Fabrication and Cytotoxic activity of Clofarabine Nano Emulzion Decorated with Chitosan on MCF-7 cell linings
Annapurna Anusha Kotwala1, Komala. M2*
1,2Department of Pharmaceutics, School of Pharmaceutical Sciences, Vels Institute of Science Technology and Advanced Studies (VISTAS), Chennai - 600117, Tamilnadu, India.
*Corresponding Author E-mail: anusshah.seethala@gmail.com, komala.pharmacy@gmail.com
ABSTRACT:
One antineoplastic analogue of purine nucleosides from the second generation is clofarabine. This study aimed to produce clofarabine-loaded chitosan nanoparticles as a possible drug delivery vehicle for anticancer11,12. The design and development of innovative drug delivery systems, mostly based on nanotechnology, has become necessary due to the constraints associated with the conventional treatment of cancer. Chitosan-based nanoparticles have caught the interest of researchers as one of the many innovative medication delivery strategies for the treatment of cancer. Chitosan is a polycationic polymer that has several properties, including biocompatibility, biodegradability, and non-toxicity, which makes it the perfect polymer for creating drug delivery systems. Chitosan was used to cover the prepared nano-emulzion of CLOFARABINE. A two-step emulsification procedure was used in the current investigation to create chitosan-decorated multiple Nano emulzions (MNEs). A total of five MNE sample formulations were created and assessed. Entrapment efficiency (EE), stability, zeta potential, globule size, PDI and invitro drug release tests were used to characterize the produced formulations. The manufactured MNE displayed regulated drug release. The chitosan ornamentation used to create sustained release for the MNE. Additionally, studies were conducted on conjugated nanoparticles' cytotoxicity.
KEYWORDS: Nanoparticles, Clofarabine, Chitosan, Multiple Nano emulzions, encapsulation efficiency.
INTRODUCTION:
Clofarabine is intracellularly metabolized and inhibits the production of DNA1 and halts proliferation of tumour cells. The authors are producing and optimizing clofarabine Multiple Nano emulzion coated with chitosan for the first time, following a thorough review of the literature. Chitosan-based nanoparticles have caught the interest of researchers as one of the many innovative medication delivery strategies for the treatment of cancer.
Additionally, chitosan's physiochemical properties can be enhanced through chemical modification due to the presence of reactive amino groups. Because of their many qualities, including their nano size, suitable pharmacokinetic and pharmacodynamic properties, non-immunogenicity, enhanced stability and increased drug loading capacity, nanoparticles coated with chitosan are most commonly utilized in the pharmaceutical industry.
MATERIALS AND METHOD:
Materials2,3
Mylan Pharmaceutical Co., Ltd. (Hyderabad, India) offered clofarabine as a gift. Both Lipoid E80 lecithin and soybean oil that are purchased from Lipoid are utilized as surfactant and oil phase respectively. A. R. Chemicals and Glassware sold propylene glycol. MilliQ ® water was used as aqueous stage. Next, additional treatment was applied to the NEs by layering of Chitosan6 which was further functionalized using Fluorescin-5(6)-isothiocyanate. N₂(C₂H₄)₃ a antifade agent was utilized to prevent the bleaching of FITC. All are brought from Sigma Aldrich.
PREFORMULATION STUDIES:
Compatibility studies:
The XRD ranges of chitosan3, chitosan/Lecithin, chitosan/Lecithin@Clofarabine and also chitosan-FTIC-Derivative/Lecithin@Clofarabine/nanoparticles were shown in Fig.1A. supports the pseudo crystalline structure of chitosan. According to peaks at 7.5° as well as 18.4°, Lecithin was added successfully to chitosan nanoparticles. As displayed in fig.1A.c and 1 A.d, the XRD evaluation validated the presence of Clofarabine and also drugs as a result of the peaks at 28.6° and also 8.8°, specifically. Fig.1B presents outcome of the FT-IR spectra, supplying additional indication of the loading of various NPs. Fig.1B a) displayes bending vibrations of Hydroxy-group as well as Amino-group at 3429 cm-1, as well as C_ H stretching and also C_O flexing vibrations at 2160 cm-1 as well as 1634 cm-1, specifically. Fig.1B b) programs reducing C _O bond frequency, validating hydrogen bonding between chitosan and also Lecithin molecules. Filling Clofarabine right into chitosan/ Lecithin N. Ps triggered a boost in the intensity of main NH3+. Connected band at 1548 cm-1, and also a brand-new wide height at 759 cm-1, connected to the C_H bond (Fig.1B c). As shown in Fig. 1B d) the 1459 centimeters-1 top is improved due to the formation of hydrogen bonding in between NH3+ and teams of chitosan and carbonyl teams of Clofarabine N.Ps. Proportionately all NanoParticles are appropriately loaded.
Viscosity study:4
To stabilize the emulzion, an attempt was made to increase the viscosity of the dispersant phase by adding Propylene glycol. The addition of Propylene glycol was expected to minimised emulzion coalescence and stabilise smaller oil Nano-droplets as a result. Various proportions of Propylene glycol were blended with surfactant and oil conc to find the optimal formulation. After several formulation trials, it was possible to optimize the process and achieve size reduction without negatively affecting the PDI (Polydispersity Index). The best formulation resulted in significantly smaller sizes for the NEs.
Fig. 1: (A) XRD patterns and (B) FTIR Images of (a) chitosan, (b) chitosan/Lecithin, (c) chitosan/Lecithin@Clofarabine,and (d) chitosan-FTIC/ Lecithin@Clofarabine-nanoparticles.
Fig 2A- Effect on Particle Size
Fig 2 B- Effect on Poly Dispersity Index
Fig.2 Effect of Different Concs of Propylene Glycol on viscosity of Nano emulzion
Method of Preparation:
Designing of Primary Emulzions:4,5
Various O/W emulzions had been fabricated by various oil % and quantity of lecithin. Soybean oil was mixed with Lipoid-E80 formerly weighted with a high precision balance in a beaker. The surfactant was dissolved in the oil phase by high-pace blender and sonicated for 15 minutes with an immersion sonicater till lecithin become absolutely solubilised. After measuring milliQ water phase we dissolved 100mg of Clofarabine. To this aqueous segment 50% Propylene glycol was added. To reap the pre-emulzion, the oil phase is delivered dropwise to the aqueous segment with mixing using an immersion sonicater for 5 minutes.The pre-emulzion practice was achieved in a massive beaker jacketed with ice, to keep temperature low and keep away from pre-emulzion overheating for the duration of sonication. The pre-emulzion was at last subjected to 2000 bar excessive strain by homogenizer for 3 turns and then in non-stop steps modality, recycling and casting off little quantities of emulzion at every 50 steps until 200 steps to discharge further tests. The tool used for size, PDI and Z-ability measurements is ZetasizerNano series with the aid of Malvern©.
Table 1. Preparation of 1° Nano emulzion (O/W)
|
Code |
Drug (mg) |
Oil Phase (%) |
Propylene Glycol (%) |
Lipoid-E80 Lecithin(g) |
Droplet Size |
PDI |
ZP (mV) |
|
F1 |
100 |
20 |
50 |
1.2 |
157 |
0.03 |
-42.1 |
|
F2 |
100 |
20 |
50 |
2.4 |
118 |
0.06 |
-37.6 |
|
F3 |
100 |
20 |
50 |
3.6 |
101 |
0.06 |
-36.2 |
|
F4 |
100 |
20 |
50 |
4.8 |
80 |
0.09 |
-36.3 |
|
F5 |
100 |
20 |
50 |
6.0 |
76 |
0.1 |
-38.1 |
Fig 3 Effect of 20% Concs of oil on particle size of Nano-emulzions
Fig 4 Effect of 20% Concs of oil on Poly dispersity index of Nano emulsion
Fabrication of 20 Nano-Emulzions:6
It's been visible in initial experiments that purified samples of chitosan are necessary for precise deposition on 10 emulzion. Chitosan was additionally functionalized with FITC. Numerous concs (0.01%, zero.02%, zero.03%, zero.05%,0.10%, zero.18%,0.25%) of Chitosan3 were devised in 0.1 M CH3 C00 Milli-Q water. Nine ml of these conc are expanded with 27ml of water phase to 36ml total and brought to pH four with inclusion of Na(OH) 6M soln . Next 9ml of 10 emulzion made up with 20% oil phase was introduced quick to chitosan solutions of different concs under robust blending for fifteen min to permit constat chitosan banding. Our findings advise that the small size and goods tability of emulzions can be achieved through adjusting the concentration of oil, lecithin, and co-surfactant.
Table 2. Preparation of Nano emulzion (O/W/O)
|
Code |
Chitosan Conc (%) |
Droplet Size (nm) |
PDI |
|
F1 |
0.01 |
172.4 |
0.06 |
|
0.02 |
180.7 |
0.02 |
|
|
0.03 |
174.2 |
0.03 |
|
|
0.05 |
173.2 |
0.05 |
|
|
0.10 |
182.9 |
0.10 |
|
|
0.18 |
199.3 |
0.18 |
|
|
0.25 |
197.6 |
0.25 |
|
|
F2 |
0.01 |
128.4 |
0.08 |
|
0.02 |
128.7 |
0.06 |
|
|
0.03 |
127 |
0.06 |
|
|
0.05 |
125 |
0.1 |
|
|
0.10 |
139.7 |
0.12 |
|
|
0.18 |
162 |
0.17 |
|
|
0.25 |
197 |
0.17 |
|
|
F3 |
0.01 |
101.9 |
0.08 |
|
0.02 |
103.4 |
0.10 |
|
|
0.03 |
102.5 |
0.10 |
|
|
0.05 |
109 |
1.12 |
|
|
0.10 |
115.5 |
0.13 |
|
|
0.18 |
126.5 |
0.18 |
|
|
0.25 |
142 |
0.20 |
|
|
F4 |
0.01 |
88.18 |
0.12 |
|
0.02 |
89.88 |
0.13 |
|
|
0.03 |
89.18 |
0.11 |
|
|
0.05 |
91.6 |
0.15 |
|
|
0.10 |
95.8 |
0.15 |
|
|
0.18 |
104.5 |
0.19 |
|
|
0.25 |
110.2 |
0.2 |
|
|
F5 |
0.01 |
83.2 |
0.12 |
|
0.02 |
83.82 |
0.13 |
|
|
0.03 |
82.28 |
0.12 |
|
|
0.05 |
88.4 |
0.17 |
|
|
0.10 |
81.88 |
0.12 |
|
|
0.18 |
95.4 |
0.18 |
|
|
0.25 |
108 |
0.21 |
Fig 5 TEM- 10 Nano emulzions
Fig 6 TEM- Chitosan 20 Nano emulzions
RESULTS AND DISCUSSION:
Conductivity studies: 7
Electrical Conductivity measurements were performed categorise if emulsion is oil in water or vice versa. High conductivity worth was recorded for both empty and also medication-loaded formulas confirming that both solutions were o/w type. By this Phase inversion temperature can be reported which has promising role for storage parameters.
Fig 7 - Conductivity test for Nano Emulsion
Fig 8 - PIT of Clofarabine-NE
Stability of Primary Emulzions
Each emulzion was carefully analyzed to determine its stability over time. This was done by measuring the size of the droplets and calculating the Polydispersity Index (PDI), which indicates the uniformity of droplet sizes within the emulzion. When examining the emulzions at room temperature, it was observed that the last two emulzions remained relatively stable. However, the first three emulzions underwent significant changes, which was expected due to the difference in droplet sizes. Larger droplets are more flexible and tend to merge (coalesce) more easily, leading to instability in the emulzion. Interestingly, when the emulzions were stored at a lower temperature of 4°C, even the first three emulzions gained stability. Along with reduce the size of the Nano emulzions (NEs), an attempt was made to increase the viscosity of the dispersion medium.
Fig 9 - Stability Evaluation of Nano emulzions - Globule Size
Fig 10 - Stability Evaluation of Nano emulzions- PDI
DLS of Secondary Emulzions:
Different concs of Chitosan were added to Clofarabine Nano emulzions. The outcoe of conc of chitosan on globule size, polydispersity index and ZPI of emulzion were studied.The capability of 20 emulzions to remain stable rely on the conc of the polymer used for coating. By boosting the polymer conc, emulzion steadiness improved, possibly by appropriate covering of the emulzions by the polymer.F5 formulation can be optimized one with 0.1% chitin conc.
Fig 11 Effect of Chitin Conc on Particle-size-variation and PDI in 28-Day study of different NE-Formulations re-dispersed through high pressure Homogenizer Fig 15 A- Optimized NE(F5)
Viscosity Measurement:6,7
The viscosity of the formulations was assessed via Brookfield at 30°C. Dynamic Viscosity was found to be 15.22±2.97, 18.22±2.97, 20.22±3.12, 23.22±3.12, 28.22±6.97 for F1 to F5 formulations respectively
Drug Content and Entrapment Efficiency (%) Determination:6,7
The drug content of MNE was studied by solubilizing 2g of prepartion in ten ml Gomori buffer of pH 5.5. It is centrifuged at 14,000rpm for 30min. Supernatant solution is then checked spectrophotometrically at 262 nm. Drug content is found to be 82±3.12%, 91±2.02%, 90±2.02%, 93±2.02%, 97±2.31% for F1 to F5 formulations respectively. Entrapment Efficiency (%) was found to be 89.19±1.52%, 92.11±2.03%, 93.31± 1.12%, 95.84±1.72%, 97.42±2.09% for F1 to F5 formulations respectively.
Fig 12 Globule size distribution by DLS for chitosan MNE - F1 to F5
Fig 13 -Potential (mV) analysis on NE-Formulation#5-Secondary Emulzion at different chitosan levels in (% wt) from0%-to-0.2
%
Reproducibilty
Among the several tests accomplished to enhance the preparation approach in regards to formulation and devices, it was additionally tested for the reproducibility. In the process of reproducibilty study we,followed the average dimension and PDI.As it is possible to see from the chart, there is a best reproducibility both in regards to dimension and also PDI.
Fig. 14 Reproducibility of Multiple nano emulzion- Particle Size
Fig. 15 Reproducibility of Multiple nano emulzion- PDI
In vitro Drug Release:8,9
Invitro dg release profile of Clofarabine Nano emulzion was reviewed utilizing a dialysis bag technique. The medication quantity, and cumulative percent liberation were quantified using HPLC analysis approach
Fig 16: In vitro release profile of Clofarabine
Drug Release Kinetics:
The optimized formulation was exposed to drug release kinetic studies. F5 formulation exhibited the best fit with Higuchi model. It exhibited the R2 of 0.9343. The 'n' value is 0.89 revealing Case II transport.
Table 3 Kinetics data
|
Kinetic Model |
R² |
|
Zero order Kinetics |
0.854 |
|
First order Kinetics |
0.8735 |
|
Higuchi |
0.9343 |
|
Hixson Crowell |
0.895 |
|
Korsmeyer -Peppas |
0.9014 |
Cytotoxicity Study: 11,12
Anticancer 12 property of Clofarabine Nano emulzion coated with chitosan was estimated in MCF-7 cell lines.
Microscopic images of MCF cells after 6-h incubation with the FITC-conjugated-Chitosan stabilized Clofarabine nanoemulsions (top) and plain Primaracy nanoemulsions (bottom)
Fig 17 Cytotoxic Studies on MCF-7-Cell Lines
CONCLUSION:
With this study, 5 chitosan-decorated M ultipleNano emulzions are fabricated and evaluated. Amongst 5 formulations F5 formula turned into selected as first-rate formula. We observed that the quantity of polymer needed to cover the emulzions became approximately 0.1% wt. The organized system was characterized for parameters which include Globule size, PDI, ZP, entrapment performance (EE) and Invitro drug launch studies. F5 Nano emulzions showed very less Globule size of 81.8 nm, less Poly Dispersity index 0.12 and high electrokinetic potential. The Drug content observed to be 97 ± 2.31% and Entrapment performance was ninety 97.42± 2.0%. The formulated MNE showed controlled launch of drug. Around 100% of the medication released in 72hrs. Clofarabine has high dividing co-efficient and a low Log-P worth. This was attributed to the chitosan ornament around multiple emulzions. Drug release kinetics showcased Case II transport. Moreover, absorption of fabricated NanoParticles in most tumor lining has been explored. MNE decorated with chitosan confirmed suitable cytotoxic belongings.
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Received on 03.05.2024 Revised on 23.09.2024 Accepted on 26.11.2024 Published on 10.04.2025 Available online from April 12, 2025 Research J. Pharmacy and Technology. 2025;18(4):1507-1514. DOI: 10.52711/0974-360X.2025.00216 © RJPT All right reserved
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