(W1130-03-16) Design, Physicochemical Characterization and In Vitro Cytotoxicity of Cisplatin-Loaded PEGylated Chitosan Injectable Nano / Sub-micron Crystals

Purpose: The study aimed to develop novel cisplatin-loaded PEGylated chitosan injectable nano / sub-micron crystals and to determine their physicochemical properties and cytotoxicity efficacy of the formulated nano nanoparticles.

Methods: Cisplatin-loaded PEGylated chitosan nanoparticles were prepared using an ionic gelation technique. Initially, 1% w/v chitosan polymer was prepared in 1% v/v glacial acetic acid. The chitosan gel was kept overnight to stabilize. Then, a 1% w/v cisplatin solution was prepared in phosphate-buffered saline at pH 7.4. PEG (50% w/v) was used as the grafting polymer for the PEGylation of the chitosan polymer. Formalin solution (0.25% v/v) was used as the cross-linker to the chitosan-based on ionic attraction. Formaldehyde also reacted with PEG because PEG has a free OH group. The pH of nanoparticle reaction mixture was measured by Oakton pH 700, a benchtop meter (Oakton Instruments, Vernon Hills, IL, USA). The lyophilization was performed by using Millrock BT85 desktop freeze dryer (Millrock Technology, USA). The nanoparticles were physically characterized by measuring their zeta potential (ZP) in millivolts (mV), their conductivity in milli siemens per centimeter (mS/cm), their pH, their size in nanometers (d.nm and z. d.nm), and their polydispersity index (PDI). The morphological features and particle size of the formulated nanoparticles were studied using a high-resolution scanning electron microscopy study. Lyophilized PEGylated nanoparticles were analyzed by means of the DSC method to ascertain the enthalpy changes that occurred because of changes in the physical and chemical properties of the samples. A thermogravimetric analyzer (TGA 8000, Perkin Elmer, Waltham, MA, USA) was utilized to determine the thermal stability of the lyophilized nanoparticles. X-ray diffraction (XRD) was utilized so that the crystalline structures of lyophilized PEGylated nanoparticle powder samples could be investigated. The functional groups of the samples were analyzed by using Bruker FT-IR spectroscopy, USA. The release of cisplatin from PEGylated chitosan nanoparticles were determined by establishing a standard curve analyzed using UV/visible spectroscopy at 265 nm, and the release pattern was determined by extrapolating the optical density against cisplatin concentration. The encapsulation efficiency (EE) and drug loading (DL) were calculated using an equation. The MTT assay was performed to evaluate the In vitro cytotoxicity activity using human breast cancer cells from the MCF-7 ATCC strain.

Results: The optimal batch of cisplatin-loaded PEGylated chitosan nanoparticles had a + 49.9 mV zeta potential, PDI of 0.347, and % PDI of 58.9. The Nanoparticle zeta size was 741.4 z. d.nm, the size in diameter was 866.7 ± 470.5 nm, and nanoparticle conductivity in colloidal solution was 0.739 mS/cm. Differential scanning calorimetry (DSC) revealed that cisplatin-loaded PEGylated chitosan nanoparticles had sharp endothermic peaks at 168.6 °C. The thermogravimetric analysis (TGA) showed the weight loss of cisplatin-loaded PEGylated chitosan nanoparticles, which was 95% at 262.76 °C. XRD investigation on cisplatin-loaded PEGylated chitosan nanoparticles exhibited distinct peaks at 2h as 9.7°, 20.4°, 22.1°, 25.3°, 36.1°, 38.1°, 39.5°, 44.3°, and 64.5°, confirming crystalline structure. The 1 H NMR analysis showed the fingerprint region of cisplatin-loaded PEGylated chitosan nanoparticles as 0.85, 1.73, and 1.00 ppm in the proton dimension, and de-shielded proton peaks appeared at 3.57, 3.58, 3.58, 3.59, 3.65, 3.67, 3,67, 3,67, 3.70, 3.71, 3.77, 3.78 and 4.71 ppm. The 13C NMR spectrum showed specified peaks at 63.18, 69.20, and 70.77 ppm. The FT-IR spectra of cisplatin-loaded PEGylated nanoparticles show the existence of many fingerprint regions at 3186.52, 2931.68, 1453.19, 1333.98, 1253.71, 1085.19, 1019.60, 969.98, 929.53, 888.80, 706.13, and 623.67 cm ¹. The drug release kinetics of cisplatin-loaded PEGylated chitosan nanoparticles showed zero order kinetics with 48% of drug release linearity fashion which has R2 value of 0.9778. Studies on the MCF-7 ATCC human breast cancer cell line in vitro revealed that the IC50 value was 82.08 µg /mL.

Conclusion: Cisplatin-loaded PEGylated chitosan nanoparticles in an injectable dosage form can be successfully formulated using an ionic gelation technique. This study demonstrated that a low concentration of formaldehyde solution was able to link chitosan and PEG successfully. The formulation will serve as a good therapeutic agent to deliver cisplatin safely. The analysis of cisplatin-loaded PEGylated chitosan nanoparticles' physicochemical properties demonstrated that these particles have substantial properties as injectable formulations. The cisplatin-loaded PEGylated chitosan nanoparticles exhibited lower cytotoxicity on MCF-7 ATCC human breast cancer cells. Further studies can be established to assess the in vivo performance and PK parameters and cisplatin dose optimization. The results obtained in this study are promising to develop a novel anticancer drug delivery system through PEGylation process of chitosan.

References: Sultan, M.H., Moni, S.S., Alqahtani, S.S., Bakkari, M.A., Alshammari, A., Almoshari, Y., Alshahrani, S., Madkhali, O. and Mohan, S., 2023. Design, physicochemical characterisation, and in vitro cytotoxicity of cisplatin-loaded PEGylated chitosan injectable nano/sub-micron crystals. Saudi Pharmaceutical Journal.

Acknowledgements: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors extend their appreciation to the Deputyship for research and innovation. Ministry of education in Saudi Arabia for funding this research work through the project number ISP22-3.


Cytotoxicity study. Dose-response curve of lyophilized cisplatin-loaded PEGylated chitosan nanoparticles of ideal batch against MCF-7 breast cancer cells

n vitro release profile. Release kinetics of lyophilized cisplatin-loaded PEGylated chitosan nanoparticles of the ideal batch.

Scanning electron micrograph study of batch 1 (A) Cisplatin-loaded PEGylated chitosan nanoparticles of batch 1 under 8,000 × magnification (B) Cisplatin-loaded PEGylated chitosan nanoparticles of batch 1 under 20,000 × magnification.