1. IntroductionPancreatic ductal adenocarcinoma (PDAC) is the most invasive form of pancreatic cancer. It is currently the fourth most common cancer that results in death, and, by 2030, it is predicted to overtake lung and colon cancer as the second most lethal disease in the United States. In 2021, there were 48,220 deaths from pancreatic cancer, with 60,430 new cases reported by the American Cancer Society [1]. PDAC is not responsive to standard chemotherapy, surgery, targeted therapy, or immunotherapy; these treatments only provide modest-to-marginal additional improvements [2]. The current treatment strategies include albumin-bound paclitaxel (Abraxane), gemcitabine, 5-fluorouracil, irinotecan, single-agent therapy, and combinations of chemotherapy agents such as folfirinox [3,4].The first-line treatment for pancreatic cancer is still gemcitabine (Gem), a DNA synthesis inhibitor. Even though therapeutic applications of Gem have demonstrated significant value, over time, its efficacy declines. This is mostly due to its short half-life, poor bioavailability, inadequate cellular absorption, and rapid degradation [5]. Due of its hydrophilicity, Gem needs transporter proteins to permeate across the cell membrane. There is proof that reduced cellular absorption caused by low nucleoside transporter proteins predicts the effectiveness of Gem [6]. Due to its strong affinity and efficiency to transport Gem, hENT1 is regarded as the major Gem carrier among the equilibrative nucleoside transporters [7]. In pancreatic ductal adenocarcinoma, the low expression of the human equilibrative nucleoside transporter-1 (hENT1) may lead to Gem resistance (PDAC) [8,9]. For overcoming this issue, the researcher developed Gemcitabine Elaidate (Gem Elaidate), which is chemically synthesized by covalently linking Gem and elaidic acid. The developed Gem Elaidate does not require hENT1 and can cross the biological membrane independently [9,10].The Protein kinase C (PKC) family contains numerous isozymes with different roles in pancreatic cancer signaling pathways and commonly comprises various PKCs such as PKC-α, βI, βII and γ. Amongst these, PKCβ expression was observed to be substantially elevated in pancreatic ductal carcinoma [11]. The overexpression of PKC is directly correlated with the tumorigenicity of pancreatic cancer in vivo. PKC has been demonstrated to increase pancreatic cancer cell line proliferation and metastasis by downregulating PTEN and it has a role in the development of drug resistance in pancreatic cancer [12]. Most importantly, PKC triggers human endothelial cells’ angiogenic activity and produces VEGF, which in turn prompts it to release consistently through an autocrine positive feedback loop. Therefore, combining PKC inhibitor with an anticancer agent could be a new therapeutic strategy to deal with pancreatic tumors [13,14]. For improved cellular absorption and increased anticancer activity in PDAC, we employed Palmitoyl-DL-carnitine chloride (PC) as a Protein kinase C (PKC) inhibitor in the current investigation [15].A reimagining is required for effectively delivering Gem Elaidate, which can be fulfilled with the help of the nanomedicine platform that helps to overcome the issues related to conventional drug formulations. Gem Elaidate, a lipophilic derivative, has very low water solubility. Therefore, formulating an oral formulation is problematic [16]. Due to their inadequate tumor site targeting, traditional chemotherapeutics were always associated with serious side effects in cancer treatment. PEGylated stealth liposomes are frequently employed as a nanocarrier to deliver anticancer medications because they evade the immune system and reticuloendothelial system (RES) detection and extend blood circulation [17,18,19]. Furthermore, nanoliposomes tend to aggregate in tumor tissue because of EPR-mediated passive tumor targeting, thus improving the efficacy of drug delivery [20]. Considering the above, liposomes are the appropriate choice to co-deliver Gem Elaidate and PC. The tumor-specific delivery of anticancer drugs is greatly influenced by the surface properties of liposomes. Cationic liposomes have been shown in prior research to accumulate more in tumor vasculature, improving the intratumoral delivery of chemotherapeutic agents [17,18]. Additionally, lactate production from anaerobic glycolysis is the cause of the acidic pH of solid tumors and an anionic charge is due to the presence of flipped phosphatidylserine and interstitial hyaluronic acid. Therefore, the cationic charge provided by PC to nanoliposomes and the anionic charge of vascular endothelial cells interact electrostatically, thereby improving cell penetration [21]. The purpose of this research is to develop PKC inhibitor-anchored Gem Elaidate-loaded PEGylated nanoliposomes (PGPLs) and to investigate their anticancer activity in 2D and 3D models of pancreatic cancer. 4. DiscussionIn current clinical practice, Gem is an FDA-approved chemotherapeutic agent for treating pancreatic, breast, lung, and ovarian cancer alone or in combination therapy. Despite all the advancements in Gem research, it is not therapeutically efficient due to its poor pharmacokinetics profile [33]. Gem is rapidly metabolized when administered systemically; consequently, frequent dosing is required to achieve the therapeutic benefit, leading to adverse effects. Along with that, Gem encounters several sequential obstacles, hindering the use of Gem as a hydrophilic molecule, with low bioavailability, poor cellular uptake, and equilibrative nucleoside transporter-dependent entry [34]. We portrayed the anticancer efficacy of Gem Elaidate in a pancreatic cancer cell line, which is synthesized using lipophilic Gem by covalently linking with the elaidic acid to overcome the obstacles of Gem.Nano-liposomal formulations enable the incorporation of chemotherapeutic drugs with distinct chemical characteristics, increasing anti-tumoral potency, while limiting adverse effects on normal tissues. Conventional liposomes have poor stability and are cleared rapidly by the spleen and liver. Furthermore, transforming liposomes to stealth liposomes (PEGylated Liposomes) using DSPE-PEG2000 not only avoids the reticuloendothelial system (RES) uptake but also prolongs circulation time, improving the stability of the formulated liposomes [35]. For instance, PEGylation of the doxorubicin liposome improved its half-life from minutes to hours. As per Han et al., the encapsulation of paclitaxel in long-circulating liposomes prolongs its biological half-life, leading to improved therapeutic efficacy in vivo [36,37]. In addition, in this manuscript, we investigated Palmitoyl-DL-carnitine chloride (PC) as a Protein kinase C (PKC) inhibitor, which also provides a cationic surface charge to nanoliposomes that increases the cellular uptake and enhances the anticancer potency.PGPLs were successfully formulated using the modified hydration method to yield a particle size of 81.78 ± 2.31 nm and a PDI less than 0.2. Alavi et al. mentioned that, with the small size of liposomes (400 nm), passive targeting can be achieved by enhanced permeation and retention (EPR), which can help accumulate the drug around the tumor tissue, while minimizing damage to healthy surrounding tissues [38]. Protein kinase C inhibitor–palmitoyl carnitine (PC) is lipophilic in nature, having a lipophilic palmitoyl chain within the lipid bilayer and carnitine head on the outer surface of the liposome, which is responsible for providing a cationic charge, while the tumor vasculatures are negatively charged because of the presence of phosphatidylserine on the surface of the cancerous blood vessels; therefore, cationic liposomes (PGPLs) would preferentially bind there and facilitate its permeation into cells. The zeta potential of PGPLs confirmed the cationic charge imparted by PC. A similar liposomal batch was prepared in the absence of PC. It was found that the particle size of PGPLs without PC is more than 130 nm and the PDI was more than 0.30, while the zeta potential was measured as −46.4 ± 12.8. A previous report posits that Protein kinase C inhibitor–palmitoyl carnitine (PC) in the lipid bilayer results in the reduction in particle size while improving the poly dispersity index of nanocarriers, which is reflected in our results. The stability study of PGPLs showed no significant change in the size, PDI and zeta potential of the prepared liposomes for two months at 4 °C storage.The results of the in vitro cytotoxicity study of PGPLs in two human pancreatic cancer cells (MIA PaCa-2 and BxPC-3) were promising. In comparison to Gem and PC, PGPLs and Gem Elaidate had lower IC50 values in the cytotoxicity assay, indicating that Gem Elaidate is the cause of the cytotoxicity in PGPLs. Fu et al. found similar findings when they calculated the IC50 values for PC in Vemurafenib-resistant melanoma cell lines (A375R and SK-MEL-28 R) as 23 μM and 26 μM [15]. The IC50 of palmitoyl carnitine in the hepatic cancer cell line was reported by Sonja et al. to be 76 μM, indicating that PC alone lacks the potency to be a cytotoxic agent but can be used as one at greater concentrations [39].Being a hydrophilic molecule, Gem required a transporter identified as human equilibrative nucleoside transporter-1 (hENT1) for the efficient entry into cells. On the other hand, Gem Elaidate enters independently into pancreatic cancer cells. Our quantitative cellular uptake study is in agreement to demonstrate the lower uptake of Gem compared to Gem Elaidate alone and PGPLs. In contrast, Gem Elaidate alone shows a higher uptake compared to PGPLs, which could be aided by the presence of DMSO. Thus, during 2D cell culture assays, it is possible that the drug–DMSO solution showed a higher uptake than liposomes. During in vivo studies, nanoliposomes show a much higher tumor uptake due to the biodistribution and EPR effect [40]. Our present studies give an idea regarding the quantitative cellular uptake of Gem Elaidate and PGPLs in tumor cells compared Gem.The Food and Drug Administration (FDA) suggests performing an in vitro hemolysis test for the formulations that need to be administered intravenously. As per Mourtas et al., the study results indicate that conventional lipids such as DOPC, cholesterol, and DSPE-PEG-2000 are safe to use in humans after the intravenous administration of the liposomal formulation. The standardized test method also specifies that, for the product to be safe, the percent hemolysis should not be more than 5% [41,42]. Indeed, our results depict that PGPLs, when analyzed at a Gem Elaidate concentration range of 5–20 µM, show minimum disruption of RBCs (The Extracellular Matrix (ECM) proteins of pancreatic cancer play a crucial role in tumorigenesis. The effect of a different treatment group on cancer cell migration and metastasis was evaluated using a two-dimensional (2D) surface migration assay [43]. The in vitro bridging of MIA PaCa-2 cells in the presence of Gem Elaidate (68% bridging of migration area) was observed at 100 nm, which explains its role in the inhibition of metastasis for the treatment of pancreatic cancer. Moreover, the PGPL treatment group resulted in a 46% reduction in the percentage bridging migration potential of cells compared to control cells, while only an 80% reduction in migration capability was seen when treated with Gem alone. Furthermore, the surrounding cells in the wells were not affected, with the treatment group further verifying that the reduction in the bridging area is not due to the cytotoxic effects of the drug but it is due to the inhibition of cell migration. Further, the clonogenic assay was performed to determine whether there are metastatic-resistant pancreatic progenitors to quantify their ability to proliferate and differentiate into colonies in a six-well plate. Firstly, the capacity of pancreatic cells to develop colonies was determined, and then the effect of Gem Elaidate, Gem, and PGPL treatments on the clonogenicity of MIA PaCa-2 cells was investigated. The results demonstrate that Gem Elaidate counteracts the growth and metastasis of the aggressive MIA PaCa-2 cells at a non-cytotoxic concentration (50 nM). Moreover, PGPLs significantly inhibited the formation of a number of colonies and led to a reduction in the colony area compared to Gem Elaidate. Furthermore, the percent colony formation efficiency of PGPLs reduced 3.2-fold compared to Gem Elaidate and 4.9-fold compared to Gem-treated cells. Hence, the results obtained from in vitro cytotoxicity, migration, and clonogenic assays illustrate that Gem Elaidate effectively inhibits the proliferation, migration, and metastasis of MIA PaCa-2 pancreatic cancer cells, and formulated nanoliposomes (PGPLs) further enhanced its anticancer capability. The expression of PKCα, PKCβ1, and PKCδ is elevated in PDAC, whereas that of PKCε is elevated in normal tissue. Moreover, PKCα is believed to be one of the biomarkers for the diagnosis of pancreatic cancers. On the overexpression of PKCα, the survival rate is decreased because it is directly related to the cell proliferation of pancreatic cancer cells. It was reported that PKC is essential for vasculogenic tube formation, so the inhibition of PKC can help in pancreatic cancer cell invasion [44,45]. Anti-pancreatic cancer vasculogenic mimicry agents are inadequate, so Palmitoyl-DL-carnitine chloride (PC) was explored as a Protein kinase C (PKC) inhibitor in the MIA PaCa-2 pancreatic cancer cell line. Using PC, the inhibition of vasculogenic mimicry channel formation was previously seen in melanoma cancer cell lines (A375 and A375R) using a PKC inhibitor [15,46]. Similar results were obtained for the PGPL treatment group in MIA PaCa-2 pancreatic cancer cells. From our western blot results, we found that Gem Elaidate significantly reduced the levels of anti-apoptotic protein Bcl-2, while increasing the level of apoptotic marker-cleaved caspase-3 and P53. Our findings are consistent with those of Patki et al., who claimed that Gem induces apoptosis in MIA PaCa-2 pancreatic cell lines regulating members of the Bcl-2, which is an anti-apoptotic protein [27]. According to our western blot data, the expression of cleaved caspase-3 was also found to be higher in cells treated with PGPLs than in those treated only with Gem Elaidate.

The development of 3D culture models has proven to be a valuable and versatile tool for anticancer drug screening, but also for gaining mechanistic insight into the regulation of cancer cell death and viability under conditions imitating those in the tumor microenvironment. Cell proliferation has a pivotal effect for anticancer drugs, which seems to reduce in 3D compared to 2D assay conditions; therefore, it is important to check the activity in 3D tumor spheroids rather than 2D cell culture. In the Gem Elaidate treatment group, the spheroid area reduced, indicative of cell death, until the fourth day. The growth as sustained until the tenth day of treatment compared to the control group, which showed continual growth. On the contrary, Gem, a polar molecule, has restricted uptake into the cancer cell spheroids. This, therefore, is a reason to explore a lipophilic form of Gem and entrapping Gem Elaidate in nanoliposomes that would most likely improve the anticancer activity of the Gem by protecting it against rapid metabolism and improving its cellular uptake. Furthermore, PGPL treatment reduced the spheroid area in a sustainable manner until day 10 of treatment. Using a Live/Dead Cell Assay Kit and fluorescence microscopy, the cell viability within 3D multicellular tumor spheroids was further examined. Based on the intense red fluorescence of the ethidium homodimer-1 stain compared to the green fluorescence of the calcein acetoxymethyl (AM) stain produced by the control group spheroids, our findings reveal that Gem Elaidate and PGPLs significantly increased apoptosis in the spheroids.