INTRODUCTION:

Since life began on earth today, the human being is exposed to various germs that may lead to serious or sometimes cause fatal injuries. These germs may be bacteria, fungi, or viruses. Currently, there is a serious viral disease that has infected many people around the world and it is very contagious. It is caused by the novel coronavirus. Very severe symptoms and even deadly infections have been produced named severe acute respiratory syndrome coronavirus (SARS-CoV-2) disease. Coronaviruses are enveloped viruses contain the genome of single-strand RNA, which primarily binds the mucosal surfaces after entry from the human respiratory tract to cause sever infection^1,2. The essential oils (EOs) from aromatic plants are a natural source of preservative and medicinal agents from ancient times.

It imparts flavor and aroma to pharmaceutical preparation. EOs such as myrtle and peppermint oil are contain many natural components of therapeutic agents including terpenes, terpenoids, and aromatic compounds. There are many studies indicate the powerful activity of myrtle and peppermint oils against wide species of pathogens, also, to act as anti-inflammatory, antioxidant and analgesic^3-5. The effect of myrtle and peppermint oils against wide species of pathogens is associated with their constituents and cytotoxic activity, which results in cell membrane damage after attachment to pathogens. The myrtle and peppermint oils are hydrophobic that can enter the cytoplasmic membrane and cell wall. They disrupt the structure of the phospholipid layers, fatty acid, and polysaccharide that result in membrane-permeable lead to lysis of pathogenic cell^6-8. The volatile nature of EOs and rapid decomposition by the effect of light and oxygen impart physical and chemical instability to myrtle and peppermint EOs in addition to the lipophilic properties that make difficulty in formulation, administration, and storage of prepared dosage form⁹. Nanotechnology is an approach to improve the stability and delivery of myrtle and peppermint EOs as therapeutic agents. The lipid-polymer hybrid nanocarrier pulmonary solution belongs to nanotechnology. It is an attractive delivery system and considers the double-edged sword acted as a local and systemic therapeutic system and protect from pathogens attacked for body cells^10,11. The widely accepted inhaled medications through the pulmonary route consider of first-line therapy for acute and chronic lung diseases. It has many merits such as needle-free or noninvasive delivery system, bypass the obstacles that reduce the therapeutic efficacy as the first-pass metabolism in the liver and poor gastrointestinal absorption, achieve rapid clinical response, a high therapeutic agent concentrations can reach directly to the disease site and low systemic side-effects. All these properties make the pulmonary route a fantastic site for lipid-polymer hybrid nanocarrier delivery¹². The prepared lipid-polymer hybrid nanocarrier solution composes of oleic acid and myrtle and peppermint essential EOs at ratio (2:4:4) in addition to the water-soluble chitosan polymer. The wide properties of chitosan make it more suitable for hybridization with the lipid base formulation to achieve the desired therapeutic target. The main characteristics of chitosan are cationic agent, highly basic, biocompatible, biodegradable, penetration enhancer where it facilitates both paracellular and transcellular passage of therapeutic agent. Chitosan is mucoadhesive polymer, interacts with mucus which is negatively charged bio ingredient and negatively charge of RNA genome of SARS-CoV-2 to form a complex by ionic or hydrogen bonding as well as though hydrophobic interactions. This will create protective and therapeutic activity against lung pathogens and synergistic with the therapeutic activity of myrtle and peppermint EOs^13-16. There are many methods to formulate lipid-polymer hybrid nanocarrier but associated with many disadvantages mainly cost-effective and time-consuming. The new microwave method is cheap and rapidly achieved in both small and large scale. Microwaves are a form of electromagnetic non-ionizing radiation with frequencies between 300 MHz and 300 GHz and wavelengths ranging from about one meter to one millimeter. It have three main properties that permit them to be used in the pharmaceutical industry which are, they are reflected by metal material, can pass through the glass, plastic, paper, and the same materials and they are absorbed by pharmaceutical ingredients^17-21. This study aims to prepare a colloidal dispersion system to be given by nebulization through the pulmonary route to achieve the rapid therapeutic efficacy of myrtle and peppermint EOs using a novel microwave method.

MATERIALS AND METHOD:

Materials:

The oleic acid purchased from S. J. chemicals private limited India. The peppermint oil was purchased from BAR-SUR-loop Grasse A. M Franc. The myrtle oil was purchased Hemani international KEPZ Karachi; Pakistan. Tween 80 from SD fine Chemlimited (SDFCL) Mumbai, India. The methanol and ethanol from grin land chemical comp, United Kingdom. The KH2Po4 and Na2HPo4 from Merck and Co., Inc. Germany. The water-soluble chitosan, from China. The propylene glycol from AOBA Chemie India.

Method:

The lipid polymer hybrid nanocarrier delivery system was prepared by the microwave method using a model of the Denka YMO-G30LR-30L microwave instrument. Under magnetic stirrer device at 1000rpm for 10 minutes, the hydrophobic phase ingredients which are myrtle oil, peppermint oil, and oleic acid were mixed and hydrophilic phase ingredients which are water-soluble chitosan, Tween 80, propylene glycol and distilled water was mixed according to the quantities of formulation that described in table 1. Then mixing hydrophobic phase and hydrophilic phase ingredients, then put it in microwave device for less than 15 seconds. Then incite the formulation blend adequate time using a magnetic stirrer instrument at 1000 rpm for sufficient low time (seconds to minutes) until to get a solution that is clear to the eyes that reflect colloidal features of lipid polymer hybrid nanocarrier delivery system that is obtained.

Table 1: Compositions of lipid polymer hybrid nanocarrier formulations (F1-F6).

Formulation components (%w/w)	Formulations (F1-F6)
Formulation components (%w/w)	F1	F2	F3	F4	F5	F6
Myrtle oil/ Peppermint oil/ Oleic acid (4:4:2)	10	10	10	15	20	25
Tween 80/ propylene glycol (4:1)	40	35	30	30	35	40
chitosan	0.025	0.025	0.025	0.025	0.025	0.025
Distilled water up to	100	100	100	100	100	100

Evaluation of thermodynamic stability:

The thermodynamic stability test was performed according to the following tests such as centrifugation test, heating-cooling test and freezing-thawing test. These assays were performed for accelerated stability measurement of lipid-polymer hybrid nanocarrier formulations²².

1. Centrifugation test:

Using a centrifuge device at 5000rpm, the formulas centrifuged for 30 minutes then noticed the creaming and phase separation. The measurements were achieved in three trails.

2. Heating-cooling test:

The formulas were subjected at two different temperatures, which are 45^oC and at 0^oC for not less than 48 hours for each temperature test. Three trails should be achieved.

3. Freezing–thawing test:

The formulations should be kept at temperature (-21^oC and 21^oC) for each temperature test not less than 24 hours. The assays occur in triplicate.

Characterization of the prepared lipid-polymer hybrid nanocarriers

1. The colloidal carrier size measurement

The samples of colloidal carriers were put in a 5ml cell of ABT-9000 nanolaser globule size analyzer after sonication at the 37^oC for 30 min, the mean colloidal carrier size can be obtained. The assays occur in three trail²³.

2. The index of polydispersity measurement:

The uniformity colloidal carrier size and can be obtained by a polydispersity test through the ABT-9000 nanolaser particle size instrument. The measurements were achieved in three trails. The higher value of the polydispersity index refers to the lower uniformity of the colloidal carrier system²⁴.

3. Measurement of Zeta potential:

Zeta potential measures the stability of the colloidal dispersion system. Zeta potential was determined by brook heaven device USA zeta sizer. Samples were placed in clean disposable cuvettes then recording the obtained results. The assays were performed in three trails²⁵.

4. Viscosity measurement:

The viscosity parameter of the colloidal carrier formulas was measured by NDJ-55 digital Viscometer instrument using a spindle no. 1 at 25°C. The tests occurred in triplicate²³.

Statistical analysis:

Using the analysis of variance (ANOVA) as a statistical tool to analyze the data obtained during the research. The results of the investigation were given as an average of triplicate samples were studied at level (P<0.05)^23,26.

RESULTS:

Evaluation of thermodynamic stability:

The thermodynamic outcomes indicate that all the formulations show excellent physical stability as shown in Table 2 and Figure 1.

Table (2): Thermodynamic stability data of lipid polymer hybrid nanocarrier formulations (F1-F6).

Formulation code	Evaluation parameters observation
Formulation code	Centrifugation	Heating-cooling	Freezing–thawing
F1	No change	No change	No change
F2	No change	No change	No change
F3	No change	No change	No change
F4	No change	No change	No change
F5	No change	No change	No change
F6	No change	No change	No change

Figure 1: A photographic picture of lipid polymer hybrid nanocarrier formulations (F1-F6).

Characterization of the prepared lipid-polymer hybrid nanocarrier formulations:

1. The colloidal carrier size measurement

The results of the average colloidal carrier size was F1 = 28.1nm, F2 = 31.5nm, F3 = 32nm, F4 = 35.3nm, F5 =36 nm and F6 = 40nm as shown in table 3. That means all the formulation present within the nanosize range. The nanosize range of studies formulations (F1-F6) is very important due to it reflects the ability of nanocarrier to achieve the required pharmacokinetics features within the body that lead to reach the therapeutic aim of scientific research. According to the Anova, significant correlativity between Tween80/propylene glycol (%w/w) as independent variable and globule size values where (p<0.05).

2. The index of polydispersity measurement

The polydispersity index (PDI) was from (0.011 to 0.03) as shown in table 3. The results of PDI indicated that nanocarrier formulations have a constrict distribution size and homogeneous.

3. Measurement of Zeta potential:

Zeta potential is an index of the nanoparticle dispersion stability. The results of the mean zeta potential scale in absolute values was (31.1mV to 33.4mV) as shown in table (3) which indicates the stability of nanocarrier formulations. There higher electrical charge greater than +/- 30 related to long term physical stability of pharmaceutical preparation²³.

Table (3): The data of characterization parameters for lipid polymer hybrid nanocarriers formulations(F1-F6).

Formulation code	Characterization parameters
Formulation code	Colloidal carrier size(nm)	Index of polydispersity	Absolute values of Zeta potential(mV)
F1	28.1	0.02	33.1
F2	31.5	0.03	32.9
F3	32	0.019	33.4
F4	35.3	0.012	32.2
F5	36	0.011	31.9
F6	40	0.014	31.1

4. Viscosity measurement:

The viscosity data of nanocarrier systems was from (202 mPa sec) to (293 mPa sec). The results show low viscosity of the formulations. The program was obtained as shown in figure 2 when data of plot shear stress against data of the rate of shear.

Figure (2): The rheogram of lipid polymer hybrid nanocarrier formulations (F1-F6).

DISCUSSION:

Since the birth of humanity to this day, there are many epidemics and deadly diseases that people are exposed to it. A long time ago several strategies have emerged to treat these diseases, including the use of plants and essential oils such as myrtle and peppermint EOs which has proven to be very effective in combating many viral, bacterial and fungal diseases. Currently, we are living a fierce attack from a deadly virus called SARS-CoV-2, which has infected thousands of people around the world. It is a disease that affects the respiratory system. It is wonderful and useful to use essential oils to combat this virus because it is widely available and cheap in most countries of the world. These volatile oils suffer from rapid waste damage when exposed to air, and they are also oily composition. So, there is difficulty in giving it to the patient through the spray to the respiratory system. Therefore these oils must be transferred to the respiratory system through the appropriate delivery system. The prepared lipid-polymer hybrid nanocarriers dispersion system shows excellent pharmaceutical properties to deliver myrtle and peppermint EOs to the pulmonary system using a new microwave method. The formulations (F1-F6) show wonderful appearance as shown in figure 1, that is free from any instability parameters such as phase separation, creaming and Ostwald ripening. The particle size is a very important parameter of lipid base hybrid nanocarriers, which influence bio-distribution, stability, encapsulation efficiency, mucoadhesion, profile of drug release and cellular internalization of active therapeutic agent²⁷. One of the most important physicochemical attributes to be taken when in vivo applications consider is cellular internalization or uptake. The small molecules and particles picking by any cell achieve by endocytosis. The two endocytosis mechanisms which are pinocytosis and phagocytosis²⁸. Pinocytosis is the mechanism of nanocarrier formulations uptake due to its presence among nanosize formulations as shown in table 3. After administration of the nanocarrier dispersion system through nebulization, the colloidal particle reach rapidly to infected lung cell. The colloidal system will release the myrtle and peppermint EOs, that attack viral cells result in disruption. The positively charge chitosan facilitate rapid attachment and penetration of the colloidal system to inside the cell through pinocytosis. Inside the infected cell, it attaches to all negative charge RNA genome of virus result in an inactive complex that is easily degraded and eliminated from the body by macrophage and other immune cells. On the other hand, nanosized hybrid carrier formulations have an attractive view for pulmonary drug delivery because many merits as targeted deposition, sustained release, bioadhesion, and reduction in dosing frequency that enhance patient compliance. The colloidal particles smaller than 150 nm are associated with delayed pulmonary clearance in comparison to coarse particles²⁹. The results of PDI indicated that colloidal dispersion systems (F1-F6) have excellent homogeneous constancy and this is important results due to that PDI of lipid-polymer hybrid nanocarriers can influence the bulk features, product activity, processability, appearance and stability of final dosage form. According to the thumb rule, zeta potential values of dispersion system are: (+/-5 mV) show fast aggregation, about (+/-20 mV) supply only short term stability, above (+/-30 mV) offers good stability and above 60 mV excellent stability. The colloidal dispersion systems (F1-F6) show excellent stability due to zeta potential values were (31.1 mV to 33.4 mV). The rheogram was shown a nonlinear relationship between shear stress and rate of shear as shown in figure 2. This indicates the presence of a non-Newtonian flow system. The prepared lipid-polymer hybrid nanocarriers (F1–F6) deform and flow immediately with applied stress this indicates pseudoplastic flow also was shown in rheological profile of formulations(F1–F6). The pseudoplastic flow provides stability and uniformity of therapeutic dose³⁰. From lipid-polymer hybrid nanocarrier formulations (F1 –F6), the selected colloidal dispersion was F6 due to this nanosize colloidal dispersion as shown in figure 3, contain greater loading quantity of myrtle and peppermint EOs. The F6 will subject further perspective analytical studies.

Figure 3: The particle size distribution for F6.

CONCLUSION:

The formulated lipid polymer hybrid nanocarrier dispersion system can be loaded by the myrtle and peppermint oil EOs to achieve efficient therapeutic efficacy against pathogenic lung diseases. The new microwave method is reliable and rapidly achieved in both small and large scale. The presence of chitosan polymer not only for stability the formulation structure but also impart therapeutic activity to the carrier system. The formulated lipid-polymer hybrid nanocarrier considered the double-edged sword, that means it can be loaded by drugs in addition to the therapeutic activity of nanocarrier components. The hybrid nanocarrier delivery system protects and stabilizes the loaded ingredients where it show non-Newtonian pseudoplastic rheological properties.

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Received on 31.03.2020 Modified on 29.05.2020

Research J. Pharm. and Tech 2021; 14(3):1233-1237.

DOI: 10.5958/0974-360X.2021.00219.5