur, Sol EFV, Nano EFV, Sol (Cur+ EFV), ECNPs, Sol (Cur + TNF) and Sol (Cur + DPV) were used and the study was carried out up to three doses.

The studies for safety analysis for the combination of drug curcumin & efavirenz is showed in Fig. 10a. The Soluble EFV and Nano-form EFV showed at least three-fold increase in the level of TNF alpha & IL-6.ECNPs showed the increased TNF alpha & IL-6 levels drastically dropped by 2.5 fold as compared to its sol formulation.

The studies for safety analysis for the combination of drug curcumin & tenofovir is showed in Fig. 10b. The Sol TNF and Nano TNF showed at least three-fold increase in the level of TNF alpha & IL-6.ECNPs showed the increased TNF alpha & IL-6 levels drastically dropped by greater than 2 fold as compared to its sol formulation.
The studies for safety analysis for the combination of drug curcumin & dapivirine is showed in Fig. 10c. Sol DPV and Nano DPV showed at least 2-3 fold increase in the level of TNF alpha & IL-6.ECNPs showed the increased TNF alpha & IL-6 levels drastically dropped by 2.5 fold as compared to its sol formulation.

[0042] Histopathology Analysis
Fig. 11A-11C shows histopathology analysis according to the present invention. Histopathological studies of cervico vaginal epithelia showed that ECNPs causes a reduced amount of toxicity in a dose dependent manner. The integrity of epithelia was found heavily damaged when the combination of sol drugs were used. In the case of ECNPs, the integrity of tissue was found to be same as that of control or very lesser extent. Tissue section pictures revealed that even at higher dose, ECNPs caused very less amount of tissue damage and at the same time soluble drug combination shows higher tissue damage. Negative (Fig. 11g) and positive controls (Fig. 11h) were treated with saline and nonoxynol-9 respectively. Left panel (Fig. 11a, c, e) represents the vaginal epithelia treated with ECNPs at first, second and third dose respectively. Right panel (Fig. 11b, d, f) represent the epithelia lining treated with Sol (Cur + EFV) at first, second and third dose respectively.

Histopathology analysis for the combination of curcumin & tenofovir studies is as shown in Fig. 11B. The tissue section pictures revealed that even at higher dose ECNPs caused very less amount of tissue damage and at the same time soluble drug combination shows higher tissue damage.

Histopathology analysis for the combination of curcumin & dapivirine studies is as shown in Fig. 11C. Tissue section pictures revealed that even at higher dose DCNPs caused very less amount of tissue damage and at the same time soluble drug combination shows higher tissue damage.

[0043] Pharmacokinetic Study Result in Vaginal Lavage
Fig. 12a-12c shows pharmacokinetic analysis for combination of nano-particles according to the present invention. The Pharmacokinetics study as shown in (Fig. 12a-12c) is done for different combination of nano particles such as curcumin & EFV, curcumin & TNF and curcumin & DPV
for soluble and nano-forms by applying an equivalent amount of ECNPs as single dose in vagina for indicated time points as shown in table 12a-12c.

Curcumin and efavirenz levels were measured separately in vaginal lavage after administration of single dose of combination drugs either in soluble or nanoform. Drug levels were observed at nine different time points up to 24 h (Fig. 12a-12c). We found enhanced PK profile (Table 12a-12c) when drugs were delivered via nano-particles. The lavage PK shows more than 3-fold increase in AUMC both in the case of efavirenz as well as curcumin. Similarly there was more than 2-fold increase in Tmax and t1/2 (Table 12a-12c). AUC values were also seen to be increased by 50% when nano-particles were used as delivery system. This PK profile suggest the higher bioavailability of drugs when given through lactoferrin nano-particles.
Values in the parenthesis designate the concentration of curcumin in milligrams per swab. The Pharmacokinetic parameters for the combination of curcumin & EFV is shown in table 12a (Fig. 12a) such as AUC: The integral of the concentration-time curve (after a single dose or in steady state). AUMC: Partial area under the moment curve between t start and t end. Cmax: The peak plasma concentration of a drug after oral administration. Tmax: Time to reach Cmax. t1/2: The time required for the concentration of the drug to reach half of its original value.

Values in the parenthesis designate the concentration of curcumin in milligrams per swab. The Pharmacokinetic parameters for the combination of curcumin & TNF is shown in table 12b (Fig. 12b) such as AUC: The integral of the concentration-time curve (after a single dose or in steady state). AUMC: Partial area under the moment curve between t start and t end. Cmax: The peak plasma concentration of a drug after oral administration. Tmax: Time to reach Cmax. t1/2: The time required for the concentration of the drug to reach half of its original value.

Values in the parenthesis designate the concentration of curcumin in milligrams per swab. The pharmacokinetic parameters for the combination of curcumin & DPV is shown in table 12c (Fig. 12c) such as AUC: The integral of the concentration-time curve (after a single dose or in steady state). AUMC: Partial area under the moment curve between t start and t end. Cmax: The peak plasma concentration of a drug after oral administration. Tmax: Time to reach Cmax. t1/2: The time required for the concentration of the drug to reach half of its original value.
[0044] Evaluation of Drug Release kinetics of ECNPs
Fig. 13a-13c shows time course analysis for combination of nano-particles according to the present invention. To investigate the extent of availability of drugs over a long period of time, the time-dependent release study was performed. The time-dependence was studied individually for the combination of drugs such as curcumin & efavirenz, curcumin & tenofovir and curcumin & dapivirine delivered in ECNPs; the results are shown in (Fig. 13a-13c). The figure represents that the concentration of curcumin and efavirenz in vaginal lavage at various time point after one (sol drugs combination) and two (ECNPs) hours of lag phase of drug administration. The results suggest high concentration in the initial stage for both the sol and nano-form, which got decreased later exponentially.

The results for the combination of curcumin & EFV nano-formulations of drugs as shown in (Fig. 13a) is the nano-formulations even after 2 hr of lag phase showed sustained and significant release of drug up to 12 hr whereas both the drugs in the sol form, showed fast reduction (at 4hr) in the concentration. We have indeed detected the presence efavirenz and curcumin up to 8–12 h, when drugs were given in nanoformulation but in case of the soluble form, the drugs were eliminated in 4–6 hrs.

The results for the combination of curcumin & TNF nano-formulations of drugs as shown in Fig. 13b is nano-formulations even after 2hrs for curcumin and 2.5hrs for TNF of lag phase showed sustained and significant release of drug up to 12 hr whereas both the drugs in the sol form, showed fast reduction (at 4hr) in the concentration. We have indeed detected the presence TNF and curcumin up to 8–12 h, when drugs were given in nano-formulation but in case of the soluble form, the drugs were eliminated in 4–6 hrs.

The results for the combination of curcumin & DPV nano-formulations of drugs as shown in Fig. 13c is nano-formulations even after 2.5hrs for DPV and 2hr for curcumin of lag phase showed sustained and significant release of drug up to 12 hr whereas both the drugs in the sol form, showed fast reduction (at 4hr) in the concentration. We have indeed detected the presence DPV and curcumin up to 8–12 h, when drugs were given in nano-formulation but in case of the soluble form, the drugs were eliminated in 4–6 hrs.
[0045] In one embodiment the size of nano particles 20-26nm (lacto) and 40-70nm (ARV lacto), E.E% ˜ 58% to 61%, IR results showed drugs are chemically stable. Maximum drug release was observed at pH-5 in pH dependent drug release. Optimum drug release showed in vaginal simulated fluid (pH 4.5) and antiviral activity was enhanced in combinational nano-forms, this gives the advantage of using drugs in combination with the nano-form there by reducing the dosage leading to the prevention of unwanted side effects and toxicities. Due to no toxicity to lactobacillus exhibited by all forms of drugs there may not to be any threat to vaginal micro flora which is considered as a safety Microbicide and Curcumin showed effective spermicidal activity in both soluble nano-forms at 200µM concentration.

[0046] In other embodiment the Bioavailability studies shown that in nano-formulation can treate maximum amount of drugs present in the vaginal lavage, vagina tissue and very low concentrations of drugs were observed in systemic circulation. The integrity of epithelia was found heavily damaged when the combination of soluble drugs were used. In case of ECNPs, the integrity of tissue was found to be same as that of control or very lesser extent. We found enhanced PK profile when drugs were delivered via nano-particles, it suggests that the higher bioavailability of drugs when given through lactoferrin nano-particles. In time course assay drugs were present up to 8-12hrs in nano-form treated but in case of soluble treated drugs were eliminated in 4-6hrs.

[0047] A detailed description of the above embodiment of the present invention, is only for ease of understanding and cited examples should not be construed as limiting the scope of the invention. Any person skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

,CLAIMS:1. A composition to treat sexually transmitted infections and mediated inflammations in vagina, wherein the composition comprises of:
a lactoferrin;
curcumin of 20mg and efavirenz (EFV) of 10mg;
curcumin of 20mg and tenofovir (TFV) of 10mg; and
curcumin of 20mg and dapivirine (DPV) of 10mg;

2. A method of preparing a composition to treat sexually transmitted infections and mediated inflammations in vagina, wherein the method comprises steps of:
dissolving 40mg of lactoferrin in 500µl ice cold PBS pH7.4;
dissolving 20mg of curcumin in 100µl of methanol and/or Dimethyl sulfoxide (DMSO) to prepare a drug mixture;
dissolving varying concentrations (5,10,15 and 20mg) of efavirenz or tenofovir or dapivirine in 100µl of methanol and/or DMSO to prepare a drug mixture;
adding the drug mixture to lactoferrin and incubated on ice for one hour;
adding slowly the whole mixture into a 60ml plastic container containing 25ml of ice cold olive oil followed by gentle manual vortexing;
keeping the container on ice at 4oC and sonicated using the narrow titanium probe for 15 minutes at an interval of 5 min;
transferring the whole mixture after sonication to 50ml oak ridge centrifuge tube;
freezing the mixture in liquid nitrogen for 15 mins;
thawing the tube on ice for four hours after freezing for 15 mins;
centrifuging the nano-particles tube at 600rpm for 10 minutes at 4oc;
discarding supernatant (olive oil) and save the nano-particles pallet;
washing the nano-particles pallet thrice with ice cold diethyl ether to remove any oil traces;
suspending the nano-particle pallet finally in PBS and used for further study;
and
storing the nano-particle pallet at 4oc for long term.
3. The composition according to claim 2, characterized to prepare the lactoferrian nano-particles with combination of curcumin and efavirenz using sol-oil chemistry method and used as a controlled delivery system through vaginal route as a microbicide.

4. The composition according to claim 2, characterized to comprise nano-particles of lactoferrin loaded with 94% pure curcumin and 98% pure efavirenz or tenofovir or dapivirine.

5. The method according to claim 2, wherein the characterization of nano-particles are studied through one or more ways, including:
a field emission scanning electron microscope (FE-SEM);
a atomic force microscope (AFM);
a transmission electron microscopy (TEM); and
a fourier transform infrared spectroscopy (FTIR).

6. The method according to claim 2, wherein the lactoferrin loaded curcumin and EFV nano-particles serve as a highly effective and efficient multi-protective and microbicide formulation.

7. A process to estimate the pH dependent drug release from nano-particles, where in the method comprises steps of:
adding nanoparticle with the PBS (Phosphate Buffer Saline);
incubating for two hours on rocker at room temperature;
adding 30% silver nitrate to resulting mixture to precipitate the protein;
adding 1ml methanol to the mixture;
collecting supernatant for analysing; and
estimating the drug release for the present drug.

8. The process according to claim 7, wherein the pH dependent drug release is analysed for more than include but not limited combination of nano-particles, wherein the nanoparticle include:
curcumin;
efavirenz;
tenofovir;
dapivirine;
combination of curcumin and efavirenz;
combination of curcumin and tenofovir;
combination of curcumin and dapivirine;

9. A process of cellular localization for understanding protein function of nano-particles, wherein the process includes;
selecting 0.2 million cells;
seeding 0.2 million of cells in twelve well plates with SFM (Serum Free Medium);
placing the supplements for four hours at 370 and 5% CO2;
treating the cells with 10µm concentration of soluble and nano-formulation for one hour, two hour, four hour and eight hour;
washing and replacing the cells with fresh media after each time point;
and;
observing the protein function of each cell under confocal microscopy.

10. A process for analysing spermicidal activity of nano-particles in both soluble and nano-forms, wherein the process includes;
selecting 0.2 million sperms;
seeding 0.2 million sperms in 96 well plates with M2 media;
placing the plates at 370 and 5% Co2;
treating the sperms at varying concentrations of soluble and nano-formulation for two hours;
washing and replacing the cells with fresh media for every two hours;
adding 20ml of 5mg/ml MTT and incubated for two hours;
and;
removing the media and dissolving the precipitate in 200µl.

11. The spermicidal activity according to claim 10, wherein is observed at 570nm, curcumin showed spermicidal activity at 200µm in both soluble and nano forms.

12. A process for analysing an anti HIV-1 activity for different combinations of nano-particles in both soluble and nano-forms, wherein the process includes;
selecting 0.5m Supt1 cells;
seeding 0.5m Supt1 cells with RPM1 1640 + 0.1% FBS;
challenging the cells with HIV-1 of 20 ng/ml for twelve hours;
incubating the cells with drugs of soluble and nano-forms;
replacing with fresh media with 10% FBS after twelve hours and incubated for 96 hours;
and;
measuring virus titer using P24 ELISA assay after 96 hours.

13. The anti HIV-1 activity according to claim 12, characterized to be analysed in different combinations of nano-particles include but not limited to:
combination of curcumin + efavirenz;
combination of curcumin + tenofovir; and
combination of curcumin + dapivirine.

14. The anti HIV-1 activity according to claim 12, wherein the combination of different nano-particles are found to be improved and/or intact even after 50% reduction in dose.

15. A process for conducting lactobacillus viability test by using disc method for different combinations of nano-particles in both soluble and nano-forms, wherein the process includes;
culturing the lacto spores in nutrient broth at 370c for 24 hours;
inoculating of particular bacteria were streaked on the MHA (Muller Hinton Agar) plates using a sterile inoculation loop;
placing the drugs on sterile disc on MHA plates;
and;
incubating the inoculated plates at 370 c for 24 hours.

16. The lactobacillus viability test according to claim 15, characterized to place the drugs of 1mg, 2mg and 5mg on the MCA plate in both soluble and nano-forms in single and combination forms, wherein the drugs include but not limited to:
curcumin;
efavirenz (EFV);
tenofovir (TNF); and
dapivirine (DPV).

17. The lactobacillus viability test according to claim 15, wherein the studies show no bacterial growth inhibition in both soluble and nano-forms of drugs.