HPLC METHOD DEVELOPMENT AND VALIDATION FOR METFORMIN AND PHENYL BUTYRIC ACID AND IN VITRO EVALUATION OF THEIR PEGYLATED LIPOSOMAL FORMULATION AGAINST COLORECTAL CANCER

HUSSEIN S. JANABI; ZAINAB LAFI; SINA MATALQAH

doi:10.22159/ijap.2025v17i3.53441

Authors

HUSSEIN S. JANABI Pharmacological and Diagnostic Research Center, Faculty of Pharmacy, Al-Ahliyya Amman University, Jordan. College of Pharmacy, Uruk University, Baghdad, Iraq
ZAINAB LAFI Pharmacological and Diagnostic Research Center, Faculty of Pharmacy, Al-Ahliyya Amman University, Jordan. Department of Pharmaceutics and Pharmaceutical Technology, Al-Ahliyya Amman University, Amman, Jordan https://orcid.org/0000-0003-2847-2416
SINA MATALQAH Pharmacological and Diagnostic Research Center, Faculty of Pharmacy, Al-Ahliyya Amman University, Jordan. Department of Pharmaceutics and Pharmaceutical Technology, Al-Ahliyya Amman University, Amman, Jordan https://orcid.org/0000-0002-2101-3420

DOI:

https://doi.org/10.22159/ijap.2025v17i3.53441

Keywords:

HPLC validation, Liposomes, Combination, Nanoparticles, Colorectal cancer

Abstract

Objective: This study aimed to develop a PEGylated liposomal formulation combining Metformin (MET) and Phenyl Butyric Acid (PBA) for colorectal cancer treatment. Current colorectal cancer therapies face limitations such as poor drug solubility, rapid clearance, and systemic toxicity. Combining MET and PBA in liposomes could potentially overcome these limitations by enhancing drug stability, prolonging circulation time, and improving targeted delivery.

Methods: A High-Performance Liquid Chromatography (HPLC) method was optimized and validated for the simultaneous quantification of MET and PBA, achieving calibration curve R² values above 0.99. Liposomal formulations were prepared, and encapsulation efficiencies were evaluated. Sustained drug release was studied over 72 h at 37 °C. Cytotoxicity assays were conducted using colorectal cancer cell lines HCT116 and HT29. Additionally, colony formation assays assessed the formulation's long-term effects on cell proliferation.

Results: The liposomal formulation showed encapsulation efficiencies of 48% for Lip-MET and 56.8% and 83.9% for Lip-MET-PBA. Sustained release profiles were observed, with 60% of MET and 55% of PBA released over 72 h. Cytotoxicity assays revealed that Lip-MET-PBA significantly reduced cell viability in HCT116 and HT29 cell lines, with IC50 values of 0.65 mmol and 0.82 mmol, respectively, compared to MET alone (1.1 mmol and 1.3 mmol). Colony formation assays demonstrated a concentration-dependent reduction in colony numbers, highlighting the enhanced anticancer efficacy of the combination. Statistical analysis showed significant differences (p<0.05) in cell viability and colony formation between the liposomal formulation and the free drugs.

Conclusion: This is the first PEGylated liposomal formulation combining MET and PBA, offering a promising strategy for colorectal cancer therapy. The formulation combines sustained release, improved cytotoxicity, and a significant impact on colony formation, warranting further in vivo investigations.

References

El Zarif T, Yibirin M, DE Oliveira Gomes D, Machaalani M, Nawfal R, Bittar G. Overcoming therapy resistance in colon cancer by drug repurposing. Cancers (Basel). 2022;14(9):2105. doi: 10.3390/cancers14092105, PMID 35565237.

Cappell MS. Pathophysiology clinical presentation and management of colon cancer. Gastroenterol Clin North Am. 2008;37(1):1-24. doi: 10.1016/j.gtc.2007.12.002, PMID 18313537.

Kraft JC, Freeling JP, Wang Z, HO RJ. Emerging research and clinical development trends of liposome and lipid nanoparticle drug delivery systems. J Pharm Sci. 2014;103(1):29-52. doi: 10.1002/jps.23773, PMID 24338748.

Lafi Z, Bustanji Y. Formulation and functionalization of aptamer guided pH-sensitive liposomes for targeted drug delivery of echinomycin cyclodextrin complex into cancer cells: [dissertion] faculty of pharmacy. University of Jordan; 2020.

Munef A, Lafi Z, Shalan N. Investigating anti-cancer activity of dual loaded liposomes with thymoquinone and vitamin C. Ther Deliv. 2024;15(4):267-78. doi: 10.4155/tde-2023-0140, PMID 38449422.

Matalqah S, Lafi Z, Asha SY. Hyaluronic acid in nanopharmaceuticals: an overview. Curr Issues Mol Biol. 2024;46(9):10444-61. doi: 10.3390/cimb46090621, PMID 39329973.

Niu G, Cogburn B, Hughes J. Preparation and characterization of doxorubicin liposomes. Methods Mol Biol. 2010;624:211-9. doi: 10.1007/978-1-60761-609-2_14, PMID 20217598.

Makwana V, Karanjia J, Haselhorst T, Anoopkumar Dukie S, Rudrawar S. Liposomal doxorubicin as targeted delivery platform: current trends in surface functionalization. Int J Pharm. 2021;593:120117. doi: 10.1016/j.ijpharm.2020.120117, PMID 33259901.

Lafi Z, Alshaer W, Gharaibeh L, Alqudah DA, AlQuaissi B, Bashaireh B. Synergistic combination of doxorubicin with hydralazine and disulfiram against MCF-7 breast cancer cell line. Plos One. 2023;18(9):e0291981. doi: 10.1371/journal.pone.0291981, PMID 37768997.

Shade CW. Liposomes as advanced delivery systems for nutraceuticals. Integr Med (Encinitas). 2016;15(1):33-6. PMID 27053934.

Morales DR, Morris AD. Metformin in cancer treatment and prevention. Annu Rev Med. 2015;66:17-29. doi: 10.1146/annurev-med-062613-093128, PMID 25386929.

Saraei P, Asadi I, Kakar MA, Moradi Kor N. The beneficial effects of metformin on cancer prevention and therapy: a comprehensive review of recent advances. Cancer Manag Res. 2019;11:3295-313. doi: 10.2147/CMAR.S200059, PMID 31114366, PMCID PMC6497052.

Marmol JM, Carlsson M, Raun SH, Grand MK, Sorensen J, Lang Lehrskov L. Insulin resistance in patients with cancer: a systematic review and meta-analysis. Acta Oncol. 2023 Apr;62(4):364-71. doi: 10.1080/0284186X.2023.2197124, PMID 37042166.

Mohamed Tawfik S. Repurposing metformin and antifolates for the treatment of hepatocellular carcinoma. [Dessertion] American University in Cairo; 2022.

Jang SK, Hong SE, Lee DH, Kim JY, Kim JY, YE SK. Correction to: inhibition of mTORC1 through ATF4-induced REDD1 and sestrin2 expression by metformin. BMC Cancer. 2021;21(1):881. doi: 10.1186/s12885-021-08584-z, PMID 34340683.

ZI F, ZI H, LI Y, HE J, Shi Q, Cai Z. Metformin and cancer: an existing drug for cancer prevention and therapy. Oncol Lett. 2018 Jan;15(1):683-90. doi: 10.3892/ol.2017.7412, PMID 29422962, PMCID PMC5772929.

Morale MG, Tamura RE, Rubio IG. Metformin and cancer hallmarks: molecular mechanisms in thyroid prostate and head and neck cancer models. Biomolecules. 2022;12(3):357. doi: 10.3390/biom12030357, PMID 35327549.

Zhang W, Zhang SL, HU X, Tam KY. Phenylbutyrate inhibits pyruvate dehydrogenase kinase 1 and contributes to its anti-cancer effect. Eur J Pharm Sci. 2017 Dec 15;110:93-100. doi: 10.1016/j.ejps.2017.04.018, PMID 28450154.

Rivera CN, Smith CE, Draper LV, Watne RM, Wommack AJ, Vaughan RA. Physiological 4-phenylbutyrate promotes mitochondrial biogenesis and metabolism in C2C12 myotubes. Biochimie. 2024 Apr;219:155-64. doi: 10.1016/j.biochi.2023.11.009, PMID 38008282.

DE Pablo S, Rodriguez Comas J, Diaz Catalan D, Alcarraz Vizan G, Castano C, Moreno Vedia J. 4-phenylbutyrate (PBA) treatment reduces hyperglycemia and islet amyloid in a mouse model of type 2 diabetes and obesity. Sci Rep. 2021;11(1):11878. doi: 10.1038/s41598-021-91311-2, PMID 34088954.

Allateef A, Shalan N, Lafi Z. Anticancer activity of liposomal formulation co-encapsulated with coumarin and phenyl butyric acid. J Appl Pharm Sci. 2024;14(11):208-15. doi: 10.7324/JAPS.2024.181335.

Fahdawi A, Shalan N, Lafi Z, Markab O. Analytical approaches for assessing curcumin and nicotinamide Co-encapsulated in liposomal formulation: UV spectrophotometry and HPLC validation. Jordan J Pharm Sci. 2024;17(3):468-80. doi: 10.35516/jjps.v17i3.2359.

Alshaer W, Zraikat M, Amer A, Nsairat H, Lafi Z, Alqudah DA. Encapsulation of echinomycin in cyclodextrin inclusion complexes into liposomes: in vitro anti-proliferative and anti-invasive activity in glioblastoma. RSC Adv. 2019;9(53):30976-88. doi: 10.1039/C9RA05636J, PMID 35529392.

Alrubaye MA, Lafi Z, Abu Hajleh MN, Abuamara TM, Mehdawi A. Co-encapsulation of coumarin and Q10 into solid lipid nanoparticles for wound healing. J Drug Deliv Sci Technol. 2025 Mar;105:106645. doi: 10.1016/j.jddst.2025.106645.

Abu Saleem E, Lafi Z, Shalan N, Alshaer W, Hamadneh I. Formation and evaluation of doxorubicin and cromoglycate metal-organic framework for anti-cancer activity. Nanomedicine (Lond). 2025 Mar;20(5):467-79. doi: 10.1080/17435889.2025.2459059, PMID 39888613, PMCID PMC11875491.

Nsairat H, Alshaer W, Lafi Z, Ahmad S, Al Sanabrah A, El Tanani M. Development and validation of reversed-phase HPLC method for simultaneous quantification of fulvestrant and disulfiram in liposomes. Bioanalysis. 2023 Dec;15(23):1393-405. doi: 10.4155/bio-2023-0137, PMID 37847056.

Shukla SK, Kulkarni NS, Chan A, Parvathaneni V, Farrales P, Muth A. Metformin encapsulated liposome delivery system: an effective treatment approach against breast cancer. Pharmaceutics. 2019 Oct 28;11(11):559. doi: 10.3390/pharmaceutics11110559, PMID 31661947, PMCID PMC6920889.

Vozgirdaite D, Herve Aubert K, Uzbekov R, Chourpa I, Allard Vannier E. Design optimization characterization and in vitro evaluation of metformin loaded liposomes for triple-negative breast cancer treatment. J Liposome Res. 2024 Dec;34(4):547-61. doi: 10.1080/08982104.2024.2321528, PMID 38459750.

Nounou MM, El Khordagui LK, Khalafallah NA, Khalil SA. In vitro release of hydrophilic and hydrophobic drugs from liposomal dispersions and gels. Acta Pharm. 2006 Sep;56(3):311-24. PMID 19831280.

Biltonen RL, Lichtenberg D. The use of differential scanning calorimetry as a tool to characterize liposome preparations. Chem Phys Lipids. 1993;64(1-3):129-42. doi: 10.1016/0009-3084(93)90062-8.

Divakar P, Kumar D, Praveen C, Sowmya C, Reddy CS. Formulation and in vitro evaluation of liposomes containing metformin hydrochloride. Int J Res Pharm Biomed Sci. 2013;4(2):479-85.

Chen YH, Yang SF, Yang CK, Tsai HD, Chen TH, Chou MC. Metformin induces apoptosis and inhibits migration by activating the AMPK/p53 axis and suppressing PI3K/AKT signaling in human cervical cancer cells. Mol Med Rep. 2021 Jan;23(1):88. doi: 10.3892/mmr.2020.11725, PMID 33236135, PMCID PMC7716426.

Chen D, Liu X, LU X, Tian J. Nanoparticle drug delivery systems for synergistic delivery of tumor therapy. Front Pharmacol. 2023 Feb 16;14:1111991. doi: 10.3389/fphar.2023.1111991, PMID 36874010, PMCID PMC9978018.

Rajendran V, Jain MV. In vitro tumorigenic assay: colony-forming assay for cancer stem cells. Methods Mol Biol. 2018;1692:89-95. doi: 10.1007/978-1-4939-7401-6_8, PMID 28986889.

Al Kabariti AY, Arafat BT, Oriquat GA, Mozna P, Jaidy H, Rehmani A. In vitro and in vivo evaluation of dark chocolate as an age-appropriate oral matrix. Eur J Pharm Sci. 2024 Jan 1;192:106646. doi: 10.1016/j.ejps.2023.106646, PMID 37989467.

Al Daghistani HI, Zein S, Abbas MA. Microbial communities in the dead sea and their potential biotechnological applications. Commun Integr Biol. 2024 Jun 23;17(1):2369782. doi: 10.1080/19420889.2024.2369782, PMID 38919836, PMCID PMC11197920.