DESC:A FORMULATION COMPRISING NANO-SCALE ANTI-CANCER COMPOUND
TECHNICAL FIELD
[0001]Embodiments are generally related to the field of pharmaceuticals. Embodiments are also related to pharmaceutical compositions. Embodiments are further related to the process of preparing a pharmaceutical composition.
BACKGROUND OF THE INVENTION
[0002]Cancer is the most dreadful disease affecting all age groups throughout the world. The advent of nanotechnology in this era has embarked on a new scenario in the field of cancer medicine. The major impact of nanotechnology in medicine is due to its versatile properties such as easy permeation through blood vessels without differentiation by host defence mechanism, improved therapeutic efficacy, prolonged half-life, controlled and sustaining release, improved bioavailability, enhanced site specificity, reduced dosage concentration and less or no toxic side effects.
[0003]Also, researches have recently demonstrated that the therapeutic potential of the polyphenolic components from spices and their oils have potent cytotoxic and antimicrobial activity as well. These spices are widely produced in many countries, especially in India. The easy availability of these products and the bio-based approach without using highly sophisticated techniques could bring about a major breakthrough in cancer treatment.
[0004]The current work involves nano-scaled formulation of Indian celery oil using a simple microemulsion technique. The formulation involves the use of three components, i.e., oil, surfactant and water, at specified concentrations. The droplets produced are in the nano size range of less than 10 nm, and demonstrate potent anticancer activity in human cancer cell lines. The ease of preparation, easy availability, bio-based approach and the utilization of nanotechnology would constitute a major breakthrough in treating various types of cancer more effectively at low-dosage concentrations with limited or no side effects. Moreover, this product, when commercialized, could easily reach common people with needs due to its cost effectiveness.
summary of the Invention
[0005]The following summary is provided to facilitate an understanding of some of the innovative features unique to the disclosed embodiment and is not intended to be a full description. A full appreciation of the various aspects of the embodiments disclosed herein can be gained by taking the entire specification, claims, drawings, and abstract as a whole.
[0006]One aspect of the disclosed embodiments is to provide a formulation for a nano scaled anti cancer compound.
[0007]Another aspect of the disclosed embodiments to is to provide a method for preparing the formulation for a nano-scaled anti cancer compound.
[0008]The aforementioned aspects and other objectives and advantages can now be achieved as described herein. The invention involves a bio-based formulation prepared using nanotechnology. The ease of preparation, optical clarity, nano-sized droplets and cost effectiveness could be of great advantage in the pharmaceutical industry. The formulation shows promising results against certain human cancer cell lines. This nano formulation with good aqueous solubility is biocompatible with minimum or no side effects as compared to other conventional chemotherapeutic treatments, which, in turn, could comply with the patient’s needs.
[0009]The composition involves a mixture of Indian celery (Apium spp) oil, non ionic surfactant and water. The formulations are prepared at varying oil-to-surfactant ratios using microemulsion technique. The optimized formulation was formed by keeping the oil concentration as constant (4% to 6%). The optimized formulation was selected based on its good stability, optical clarity, droplets in nanometer range, low polydispersity index and reduced surfactant concentration (to reduce adverse effects due to gastrointestinal irritation). The optimized formulation showed droplets in the nanometer range with a low polydispersity index as demonstrated through Dynamic Light Scattering technique. The turbidity measurements through UV-Spectroscopy demonstrated relatively weak scattering from this optically clear formulation. The droplet size and morphology was further confirmed through Transmission Electron Microscopy. The optimized formulation was found stable at 25°C, 45°C and 4°C. Based on the stability, the Indian celery (Apium Graveolens) oil to surfactant ratio (tween 20) of 1:6, 1:7, 1:8, 1:9, and 2:8 and Indian celery (Apium Graveolens) oil to surfactant ratio (tween 80) of 1:6, 1:7, 1:8 and 1:9 were found to be stable. Moreover, this system is of oil-in-water type with low viscosity, and hence, usable as oral formulation. The celery extract with anti-gastric/ulcer property and reduced surfactant concentration would lower the effects due to gastric problems.
[0010]This low viscous and transparent formulation shows promising results against human cervical cancer cell lines (SiHa) and human colon cancer cell lines (HCT-116 wild type) as demonstrated through MTT assay and Annexin V-FITC assay. Further, this optimized formulation can be developed as an effective anti-cancer therapy after screening against various other human cancer cell lines. The optimized formulation also has potent antimicrobial activity.
[0011]The process proposed herein is an exemplary demonstration of working of the proposed concept of a method for preparing a formulation of a nano-scaled anti-cancer compound. The standards and operating protocol for the same can be standardized and adapted by a person skilled in the art to achieve the same or enhanced results.
Brief description of drawings
[0012]The accompanying figures, in which like reference numerals refer to identical or functionally-similar elements throughout the separate views and which are incorporated in and form a part of the specification, further illustrate the present invention and, together with the detailed description of the invention, serve to explain the principles of the present invention.
[0013]FIG. 1 illustrates a flow diagram for preparation of a nano-scaled anti-cancer compound.
[0014]FIG. 2 illustrates seed oil of Apium spp formulation using tween 20 as surfactant a) left to right; 1:1 to 1:9 and b) left to right; 1:9 to 9:1.
[0015]FIG. 3 illustrates seed oil of Apium spp formulation using tween 80 as surfactant a) left to right; 1:1 to 1:9 and b) left to right; 1:9 to 9:1
[0016]FIG 4. (a) Ternary phase diagram constructed using celery oil, tween 20 and water shows O/W microemulsion region with (left) varying oil: surfactant of 1:1 to 1:9 and (right) varying oil: surfactant of 1:9 to 9:1. (b) Ternary phase diagram constructed using celery oil, tween 80 and water shows O/W microemulsion region with (left) varying oil: surfactant of 1:1 to 1:9 and (right) varying oil: surfactant of 1:9 to 9:1.
detailed description
[0017]The particular values and configurations discussed in these non-limiting examples can be varied and are cited merely to illustrate at least one embodiment and are not intended to limit the scope thereof.
[0018]The embodiments now will be described more fully hereinafter with reference to the accompanying drawings, in which illustrative embodiments of the invention are shown. The embodiments disclosed herein can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Like numbers refer to like elements throughout. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
[0019]The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
[0020]Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
[0021]The formulation of the nano-scaled anti-cancer compound can be achieved as disclosed herein. The composition involves a mixture of Indian celery (Apium spp) oil, non ionic surfactant and water. The formulations are prepared at varying oil-to-surfactant ratios using microemulsion technique. The optimized formulation was formed by keeping the oil concentration as constant (4% to 6%). The optimized formulation was selected based on its good stability, optical clarity, droplets in nanometer range, low polydispersity index and reduced surfactant concentration (to reduce adverse effects due to gastrointestinal irritation).
[0022]The process of preparing the formulation of the nano-scaled anti-cancer compound can be achieved as disclosed herein. The various formulations were prepared by varying oil-to-surfactant ratios from 1:1 to 1:9 and 1:9 to 1:1 respectively to find the presence of microemulsion existence region. The non-ionic surfactants used were Tween 20 and Tween 80. Tween 20 was found easily miscible with celery oil. Water is added drop-wise to the oil-surfactant mixture by constant stirring at 500 rpm under laboratory conditions and the clear emulsion was formed spontaneously at optimized concentrations of the formulation.
[0023]The following is the best embodiment to perform the invention. The celery oil concentration of was 5%. The best oil-surfactant ratio was chosen based on reduced amount of surfactant utilized, optical clarity, low viscosity, thermodynamic stability and nano-sized droplets obtained with minimum polydispersity index.
[0024]Microemulsions were prepared using a conventional titration technique by titration of oil and surfactant against aqueous phase at laboratory temperature. Different ratios of oil and surfactant (1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 and 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9) were prepared initially. Water was added drop by drop to the mixture using a magnetic stirrer to allow equilibration. The stirring rate was maintained at 500 rpm to prepare all the samples. Visual changes in the sample from turbid to transparent and vice versa were observed to determine the endpoint of the compositions. The presence of milky white or turbid mixtures that eventually phase separated within a few hours was termed as biphasic. Clear, transparent, and low-viscous mixtures that formed within a few seconds with no phase separation were termed as monophasic. From the endpoint compositions of the titrated samples, various concentrations of the components (celery seed oil, surfactant (tween 20/tween 80), and water) were plotted on triangular coordinates, to construct the phase diagram. Only the area covered by the monophasic region was shaded and termed as the microemulsion existence region. All experiments were done in triplicate, to ensure reproducibility.
[0025]The stability of the different formulations under extreme stress conditions was examined. Initially, all formulations were subjected to centrifugation at 3500 rpm for 30 min to assess physical stability. Those formulations with no phase separation, indicating long-term stability, were taken for further checking. The samples that passed centrifugation were checked at 4, 25 and 45 °C for a period of one month. This study was performed to check the effect of temperature variations on the stability of microemulsion formulations. Formulations that passed the stress tests were taken for further optimization and utilized for application studies.
[0026]The cytotoxicity was carried out by MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay for investigating changes in mitochondrial/non-mitochondrial dehydrogenase activity. In brief, cells (SiHa and HCT-116 wild type respectively) were seeded in 96-well plates (10, 000 cells/well) and cultured in DMEM containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2 for 48?h.
[0027]The optimized celery oil based microemulsion system (A6 formulation) was diluted with DMEM to various concentrations (0.1, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0 and 10 µL/mL respectively) and added. A surfactant: water mixture was used as a negative control. Exposure periods of 48?h were chosen for determining the in vitro cytotoxicity. After incubation, the supernatant was removed, and MTT solution (5?mg/mL) was also added to each well, 30?min prior to the end of the experiment and incubated for 3 h. Water-insoluble dark blue formazan crystals formed in viable cells were solubilized in DMSO and the absorbance was measured at 570?nm using a microplate reader. Those cells that have lost their viability cannot do so. The percentage of cell viability was determined by comparing the absorbance values obtained from control and treated cells. The cytotoxicity was expressed as the concentration of sample that inhibited 50% of cell growth (IC50). This is a high throughput screening technique that measures cell viability using a calorimetric reaction.
[0028]The Annexin V-FITC Early Apoptosis Detection kit was used to detect early apoptotic cells within a cell population through flow cytometry. The HCT-116 wild type cells (1 x 105 to 106 cells) were plated onto 75-cm2 flasks and cultured overnight; this was followed by incubation with IC50 values of the optimized formulation for 48 h. The cells of interest are collected through centrifugation and washed cells with ice-cold culture medium or PBS. The cells were then resuspended to 105 - 106 cells/ml with Annexin V Binding Buffer. An aliquot of 96 µL cell suspension was taken in an assay tube. 1 µL Annexin V-FITC conjugate and 12.5 µL Propidium Iodide (PI) solution were added to each 96 µL cell suspension. Then, allow cells to incubate 10 min on ice in the dark. The cell suspension was diluted to a final volume 250 µL/assay with ice-cold 1X Annexin V Binding Buffer and was immediately analyzed using a flow cytometer with in-built software.
[0029]The nano-scaled system was formulated using Indian celery seed oil, non-ionic surfactant Tween 20 and water using simple microemulsion technique. This small molecule surfactant gets adsorbed onto the emulsion droplet surface more rapidly than high molecular weight surfactants. Hence, this surfactant is comparatively more effective in minimizing the droplet size than other polymers. The concentration of celery seed oil (5% v/v) was fixed for all the formulations. The organic phase was prepared by mixing oil and surfactant in different ratios (v/v) as mentioned, and the organic phase was then added drop wise to water. The reaction was carried out using a magnetic stirrer at 500 rpm at laboratory conditions. Emulsions were formed spontaneously and characterized. The formulations using tween 20 as surfactant are shown in Table 1, and the respective images in Fig. 2. The formulations using tween 80 as surfactant are shown in Table 2, and their images in Fig 3. All the images were taken after 24 h of preparation. It is seen that the formulation codes A5 and A11 were translucent in nature when prepared spontaneously. But after 24 h of preparation this was changed to transparent in nature.
Table 1. Celery Seed Oil formulations with Tween 20 as surfactant
Formulation Code (O:S) Celery Oil Tween 20 Water Appearance
A1 (1:1) 5 5 90 Milky White
A2 (1:2) 5 10 85 Milky White
A3 (1:3) 5 15 80 Turbid
A4 (1:4) 5 20 75 Turbid
A5 (1:5) 5 25 70 Translucent to Transparent
A6 (1:6) 5 30 65 Clear and Transparent
A7 (1:7) 5 35 60 Clear and Transparent
A8 (1:8) 5 40 55 Clear and Transparent
A9 (1:9) 5 45 50 Clear and Transparent
A10 (2:8) 10 40 50 Clear and Transparent
A11 (3:7) 15 35 50 Translucent to Transparent
A12 (4:6) 20 30 50 Milky White
A13 (5:5) 25 25 50 Milky White
A14 (6:4) 30 20 50 Milky White
A15 (7:3) 35 15 50 Milky White
A16 (8:2) 40 10 50 Milky White
A17 (9:1) 45 5 50 Milky White

Table 2. Celery Seed Oil formulations with Tween 80 as surfactant
Formulation Code (O:S) Celery Oil Tween 80 Water Appearance
B1 (1:1) 5 5 90 Cloudy
B2 (1:2) 5 10 85 Cloudy
B3 (1:3) 5 15 80 Cloudy
B4 (1:4) 5 20 75 Turbid
B5 (1:5) 5 25 70 Turbid
B6 (1:6) 5 30 65 Clear and Transparent
B7 (1:7) 5 35 60 Clear and Transparent
B8 (1:8) 5 40 55 Clear and Transparent
B9 (1:9) 5 45 50 Clear and Transparent
B10 (2:8) 10 40 50 Milky White
B11 (3:7) 15 35 50 Milky White
B12 (4:6) 20 30 50 Milky White
B13 (5:5) 25 25 50 Milky White
B14 (6:4) 30 20 50 Milky White
B15 (7:3) 35 15 50 Milky White
B16 (8:2) 40 10 50 Milky White
B17 (9:1) 45 5 50 Milky White
[0030]The phase diagram gives a better understanding about the phase behavior of a defined mixture and its varying compositions. Ternary phase diagrams were constructed using Celery oil as the oil phase, Tween 20 with a HLB value of 16.7 (Fig. 4 a), or Tween 80 with a HLB value of 15.0 (Fig. 4 b) as surfactants, and water as the aqueous phase. The different concentrations using either Tween 20 (Table 1) or Tween 80 (Table 2) as surfactants in formulating an optimized microemulsion are shown clearly. It is seen that at a lower concentration, phase separation occurs easily within 24 h of preparation. At this concentration, the samples are usually milky white and turbid, which is called the biphasic region. With an increase in the surfactant concentration, clear and transparent formulations are formed with no phase separation. This implies the presence of a microemulsion area in such concentrations and this domain is called the microemulsion existence region. On varying the oil-surfactant ratio as 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, and 1:9, the existence of a larger microemulsion region (shaded area) is seen with Tween 20 (Fig. 4a (left)) as compared to Tween 80 (Fig. 4b (left)). This is because Tween 20 is easily miscible with Celery oil as compared to Tween 80, and the difference in the HLB value of the surfactants is significant. On the other hand, on varying the oil-surfactant ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1, both the surfactants, Tween 20 (Fig. 4a (right)) and Tween 80 (Fig. 4b (right)), exhibited only a small microemulsion area that is shaded or dotted in black.
[0031]The selected formulations that were clear and transparent as obtained from phase diagram studies were subjected to stability studies such as centrifugation and other stress conditions. Other formulations that phase separated within 24 h of preparation were not considered for stability studies. Among the formulations that were clear and transparent, A5 and A11 formulations did not pass through centrifugation test, and thus, were disregarded from further characterization. Table 3 clearly shows that the formulations A6 to A10 and B6 to B9 passed through all stress conditions, and these were chosen for further study. The tween 20 based formulations were selected for further physico-chemical characterization in comparison with tween 80 based formulations due to the following reasons. The tween 20 based formulations demonstrated a larger microemulsion existence area. They formed clear emulsions spontaneously with much ease due to low viscosity and easy miscibility of tween 20 with celery oil. These factors could be of interest for a pharmaceutical preparation.
[0032]The selected formulations that were clear and transparent as obtained from phase diagram studies were subjected to stability studies such as centrifugation and other stress conditions. Other formulations that phase separated within 24 h of preparation were not considered for stability studies. Among the formulations that were clear and transparent, A5 and A11 formulations did not pass through centrifugation test, and thus, were disregarded from further characterization. Table 3 clearly shows that the formulations A6 to A10 and B6 to B9 passed through all stress conditions, and these were chosen for further study. The tween 20 based formulations were selected for further physico-chemical characterization in comparison with tween 80 based formulations due to the following reasons. The tween 20 based formulations demonstrated a larger microemulsion existence area. They formed clear emulsions spontaneously with much ease due to low viscosity and easy miscibility of tween 20 with celery oil. These factors could be of interest for a pharmaceutical preparation.
Table 3. Observations of stability studies of Group A and B formulations
Formulation Oil: Surfactant Centrifugation 4o C 25o C 40o C
A5 1:5 X - - -
A6 1:6 v v v v
A7 1:7 v v v v
A8 1:8 v v v v
A9 1:9 v v v v
A10 2:8 v v v v
A11 3:7 X - - -
B6 1:6 v v v v
B7 1:7 v v v v
B8 1:8 v v v v
B9 1:9 v v v v

[0033]The cytotoxic effect of the optimized nano-scaled system (A6 formulation) was evaluated using MTT assay. The MTT test results revealed that the formulation expressed cytotoxicity against HCT-116 wild type (colon) and SiHa (cervical) cancer cell lines.
[0034]In the case of HCT-116 wild type cell lines, 36-40% and 59-63% cell death was observed after treatment (48 h) with 1.4 µL/mL and 1.6 µL/mL concentration of optimized formulation respectively. So, the IC50 value of our optimized system was found to be in the range of 1.4 – 1.6 µL/mL concentration for colon (HCT-116 wild type) cancer cell line.
[0035]Whereas, in the case of SiHa cell line, 40-45% and 59-64% cell death was observed after treatment (48 h) with 1.4 µL/mL and 1.6 µL/mL concentration of our optimized formulation respectively. So, the IC50 value of our optimized system was also found to be in the range of 1.4 – 1.6 µL/mL concentration for cervical (SiHa) cancer cell line. The effect of surfactant: water mixture was also checked as a vehicle control. In both cases, the vehicle control did not play any role in causing cytotoxicity.
[0036]The results above showed that celery oil based nano-scaled emulsion system inhibited HCT-116 wild type and SiHa cell survival and proliferation. Cell apoptosis was investigated with respect to its involvement in this effect for colon cancer cell line. In this assay, Annexin V-FITC binds to cell surfaces expressing phosphatidylserine, an early apoptosis marker. The cells stained with Propidium iodide, a non-permeable DNA dye, indicate necrotic cells. Cells stained with both PI and Annexin V-FITC demonstrates later stage apoptosis and early necrosis. Celery oil based system induced both early (Annexin V+/PI-) and late (Annexin V+/PI+) apoptosis in HCT-116 wild type cells. Interestingly, we could also notice that our nano-scaled system induced necrotic HCT-116 wild type cell death (Annexin V-/PI+). This assay indicates that direct apoptosis has a major contribution towards the celery oil based nano-scaled system-induced cytotoxicity in HCT-116 wild type cells. Though, slight necrosis is observed, it was not found significant.
[0037]This low viscous and transparent formulation shows promising results against human cervical cancer cell lines (SiHa) as demonstrated through MTT assay. Further, this optimized formulation can be developed as an effective anti cancer therapy after screening against various other human cancer cell lines. The optimized formulation is also a potent antimicrobial agent.
[0038]The process proposed herein is an exemplary demonstration of working of the proposed concept of a method for preparing a formulation of a nano-scaled anti-cancer compound. The standards and operating protocol for the same can be standardized and adapted by a person skilled in the art to achieve the same or enhanced results.
[0039]It will be appreciated that variations of the above-disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Also that various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the field.
,CLAIMS:I/We claim
1.A nano-scale anti-cancer compound formulation comprising,
seed oil of  Apium spp,
non-ionic surfactant, and
water.
2. The anti cancer formulation as claimed in claim 1, wherein; the Apium spp comprises  Apium Graveolens, Apium annuum, Apium australe, Apium bermejoi, Apium fernandezianum, Apium filiforme, Apium inundatum, Apium insulare, Apium leptophyllum, Apium nodiflorum, Apium prostratum, Apium repens
3.The anti cancer formulation as claimed in claim 1, wherein; the non ionic surfactant comprises tween 20, tween 80 or a combination thereof.
4.A method of preparing a nano-scale anti-cancer compound formulation, steps comprising,
preparing an organic phase using seed oil from Apium spp and a non ionic surfactant,
and adding the organic phase drop wise to water,
stirring the content using a magnetic stirrer at 400-600 rpm to obtain nano scaled emulsion spontaneously, which has anti cancer activity.
5.The method as claimed in claim 4, wherein; the Apium spp comprises  Apium Graveolens, Apium annuum, Apium australe, Apium bermejoi, Apium fernandezianum, Apium filiforme, Apium inundatum, Apium insulare, Apium leptophyllum, Apium nodiflorum, Apium prostratum, Apium repens
6.The method as claimed in claim 4, wherein; the non-ionic surfactant comprises tween 20, tween 80 or a combination thereof.
7.The method as claimed in claim 4, wherein; the seed oil composition of Apium spp is 4-6% v/v.
8.The method as claimed in claim 4, wherein; the organic phase is prepared by mixing different ratios of seed oil of Apium and non ionic surfactant, and choosing the emulsion formulation which is transparent immediately after preparation.