INTRODUCTION:

The most significant alternatives for the topical delivery of drugs are self-assembled colloidal lipid systems such as liposomes, emulsion, solid lipid nanoparticles(SLN), phytosomes and nanostructured lipid carriers (NLCs) [1]. However, the traditional liposomes and emulsions have some disadvantages including, toxicity from organic solvent residue based on the preparation method, physical and chemical instability, having to undergo lengthy, multistep processes, and non-reproducible drug release [2,].

SLN consists of an aqueous dispersion of solid lipid (0.1 - 30% w/w), they may be further stabilized with the addition of 0.5 – 5% (w/w) surfactant [3]. Although thought the SLN appeared to offer several potential uses in drug delivery and research. There are also major drawbacks to drug release during storage, due to the drug’s lower solubility in the solid lipid, and the loading capacity has been frond lower [4]. The developed NLCs are promising nanocarriers that were able to overcome the restrictions, the nanometer-sized particles of NLCs allow them to be in an intimate interface with the skin layers, enhancing the drug penetration of the epidermis [5]. Most of the lipids used as excipients in the development of NLSs have been considered safe and biodegradable providing them less harmful to the skin, solid lipids are responsible for the sustained drug release from the bloodstream. In addition to delivering irritant-causing drugs at high concentrations, it is useful for limiting systemic drug absorption [6]. Following topical applications, the lipid film development gives rise to occlusive features, which enhance the skin permeation ability and retain moisture. NLCs can be an effective delivery for light-sensitive drugs because of their inherent benefit of scattering light due to structural deficiencies [7].

Naringenin belongs to the flavanone class of flavonoids and is abundantly present in citrus fruit. It functions as an anti-inflammatory, anti-microbial, anti-diabetic, and anticancer drug in addition to other therapeutic treatments. It was studied whether naringenin has been found antimicrobial effects on harmful bacteria such as L. monocytogenes, E. coli, and S. aureus pathogens was investigated. It is also used as an antioxidant it helps in reducing the effect of free radicals and reactive oxygen species (ROS), [8] Itis also indicated in the treatment of damaged skin. Despite its many advantages,naringenin has some drawbacks being a strong irritant that causes skin redness and peeling. Furthermore, light and oxidation can cause it to degrade [9].

The objective of the present study was to investigate and develop NLCs as the carrier for naringenin. The reported literature regarding the study of naringenin-loaded NLCs is limited.Hence, the optimized formulations were carried out with respect to thecharacterization of particle size, release study, stability studies and In Vivo skin irritancy studies.

MATERIAL AND METHOD:

Preparation and optimization of NLC

The naringenin-loaded NLCs were prepared by the “Melt emulsification method” The solid and liquid lipids are heated and mixed. Then the drug was added in an organic phase then theorganic phase was added in an aqueous phase containing the surfactant and stirred to form a coarse emulsion. Then high- pressure homogenization is subsequently applied for the formation of NLCs. This method used materials and quantity is mentioned in table no. 1.

Selection of Lipids

Lipid screening was carried out to investigate the suitable lipid for the incorporation of naringenin in NLCs. The liquid lipids are selected oleic acid and castor oil for screening. Naringenin excess was dispersed in the liquid lipid and shaken in a water bath shaker (Remi, Mumbai, India) for 24 hours at 25± 2˚C. After 24 h each saturated sample was centrifuged for 10 min at 5000rpm. The supernatant was diluted and the amount of naringenin was solubilized and analyzed by spectrophotometrically [10].

In the hot melt micro emulsification method [11], the hot liquid phase containing naringenin was poured into the hot aqueous phase under stirring at 3000 rpm to obtain the microemulsions it was stabilized by stirring for 15 min at 60-65 ˚C, then poured into ice cold water (4-5˚C) dropwise under the continuous stirring at room temperature. From NLCs liquid vesicles are higher due to the high ratio of cold water to the microemulsions (5:1).

In the hot melt probe sonicator method [12] the hot liquid melt containing Naringenin was added dropwise to the hot aqueous solution and Tween 80 surfactant. Then it was sonicated using probe sonication (VC 130, Sonics and Materials Inc., CT, USA) for 15 min at 40% amplitude. The hot sonicated dispersion was allowed to drop at room temperature under mild stirring (1000 rpm).They produced a dispersion of naringenin NLCs which was lyophilized to obtain dried NLCs.In the study, the data are reported as mean value ± standard deviation (SD, N=3).

Formulation design

Statistical models are extensively used in diversified areas to strengthen the art of drug formulation. The response surface method (with a 3-level factorial design and quadratic model) was employed to study the effect of selected parameters. The ratio of Naringenin: lipid {D:L ratio, X1}, solid lipid: liquid lipid {steric acid: oleic acid X2}, surfactant {X3}, stirring speed, Temperature

Table 1: Formulation pattern with processing parameters

Formulation (code)	Drug:Lecithin (X1)mg	Steric acid: oleicacid (X2) mg	Surfactant (ml) (X3)	Speed of magnetic stirrer (rpm)	Temp (^oC)
NLC1	2:1 (10:5)	5:3 (150:90)	0.5	1500	40
NLC2	2:1 (10:5)	10:3 (300:90)	0.5	1500	50
NLC3	2:1 (10:5)	5:1 (450:90)	0.5	1500	60
NLC4	1:2 (10:10)	5:6 (150:180)	1	2000	40
NLC5	1:1 (10:10)	5:3 (300:180)	1	2000	50
NLC6	1:1 (10:10)	5:2 (450:180)	1	2000	60
NLC7	2:3 (10:15)	5:9 (150:270)	1.5	2500	40
NLC8	2:3 (10:15)	10:9 (150:270)	1.5	2500	50
NLC9	2:3 (10:15)	10:9 (300:270)	1.5	2500	60

Characterization of NLCs

The particle size range of the nanoparticle dispersion was measured by the dynamic light scattering method [13] using a Malvern Nano ZetaSizer (Malvern Instrument Ltd., UK) yielding the diameter of the bulk population (Z-Ave) and the polydispersity index (PDI).The Zeta potential was also measured using the same instrument based on Laser Doppler Electrophoresis with water as the dispersion medium of pH7.0±0.2.

Scanning Electron Microscope (SEM)

The average diameter and particle morphology were studied using a Scanning Electron Microscope(SEM, Zeiss International,Germany). Sample aqueous dispersion of NLCs was spread on a thin carbon film holder. Then placed inside the vacuum column of the microscope and the vacuum was created by pumping the air out of the chamber. Then samples were spread on the carbon slides and left to dry for 30 min before being placed in the microscopic chamber for analysis.

Entrapment Efficiency

The entrapment efficiency was performed by centrifugation, the aqueous dispersion of NLCs (2 ml) at 5000 rpm for 20 min. the supernatant was decanted and subsequently diluted with isopropyl alcohol (IPA) and the concentration ofnaringenin was measured by spectrophotometer [14].

Stability Studies:

Naringenin NLCs were subjected to two different temperatures i.e., refrigeration temperature (4±2 °C) and room temperature (27±2 °C) for a period of 4 weeks and then carried out the stability studies. The sample was done at the end of each week and entrapment efficiency and particle size of naringenin-loaded NLCs stability were analyzed [15,16].

FT-IR analysis:

The important pre-formulation requirement is said to be the compatibility between the drug and the excipients, which provides information about the stability of the prepared formulation. The individual components, the NLCs formulation and the anhydrous physical mixture were studied by the FTIR [17,18].

In vitro drug release studies

In vitro release studies of naringenin-loaded NLC were performed in a thermostatic water bath at 37 ᵒC using the horizontal agitator (120 rpm). The volume of naringenin NLC formulation was suspended in pH 7.4 phosphate buffer (PBS) containing % 0.5 w/v. at a given time samples were centrifuged at 12000 rpm, and 4 ᵒC for 1 h, and the supernatant were withdrawn completely, then the supernatant was analyzed using a spectrophotometer (Shimadzu 1800)[19].

RESULTS AND DISCUSSIONS:

Selection of liquid and solid lipids:

In the preparation of NLCs, the lipids were selected depending on the two parameters, the liquid lipid which had a good ability to dissolve the drug and the solid lipid which was able to form a homogeneous mixture. The solubility of naringenin was observed in oleic acid and castor oil. However, naringenin showed a higher solubility in oleic acid (0.72g/100 ml). The solid lipids included stearic acid, palmitic acid, and beeswax, they were visually observed for homogeneity and no separation of layers upon congealing and cooling. Palmitic acid and beeswax showed separation into two layers which were confirmed by the immiscibility of the smear under the microscope. Stearic acid (0.11 g/100 ml) on the other hand showed good homogeneity and uniformity in the smear[20].

Characterization of Naringenin-loaded NLCs:

Particle size distribution of the Naringenin-loaded NLCs was determined by dynamic light scattering using a (Malvern Nano ZetaSizer).The results obtained in vesicular size analysis indicated that the size of the NLCs prepared by the microemulsion technique was greater compared to the size of the NLCsprepared by the sonication method.It was found that the average size (z-ave) of the NLCs without sonication (Formulation 2: Stearic acid: Oleic acid: Cholesterol, 8:2:0.5) was approximately 2156nm with a PDI value of 0.891, whereas the average particle size of the sonicated NLC(Formulation 6- Stearic acid: Oleic acid: Cholesterol; 8:2:0.5) was approximately 762 nm with a PDI value of 0.483. The physical stability of NLC can be evaluated by measuring the Zeta potential. The zeta potential of the NLC prepared without sonication was -25.35 mV whereas the NLC prepared with sonication was -28.43 mVshown in Fig.1.The potential indicatesparticle dispersions have good stability. The PDI was found to be 0.483 for the NLC prepared by probesonication-based method which indicated good dispersion of uniformly sized lipid vesicles(Table 1).

Fig. 1. Zeta Potential of Naringenin-NLC

Scanning Electron Microscope (SEM)

The surface morphology of naringenin NLCs was visualized by scanning electron microscopy (SEM). The NLCs were of spherical shape as depicted in Fig. 2. The entrapment efficiency in all the formulations was seen to be maintained within the limits of 94-98%.Increasing the stearic acid concentration had little effect on the entrapment efficiency. The entrapment efficiency was increased to a range of 97-98% in the presence of cholesterolwhichindicated the cholesterol was responsible for the increased particle stability.

Fig. 2.SEM image of Naringenin-NLC

FT-IR analysis:

The FTIR studies were carried out for the pure Naringenin, stearic acid, and the NLCs with and without cholesterol to know the possible interactions between naringenin, lipids, surfactants and excipients if any shown spectra in Fig. 3 (a) and (b). The principle peaks of the pure drug naringeninwere observed at 2347.91 cm-1 (-C=O) stretching and 3661.19 cm-1 (-O-H) whereas the peaks for stearic acid were observed at2347.91 cm-1 (-C=O), 3663.12 cm-1 (-O-H) and 2913.91 cm-1 (-C-H) stretching. The NLCformulations with and without cholesterol showed the naringenin acid peaks at 2347.91 cm-1 and 3662.16 cm-1. From these spectra, it was seen that there were no noticeable changes in the signal peaks of the drug and the excipients when compared with the formulations. Hence, the FTIR spectra indicated no interactions between naringenin and the formulation excipients.

Fig. 3 a. FT-IR spectra of naringenin

Fig. 3.b. FT-IR spectra of naringenin-NLC

In vitro drug release studies

In vitro release studies of naringenin were performed, PBS buffer containing 0.5% naringenin (w/v), pH 7.4 was used as the release medium for maintaining sink condition because of low water solubility of naringenin. The release of naringenin from the NLC formulation was evaluated by a high initial release that was followed bymore sustained release behavior shown in Fig. 4.

Fig. 4. Fig. Drug release of naringenin-NLC

Stability studies:

The stability of the NLCs is a major consideration in all steps of their production and application from process steps to storage and delivery. A stable dosage form maintains its physical integrity and does not adversely influence the chemical integrity of the active ingredient during its shelf life. The stability of the vehicles is the major determinant of the stability of the formulations. Hence, a study was carried out to evaluate drug entrapment at room temperature(27±2 °C) and refrigeration temperature (4±2 °C) for a period of 4 weeks. According to the data(not shown), formulations stored at refrigeration temperature and room temperature showed no significant loss in the entrapped drug content and no changes in the particle size, after a period of 4 weeks. Shown in Fig.5. This showed that the NLC formulations were highly stable at refrigerationand room temperature [21].

Fig. 5.Stability study of naringenin-NLC in different temperature

CONCLUSION:

Our research will concentrate on the development of nanostructured lipidcarriers(NLC)fortopicalapplications. The release rate of the drug can be controlled due to the solid matrix of lipid polymer hybrid nanoparticles and their special structure provides flexibility to achieve the desired prolonged releaseof lipid polymer hybrid nanoparticles prepared from biocompatible lipids. Further, their lipid nanoparticle will develop for better physical stability and prolong drug release. This is one of the best carriers for topical drug delivery systems. The lipid nanoparticle will formulate for better acceptability and stability. The formulation is expected to be more effective and less side effects by reducing the administration of naringenin repeatedly.

REFERENCES:

1. Dange SM, Kamble MS, Bhalerao KK, Chaudhari PD, Bhosale A V., Nanjwade BK, et al. Formulation and evaluation of VLF nanostructured lipid carriers. J Bionanoscience. 2014;8(2):81–9

2. Qadir A, Aqil M, Ali A, Warsi MH, Mujeeb M, Ahmad FJ, Ahmad S, Beg S. Nanostructured lipidic carriers for dual drug delivery in the management of psoriasis: systematic optimization, dermatokinetic and preclinical evaluation. Journal of Drug Delivery Science and Technology. 2020 Jun 1;57:101775.

3. Jangde RK, Khute S, Bhardwaj H, Yadav K. Development of Nanoformulations forTargeted Transport of CNS Drugs with Greater Pharmacological Activity via the Nose in tothe Brain. Int. J Drug Dev. Res. 2022; 12(14): 984.

4. Jangde R, Elhassan GO, Khute S, Singh D, et al. Hesperidin-loaded lipid polymer hybridnanoparticles for topical delivery of bioactive drugs. Pharm. 2022; 15(2):211.

5. Elmowafy M, Al-Sanea MM. Nanostructured lipid carriers (NLCs) as drug delivery platform: Advances in formulation and delivery strategies. Saudi Pharmaceutical Journal. 2021 Sep 1;29(9):999-1012.

6. Pooja Sahu, Rajendra Jangade. An Updated Review on Mechanism of Novel Carrier System for Wound Healing. Research J. Topical and Cosmetic Sci. 10(2): July- Dec. 2019 page 65-78.

7. Rajendra Kumar Jangde, Rabsanjani, Sulekha Khute. Design and Development of Ciprofloxacin Lipid Polymer Hybrid Nanoparticle by Response Surface Methodology. Research J. Pharm. and Tech. 2020; 13(7): 3249-3256.

8. Jangde R, Singh D. Preparation and optimization of quercetin-loaded liposomes for wound healing, using response surface methodology. Artificial cells, nanomedicine, and biotechnology. 2016 Feb 17;44(2):635-41.

9. Kumar RP, Abraham A. PVP-coated naringenin nanoparticles for biomedical applications–In vivo toxicological evaluations. Chemico-biological interactions. 2016 Sep 25;257:110-8.

10. Bhia M, Motallebi M, Abadi B, Zarepour A, Pereira-Silva M, Saremnejad F, Santos AC, Zarrabi A, Melero A, Jafari SM, Shakibaei M. Naringenin nano-delivery systems and their therapeutic applications. Pharmaceutics. 2021 Feb 23;13(2):291.

11. Maity S, Mukhopadhyay P, Kundu PP, Chakraborti AS. Alginate coated chitosan core-shell nanoparticles for efficient oral delivery of naringenin in diabetic animals—An in vitro and in vivo approach. Carbohydrate Polymers. 2017 Aug 15;170:124-32.

12. Smruthi MR, Nallamuthu I, Singsit D, Anand T. Toxicological evaluation of PLA/PVA-naringenin nanoparticles: In vitro and in vivo studies. OpenNano. 2022 Jul 1;7:100061.

13. Cordenonsi LM, Sponchiado RM, Bandeira JR, Santos RC, Raffin RP, Schapoval EE. Polymeric nanoparticles loaded naringin and naringenin: effect of solvent, characterization, photodegradation and stability studies. Drug Analytical Research. 2020 Dec 18;4(2):64-71.

14. Mukherjee S, Ray S, Thakur RS. Solid lipid nanoparticles: a modern formulation approach in drug delivery system. Indian journal of pharmaceutical sciences. 2009 Jul;71(4):349.

15. Garud A, Singh D, Garud N. Solid lipid nanoparticles (SLN): method, characterization and applications. International Current Pharmaceutical Journal. 2012 Oct 3;1(11):384-93.

16. Garcês A, Amaral MH, Lobo JS, Silva AC. Formulations based on solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) for cutaneous use: A review. European Journal of Pharmaceutical Sciences. 2018 Jan 15;112:159-67.

17. Jain S, Jain S, Khare P, Gulbake A, Bansal D, Jain SK. Design and development of solid lipid nanoparticles for topical delivery of an anti-fungal agent. Drug delivery. 2010 Aug 1;17(6):443-51.

18. Ridolfi DM, Marcato PD, Justo GZ, Cordi L, Machado D, Durán N. Chitosan-solid lipid nanoparticles as carriers for topical delivery of tretinoin. Colloids and Surfaces B: Biointerfaces. 2012 May 1;93:36-40.

19. Orgul D, Eroglu H, Hekimoglu S. Formulation and characterization of tissue scaffolds containing simvastatin-loaded nanostructured lipid carriers for the treatment of diabetic wounds. Journal of Drug Delivery Science and Technology. 2017 Oct 1;41:280-92.

20. Liu M, Wen J, Sharma M. Solid lipid nanoparticles for topical drug delivery: mechanisms, dosage form perspectives, and translational status. Current Pharmaceutical Design. 2020 Aug 1;26(27):3203-17.

21. Jangde R, Singh D. Compatibility studies of quercetin with pharmaceutical excipients used in the development of novel formulation. Research Journal of Pharmacy and Technology. 2014;7(10):1101-5.

Received on 16.12.2022 Modified on 15.02.2023

Research J. Pharm. and Tech 2023; 16(5):2572-2576.

DOI: 10.52711/0974-360X.2023.00422