Nikita Verma*

University Institute of Pharmacy, Pt. Ravishankar Shukla University, Amanaka, Raipur, C.G. Pin-492010,

*Corresponding author: nikitaverma510@gmail.com

[Received: 18 January 2019; Revised version: 1 March 2019; Accepted: 15 March 2019]

Abstract. As a disease skin cancer has obtained different characteristics over the decades. Solar radiation that contains ultraviolet ray is the prime cause of skin cancer. In this present research, the nano-precipitation method was applied for preparing Quercetin loaded Nanoparticle (Qu-Nps) with much enhanced loading properties and improves incorporation of corresponding drugs. At the same time, the Quadratic model that takes help of the Response Surface Method was applied to observe the effects of some specific parameters maintained in the development of nanoparticle. Here, the sonication time was 20 min and delivery system F₆ (with Drug: Polymer ratio of 1:45) provided optimum drug entrapment ability which is 70%. The optimized formulation for average size was almost 102.39 ∓7.64 nm with zeta potential diameter averaging -28.43mV. Quercetin is a dietary flavonoid possessing multidimensional properties that is used in various other diseases including viral infection, bacterial infection, diabetes mellitus, and cancer. All outcomes support the view that Quercetin loaded nanoparticles (Qu-Nps) has high entrapment and drug loading abilities.

Keywords: skin cancer, quercetin, quercetin loaded nanoparticle (Qu-Nps), drug delivery.

Introduction

In the human body, skin is the largest organ. It remains exposed to solar radiation including ultraviolet rays and a long-term exposure could result in abnormal growth of cells which is the main cause of skin cancer (Orazio et al, 2013). There are several types of electromagnetic waves are present in the solar rays among which ultraviolet (UV) radiation and infrared radiation are the primary constituents. The UV-radiation within the range of 280-320 nm (known as UVB) has the ability to change the biological features of the cells in the human body. UVB can damage DNA by creating free radicals in the cells and promoting abnormal growth of cells (Gupta et al., 2014). These days, various drugs are available in the market that can protect the skin from such damage and also arresting abnormal growth of cells. But, due to constant wear and tear of the keratin layer of skin, lipophilicity of skin membranes, development of various enzymes and several other adversities drug delivery to the skin has become a tough task (Gupta et al., 2016). There are some traditional therapies often used for arresting skin cell damage but those are found to be of little use as they fail to differentiate between the cancerous cells and normal skin cells. Thus, the primary target of the cancer therapy for the skin is to create a system for targeted drug delivery that could supply the required medicaments to the damages areas (Tavana et al., 2010). Quercetin - the flavonoidal drug has a natural property of creating defence against free radicals as the drug has a high antioxidant property. The antioxidant properties of Quercetin make it an appropriate drug for bacterial infection, viral infection, inflammation, oxidative damages, diabetes mellitus, several cardiovascular issues, and cancer. However, the affectivity of Quercetin in these diseases is not equally-established due to insufficient evidence. This research is a continuation of the studies that have been undergoing for the invention of targeting drug delivery process for skin cancer. We have prepared nanoparticles loaded with Quercetin which may be appropriate for skin cancer. It can be effectively applied in the damaged areas for the supply of Quercetin to the affected cells. It can also reduce the all negative effects that the targeted drug delivery systems often cause (M.martinez et al., 2015).

Experimental Detail, Methods, Materials

We received the supply of Quercetin (Qu) Sigma Aldrich, the USA. Tween-80, Phospholipids, and Chitosan were bought from Himedia Laboratories of Mumbai, India. Soya was used as a source of Phosphatidyl Choline (soya contains 98% of the compound). Phosphate-buffered Saline (PBS) with pH 5.5 and 5.6 was applied to measure the release of the drug. Other consumables used in the investigation were of analytical grade.

Formulation of Quercetin Loaded Nanoparticles (Qu-Nps)

The nanoparticles used in this process were formulated through the Nanoprecipitation Method which is also known as Solvent Displacement Method. It is a simple method where nanoparticles are formed just in one step.

In this method, a volatile, water dissolving organic solvent like ethanol was used as the base. The drug and polymer were mixed with the solvent. The solution was then added through magnetic stirring to an aqueous phase (like Tween-80) that contains a stabilizing compound. The organic solvent quickly diffused to the aqueous phase which helped in the formation nanoparticles as also entrapment of the drug. The size of the nanoparticles so developed through this process ranged 100-300 nm. They developed a tapered unimodal distribution. This method is most appropriate for the preparation of lipophilic drugs. It is not suitable for water-soluble drugs formulation.

Formulation and Optimization of Nanoparticles

An upgraded nanoprecipitation technique was applied in our experiment for the formulation of Quercetin loaded nanoparticle carrier system. Specific parameters were measured on the basis of the response surface method and the quadratic model. The composition of the drug was as follows:

Polymer Ratio - {D:P ratio, X₁}

Solvent Volume - {X₂}

Sonication time - {X₃}

            The above-mentioned three were independent variables while the following were dependent variables:

Particle Size - (Y₁),

Zeta Potential - (Y₂)

Entrapment efficiency - (Y₃)

             The experimental data was tallied with the mathematically deduced data. These two results for Entrapment efficiency (EE%), Particle size (nm), and Zeta potential were agreeing with each other. The experimental outcomes of the above variable in F₆ are shown in the table below –

Optimized formulation of Qu-NPs

Observation and Results

Characterization parameters

Vesicular Size Analysis

Zetasizer instruments were used to measure the vesicular diameter of nanoparticles (Zetasizer, Malvern, UK). The nanoparticles so formed possessing uni-modal size. PDI or Polydispersity Index was used for the measurement of nanoparticles. PDI with a small range was accepted as a homogenous population. Large ranges indicate the presence of a heterogeneous population (D.D. Verma et al., 2003).

Figure 1. Size analysis by intensity.

Zeta Potential Analysis

There was a charge on the vesicles’ surface due to the presence of electrical ions. This charge determined the efficiency of the vesicular delivery system. Zeta Potential was used to indicate this charge. It was measured with the help of Zetasizer 3000 HAS (Malvern Instruments, UK). Before initiating the experiment the delivery system was diluted by pure water (Tsai et al., 2015)

Figure 2. Zeta potential of Qu-Nps

Surface Morphology Study

This study was conducted with the help of Transmission Electron Microscope (TEM). Brand Hitachi J500, H7500 was used for the study. A sample of nanoparticles was placed on a copper grid coated with carbon. Then it was stained with phosphor-tungstic acid (1% aqueous solution). It was then observed placing under TEM providing 100kV voltage (Sarwa et al., 2014). TEM images confirmed that the nanoparticles are almost sphere shaped with tapered distribution. The images further established the non-aggregation nature of the particles. The images also made it clear that the nanoparticles possessing lower levels of polydispersity and their size distribution was not uniform.  

Figure 3. Represents the TEM images of  Qu-Nps

Fourier Transform Infrared Spectroscopy (FTIR)

The compatibility aspects related to Active Pharmaceutical Ingredients (API) and excipients are revealed through FTIR. Bruker Alpha II – FTIR was used for applying Pellet method for finding the FTIR spectra of Phospholipids, Chitosan, and Quercetin. Quercetin and excipients were mixed with KBr (Shinde et al., 2015). Shimadzu Bruker Alpha -II FTIR is used to undertake the spectral scan. The frequency range was limited with 4000 to 400 cm^-1. The data so obtained confirmed the presence of Qu-Nps. In-depth analysis of FTIR revealed the characteristic bands with hydroxide group stretching for Quercetin was detected at the range 3700-300cm^-1. An aromatic ring was detected at the range 1200-900 cm^-1.  Qu-Nps showed band stretching for C=O group at 1700-1800cm^-1 range.

Figure 4. FTIR of Qu-Nps

Measurement of Entrapment Efficiency

This measurement was undertaken by an indirect process previously used by (Sayyad et al 2017). Eppendorf tubes at low temperature were used for measuring the volume of Quercetin present in each preparation of nanoparticles. A centrifugal force was developed with the help of Centrifuge (manufactured by Remi Instrument Ltd., Mumbai, India) at 14000rpm on each preparation for 30 min. After measuring the volume of Quercetin, the supernatant solution was studied for drug loading. A spectrophotometrical process was used where λ_max of 273 nm by UV-Spectrophotometer (Schimadzu Model UV-1800, Tokyo Japan) was applied followed by a dilution with phosphate buffer pH 5.5 and 5.6. Entrapment Efficiency (EE %) was then measured with the help of the following formula: EE% = (Initial Qu Concentration - Concentration of unentraped Qu) / Initial Qu Concentration × 10 –

In vitro Release Study

PBS or Phosphate Buffered Saline at 37 degree centigrade was used for the investigation of in vitro release nature of Qu-Nps. Figure 05 shows the cumulative percentage of this release profile. Physiological and acidic pH 5.5 and 5.6 respectively were applied to conduct the whole in vitro release study. The release profile marked similar kinetics in both pH conditions. The cumulative release was approximately 60% of the starting drug loading after 12 hours. As the Figure depicts, the first release of drug for Qu-Nps is almost 45% at pH 5.6 after 12 hours (Figure 5). And, at pH level 5.5, the same Figure is almost 30%. The initial release might be due to low level diffusion in the external layers of the polymeric particles. On the other hand, the release from the inner polymeric medium provided a sustainable cumulative release profile.

Figure 5. In-vitro drug release study of Qu-Nps at different pН

The ICH guidelines were thoroughly followed for the formulation of the nanoparticles. The ICH guidelines were also followed for the study of changes in the particle size and actual visualization of the particles. It formed the basis of the stability study of this research. Samples were kept at two different temperatures - 30° C and 4° C. They were observed after certain time intervals, in this research, those were 1 month, 3 month, and 6 months. These samples were observed for changes in the physical appearance like size and distribution (Muppidi et al., 2012). With an aim to ensure the stability of the formulated nanosystem, i.e. Qu-Nps, the stability investigation was conducted following the ICH guidelines as mentioned in Q1AR2. Different temperature conditions and relative humidity conditions were developed which were room temperature zone with temperature (25±2ºC and relative humidity 60±5%RH and the accelerated temperature zone with temperature 40±2ºC and relative humidity 75±5%RH. This condition was maintained for 6 months at a stretch. At distinct time gaps, zeta potential, the common particle size, and entrapment efficiency (EE %) were estimated. The samples were taken in triplicates at the time of the estimations. Results are shown in the table.  

The study shows that a minor increase in the particle size had taken place when the storage states of the samples changed from 100.57± 2.53to 109.2 ± 1.39. But Zeta potential had not changed to any significant amount. In the same condition, EE% of the optimized nanoparticles was measured at 75.48 ± 3.2%. After a six months’ storage at 4^oC EE% was found to be 71.01±0.24 % and at 25^oC it was 69.06±0.19 %. These study results make it clear that Quercetin is capable of retaining the nanoparticles for a long time. At the same time, the study outcomes show that the % EE has decreased quite a bit. So, Qu-Nps at the optimized state can be quite stable at any storage condition for a longer period of time. Stability study of optimized Qu-Nps for 6 months maintained at different temperature conditions

Discussion

In this study we have tried to take help of the approach to develop an advanced process of the targeted natural bio-active drug delivery for the skin cancer condition. Our initial challenge was to enhance the stability and solubility of Quercetin. Quercetin is a natural bio-active ingredient that has wide many applications in medicines. It is an active antioxidant. But its applications can be widened further if its limitations could be withdrawn. Its potential is restricted due to its poor solubility in water and poor stability in the physiologic mediums. Several methods have been followed till now to overcome its limitations. (Varshosaz et al. 2013) developed lipid nanoparticles of Quercetin in solid state for the treatment of hepatocellular carcinoma. These researchers compared the effect ts of three sterols possessing different features: Stigmasterol, Namelycholesterol, and stigmastanol. Their target was enhanced penetration power of Quercetin inside the cells.

In our study, we entrapped Quercetin in polymeric nanoparticle form (Qu-Nps). This enhanced its solubility in water as also in different physiologic mediums. The relationship between aqueous solubility and Zeta potential was measured as -28.43 mV. This, being lower than -30 mV ensures its higher stability. All these positive results made us optimistic about the formulation of targeted drugs for skin cancer with natural bio-active ingredients. Here, the most challenging factor was the size of the nanoparticles. We had to keep the size within 90nm to ease their entry into the circulatory system and their retention on the skin surface. We kept the nanoparticle size within the range, 102.39 ∓7.64. The cumulative release of Quercetin from the formulated Qu-Nps was found to be 77.87 ∓1.85% for at 5.6 pH level and 69.23 ∓1.54% at 5.5 pH level. The time period of measurement was 24 hours. The FTIR and TEM image studies further confirmed the actual shape and size of Qu-Nps. Overexposure to UV radiation makes the skin susceptible to enhanced release of macrophage migration inhibitory (MIF). The cargoes were developed in such a manner that Qu could work as an anti-MIF factor. Our study is just a first step in the formulation of nanosystem for immensely beneficial bio-actives. However, more studies on different parameters are required to get more optimistic results from these Nps. Macrophage-targeting will definitely open up new avenues for optimized drug targeting for skin cancer.

Acknowledgement

I pay my warm gratitude to the head of Cosmetic Lab of the University Institute of Pharmacy, Pt. Ravi Shankar Shukla University of Raipur, Head NCNR (National center for Natural Resources), Pt. R.S.U.Raipur, C.G. for providing all necessary resources for my study. I also acknowledge the contributions made by the library of Pt. Ravishankar Shukla University for giving me an opportunity to use their e-resources available through UGC-INFLIBNET.

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