Nanotechnology is the study of the controlling the matter on an atom and molecular scale. Generally nanotechnology deals with structures sized between 1-100 nanometres in at least one dimension, and involve modifying or developing materials within that size.¹ It makes the material lighter, stronger, faster, smaller and more durable. Zinc Oxide is considered to be a bio-compatible and environment friendly material. Zinc Oxide have been used as an antibacterial agent.² Zinc Oxide can be contemplated as the most dynamic due to its great variety of applications, of all the different materials that have been studied and investigated as Nano sensors. Zinc oxide nanoparticles are nanoparticles of zinc oxide (Zinc Oxide) that have diameters less than 100 nanometers.³ They have a large surface area relative to their size and high catalytic activity. The exact physical and chemical properties of zinc oxide nanoparticles depend on the different ways they are synthesized. Zinc oxide nanoparticles are used in an increasing number of industrial products such as rubber, paint, coating, and cosmetics.⁴ They have become one of the most popular metal oxide nanoparticles in biological applications due to their excellent biocompatibility, economic. Doxorubicin (DOX) was initially isolated from Streptomyces peucetius. DOX is a highly effective chemotherapeutic drug used to treat cancers and malignant haematological diseases. DOX inhibits DNA topoisomerase II activity, causing double-stranded DNA breaks (DSB). Due to its cardiotoxicity, however, DOX’s clinical usage has been severely limited.⁵

MATERIALS

Doxorubicin was obtained from Alkem Pvt Mumbai, Ethyl cellulose and eudragit procured from SD fine chemicals Mumbai. Other chemicals and the reagents used were of analytical grade.

METHODOLOGY

Compatibility Study (IR Spectroscopy)

Fourier Transform Infrared Spectroscopy (FTIR) is an important technique that provides an easy way to identify the presence of certain functional groups in an organic molecule. Functional groups have vibration frequencies that are characteristic of that functional group. These vibration frequencies fall with the infrared (IR) frequency range. As such, passing an IR signal through the organic compound causes the functional groups to vibrate at specific frequencies. In other words, an infrared signal that passes through an organic compound will be absorbed at these characteristic frequencies, which can be transformed into a unique spectrum.⁶

Formulation Development

Sol-to-Gel Method ⁷

Two beakers containing 50ml of deionized water in each of them. Let's mark these beakers as 1 and 2. In the next step, we will add zinc nitrate with polymers (Ethyl cellulose and Eudragit) in beaker 1 and will place a magnetic pellet within the solution and the beaker 1 is then placed on the magnetic stirrer. Solution 1 in beaker 1 is thus stirred for around 5 minutes. Similarly, we will add sodium hydroxide and Doxorubicin (drug) to beaker 2 with a magnetic pellet in it and will place it on the magnetic stirrer. Solution 2 is also stirred for around 5 minutes. The solution placed in the beaker gets stirred fast due to the rotating magnetic field generated. Solution 2 consisting of sodium hydroxide is then added to solution 1 consisting of zinc nitrate hexahydrate. While adding solution 2 into solution 1, we should add it dropwise using a syringe that allowing the solutions to mix properly and hence initiate the mechanism or reaction. Also, the entire process of stirring takes place at normal room temperature. On adding the solution 2 dropwise using a syringe, we will be able to see a slight difference in the color of the mixture. The mixture will be turning into white color slowly as we add the solution 2 dropwise. This transition of the solution from transparent to opaque shows the initiation of the chemical reaction. Thus, after the complete addition of solution 2 into solution 1 we can notice that the mixture appears cloudy. Thus, the resultant solution 3 is stirred for another 2 hours on the magnetic stirrer. After stirring it for 2 hours the solution 3 is transferred into the centrifuge tube. Solution 3 thus undergoes a process called centrifugation where the precipitate is removed or filtered out and we obtain the gel at the end of the process. Around 3 rounds of centrifugation are done. After the first round, the centrifuge tubes are filled with deionized water up to the marking and then are placed in the centrifuge machine again. After doing two rounds of centrifugation using deionized water, we will now use ethanol and centrifuge it. Finally, we will obtain the gel as the residue. Here also the entire process of centrifugation takes place at the normal room temperature i.e. 28 °C. The gel obtained as the residue of the centrifugation process is then spinned by keeping on a spinner. This is done to displace the gel stuck at the bottom of the centrifuge tube. Also while doing this a small amount of ethanol is added. Thus, the gel is then finally taken into a Petri dish and is broken using a spatula. The Petri dish containing the gel is then covered using an aluminium foil and tiny perforations are made over this aluminium foil covering the Petri dish. The Petri dish containing the gel is placed overnight to dry. Once dried the Petri dish is placed in a hot air oven for 10 minutes and is then placed in the muffle furnace at a temperature of 500°C for 3 hours. This process of heating is called calcination. After undergoing calcination the particles on the Petri dish are transferred to a mortar and are finely grinded into a powder form. The finally obtained powdered particles are then transferred into tubes and stored. To determine if the synthesized material is Zinc Oxide nanoparticles we will have to use various characterization methods.

Particle Size:

All the prepared batches of Zinc Oxide nanoparticles were viewed under microscope to study their size. Size of Nano particles from each batch was measured at different location on slide by taking a small drop of nanoparticle dispersion on it and average size of Zinc Oxide nanoparticles were determined.¹⁰            

SEM Analysis:

The morphology of NPs was studied by a scanning electron microscope. For this purpose, the sample was lyophilized and placed on aluminium stubs and the surface was coated with a layer of gold particles using a sputter coater. The shape of the NPs was determined by scanning electron microscopy (SEM) (XL30, Philips, the Netherlands) at 15 kV and 750 mA.¹¹

Zeta Potential

The zeta potential of a particle represents the overall charge of the particle and stability of the formulation. Zeta potential measurement was carried out using Zeta sizer Nano-ZS90, Malvern Instrument Ltd., UK by differential light scattering (DLS) technique. Nanoparticle samples redispersed in Milli-Q water. All measurements were carried out in triplicates at 25 °C.¹²

Drug Encapsulation Efficiency:

Lyophilized Zinc Oxide nanoparticles 50mg were dissolved in 100ml of phosphate buffer and the drug amount was determined by UV analysis. The encapsulation efficiency was determined as the mass ratio of entrapped Doxorubicin in Zinc Oxide nanoparticles to the theoretical amount of the drug used in the preparation .The entrapment of the Doxorubicin Zinc Oxide nanoparticles was expressed as loading capacity.¹³

In-Vitro Drug Release Studies:

The release studies were carried out by Franz diffusion cell. It containing 10 ml Phosphate buffer. Phosphate buffer pH 7.4 (100 ml) was placed in a 10 ml of beaker. The beaker was assembled on a magnetic stirrer and the medium was equilibrated at 37±5⁰C. Dialysis membrane was taken and one end of the membrane was sealed. After separation of non-entrapped Doxorubicin dispersion was filled in the dialysis membrane and other end was closed. The dialysis membrane containing the sample was suspended in the medium. 1ml of aliquots were withdrawn at specific intervals, filtered after withdrawal and the apparatus was immediately replenished with same quantity of fresh buffer medium.¹⁴

Percentage of drug release was determined using the following formula.

Perentage drug release= DaDt ×100         

Where, Dt = Total amount of the drug

             Da = The amount of drug released

Drug Release Kinetics:¹⁵

The models used were zero order (equation 1) First order (equation 2) and Higuchi model (equation 3) and Korsmeyer Peppas model (equation 4).  

I) Zero Order Kinetics:

R =     Ko t                                              -- (1)

R=cumulative percent drug 

Ko=zero order rate constant

Ii) First Order Kinetics

log C = log Co –K ₁ t /2.303                 -- (2)

Where C = cumulative percent drug 

K ₁= first order rate constant

iii) Higuchi Model

R = K _H   t ^0.5                                            -- (3)

Where R = cumulative percent drug

K _{H  =} Higuchi model rate constant

iv) Korsmeyer Peppas Model:

M t / M ?   = K _k t ⁿ  

log M t / M ? = log K _{k  +}n log t        -- (4)

Where K _{k  =} Korsmeyer Peppas rate constant

‘M t / M ?’   is the fractional drug, n = diffusional exponent, which characterizes the mechanism of drug. The obtained regression co-efficient (which neared 0.999) was used to understand the pattern of the drug from the Zinc Oxide nanoparticles.

Stability Studies:¹⁶

Selected Formulation was subjected to stability studies as per ICH guidelines. Following conditions were used for Stability Testing.

1. 25⁰C/60% RH analysed every month for period of three months.

2. 30⁰C/75% RH analysed every month for period of three months.

3. 40⁰C/75% RH analysed every month for period of three months.                                                  

RESULTS AND DISCUSSION

Drug-Excipient Compatibility Studies (FT-IR)

The compatibility between the drug and the selected lipid and other excipients was evaluated using FTIR peak matching method. There was no appearance or disappearance of peaks in the drug-lipid mixture, which confirmed the absence of any chemical interaction between the drug, lipid and other chemicals.

The surface characteristic of prepared crystal was studied by SEM (ZEISS Electron Microscope, EVO MA 15). Powder samples was mounted onto aluminum stub using double sided adhesive tape and sputter coated with a thin layer of gold at 10 Torr vacuum before examination. The specimens were scanned with an electron beam of acceleration potential of 20 kV and the images were collected as secondary electron mode.

Zeta potential is a measure of charge present on the vesicle surface. It was determined by using phase analysis light scattering with Malvern zetasizer at field strength of 20V/cm in distilled water and based on electrophoretic mobility of charged particles present in the nano carrier system. Charged particles were attracted to the electrode with the opposite charge when an electric field is applied. 

Particle Size

Characterization of Zinc Oxide Nanoparticles of Doxorubicin

The drug entrapment efficiency of all 8 formulations was evaluated. From the F8 formulation showed maximum drug entrapment efficiency 79.68 % compared to other formulations.  The zeta potential or the change on the surface of colloidal particles in Doxorubicin Zinc Oxide nanoparticles was measured by electrophoretic light scattering mode using Zetasizer nano ZS. The particle charge of Doxorubicin Zinc Oxide nanoparticles were quantified at 25^o C. The samples were diluted approximately with the deionized water for the measurements of particle size.

In Vitro Drug Release Studies

Drug Release Kinetics

The release kinetics for all the prepared Zinc Oxide nanoparticles was evaluated to determine the release behaviour of Doxorubicin from the prepared Zinc Oxide nanoparticles. The release data were analyzed with zero-order kinetic, first-order kinetic, and Korsmeyer–Peppas kinetic models, as well as the Higuchi kinetic model. It was revealed that the release data from Zinc Oxide nanoparticles fit to Higuchi kinetic model with the highest (r) value, while for free Doxorubicin Zinc Oxide nanoparticles,  the release data fit the zero order kinetic model.

In Vitro Drug Release Kinetics

First Order Kinetics

       
           
       
Fig-8: First Order Kinetics of Optimized Formulation

There was no significant change in physical and chemical properties of the Zinc Oxide nanoparticles of formulation F-8 after 90 days. Parameters quantified at various time intervals were shown.