Formulation & Development of Nanocarrier for Combination Delivery for Skin Cancer

Cancer

Cancer is a broad term given to a group of diseases all involving unregulated growth of cells. Cancer originates in a normal cycle of cell division when a cell can turn cancerous and divide infinitely. Lymphatic system and bloodstream can also spread the cancer to distant parts of the body. Two types of genes are affected: Oncogenes and Tumor suppressor genes. Oncogenes are those genes that stimulate reproduction and growth of the cells. Tumor suppressor genes are those genes that obstruct the survival and division of the cells. Tumor formation can take place due to the inappropriate over-expression of normal oncogenes, formation of novel oncogenes, or because of under-expression or deactivating of tumor suppressor genes (figure 1.1). Characteristically, many genes are required to be altered in order to convert a normal cell into a cancerous one (1). With over 10 million new cases per year worldwide, cancer remains a difficult disease to treat and a significant cause for morbidity and mortality. Conventional anti-cancer therapies come with a number of drawbacks and side effects. Conventional therapy for cancer includes treatment with cytotoxic drugs (eg. doxorubicin, paclitaxel etc), radiation therapy and surgical removal of tumors. Cytotoxic drugs cause severe side effects because the mechanisms which they inhibit are common to both cancer cells and normal cells, such as ligation – relegation reaction during cell division. Thus tumor cells and normal cells both are killed by anti-cancer drugs. Bone marrow, gastrointestinal mucosa, hair follicles and gonads are among the tissues most sensitive to chemotherapy. Chemotherapeutic agents are also highly teratogenic (2). Development of resistance is another major drawback of chemotherapy. Higher doses are required to kill resistant tumor cells which further worsen the problem of unwanted side effects. Radiation therapy is successful only when tumor is localized and well defined. For surgical removal also, the tumour must be well confined. Surgery is also often not possible when the tumour is bounded by delicate and complex tissues, like brain. The top 5 most common cancers are lung, breast, colorectal, prostate and stomach with 12.3%, 12.3%, 10.6%, 7.5% and 6.1% incidences of all cancer cases

Melanoma

Melanin is the pigment which imparts color to skin and hair, and protects skin from the damage of ultraviolet radiations. Melanocytes are the specialized cells that are known to produce melanin. The cancerous growth of melanocytes is termed as Melanoma (3). Skin cancer, including malignant melanoma and non- melanoma skin cancer, is the most common cancer in Caucasian population (4). Melanoma is the most aggressive and deadly type of skin cancer. Though it accounts for only 4% of all skin cancers, but it causes the highest number of skin cancer- related deaths worldwide. The increase in incidence of Primary Cutaneous Melanoma in Caucasian populations has been constantly rising and is reported to double up every 10– 14 years (5). The highest increase is reported in men above the age of 55 years and women of all age groups (6). The estimated number of new cases of melanoma in 2017 is 87,110, which accounts for 5.2% of all new cancer cases. Estimated deaths in 2017 are 9,730. This number of melanoma cases is perhaps even higher than mentioned as the National Cancer Registries has reported that the incidences are underestimated in few countries (7). Moreover, after lung cancer and breast cancer, melanoma is the third most common reason of brain metastases. Melanoma spreads to the brain in upto 75 % of melanoma patients. Brain metastases cause death in 95 % of the cases (8). Till date, only four drugs have been approved for the treatment of melanoma, which are, dacarbazine, interleukin-2, ipilimumab and vemurafenib. Continuous efforts have been made to develop new drugs and new treatment strategies, but not many of them have given much hope. Dacarbazine was the first drug which was approved for melanoma treatment in 1975, and it still remains the gold standard chemotherapeutic drug against melanoma; although ipilimumab and vemurafenib have shown promising results in last few years. Recent experiments and results present the combinatorial approach as a promising one for treatment of aggressive melanomas, and so combining dacarbazine or other chemotherapeutic drugs with new agents seems to hold the potential that scientists have been looking for since decades (9). Though the better understanding of pathophysiology of melanoma has led to the development of new drugs which target specific pathways such as MAPK (mitogen- activated protein kinase) pathway, there success is limited due to the resistance of melanoma cells and higher toxicities of these drugs (10).

Prevalence and Epidemiology

The occurrence of melanoma has been growing among all age groups; more than 600% increase is reported in young adults between 1970 and 2009. In United States of America, melanoma is reportedly the sixth most common cancer in men and women, and the second most common cancer in women having age between 20 and 29 years At current rates, 1 in 27 white men and 1 in 42 white women are expected to develop invasive melanoma in their lifetime. In United Kingdom (UK) also, the number of incidences are on rise. Also, it is found to be most prevalent in age group 65-69 years Most melanomas arise in previously normal skin, and only 20% to 30% arise from pre- existing nevi. Although there are several genetic syndromes that predispose individuals to melanoma, these account for less than 15% of melanomas. These syndromes include xeroderma pigmentosa and familial atypical mole-melanoma syndrome (called dysplastic nevus syndrome. In the evidence of epidemiologic studies, it is shown that solar radiation exposure is the main cause of cutaneous melanoma (11). This relationship is further sustained by anatomical differences in gender, race, latitude of residence, and migration. In men, the most common site of melanoma occurence is upper back; in women, the most common sites include upper back and lower parts of legs. According to studies, people who have migrated to countries having more of ambient solar radiations have higher risk of developing melanoma than people who did not migrate. Similarly, melanoma frequency and mortality rate in whites are inversely related to distance from the equator. Differences in race are also found to exist. The lesser incidences of melanoma in highly pigmented people is a result of the protective function of melanin and smaller number of nevi (12).

Causes and Risk Factors

The core risk factors of melanoma are phenotype (blue eyes, fair complexion, and blond or red hair), cutaneous response towards exposure to sun (freckling, tanning inability, sunburn sensitivity), blistering sunburn history, strong recurrent sun exposure, genetic susceptibility, subtypes and amount of nevi (atypical nevi or giant melanocytic nevi), immunosuppression and history of melanoma. According to genetic investigations, 50% of familial melanomas and 25% of sporadic melanomas may be a result of mutations in the tumor suppressor protein p16. Chromosome 9p21 has been identified as the familial melanoma gene in linkage studies. Familial melanoma accounts for 8% to 12% of all melanoma cases. The inheritance mode is supposed to be polygenic. The cumulative risk of developing cutaneous melanoma in persons with a history of familial melanoma is estimated to be 50% by age 50.

MATERIAL & METHODS

Preparation of Phosphate Buffer Saline (PBS) pH 7.4 (IP 2010)

2.38 gm of disodium hydrogen phosphate, 0.19 gm of potassium dihydrogen phosphate and 8.0 gm of sodium chloride were dissolved in sufficient water and the volume was made up to 1000 ml with water.

Dialysis Membrane Activation

Glycerine was removed by washing in running water for 3 hours. To remove Sulphur compounds, membrane was treated with 0.3% w/v solution of sodium sulfide at 80°C for one minute. Then it was washed with hot water (60°C) for 2 minutes followed by acidification with 0.2% (v/v) sulfuric acid, and rinsed with hot water to remove the acid.

Dacarbazine

Qualitative Analysis Physical Properties

Dacarbazine was inspected visually to check the color, appearance and physical characteristics.

UV spectral analysis

Accurately weighed quantity (10 mg) of Dacarbazine was dissolved in water in the volumetric flask and volume was made up to 100 ml to achieve the concentration of 100 µg/ml. 0.5 ml of above solution was diluted to 10 ml with water in 10 ml volumetric flask (5 µg/ml). The resulting solution was then scanned for UV-Vis absorption using UV-Vis spectrophotometer in the range of 200-400 nm and λmax was determined.

Melting Point Determination

Fine powder of Dacarbazine was filled in a clean and dry capillary of uniform diameter across the length by tapping it slowly over powder bed till whole length was packed with compact column 4-6 mm high. The capillary tube was then inserted into melting point apparatus along with thermometer. The temperature at which the drug transitioned into the liquid state was recorded.

Quantitative Analysis By UV

Sample of Dacarbazine was scanned for the absorption spectrum in the region of 200- 400 nm in different solvents and the maximum wavelength was determined in respective solvents. Calibration curves were drawn.

In distilled water

Stock solution having concentration 100µg/ml was prepared by dissolving 10 mg Dacarbazine in 100ml distilled water. Stock solution was then further diluted to get the solutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 µg/ml. The solutions were scanned to determine λmax using distilled water as blank. Different sample concentrations were analyzed by UV spectrophotometer at λmax and the absorbance was noted. Readings were taken in triplicates to ensure precision and accuracy. Calibration curve was drawn using MS-Excel 2010.

In PBS pH 7.4

Stock solution having concentration 100µg/ml was prepared by dissolving 10 mg Dacarbazine in 100ml PBS (pH 7.4). Stock solution was then further diluted to get the solutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 µg/ml. The solutions were scanned to determine λmax using PBS (pH 7.4) as blank. Different sample concentrations were analyzed by UV spectrophotometer at λmax and the absorbance was noted. Readings were taken in triplicates to ensure precision and accuracy. Calibration curve was drawn using MS-Excel 2010.

In PBS 7.4: Propylene glycol (9:1)

100 µg/ml stock solution of Dacarbazine was prepared in mixture of PBS 7.4 and propylene glycol (9:1). Stock solution was suitably diluted to get concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 µg/ml. The solutions were scanned to determine λmax using blank as mixture of PBS 7.4 and propylene glycol (9:1). Different sample concentrations were analyzed by UV spectrophotometer at λmax 333 nm and the absorbance was noted. Using MS- Excel, calibration curve is drawn. Process was repeated for 3 times for precision and accuracy.

In Ethanol

100 µg/ml Stock solution of drug was prepared in Ethanol. Stock solution was diluted to get the sample solution of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 µg/ml. The solutions were scanned to determine λmax using blank as Ethanol. Different sample concentrations were analyzed by UV spectrophotometer at λmax 333 nm and the absorbance was noted. Using MS- Excel, calibration curve is drawn. Process was repeated for 3 times for precision and accuracy.

Solubility Analysis

Solubility of Dacarbazine was determined in water, phosphate buffer saline pH 7.4 and ethanol. Excess amount of the drug was added to 2 ml of water in eppendorf tube and protected from sunlight. The sealed vials were then placed in incubator shaker. The temperature was adjusted to 37°C. The sample was filtered after 48 hours and was analyzed by UV spectrophotometer. Concentration of drug was determined from the calibration curve.

Partition Coefficient

Partition coefficient of drug was calculated in Octanol and water. 50 ml each of the above-mentioned solvents were taken together into a separating funnel. 10 mg of drug was then added and the funnel was shaken at regular time intervals. After 24 hours, aliquots were withdrawn from aqueous phase, diluted appropriately and absorbance was determined. Concentration present in the aqueous phase was determined using calibration curve.

Partition coefficient is determined by: Log Po/w = Log (Coil/ Caq) at equilibrium Where, Coil is the amount of the drug present in octanol (Total Amount - Caq) Caq is the amount of the drug present in water

Eugenol

Qualitative Analysis of The Drug Sample Physical Properties

Eugenol Sample was inspected visually to check the color appearance odor and physical characteristics.

UV spectral analysis

Accurately weighed quantity (100 mg) of Eugenol was dissolved in ethanol in the volumetric flask and volume was made up to 100ml (1000 µg/ml). 0.5 ml of above solution was diluted to 10 ml with ethanol in 10 ml volumetric flask (50 µg/ml). The resulting solution was then scanned for UV-Vis absorption using UV-Vis spectrophotometer in the range of 200-400 nm and λmax was determined.

Quantitative Analysis By UV

Sample of Eugenol was scanned for the absorption spectrum in the region of 200-400 nm in different solvents and the maximum wavelength was determined in respective solvents. Then the calibration curve was drawn.

In Octanol

100 µg/ml stock solution of eugenol was prepared in octanol by dissolving 10 mg eugenol in 100 ml octanol. Stock solution was suitably diluted to get the concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 µg/ml. The solutions were scanned to determine λmax using blank as octanol. Different sample concentrations were analyzed by UV spectrophotometer at λmax 282 nm and the absorbance was noted. Using MS- Excel, calibration curve was drawn. Process was repeated for 3 times for precision and accuracy.

In Ethanol

1000 µg/ml stock solution of Eugenol was prepared in ethanol. Stock solution was suitably diluted to get concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 µg/ml. The solutions were scanned to determine λmax using blank as ethanol. Different sample concentrations were analyzed by UV spectrophotometer at λmax 281.5 nm and the absorbance was noted. Using MS- Excel, calibration curve is drawn. Process was repeated for 3 times for precision and accuracy.

In PBS pH 7.4: Propylene glycol (9:1)

1000 µg/ml stock solution of Eugenol was prepared in mixture of PBS 7.4 and propylene glycol (9:1). Stock solution was suitably diluted to get concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 µg/ml. The solutions were scanned to determine λmax using blank as mixture of PBS 7.4 and propylene glycol (9:1). Different sample concentrations were analyzed by UV spectrophotometer at λmax 281.5 nm and the absorbance was noted. Using MS- Excel, calibration curve is drawn. Process was repeated for 3 times for precision and accuracy.

Solubility Analysis

Solubility of Eugenol was determined in distilled water, PBS 7.4, ethanol. Excess amount of eugenol was added to 2 ml of water in an eppendorf tube sealed and protected from sunlight. The sealed tubes were then placed in incubator shaker. The temperature was adjusted to 37°C. The sample was filtered after 48 hrs and was analyzed by UV spectrophotometer. Concentration of eugenol was determined from the calibration curve.

Partition Coefficient

Partition coefficient of Eugenol was calculated following the same procedure as described earlier.

Drug Excipient Compatibility Studies

Compatibility of drugs and excipients is studied by performing FTIR and DSC analysis of dacarbazine, eugenol and lipid individually, and also of the physical mixture of drugs and lipid.

By FTIR Analysis

FTIR analysis of Dacarbazine, and Lipoid S100 was carried out using Shimadzu FTIR system (Kyoto, Japan) by potassium Bromide (KBr) pellet technique. Sample was dispersed and triturated with dry KBr (5% wt. of sample), ground well in mortar and pestle and KBr disk was prepared at a pressure of 1000 psig. The disk was placed in the FTIR sample holder and IR spectra in absorbance mode were obtained in the spectral region 400 to 4000 cm-1. FTIR analysis of Eugenol and physical mixture was carried out using Spectrum Two ATR- FTIR Spectrophotometer (Perkin Elmer) containing ATR diamond crystal.

By Differential Scanning Calorimetry (DSC)

Thermal investigation of Dacarbazine and Lipoid S100, and mixture was performed by using Perkin Elmer Pyris 6 DSC (MA, USA). 5 mg of the sample was placed in aluminum pan and crimped with a lid containing a pin hole which was kept in the DSC unit. Heating range was 40-300oC.

Simultaneous Estimation

Simultaneous estimation of both the drugs during loading and release studies was difficult because of their opposite nature. Also, dacarbazine could not be estimated by HPLC because it produces active metabolites, so LC-MS was a suitable estimation technique for dacarbazine; but then, eugenol could not be estimated by either HPLC or LC-MS, as it is a volatile liquid. Keeping the easy availability and several other advantages of UV spectroscopy (Dehghani Mohammad Abadi et al. 2012; Zeng et al. 2012) in mind, we developed a robust, reproducible UV method based on absorptivity measurement for the simultaneous estimation of these two drugs (Murtaza et al. 2011). UV spectrometric method for quantification of dacarbazine (Bei et al. 2010) and eugenol (Peng et al. 2015) are available separately, but we developed a method for their simultaneous determination.

Selection Of Appropriate Solvent Systems

Selecting a suitable solvent system was a challenge since both the drugs are of different nature (one hydrophilic and other lipophilic). For drug loading determination, a solvent which could dissolve both dacarbazine and eugenol as well as lipid (the entire drugs loaded liposome) was needed. For the release study, the solvent had to be appropriate which could dissolve both the drugs but not the lipid. So, various solvent systems like PBS + Triton X, PBS + methanol, PBS + Tween 80, and PBS + propylene glycol were tried to select an appropriate solvent with good suitability and stability.

Stock Solutions Dacarbazine

Stock solution of dacarbazine (100 μg/ml) was prepared by dissolving 100 mg dacarbazine in 1000 ml PBS (pH 7.4) : propylene glycol (9:1) in 1000 ml volumetric flask with vigorous shaking. This stock solution was diluted further to obtain working solutions of different concentrations (1-10 μg/ml).

Determination of absorbance maximum (λmax):

Dacarbazine solution of 5 μg/ml concentration was prepared by appropriately diluting the stock solution. This solution was scanned in UV spectrophotometer in the UV range (200– 400 nm) to determine the λmax of the dacarbazine which was found to be 331 nm (denoted as λ1) for this solvent system.

Eugenol

Stock solution of eugenol (1000 μg/ml) was prepared by dissolving 100 mg eugenol in 100 ml of PBS (pH 7.4): propylene glycol (9:1) in 100 ml volumetric flask with vigorous shaking. This stock solution was diluted further to obtain working solutions of different concentrations (10-100 μg/ml).

Determination of absorbance maximum (λmax):

Eugenol solution of 50 μg/ml concentration was prepared by appropriately diluting the stock solution. This solution was scanned in UV spectrophotometer in the UV range (200– 400 nm) to determine the λmax of the eugenol which was found to be 281.5 nm (denoted as λ2) for this solvent system.

Method Development

Following the method developed by Murtaza et al. (Murtaza et al. 2011), the concept of Absorptivity is employed to develop a method for simultaneous estimation of both these drugs. The absorptivity (a) is extinction coefficient, which is calculated using following equation:

A = a.C

a = A/C……….            (5.1)

Where, a → Absorptivity, A → Absorbance, and C → Concentration (mg/100 ml)

Using equation (1), absorptivities of Dacarbazine and Eugenol were calculated at both λ1 (331nm) and λ2 (281.5nm), for which, the absorbance of any three dilutions (n=3) of Dacarbazine was determined at λ1 (331nm). Then the values of absorbance were divided with corresponding concentrations to calculate the absorptivity of Dacarbazine at λ1. Average of three absorptivity values was taken and denoted as ad1, where ‘a’ denotes absorptivity, ‘d’ denotes dacarbazine and ‘1’ denotes λ1 i.e. 331 nm, meaning, absorptivity of dacarbazine at λ1. Same way, absorbance of dacarbazine dilutions (n=3) was determined at λ2 also, i.e. 281.5nm, and ad2 was calculated, where ‘a’ denotes absorptivity, ‘d’ denotes dacarbazine and ‘2’ denotes λ2 i.e. 281.5 nm, meaning, absorptivity of dacarbazine at λ2. Same process was repeated for eugenol, that is, absorbance of three dilutions of eugenol was determined at λ1 (331nm) and λ2 (281.5nm) separately and ae1 and ae2 were calculated, where ‘a’ denotes absorptivity, ‘e’ denotes eugenol, ‘1’ denotes λ1 (331nm) and ‘2’ denotes λ2 (281.5nm). Next, the method involves the solving of following two simultaneous equations derived from equation (5.1) in order to determine the concentrations of both drugs in unknown samples.

A1 = ad1Cd + ae1Ce    (5.2)

A2 = ad2Cd + ae2Ce    (5.3)

A1 → Absorbance of test sample at λ1 A2 → Absorbance of test sample at λ2 ad1 → Absorptivity of Dacarbazine at λ1 ad2 → Absorptivity of Dacarbazine at λ2 ae1 → Absorptivity of Eugenol at λ1

ae2 → Absorptivity of Eugenol at λ2

Cd → Concentration of Dacarbazine (to be determined)

Ce → Concentration of Eugenol (to be determined)

For the determination of loading of drugs in the liposomes, the entire process was developed with Ethanol as solvent. Absorbance maximum of dacarbazine in ethanol (λ1 ) was found to be 333 nm; absorbance maximum of eugenol in ethanol (λ2 ) was found to be 282.5 nm. So, absorptivities of dacarbazine and eugenol (in ethanol) were measured at λ1and λ2 separately as described above.

Method Validation

For the validation of the developed method, the standard stock mixture solution of both the drugs was prepared where 100 mg of dacarbazine and 1000 mg of eugenol was dissolved in 1000 ml of the selected solvent system to obtain dacarbazine concentration of 100 μg/ml and eugenol concentration of 1000 μg/ml. This stock solution was then suitably diluted to obtain different concentrations of the drugs in the mixture.

Linearity and Range

To determine the linearity and range of both the drugs, the stock solutions of individual drugs were diluted and used. Dilutions of Dacarbazine stock solution were prepared in the range 1-10 μg/ml and calibration curve was plotted between concentration and absorbance at 331 nm and 281.5 nm separately. Similarly, dilutions of Eugenol stock solution were prepared in the range 10-100 μg/ml and calibration curve was plotted at 331 nm and 281.5 nm separately.

Accuracy

To determine the accuracy of the developed method, three dilutions of the stock mixture solution were prepared. These solutions of known concentrations were then analyzed by the developed method as unknown samples. To further check the accuracy of the developed method, the pre-analyzed sample was separately spiked with extra 50%, 100% and 150% of the drugs concentrations and the mixtures were again analyzed by the developed method.

Precision

To check the precision of the developed method, three different dilutions of stock mixture solution were made and the precision (intra-day and inter-day precision) of the method was assessed by determining the concentrations of both the drugs in the mixture.

Intra-day Precision (Repeatability):

Repeatability of the method was assessed by determining the concentrations of dacarbazine and eugenol in three different dilutions of stock mixture solution at three different times a day.

Inter-day (Intermediate) Precision:

Intermediate precision of the method was assessed by determining the concentrations of dacarbazine and eugenol in three different dilutions of stock mixture solution for three consecutive days.

Limit of Detection

The limit of detection (LoD) is the lowest amount of analyte in a sample which can be detected but not necessarily quantified (Murtaza et al. 2011). LoD is calculated using equation:

LoD = 3.3 × N/B

where ‘N’ is standard deviation of the peak areas of the drugs (n = 3), taken as a measure of noise, and ‘B’ is the slope of the corresponding calibration curve (Jain et al. 2011).

Limit of Quantification

The limit of quantitation (LoQ) is the lowest amount of analyte in a sample which can be quantified with appropriate precision and accuracy (Murtaza et al. 2011). LoQ is calculated using equation:

LoQ = 10 × N/B            where ‘N’ and ‘B’ mean same as above

RESULTS AND DISCUSSION

Dacarbazine

Qualitative Analysis Physical Properties

Dacarbazine was a very slightly pinkish white powder. It was crystalline in nature.

UV Spectral Analysis

The λmax of dacarbazine was found to be 331.0 nm in distilled water (figure 5.1). The reported value of the λmax of dacarbazine in distilled water is 333 nm. The absorbance of dacarbazine solution of concentration 5 µg/ml was found to be 0.484.

Figure 1: UV Spectrum of Dacarbazine in distilled water

Figure 2: Calibration Curve of Dacarbazine in Distilled Water In PBS (pH 7.4)

Figure 3: Calibration Curve of Dacarbazine in PBS (7.4)

Figure 4: Calibration Curve of Dacarbazine in PBS (7.4) : Propylene Glycol (9:1)

Figure 5: Calibration Curve of Dacarbazine in Ethanol

Figure 6: UV Spectrum of Eugenol in Ethanol

Figure 7: Calibration Curve of Eugenol in Octanol

Figure 8: Calibration Curve of Eugenol in Ethanol

Figure 9: Calibration Curve of Eugenol in PBS 7.4: Propylene Glycol (9:1)

Figure 10: FTIR of Dacarbazine

Wavelength		Functional Group
Reported	Observed
3399	3408.33	NH2
3263	3272.69	NH2
3162	3159.54	NH (H bonded)
2985	2988.99	CH3
1655	1648.85	C=O
1606	1601.86	(NH2)+(CO)
1536	1542.89	(C-C)+(NH)
1474	1479.57	(CH3)+(NN)
1435	1432.71	(CH3)+(NN)
961	952.13	(NH)-(NCN)
682	680.90	OCNH2

Figure 11: FTIR of Eugenol

Wavelength		Functional Group
Reported	Observed
3513	3519	OH
3003	3077	CH2
3003	3004	CH3
2972	2975	CH2
2939	2939	CH3
2906	2906	CH3
2842	2842	CH2
1637	1638	C=C
1463	1462	CH2
1366	1366	COH
1205	1205	COH
993	995	C-H

Figure 12: FTIR of Lipoid S100

Figure 13: Drugs-Excipients Compatibility by FT-IR

Figure 14: DSC of Dacarbazine

Figure 15: DSC of Lipoid S100

Figure 16: Drugs-Excipients Compatibility by DSC

(i)

(ii)

Figure 17: Method of Preparation of dual loaded Nanoliposomes

Treatment	M1 Events
Blank	26
Rh-S	303
Rh-L	3463
Rh-L-C	7474

Figure 26: Cell Uptake in B16F10 Cells

Treatment	M1 Events
Blank	25
Rh-S	95
Rh-L	401
Rh-L-C	250

Figure 27: Phagocytic Engulfment by RAW 264.7 Cells

7.1.1.   Haemolytic Study

Haemolytic test was performed for DELC using blood from healthy mice. Supernatant of the RBCs having 100% and 0% haemolysis showed absorbance value of 0.234 and 0.002 respectively at 541 nm. Supernatant of RBCs treated with DELC showed absorbance value of 0.006. On calculating, it was found that DELC caused 1.709% haemolysis at a dose of 0.2 mg. Hence, it can be concluded that our formulation is minimally toxic to the RBCs and thus can be safely administered through i.v. route. The competence of a formulation can be ascertained only in in-vivo models. Animal studies give a clear picture of the utility and applicability of the formulation in actual diseased conditions

SUMMARY & CONCLUSION

Keeping the highly resistant and aggressive nature of melanoma in mind, dacarbazine and eugenol loaded liposomes were successfully developed for a combinatorial approach against melanoma. The QbD approach enabled us to synthesize the said anti-melanoma formulation with optimum parameters in the most logical manner. Applying this QbD approach at two levels further made the process easier to reproduce, and more cost- effective. Surface functionalization of the formulation made the entire therapy more targeted to spare normal body cells from unwanted toxicity. In-vitro characterization of the nanoliposomes ascertained the utility of the QbD application. This work is a good example and illustration of the successful application and value of the QbD approach in the development of effective pharmaceutical nanoformulations. The performance of the formulation as an anti- melanoma agent was assessed by cell line studies. Combining eugenol with dacarbazine resulted in much higher anti-melanoma activity of the formulation. This enhancement is supposed to be due to the inhibition of the anti- apoptotic protein survivin, which is overexpressed in the melanoma cells, and makes them resistant towards apoptosis. Including Eugenol has supposedly resulted in downregulation of survivin protein, consequent to which, dacarbazine could perform its function to its maximum potential. In addition to increased apoptosis and cytotoxicity, this combination also promises to inhibit the metastatic potential of the melanoma. Further, HA coating was found to enhance the uptake of the liposomes in B16F10 cells and reduce the phagocytic engulfment by macrophages. Pharmacodynamics study showed significant difference in the tumor volumes after 10 days of the treatment with DELC and Dacarbazine Solution (DS). Results of biodistribution study supported the results of cell uptake study by revealing more concentration of drug from DELC in blood and tumor, and considerably less amount in the liver. Histological analysis of tumor sections showed that there was significantly more necrosis in the DELC treated group. In lungs, metastasis could be seen in the control and dacarbazine solution group, but not in DELC group. Liver toxicity was also not observed in the DELC group, while hemorrhage was seen in DS group. DELC was also found to be safe for blood cells and thus suitable for i.v. administration. Conclusively, combining eugenol with the dacarbazine results in better therapeutic outcomes in the treatment of melanoma and thus can be a hope against this resistant, aggressive and deadly cancer. Thus, the combination of dacarbazine and eugenol holds the promise of overcoming the resistance of melanoma cells and challenges of anti-melanoma therapies.

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