Development of novel pyrimidine nucleoside analogs as potential anticancer agents: Synthesis, characterization, and In-vitro evaluation against pancreatic cancer.

The present study proposed modification of 5-FU by conjugation with an acyl chloride and a 5-membered heterocyclic ring to improve its in-vitro cytotoxicity and metabolic stability. XYZ-I-71 and XYZ-I-73 were synthesized by introducing a tetrahydrofuran ring on 5-fluorocytosine (a precursor of 5-FU) and conjugation with octanoyl chloride and lauroyl chloride, respectively. The structure of the synthesized compounds was validated using NMR and micro-elemental analysis. The antiproliferative activity of the analogs was determined against MiaPaCa-2, PANC-1, and BxPC-3 pancreatic cancer cells. The analog's stability in human liver microsomes was quantified by HPLC. We found that the XYZ-I-73 (IC50 3.6 ± 0.4 µM) analog was most effective against MiaPaCa-2 cells compared to XYZ-I-71(IC50 12.3 ± 1.7 µM), GemHCl (IC50 24.2 ± 1.3 µM), Irinotecan (IC50 10.1 ± 1.5 µM) and 5-FU (IC50 13.2 ± 1.1 µM). The antiproliferative effects of this analog in Miapaca-2 cells is evident based on it having a 7-fold,3-fold, and 4-fold increased cytotoxic effect over Gem-HCl, Irinotecan, and 5-FU, respectively. On the other hand, XYZ-I-71 exhibited a 2-fold increased cytotoxic effect over Gem-HCl but a comparable cytotoxic effect to 5-FU and Irinotecan in MiaPaCa-2 cells. A similar trend of higher XYZ-I-73 inhibition was observed in PANC-1 and BxPC-3 cultures. For 48-h MiaPaCa-2 cell migration studies, XYZ-I-73 (5 µM) significantly reduced migration (# of migrated cells, 168 ± 2.9), followed by XYZ-I-71(315±2.1), Gem-HCl (762±3.1) and 5-FU (710 ± 3.2). PARP absorbance studies demonstrated significant inhibition of PARP expression of XYZ-I-73 treated cells compared to 5-FU, GemHCl, and XYZ-I-71. Further, BAX and p53 expressions were significantly increased in cells treated with XYZ-I-73 compared to 5-FU, GemHCl, and XYZ-I-71. In-vitro, metabolic stability studies showed that 80 ± 5.9% of XYZ-I-71 and XYZ-I-73 remained intact after 2 h exposure in liver microsomal solution compared to 5-FU. The XYZ-I-73 analog demonstrated a remarkable cytotoxic effect and improved in-vitro metabolic stability over the selected standard drugs and may have potential anticancer activity against pancreatic cancer.

Florida-California Cancer Research, Education and Engagement (CaRE2) Health Equity Center: Structure, Innovations, and Initial Outcomes.

INTRODUCTION: The Florida-California Cancer Research, Education, and Engagement (CaRE2) Health Equity Center is a triad partnership committed to increasing institutional capacity for cancer disparity research, the diversity of the cancer workforce, and community empowerment. This article provides an overview of the structure, process innovations, and initial outcomes from the first 4 years of the CaRE2 triad partnership. METHODS: CaRE2 serves diverse populations in Florida and California using a "molecule to the community and back" model. We prioritize research on the complex intersection of biological, environmental, and social determinants health, working together with scientific and health disparities communities, sharing expertise across institutions, bidirectional training, and community outreach. Partnership progress and outcomes were assessed using mixed methods and four Program Steering Committee meetings. RESULTS: Research capacity was increased through development of a Living Repository of 81 cancer model systems from minority patients for novel cancer drug development. CaRE2 funded 15 scientific projects resulting in 38 publications. Workforce diversity entailed supporting 94 cancer trainees (92 URM) and 34 ESIs (32 URM) who coauthored 313 CaRE2-related publications and received 48 grants. Community empowerment was promoted via outreaching to more than 3000 individuals, training 145 community cancer advocates (including 28 Community Scientist Advocates), and publishing 10 community reports. CaRE2 members and trainees together have published 639 articles, received 61 grants, and 57 awards. CONCLUSION: The CaRE2 partnership has achieved its initial aims. Infrastructure for translational cancer research was expanded at one partner institution, and cancer disparities research was expanded at the two cancer centers.

Biological evaluation of novel gemcitabine analog in patient-derived xenograft models of pancreatic cancer.

Gemcitabine (Gem) has been a standard first-line drug for pancreatic cancer (PCa) treatment; however, Gem's rapid metabolism and systemic instability (short half-life) limit its clinical outcome. The objective of this study was to modify Gem into a more stable form called 4-(N)-stearoyl-gemcitabine (4NSG) and evaluate its therapeutic efficacy in patient-derived xenograft (PDX) models from PCa of Black and White patients.Methods 4NSG was synthesized and characterized using nuclear magnetic resonance (NMR), elemental analysis, and high-performance liquid chromatography (HPLC). 4NSG-loaded solid lipid nanoparticles (4NSG-SLN) were developed using the cold homogenization technique and characterized. Patient-derived pancreatic cancer cell lines labeled Black (PPCL-192, PPCL-135) and White (PPCL-46, PPCL-68) were used to assess the in vitro anticancer activity of 4NSG-SLN. Pharmacokinetics (PK) and tumor efficacy studies were conducted using PDX mouse models bearing tumors from Black and White PCa patients.Results 4NSG was significantly stable in liver microsomal solution. The effective mean particle size (hydrodynamic diameter) of 4NSG-SLN was 82 ± 6.7 nm, and the half maximal inhibitory concentration (IC50) values of 4NSG-SLN treated PPCL-192 cells (9 ± 1.1 µM); PPCL-135 (11 ± 1.3 µM); PPCL-46 (12 ± 2.1) and PPCL-68 equaled to 22 ± 2.6 were found to be significantly lower compared to Gem treated PPCL-192 (57 ± 1.5 µM); PPCL-135 (56 ± 1.5 µM); PPCL-46 (56 ± 1.8 µM) and PPCL-68 (57 ± 2.4 µM) cells. The area under the curve (AUC), half-life, and pharmacokinetic clearance parameters for 4NSG-SLN were 3-fourfold higher than that of GemHCl. For in-vivo studies, 4NSG-SLN exhibited a two-fold decrease in tumor growth compared with GemHCl in PDX mice bearing Black and White PCa tumors.Conclusion 4NSG-SLN significantly improved the Gem's pharmacokinetic profile, enhanced Gem's systemic stability increased its antitumor efficacy in PCa PDX mice bearing Black and White patient tumors.

Evaluation of Anticancer Activity of Zhubech, a New 5-FU Analog Liposomal Formulation, against Pancreatic Cancer.

Pancreatic cancer is projected to be the second leading cause of cancer-related death by 2030 in the US. The benefits of the most common systemic therapy for various pancreatic cancers have been masked by high drug toxicities, adverse reactions, and resistance. The use of nanocarriers such as liposomes to overcome these unwanted effects has become very popular. This study aims to formulate 1,3-bistertrahydrofuran-2yl-5FU (MFU)-loaded liposomal nanoparticles (Zhubech) and to evaluate itsstability, release kinetics, in vitro and in vivo anticancer activities, and biodistribution in different tissues. Particle size and zeta potential were determined using a particle size analyzer, while cellular uptake of rhodamine-entrapped liposomal nanoparticles (Rho-LnPs) was determined by confocal microscopy. Gadolinium hexanoate (Gd-Hex) was synthesized and entrapped into the liposomal nanoparticle (LnP) (Gd-Hex-LnP), as a model contrast agent, to evaluate gadolinium biodistribution and accumulation by LnPs in vivo using inductively coupled plasma mass spectrometry (ICP-MS). The mean hydrodynamic diameters of blank LnPs and Zhubech were 90.0 ± 0.65 nm and 124.9 ± 3.2 nm, respectively. The hydrodynamic diameter of Zhubech was found to be highly stable at 4 °C and 25 °C for 30 days in solution. In vitro drug release of MFU from Zhubech formulation exhibited the Higuchi model (R2 value = 0.95). Both Miapaca-2 and Panc-1 treated with Zhubech showed reduced viability, two- or four-fold lower than that of MFU-treated cells in 3D spheroid (IC50Zhubech = 3.4 ± 1.0 µM vs. IC50MFU = 6.8 ± 1.1 µM) and organoid (IC50Zhubech = 9.8 ± 1.4 µM vs. IC50MFU = 42.3 ± 1.0 µM) culture models. Confocal imaging confirmed a high uptake of rhodamine-entrapped LnP by Panc-1 cells in a time-dependent manner. Tumor-efficacy studies in a PDX bearing mouse model revealed a more than 9-fold decrease in mean tumor volumes in Zhubech-treated (108 ± 13.5 mm3) compared to 5-FU-treated (1107 ± 116.2 mm3) animals, respectively. This study demonstrates that Zhubech may be a potential candidate for delivering drugs for pancreatic cancer treatment.

Review of genetic and pharmacogenetic differences in cytotoxic and targeted therapies for pancreatic cancer in African Americans.

Pancreatic ductal adenocarcinoma (PDAC) is currently the third leading cause of cancer mortality and the incidence is projected to increase by 2030. Despite recent advances in its treatment, African Americans have a 50-60% higher incidence and 30% higher mortality rate when compared to European Americans possibly resulting from differences in socioeconomic status, access to healthcare, and genetics. Genetics plays a role in cancer predisposition, response to cancer therapeutics (pharmacogenetics), and in tumor behavior, making some genes targets for oncologic therapeutics. We hypothesize that the germline genetic differences in predisposition, drug response, and targeted therapies also impact PDAC disparities. To demonstrate the impact of genetics and pharmacogenetics on PDAC disparities, a review of the literature was performed using PubMed with variations of the following keywords: pharmacogenetics, pancreatic cancer, race, ethnicity, African, Black, toxicity, and the FDA-approved drug names: Fluoropyrimidines, Topoisomerase inhibitors, Gemcitabine, Nab-Paclitaxel, Platinum agents, Pembrolizumab, PARP-inhibitors, and NTRK fusion inhibitors. Our findings suggest that the genetic profiles of African Americans may contribute to disparities related to FDA approved chemotherapeutic response for patients with PDAC. We recommend a strong focus on improving genetic testing and participation in biobank sample donations for African Americans. In this way, we can improve our current understanding of genes that influence drug response for patients with PDAC.

Synthesis, characterization, and anticancer evaluation of 1,3-bistetrahydrofuran-2yl-5-FU as a potential agent for pancreatic cancer.

The failure of current chemotherapeutic agents for pancreatic cancer (PCa) makes it the most aggressive soft tissue tumor with a 5-year survival of slightly above 10% and is estimated to be the second leading cause of cancer death by 2030. OBJECTIVE: The main aim was to synthesize, characterize and evaluate the anticancer activity of 1,3-bistetrahydrofuran-2yl-5FU (MFU). METHODS: MFU was synthesized by using 5-fluorouracil (5-FU) and tetrahydrofuran acetate, and characterized by nuclear magnetic resonance (NMR), micro-elemental analysis, high-performance liquid chromatography (HPLC), and liquid chromatography with mass spectrophotometry (LC-MS). MFU and Gemcitabine hydrochloride (GemHCl) were tested for antiproliferative activity against MiaPaca-2 and Panc-1 cell lines. RESULTS: The half-minimum inhibitory concentration (IC50) of MFU was twice lower than that of GemHCl when used in both cell lines. MiaPaca-2 cells (MFU-IC50 = 4.5 ± 1.2 µM vs. GemHCl-IC50 = 10.3 ± 1.1 µM); meanwhile similar trend was observed in Panc-1 cells (MFU-IC50 = 3.0 ± 1 µM vs. GemHCl-IC50 = 6.1 ± 1.03 µM). The MFU and GemHCl effects on 3D spheroids showed a similar trend (IC50-GemHCl = 14.3 ± 1.1 µM vs. IC50-MFU = 7.2 ± 1.1 µM) for MiaPaca-2 cells, and (IC50-GemHCl = 16.3 ± 1.1 µM vs. IC50-MFU = 9.2 ± 1.1 µM) for Panc-1 cells. MFU significantly inhibited clonogenic cell growth, and induced cell death via apoptosis. Cell cycle data showed mean PI for GemHCl (48.5-55.7) twice higher than MFU (24.7 to 27.9) for MiaPaca-2 cells, and similarly to Panc-1 cells. The in-vivo model showed intensely stained EGFR (stained brown) in all control, GemHCl and MFU-treated mice bearing subcutaneous PDX tumors, however, HER2 expression was less stained in MFU-treated tumors compared to GemHCl-treated tumors and controls. Mean tumor volume of MFU-treated mice (361 ± 33.5 mm3) was three-fold lower than GemHCl-treated mice (1074 ± 181.2 mm3) bearing pancreatic PDX tumors. CONCLUSION: MFU was synthesized with high purity and may have potential anticancer activity against PCa.

Multi-disciplinary Approach for Drug and Gene Delivery Systems to the Brain.

Drug delivery into the brain has for long been a huge challenge as the blood-brain barrier (BBB) offers great resistance to entry of foreign substances (with drugs inclusive) into the brain. This barrier in healthy individuals is protective to the brain, disallowing noxious substances present in the blood to get to the brain while allowing for the exchange of small molecules into the brain by diffusion. However, BBB is disrupted under certain disease conditions, such as cerebrovascular diseases including acute ischemic stroke and intracerebral hemorrhage, and neurodegenerative disorders including multiple sclerosis (MS), Alzheimer's disease (AD), Parkinson's disease (PD), and cancers. This review aims to provide a broad overview of present-day strategies for brain drug delivery, emphasizing novel delivery systems. Hopefully, this review would inspire scientists and researchers in the field of drug delivery across BBB to uncover new techniques and strategies to optimize drug delivery to the brain. Considering the anatomy, physiology, and pathophysiological functioning of the BBB in health and disease conditions, this review is focused on the controversies drawn from conclusions of recently published studies on issues such as the penetrability of nanoparticles into the brain, and whether active targeted drug delivery into the brain could be achieved with the use of nanoparticles. We also extended the review to cover novel non-nanoparticle strategies such as using viral and peptide vectors and other non-invasive techniques to enhance brain uptake of drugs.

Enhancing efficacy of gemcitabine in pancreatic patient-derived xenograft mouse models.

Gemcitabine (Gem), a nucleoside analog, is a preferred choice of treatment for pancreatic cancer (PCa) and often used in combination therapy against wide range of solid tumors. It is known to be rapidly inactivated in blood by cytidine deaminase. The objective of the study was to improve the systemic stability and anticancer activity of modified Gem termed 4-N-stearoylGem (4NSG) In this study, the IC50 values of 4NSG treated MiaPaCa-2 and primary pancreatic cancer (PPCL-46) cultures were significantly lower when compared with gemcitabine hydrochloride (GemHCl) treated cultures. In acute toxicity study, liver enzyme level of aspartate aminotransferase (AST) of the control mice was not significantly different from AST levels of 4NSG and GemHCl treated mice. However, alanine aminotransferase (ALT) level of control mice (67 ± 5 mUnits/mL) was significantly lower compared with ALT levels of GemHCl (232 ± 28 mUnits/mL) and that of 4NSG (172 ± 22 mUnits/mL) (p < 0.0001). More importantly, ALT level of 4NSG was lower than ALT level of GemHCl (p < 0.05). Although ALT levels were elevated, pathological images of liver and kidney tissues of control, GemHCl and 4NSG treated mice revealed no architectural changes and no significant change in mice weight was observed during treatment. The bioavailability (AUC) of 4NSG was 3-fold high and significantly inhibited the tumor growth as compared with equivalent dose of GemHCl. Immunohistochemical staining revealed that 4NSG significantly inhibited the expression vascular endothelial growth factor (VEGF) receptor. The study is unique because it established, for the first time, enhanced anticancer activity of 4NSG against pancreatic patient-derived xenograft (PDX) mouse model and PPCL-46 cells compared with Gem. 4SGN enhanced pharmacokinetic profile and improved the therapeutic efficacy of the standard-of-care Gem. Lastly, 4GSN showed a remarkable tumor growth inhibition and revealed significant antiangiogenic activity in 4GSN treated pancreatic PDX tumor.

Application of smart solid lipid nanoparticles to enhance the efficacy of 5-fluorouracil in the treatment of colorectal cancer.

5-Fluorouracil (5-FU) is a standard treatment option for colorectal cancer (CRC) but its rapid metabolism and systemic instability (short half-life) has hindered its therapeutic efficacy. The objective of this study was to develop a novel drug delivery system, solid lipid nanoparticle (SLN), capable of delivering high payload of 5-FU to treat CRC. The rational was to improve 5FU-nanocarrier compatibility and therapeutic efficacy. The SLN-loaded 5-FU was developed by utilizing a Strategic and unique Method to Advance and Refine the Treatment (SMART) of CRC through hot and cold homogenization approach. The SLN was made of unique PEGylated lipids and combination of the surfactants. Cytotoxicity studies, clonogenic assay, flow cytometry and confocal imaging were conducted to evaluate the effectiveness and cellular uptake of 5FU-SLN4 in HCT-116 cancer cells. Pharmacokinetic (PK) parameters and receptor expressions were determined while tumor efficacy studies were conducted on mouse bearing subcutaneous HCT-116 cancer. Among the all the formulations, 5FU-SLN4 was the most effective with particle size of was 263 ± 3 nm, zeta potential was 0.1 ± 0.02 and entrapment efficiency of 81 ± 10%. The IC50 value of 5FU-SLN4 (7.4 ± 0.02 µM) was 2.3 fold low compared with 5-FU (17.7 ± 0.03 µM). For tumor efficacy studies, 5FU-SLN4 significantly inhibited tumor growth in comparison to 5-FU while area-under plasma concentration-time curve (AUC) of 5FU-SLN4 was 3.6 fold high compared with 5-FU. HER2 receptors expression were markedly reduced in 5-FU-SLN4 treated mice compared with 5FU and liver and kidney tissues showed no toxicity at dose of 20 mg/kg. 5FU-SLN4 was highly cytotoxic against HCT-116 cells and significantly inhibited subcutaneous tumor growth in mice compared with 5-FU. This emphasizes the significance of developing a smart nano-delivery system to optimize the delivery efficiency of anticancer drugs to tumors.

Comparative study on contrast enhancement of Magnevist and Magnevist-loaded nanoparticles in pancreatic cancer PDX model monitored by MRI.

BACKGROUND: The aim of this study was to compare contrast enhancement of Magnevist® (gadopentate dimeglumine (Mag)) to that of PEGylated Magnevist®-loaded liposomal nanoparticles (Mag-Lnps) in pancreatic cancer patient-derived xenograft (PDX) mouse model via magnetic resonance imaging (MRI). METHODS: Mag-Lnps formulated by thin-film hydration and extrusion was characterized for the particle size and zeta potential. A 21.1 T vertical magnet was used for all MRI. The magnet was equipped with a Bruker Advance console and ParaVision 6.1 acquisitions software. Mag-Lnps phantoms were prepared and imaged with a 10-mm birdcage coil. For in vivo imaging, animals were sedated and injected with a single dose (4 mg/kg) of Mag or Mag-Lnps with Mag equivalent dose. Using a 33-mm inner diameter birdcage coil, T 1 maps were acquired, and signal to noise ratio (SNR) measured for 2 h. RESULTS: Mag-Lnps phantoms showed a remarkable augmentation in contrast with Mag increment. However, in in vivo imaging, no significant difference in contrast was observed between Mag and MRI. While Mag-Lnps was observed to have fairly high tumor/muscle (T/M) ratio in the first 30 min, free Mag exhibited higher T/M ratio over the time-period between 30 and 120 min. Overall, there was no statistically significant difference between Mag and Mag-Lnp in rating MR image quality. Low payload of Mag entrapment by Lnps and restricted access of water (protons) to Mag-Lnps may have affected the performance of Mag-Lnps as an effective contrast agent. CONCLUSION: This study showed no significance difference in MRI contrast between Mag and Mag-Lnp pancreatic cancer PDX mouse models.

Cytotoxic effects of gemcitabine-loaded solid lipid nanoparticles in pancreatic cancer cells.

This study investigated the cytotoxic effects of gemcitabine-loaded solid lipid nanoparticle (Gem-SLN) on the patient-derived primary pancreatic cancer cell lines (PPCL-46) and MiaPaCa-2. Different SLN formulations were prepared from glyceryl monostearate (GMS), polysorbate 80 (Tween® 80) and poloxamer 188 (Pol 188) as surfactants using a cold homogenization method. Gem-SLN was characterized for particle size and charge distribution, entrapment efficiency and loading capacity. Fourier Transform Infra-Red (FTIR) spectroscopy was used to verify Gem and SLN interaction while differential scanning calorimetry (DSC) was used to acquire thermodynamic information on Gem-SLN. Cytotoxicity studies was conducted on PPCL-46 cells and Mia-PaCa-2 cells. Among the different Gem-SLN formulations prepared, Gem-SLN15 was selected based on entrapment efficiency (EE) of Gem, loading efficiency of Gem, cytotoxicity and rate of Gem release. Growth inhibition of Gem-SLN15-treated PPCL-46 culture (IC50 (2D) =27± 5 µM; IC50 (3D) = 66 ± 2 µM) was remarkably higher than gemcitabine hydrochloride (GemHCl)-treated PPCL-46 culture (IC50 (2D) =126±3 µM; IC50 (3D) =241±3 µM). Similar trend of higher Gem-SLN15 inhibition in MiaPaCa-2 culture was found (IC50 (2D) =56±16 µM; IC50 (3D) =127±4 µM) compared with GemHCl-treated Mia-PaCa-2 culture (IC50 (2D) =188±46 µM; IC50 (3D) =254±52 µM). The anticancer activity of Gem-SLN15 was significantly more effective than GemHCl in PPCL-46 compared to Mia-PaCa-2 cancer cells. Schematic diagram for preparation of Gem-SLN through cold homogenization and methods for characterization and in-vitro studies.

Pancreatic Cancer Related Health Disparities: A Commentary.

We summarize the risk factors that may significantly contribute to racial disparities in pancreatic cancer, which is now the third leading cause of cancer deaths and projected to be second around 2030 in 12 years. For decades, the incidence rate of pancreatic cancer among Blacks has been 30% to 70% higher than other racial groups in the United States and the 5-year survival rate is approximately 5%. Diabetes and obesity have been identified as potentially predisposing factors to pancreatic cancer and both are more common among Blacks. Smoking continues to be one of the most important risk factors for pancreatic cancer and smoking rates are higher among Blacks compared to other racial groups. The overall risk of pancreatic cancer due to changes in DNA is thought to be the same for most racial groups; however, DNA methylation levels have been observed to be significantly different between Blacks and Whites. This finding may underlie the racial disparities in pancreatic cancer. Identification and prevention of these factors may be effective strategies to reduce the high incidence and mortality rates for pancreatic cancer among Blacks.

Evaluation of in-vitro cytotoxic effect of 5-FU loaded-chitosan nanoparticles against spheroid models.

PURPOSE: The studies investigate the anticancer activity of 5-fluorouracil (5-FU)-hyaluronidase (Hase) enzyme-loaded chitosan nanoparticles (5-FUChnps) using three-dimensional (3D) spheroid HCT-116 culture. Hase-loaded nanoparticles (Chnps) have recently been used to improve the efficacy of chemotherapeutic drugs for cancer treatment. It has been found that administration of Hase improves tumor vessel densities and increase perfusion within tumor. METHODS: Particle size and zeta potential of the nanoparticles were determined using a particle size analyzer while Fourier transform infrared (FTIR) was used to investigate the interactions among the various components making up the chitosan nanoparticles. Cytotoxic effects of 5-FU and 5FUchnps against dimensional (2D) and 3D spheroid cultures were assessed using trypan blue assay. RESULTS: Low molecular weight chitosan (ChiL) nanoparticle size was determined to between 215 to 670 nm while medium molecular weight chitosan (ChiM) nanoparticle size ranged from 151 to 778 nm. All 5FUChnps exhibited a zeta potential value ranging from +4 to +37 mV. Among the 16 formulations prepared, formulation #7 (5-FUChnps7) was selected for the in-vitro cytotoxic studies based on its high 5-FU entrapment efficiency (59%). 5FUchnps treated 3D HCT-116 culture exhibited significant growth inhibition compared with 5-FU treated groups. Further, spheroids with significant growth inhibition exhibited high spheroid volume and non-spherical shapes. CONCLUSION: 5-FUChnps were significantly cytotoxic to the 3D HCT-116 cultures than that of the free 5-FU. Chnps proved to have the ability to deliver and improve the anticancer activity of 5-FU in 3D HCT-116 culture studies.

Smart thermosensitive liposomes for effective solid tumor therapy and in vivo imaging.

In numerous studies, liposomes have been used to deliver anticancer drugs such as doxorubicin to local heat-triggered tumor. Here, we investigate: (i) the ability of thermosensitive liposomal nanoparticle (TSLnp) as a delivery system to deliver poorly membrane-permeable anticancer drug, gemcitabine (Gem) to solid pancreatic tumor with the aid of local mild hyperthermia and, (ii) the possibility of using gadolinium (Magnevist®) loaded-TSLnps (Gd-TSLnps) to increase magnetic resonance imaging (MRI) contrast in solid tumor. In this study, we developed and tested gemcitabine-loaded thermosensitive liposomal nanoparticles (Gem-TSLnps) and gadolinium-loaded thermosensitive liposomal nanoparticles (Gd-TSLnps) both in in-vitro and in-vivo. The TSLnps exhibited temperature-dependent release of Gem, at 40-42°C, 65% of Gem was released within 10 min, whereas < 23% Gem leakage occurred at 37°C after a period of 2 h. The pharmacokinetic parameters and tissue distribution of both Gem-TSLnps and Gd-TSLnps were significantly greater compared with free Gem and Gd, while Gem-TSLnps plasma clearance was reduced by 17-fold and that of Gd-TSLpns was decreased by 2-fold. Area under the plasma concentration time curve (AUC) of Gem-TSLnps (35.17± 0.04 µghr/mL) was significantly higher than that of free Gem (2.09 ± 0.01 µghr/mL) whereas, AUC of Gd-TSLnps was higher than free Gd by 3.9 fold high. TSLnps showed significant Gem accumulation in heated tumor relative to free Gem. Similar trend of increased Gd-TSLnps accumulation was observed in non-heated tumor compared to that of free Gd; however, no significant difference in MRI contrast enhancement between free Gd and Gd-TSLnps ex-vivo tumor images was observed. Despite Gem-TSLnps dose being half of free Gem dose, antitumor efficacy of Gem-TSLnps was comparable to that of free Gem(Gem-TSLnps 10 mg Gem/kg compared with free Gem 20 mg/kg). Overall, the findings suggest that TSLnps may be used to improve Gem delivery and enhance its antitumor activity. However, the formulation of Gd-TSLnp needs to be fully optimized to significantly enhance MRI contrast in tumor.

Pharmacokinetic, biodistribution and therapeutic efficacy of 5-fluorouracil-loaded pH-sensitive PEGylated liposomal nanoparticles in HCT-116 tumor bearing mouse.

The objective of the study was to investigate the pharmacokinetics and efficacy of 5-FU entrapped pH-sensitive liposomal nanoparticles with surface-modified anti-epidermal growth factor receptor (EGFR) antibody (pHLNps-5-FU) delivery system. Cytotoxicity of 5-FU and pHLNps-5-FU was determined in vitro against HCT-116 cells. The biodistribution and pharmacokinetic parameters of the administered 5-FU and pHLNps-5-FU as well as efficacy of 5-FU and pHLNps-5-FU were determined in HCT-116 subcutaneous mouse model. Mean size of pHLNp-5-FU was 164.3 ± 8.4 nm with entrapment efficiency (E.E) of 54.17%. While cytotoxicity of 5-FU and pHLNps-5-FU showed a strong dose-dependent, pHLNps-5-FU proved to be more effective (2-3 fold high) than that of 5-FU against HCT-116 cells. Pharmacokinetic study showed a prolonged plasma circulation of pHLNps-5-FU and a more significant body exposure while accumulation of pHLNps-5-FU in tumor was significantly higher than that of free 5-FU. Further, the efficacy of pHLNps-5-FU, was greater than free 5-FU at equivalent 5-FU dose. The study suggests that pHLNps may be an effective drug delivery system to enhance the anticancer activity of 5-FU against colorectal tumor growth.

Cytotoxicity of 5-fluorouracil-loaded pH-sensitive liposomal nanoparticles in colorectal cancer cell lines.

5-Fluorouracil (5-FU) is widely used in cancer therapy, either alone or in combination with other anti-cancer drugs. However, poor membrane permeability and a short half-life (5-20 min) due to rapid metabolism in the body necessitate the continuous administration of high doses of 5-FU to maintain the minimum therapeutic serum concentration. This is associated with significant side effects and a possibility of severe toxic effects. This study aimed to formulate 5-FU-loaded pH-sensitive liposomal nanoparticles (pHLNps-5-FU) and evaluate 5-FU release characteristics and anti-cancer effect of pHLNps-5-FU. Particle size and zeta potential were determined using a particle size analyzer. The release patterns of pHLNps-5-FU formulations were evaluated at 37°C at pH 3, 5, 6.5, and 7.4, while drug release kinetics of 5-FU from a pHLNp3-5-FU formulation were determined at pH 3 and 7.4 at different time points (37°C). Cell viability and clonogenic studies were conducted to evaluate the effectiveness of pHLNps-5-FU against HCT-116 and HT-29 cell lines while cellular uptake of rhodamine-labeled pHLNps-5-FU was determined by flow cytometry and confocal imaging. The average sizes of the pHLNp1-5-FU, pHLNp2-5-FU and pHLNp3-5-FU liposomes were 200nm ± 9.8nm, 181.9 nm ± 9.1 nm, and 164.3 nm ± 8.4 nm respectively. In vitro drug release of 5-FU from different pHLNps-5-FU formulations was the highest at pH 3.8. Both cell lines treated with pHLNps-5-FU exhibited reduced viability, two- or three-fold lower than that of 5-FU-treated cells. Flow cytometry and confocal imaging confirmed high uptake of rhodamine-labeled pHLNps-5-FU in both cell lines. The drug release profile of the chosen pHLNp3-5-FU formulation was optimal at pH 3 and had the poorest release profile at pH 7.4. The release profile of pHLNp3-5-FU showed that 5-FU release was two-fold higher at pH 3 than that at pH 7.4. This study demonstrates that pHLNp3-5-FU may be a potential candidate for the treatment of colorectal cancer.

In vitro and in vivo antitumor activity of gemcitabine loaded thermosensitive liposomal nanoparticles and mild hyperthermia in pancreatic cancer.

The study was designed to explore the feasibility of increasing the delivery of gemcitabine-HCL (Gem), a poor membrane permeable and short half-life drug, through PEGylated thermosensitive liposomal nanoparticles (TSLnps) delivery system followed by mild hyperthermia (mTH) at 42°C. In vitro release pattern of Gem-TSLnps showed a significant Gem release (60%, p<0.01) at 42°C compared to that released at 37°C (29%). Cell viability and clonogenic assay demonstrated significant inhibition of MiaPaCa-2 cells growth by Gem-TSLnps + mHT compared to Gem alone. Further, IC50 value of Gem treated cells was (0.077µM) 1.2 fold higher compared to that treated with Gem-TSLnps + mHT (0.063 µM). mHT treated cells showed moderate inhibition of cell growth compared to controls. For cellular uptake studies, flow cytometric analysis and confocal imaging revealed higher uptake of Rho-TSLnps compared to Rho-PE or untreated cells. Tumor volume of mice treated with Gem alone was 1.8 fold higher compared to the group treated with Gem-TSLnps + mHT. Further, tumor regression of Gem-TSLnps + mHT treated group was significantly higher (p<0.01) compared to Gem-TSLnps or Gem. No significant elevated liver enzymes were observed when serum alanine aminotransferase (ALT) or aspartate aminotransferase (AST) level of control group was compared to that of Gem or Gem-TSLnps+mHT treated groups. However, serum level of alkaline phosphatase (ALP) of Gem or Gem-TSLnps+ mHT treated group was significantly elevated (p<0.05) when compared to the control group. In conclusion, TSLnps increased the delivery of Gem to tumor cells and also enhanced significantly the antitumor activity of Gem when combined with heat.

Cytotoxicity of gemcitabine-loaded thermosensitive liposomes in pancreatic cancer cell lines.

Gemcitabine (GEM) is currently the standard option for the treatment of pancreatic cancer but its short half-life and rapid metabolism has caused for new modality for delivery of GEM. The purpose of this study was to formulate GEM loaded PEGylated thermosensitive liposomal nanoparticles (GEM-TSLnps) to increase residence time and deliver high payload of GEM to pancreatic cancer cells using mild hyperthermia (mHT). The GEM-TSLnps were formulated by thin film hydration. The cytotoxic effects of GEM and GEM-TSLnps were evaluated against human pancreatic cancer cell lines. In vitro release of GEM by TSLnps was determined at temperatures from 26°C through to 50°C. Cell viability studies, clonogenic assay, flow cytometry and confocal imaging were performed on pancreatic cancer cell lines using GEM and GEM-TSLnps + mHT. The GEM-TSLnp size was determined to be 216.10 ± 0.57 nm with entrapment efficiency of 41.10 ± 2.0%. GEM release from TSLnps was sharply increased at 42°C (60%) than at 37°C (25%), (p<0.01). In vitro cytotoxicity of GEM-TSLnps + mHT treated pancreatic cancer cell lines was significantly higher than GEM treated. The IC50 values for PANC-1, MiaPaCa-2 and BxPC-3 cells GEM-TSLnps + mHT treated were 1.2 to 3.5 fold-higher than GEM treated. Among the cell lines, GEM-TSLnps + mHT treated PANC-1 and MiaPaCa-2 cells show significantly reduced reproductive viability compared with the GEM treated cells. Flow cytometric and confocal images revealed high Rho-TSLnps cellular uptake. Our findings suggest that GEMTSLnps+ mHT can significantly enhance cytotoxic effect of GEM and could serve as a new chemotherapy modality for delivering GEM.

Engineering theranostic nanovehicles capable of targeting cerebrovascular amyloid deposits.

Cerebral amyloid angiopathy (CAA) is characterized by the deposition of amyloid beta (Aß) proteins within the walls of the cerebral vasculature with subsequent aggressive vascular inflammation leading to recurrent hemorrhagic strokes. The objective of the study was to develop theranostic nanovehicles (TNVs) capable of a) targeting cerebrovascular amyloid; b) providing magnetic resonance imaging (MRI) contrast for the early detection of CAA; and c) treating cerebrovascular inflammation resulting from CAA. The TNVs comprised of a polymeric nanocore made from Magnevist (MRI contrast agent) conjugated chitosan. The nanocore was also loaded with cyclophosphamide (CYC), an immunosuppressant shown to reduce the cerebrovascular inflammation in CAA. Putrescine modified F(ab')2 fragment of anti-amyloid antibody, IgG4.1 (pF(ab')24.1) was conjugated to the surface of the nanocore to target cerebrovascular amyloid. The average size of the control chitosan nanoparticles (conjugated with albumin and are devoid of Magnevist, CYC, and pF(ab')24.1) was 164±1.2 nm and that of the TNVs was 239±4.1 nm. The zeta potential values of the CCNs and TNVs were 21.6±1.7 mV and 11.9±0.5 mV, respectively. The leakage of Magnevist from the TNVs was a modest 0.2% over 4 days, and the CYC release from the TNVs followed Higuchi's model that describes sustained drug release from polymeric matrices. The studies conducted in polarized human microvascular endothelial cell monolayers (hCMEC/D3) in vitro as well as in mice in vivo have demonstrated the ability of TNVs to target cerebrovascular amyloid. In addition, the TNVs provided contrast for imaging cerebrovascular amyloid using MRI and single photon emission computed tomography. Moreover, the TNVs were shown to reduce pro-inflammatory cytokine production by the Aß challenged blood brain barrier (BBB) endothelium more effectively than the cyclophosphamide alone.

Development of a novel approach to enhance the solubility of ftibamzone formulation.

Ftibamzone (FBZ) is known to be effective against herpes simplex virus that causes genital herpes but poor solubility of FBZ has reduced its therapeutic efficacy. We investigated water-soluble complexes of various nanoparticles with FBZ to improve its solubility as well as increase its absorption. Using phase-solubility technique, we measured formation constant (K1:1 and K1:2) values at room temperature in pH 7 buffer. Solubility was determined by dissolving FBZ or FBZ-entrapped nanoparticles in phosphate buffers and pH adjusted to different pH range (2-12). The solutions were then equilibrated for 24 hours and then filtered and analyzed using HPCL. Nanoparticles were formulated using nanoprecipitation technique and cellular uptake of nanoparticle was determined by confocal microscope. No significant FBZ solubility was observed from pH 2 to 10 however we did notice a rapid increase in solubility from pH of 10 to 12 with FBZ solubility of 950 µg/ml. Our log D against pH profile revealed that FBZ is characteristic of an acid drug since unionized group was dominant at low pH. FBZ interaction with methyl-ß-cyclodextrin (mßCD) complexation/nanoparticles showed a greater solubility of FBZ compared with FBZ alone while complexation constants were determined to be K1:1 and K1:2 were 7.06×10-3 and 8.98×10-8 mM-1 respectively. Only FBZ-chitosan nanoparticles were toxic against MDCK cells. Study demonstrates that FBZ-PLGA nanoparticles could significantly enhance the solubility and absorption of FBZ compared with FBZ alone and has the potential to be used as an effective delivery system for the treatment of genital herpes.