Green synthesis and biological evaluation of novel 5-fluorouracil derivatives as potent anticancer agents

This study reports the formation of 5-FU co-crystals with four different pharmacologically safe co-formers; Urea, Thiourea, Acetanilide and Aspirin using methanol as a solvent. Two fabrication schemes were followed i.e., solid-state grinding protocol, in which API and co-formers were mixed through vigorous grinding while in the other method separate solutions of both the components were made and mixed together. The adopted approaches offer easy fabrication protocols, no temperature maintenance requirements, no need of expensive solvents, hardly available apparatus, isolation and purification of the desired products. In addition, there is no byproducts formation, In fact, a phenomenon embracing the requirements of green synthesis. Through FTIR analysis; for API the Nsingle bondH absorption frequency was recorded at 3409.02 cm−1 and that of single bondCdouble bondO was observed at 1647.77 cm−1. These characteristics peaks of 5-FU were significantly shifted and recorded at 3499.40 cm−1 and 1649.62 cm−1 for 5-FU-Ac (3B) and 3496.39 cm−1 and 1659.30 cm−1 for 5-FU-As (4B) co-crystals for Nsingle bondH and single bondCdouble bondO groups respectively. The structural differences between API and co-crystals were further confirmed through PXRD analysis. The characteristic peak of 5-FU at 2θ = 28.79918o was significantly shifted in the graphs of co-crystals not only in position but also with respect to intensity and FWHM values. In addition, new peaks were also recorded in all the spectra of co-formers confirming the structural differences between API and co-formers. In addition, percent growth inhibition was also observed by all the co-crystals through MTT assay against HCT 116 colorectal cell lines in vitro. At four different concentrations; 25, 50, 100 and 200 µg/mL, slightly different trends of the effectiveness of API and co-crystals were observed. However; among all the co-crystal forms, 5-FU-thiourea co-crystals obtained through solution method (2B) proved to be the most effective growth inhibitor at all the four above mentioned concentrations

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

Smart thermosensitive liposomes for effective solid tumor therapy and in vivo imaging - Fig 1

<p><b>(A)Tumor inhibition curve and ex-vivo tumor MR imaging</b>. Thermosensitive liposomal nanoparticles loaded-gadopentetic acid (Magnevist^®, a gadolinium-based MRI contrast agent) was injected intraperitoneally and excised tumor was imaged and area circled yellow indicates a higher contrast area. Gd-TSLnps, and thermosensitive liposomal nanoparticles loaded gemcitabine, Gem-TSLnps were intravenously injected through the tail vein of mice. Mild heat (mHT) was applied to tumor site containing Gem-TSLnps and tumor growth inhibition determined, <b>(B) Flow chart summarizing the various studies.</b> The diagram depicts graphical representation of studies conducted in a coherent fashion.</p

Quantitative measurement of Gd-TSLnps and Gem-TSLnps in mice organs following i.p injection of 4mg /kg of Gd or Gd-TSLnps (Gd dose equivalent, 4mg /kg); 20 mg/kg of Gem or Gem-TSLnps (Gem dose equivalent, 10 mg/kg).

<p>(A) Gd and Gd-TSLnps in tissue, (B) Gem and Gem-TSLnps in tissue. ICP-MS and HPLC were used to analyze Gd and Gem respectively. Data is expressed as mean ± S.D, n = 5 per group (p* < 0.05; p**<0.01; p***<0.001).</p

<i>Ex vivo</i> MRI scan.

<p>T₁ maps of <i>ex vivo</i> tumors acquired at 30, 60 and 90 min after i.p. injection of free Gd and Gd-TSLnps. On the left of each sample, the ROI placement for T₁ extraction is shown. The right full T₁ map is shown. The 30-min Gd-TSLnps sample showing a region with increased T₁ contrast also has the magnitude image included. **ND = not detected**</p

Pharmacokinetic profiles of Gem and Gem-TSLnps in mouse.

<p>Pharmacokinetic profiles of Gem and Gem-TSLnps in mouse.</p

Phantom images.

<p>(A): The top row show T₁ weighted images with increasing signal with increased Gd concentration at TR = 750 ms. The bottom row show the corresponding T₁ maps for each respective Gd concentration: (B) T₁ and T₂ values of varying concentrations of Gd only and Gd in TSLnps with the corresponding relaxivity values.</p

Differential scanning calorimetry (DSC).

<p>Thermogram analysis of DPPC and TLSnps.</p

Pharmacokinetic profiles of free Gd and Gd-TSLnps in mouse.

<p>Pharmacokinetic profiles of free Gd and Gd-TSLnps in mouse.</p

Plasma concentration-time curves of measured Gd-TSLnps and Gem-TSLnps.

<p>A) Plasma concentration of Gd only and Gd-TSLnps as a function of time. B) Plasma concentration of Gem only and Gem-TSLnps as a function of time. C) Log plasma concentration of Gd only and Gd-TSLnps against time. D) Log plasma concentration of Gem only and Gem-TSLnps against time. Concentration of Gd (Magnevist^®) was measured by inductively coupled plasma mass spectrometry (ICP-MS), while concentration of Gem was measured by HPLC. Data represent mean ± SD, (n = 5 per group).</p