Floxuridine

Lipophilic Prodrug of Floxuridine Loaded into Solid Lipid Nanoparticles: In Vitro Cytotoxicity Studies on Different Human Cancer Cell Lines

Daniela Chirio, Elena Peira, Luigi Battaglia, Benedetta Ferrara, Alessandro Barge,
Simona Sapino, Susanna Giordano, Chiara Dianzani, and Marina Gallarate
Università degli Studi di Torino – Dipartimento di Scienza e Tecnologia del Farmaco, via Giuria 9, 10125 Torino, Italy

Floxuridine is a very effective drug with high potency in the treatment of various tumors but its utility
is limited by its low efficiency of cellular uptake. In order to improve the floxuridine efficiency of cellu-
lar uptake, lipophilic prodrug of floxuridine (3 ,5 -distearoyl-5-fluoro-2 -deoxyuridine) was synthetized
and loaded into behenic acid nanoparticles produced by fatty acid coacervation technique. Gener-
ally, spherical shaped SLN with mean diameters below 300 nm were obtained. Distearoyl-floxuridine
was loaded in SLN with high entrapment efficiency (from 70.8 to 82.8%). In vitro cytotoxicity studies
on different human cancer cell lines (M14, HT-29 and MDA-MB231) were performed in order to test
the ability of distearoyl-floxuridine-SLN to inhibit the cancer cell growth. In MTT test distearoyl flox-
uridine SLN showed a greater efficacy than floxuridine on all cancer cell lines revealing an ef ficiency about 100 times higher. Also clonogenic assay showed a higher cytotoxicity of distearoyl-floxuridine- SLN compared to floxuridine but the difference between the formulations was only about 10 times. In conclusion, SLN proved to be a promising vehicle to increase the floxuridine efficacy in cancer therapy.
Keywords: Floxuridine, Prodrug, MTT, Clonogenic, In Vitro Cytotoxicity, Cancer.

1. INTRODUCTION
Cancer is a disease that affects millions of people in all age groups and both sexes. Most of the current chemothera- peutic agents on the market have low therapeutic index and therefore require high concentrations that consequently

FUDR is a very effective drug with high potency in the treatment of various tumors such as colorectal cancer, liver cancer and colon cancer. Although FUDR is clini- cally effective, it exhibits various side effects as a result of its low tumor selectivity, adverse effects at nontumor sitelead to high toxicity.

actions and low efficiency of cellular uptake.

Moreover, effective cancer chemotherapy depends on control of drug levels at the tumor site.
The fluoropyrimidines represent the most widely pre- scribed class of anticancer drugs worldwide.

Many studies were performed in the last years in order to improve the efficiency of cellular uptake of FUDR and reduce its toxicity. Among different strategies, the pro- drugs synthesis is the most extensively adopted.

Floxuridine (FUDR) is a fluorinated pyrimidine

Tsume et al.

synthesized dipeptide monoester prodrugs

monophosphate analogue of 5-fluorouracil (5-FU); as an antimetabolite, FUDR inhibits thymidylate synthetase, resulting in disruption of DNA synthesis and cytotoxicity. This agent is also metabolized to fluorouracil and other metabolites that can be incorporated into RNA and inhibit the utilization of preformed uracil in RNA synthesis.

using L-phenylalanyl-L-tyrosine as a dipeptide promoi- ety. FUDR prodrugs were more enzymatically stable than FUDR, exhibited 2.4- to 48.7-fold higher uptake than their parent drug in Caco-2, Panc-1, AsPC-1 cells, and cell pro- liferation assays in ductal pancreatic cancer cells (AsPC-1 and Panc-1) indicated that FUDR dipeptide prodrug was more potent than parent drug.
Landowski et al. synthesized amino acid ester prodrugs glycosidic bond metabolism. Isoleucyl prodrugs exhibited the highest chemical and enzymatic stability and enhanced the stability of the glycosidic bond of FUDR. Thymidine phosphorylase rapidly cleaved FUDR to 5-FU, whereas with the prodrugs no detectable glycosidic bond cleavage was observed. 50-L-isoleucyl and 50-L-valyl monoester prodrugs exhibited 8- and 19-fold PEPT1-mediated uptake enhancement in HeLa/PEPT1 cells, respectively. Uptake enhancement in HeLa/PEPT1 cells correlated highly with Caco-2 permeability for all prodrugs tested. Caco-2 per- meability of 50-L-isoleucyl and 50-L-valyl prodrugs was 8- to 11-fold greater compared with FUDR.
Other FUDR prodrugs synthetized to enhance the anti- cancer activity of parent drug, consist in twin drugs, com- pounds that combine two different drugs in one molecule for synergistic treatment, and that can produce stronger pharmacological effects because the twin drugs show two different pharmacological activities after they enter into cancer cells.
Huczynˇski et al. synthesized conjugates between FUDR and salinomycin (SAL), molecule able to selectiv- ity kill cancer stem cells. The conjugate obtained by ester- ification reaction between FUDR and SAL has showed a significantly higher antiproliferative activity against the drug-resistant cancer cells and lower toxicity than those of pH of is lowered by acidification, the fatty acid precipi- tates as nanoparticles owing to proton exchange between the acid solution and the soap.
After characterization, DS-FUDR SLN were tested in vitro on different human tumor cell lines such as HT29 (colon adenocarcinoma), MDA-MB231 (triple neg- ative breast cancer) and M14 (melanoma) in order to eval- uate their cytotoxicity.

2. EXPERIMENTAL DETAILS
2.1. Chemicals
Sodium behenate (Na-BA) was purchased from Nu-Chek Prep, Inc. (Elysian, U.S.A.), benzilic alcohol, octanol, stearoyl chloride, sodium bicarbonate from Fluka (Buchs, Switzerland), 80% hydrolyzed polyvinyl alcohol 9000– 10000 Mw (PVA 9000), floxuridine (FUDR), potassium chloride, 2,3-bis[2-methoxy-4-nitro-5sulphophenyl]-2H- tetrazolium-5carboxanilide, crystal violet solution, sodium sulphate from Sigma (Dorset, UK), hydrochloric acid, potassium phosphate monobasic, silica gel 60 and sul- furic acid from Merck (Darmstadt, Germany), sodium chloride, chloroform, pyridine, methanol and ethanol from Carlo Erba (Val De Reuil, France). Deionized water was obtained by a MilliQ system (Millipore, Bedford, MO).

SAL and FUDR towards normal cells. Zhang et al. syn-
thesized a novel amphiphilic twinIP:drug179.61.200.41ofFUDR andOn:ben-Wed,2.2.01 SynthesisAug2018of19:33:56DS-FUDR damustine (BdM). They associated theCopyright:prodrugs Americanstrategy Scientific Publishers
to nanotechnology, widely used in cancer therapy to solve

rapid clearance, premature degradation, low accumulation in tumors and severe multidrug resistance (MDR) of free anticancer drugs. Indeed, due to its inherent amphiphilic-

added under ice bath (Fig. 1). The mixture was stirred over night at room temperature. Afterwards, the reaction solu- tion was poured into 5 ml iced water bath and extracted

ity, the FUDR–BdM twin drug could self-assemble into three times with 7 ml CHCl3 was then washed stable and well-defined nanoparticles in water. in vitro twice with 30 ml each of 1 M H2 and studies showed that FUDR–BdM twin drug nanoparticles can overcome MDR of tumor cells and that after hydrol- ysis of FUDR–BdM twin drug nanoparticles in the cells, both FUDR and BdM can be released as active molecules to kill cancer cells, resulting in a better anticancer efficacy, because of higher expression of caspase-3 and apoptotic rate than the free drugs.
In order to overcome the limited access of FUDR to the brain, Wang et al. synthetized 3 ,5 -dioctanoyl-5-fluoro- 2 -deoxyuridine (DO-FUdR) and loaded it into solid lipid nanoparticles (SLN). The in vivo studies showed that the brain area under the concentration–time curve of DO- FUdR-SLN and DO-FUdR were 10.97- and 5.32-fold higher than that of FUDR, respectively.
In this work 3 ,5 -distearoyl-5-fluoro-2 -deoxyuridine (DS-FUDR), a lipophilic prodrug of FUDR, was syn- thetized and incorporated into SLN (DS-FUDR SLN) pre- pared by the fatty acid coacervation method. Briefly, a fatty acid alkaline salt micellar solution in the presence of an appropriate polymeric stabilizer is prepared; when the
J. Nanosci. Nanotechnol. 18, 556 –563, 2018

finally with distilled water. After drying with anhydrous Na2 SO4 , the chloroform phase was concentrated and puri- fied by flash chromatography (first eluting with 50 ml CHCl3 and then with 10 ml CH3 OH/ CHCl3 50:50). Mix- ture of CH3 OH/CHCl3 containing DS-FUDR was evapo- rated under reduced pressure.

Figure 1. DS-FUDR synthesis scheme.

Lipophilic Prodrug of Floxuridine Loaded into Solid Lipid Nanoparticles: In Vitro Cytotoxicity Studies Chirio et al.

2.3. Characterization of DS-FUDR
2.3.1. Mass Spectrometry
MS-APCI+ spectra were recorded on Micromass ZQ spec- trometer (Waters, Milford, USA) equipped with ESCI source.

2.3.2. NMR
NMR spectra were recorder on Avance 300 spectrom- eter (Bruker, Billerica, USA) operating at 7 T. Sample were dissolved in deuterated chloroform and chemical shift was referenced to the residual solvent peak (1H, d = 7 26 ppm).

2.3.3. Partition Coefficient Determination

SLN employed in in vitro cell toxicity experiments were prepared in aseptic conditions (under laminar flow airfilter starting from materials sterilized by UV or in autoclave). 5 ml samples were then centrifuged for 15 min at 62,000 g, the supernatant was removed and the precipitate was sus- pended in 0.5 ml sterile water.

2.5. SLN Characterization
SLN suspension was observed by optical microscopy (Leica DM 2500, Solms, Germany) at 1000 × magnification.
SLN shape and mean sizes were determined by scan- sion electron microscope (SEM) using Stereoscan 410 (Leica, Wetzlar, Germany). DS-FUDR-loaded SLN were

1:50 diluted in water and 15 l suspension were deposed
FUDR partition coefficient determination. 3 ml 50 mg/ml
coated with 15 nm gold layer (SCD 050 Balzers, Liechten- stein) for 30 seconds under vacuum at a current intensity of
tor funnel with 3 ml of octanol or water respectively. The
40 mA. The gold-coated particle layer was scanned using
mixtures were then shaken for 5 minutes and left to rest
the accelerating voltage scanning of 20 kV.
overnight; aqueous FUDR and octanolic DS-FUDR con-
SLN particle sizes and polydispersity indexes (PDI)
centrations before and after partitioning were determined
were determined using laser light scattering technique-LLS
by HPLC.
(Brookhaven, New York, USA). Size measurements were
P was calculated as octanol/water FUDR and DS-FUDR
obtained at an angle of 90 at 25 C using the intensity
molar concentration ratio at the equilibrium. The results
method. Size measurements were also recorded diluting
were expressed as Log P. Each experiment was repeated
samples with the grown medium used to culture MDA-
IP: 179.61.200.41 On: Wed,MB01 Aug231 cells2018to19:33:56mime the conditions under which SLN Copyright: American Scientific Publishers
2.4. DS-FUDR-Loaded SLN Preparation
Different SLN formulations were prepared (Table I) using
10 as the ratio between DS-FUDR amount in SLN and that in
Briefly, appropriate amounts of Na-BA and PVA 9000 the starting micellar solution ×100. DS-FUDR EE% deter- were dispersed in 5 ml deionized water and the mixture
was then heated under 5 min-stirring (300 rpm) just above

the Krafft point of Na-BA (75 C) to obtain a clear solu- tion (micellar solution). Different amounts of DS-FUDR benzyl alcohol solution were added to the warm micel- lar solution. A selected acidifying solution (100 l 1 M

washed twice with 1 ml ethanol:water 50:50 to eliminate adsorbed drug. The solid residue was dissolved in 0.9 ml ethanol, 0.1 ml water were then added to precipitate the lipid matrix and the supernatant obtained was injected in
Table I. SLN composition, size and EE%.

EE% was also determined after SLN gel filtration using a matrix of cross-linked of agarose (Sepharose CL 4B) as stationary phase that permits to separate DS-FUDR-loaded particles by free drug. Briefly, 1 ml SLN was introduced

SLN A SLN B SLN C SLN D SLN E SLN F
DS-FUDR (mg) 2 2 3 3 4 4 Na-BA (mg) 25 25 25 25 25 25 PVA 9000 (mg) 25 50 25 50 25 50 Benzyl alcohol ( l) 100 100 100 100 100 100 HCl 1 M ( l) 80 80 80 80 80 80 NaH2 PO4 1 M ( l) 50 50 50 50 50 50 Water (ml) 2.5 2.5 2.5 2.5 2.5 2.5 Size (nm) 291.2 ±5.6 207.3 ±2.4 269.9 ±2.7 227.9 ±5.5 2443 ±169 9249 ±428 Polidispersity index 0.189 0.169 0.140 0.201 0.413 0.427 EE% 82.8 78.0 77.5 70.8 n.d. n.d.
Chirio et al. Lipophilic Prodrug of Floxuridine Loaded into Solid Lipid Nanoparticles: In Vitro Cytotoxicity Studies

at the head of 10 ml column and eluted by gravity with hypertonic PBS buffer (8.0 g/l NaCl, 0.2 g/l KCl, 1.44 g/l Na2 HPO4 2H2 O, 0.24 g/l KH2 PO4 . Fractions of 1 ml each were collected. The opalescent fractions containing the purified SLN were concentrated under nitrogen up to 1 ml final volume. The DS-FUDR in the resultant suspen- sion was determined solubilizing 50 l SLN into 0.95 ml ethanol and analyzing it by HPLC.

2.6. HPLC Analysis
HPLC analysis was performed using a LC9 pump

The medium was changed after 72 h and cells were cultured for additional 10 days. Subsequently, cells were fixed and stained with a solution of 80% crystal violet and 20% methanol. Colonies were then photographed and counted with a Gel Doc equipment (Bio-Rad Laboratories, Milan, Italy). Then the cells were perfectly washed and 30% acetic acid was added to induce a completely disso- lution of the crystal violet. Absorbance was recorded at 595 nm by a 96-well-plate ELISA reader. Five different experiments were performed.
2.10. Statistical Analysis

2.5 125 × 4 6 mm column and a C-R5A integrator (Shimadzu, Tokyo, Japan); mobile phase: CH3 OH:H2 O 90:10 (flow rate 1 ml min ; detector: UV = 265 nm (Shimadzu, Tokyo, Japan). Retention time was 4.8 min.
The limit of quantification, defined as the lowest DS-FUDR concentration in the curve that can be mea- sured routinely with acceptable precision and accuracy, was 0.032 mol/ml; the limit of determination, defined as the lowest detection limit, was 0.015 mol/ml (signal to noise < 2.0). Data were expressed as the mean ± SD. Significance between experimental groups was determined by one-way ANOVA followed by the Bonferroni’s multiple compari- son post tests using GraphPad InStat software (San Diego, USA). Values of p ≤ 0 05 were considered statistically significant. 3. RESULTS AND DISCUSSION 5-FU is one of the oldest chemotherapeutic agents and it has played a dominant role for decades in the treatment of breast cancer and in a variety of other solid tumors. 5-FU 2.7. In vitro Cytotoxicity is widely used alone or in combination with chemother- The following human tumor cells were used: HT29 (colon adenocarcinoma), MDA-MB231 (triple negative breast cancer) and M14 (melanoma). HT29 and MDA-MB-231 from the American Type Culture Collection (ATCC; Man- 11 assas, VA), M14 from Dr. Pistoia (Gaslini Institute, Genoa, Italy). The HT29 and M14 were grown as a monolayer 12 culture in RPMI 1640 medium, MDA-MB-231 in DMEM, both of them supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mmol/l L-glutamine and peni- cillin/streptomycin (100 units/ml), at 37 C in 5% CO2 13–15 Coacervation method, the technique used to prepare FUDR-loaded SLN, expects that the drug is loaded inside 2.8. MTT Assay Cells (1 × 103/well) were seeded in 96-well plates and the Na-BA micelles which, precipitating after acidifica- tion, form drug-loaded SLN. In order to be incorporated incubated at 37 C, 5% CO , for 24 h. Then, they into micelles, the drug should have lipophilic properties. were treated with different concentrations of FUDR or DS-FUDR SLN (0.004–20 M) and with empty SLN. After 72 h incubation, viable cells were eval- uated by 2,3-bis[2-methoxy-4-nitro-5sulphophenyl]-2H- tetrazolium-5carboxanilide (MTT) inner salt reagent at 570 nm, as described by the manufacturer’s protocol. The controls (i.e., cells that had received no drug) were nor- malized to 100%, and the readings from treated cells were expressed as % of viability inhibition. Eight replicates were used to determine each data point and five different experiments were performed. 2.9. Colony-Forming Assay M14, HT29 and MDA-MB231 cells (800 per well) were seeded into six-well plates and treated with FUDR or DS-FUDR SLN (0.04–20 M) and with empty SLN. J. Nanosci. Nanotechnol. 18, 556 –563, 2018 FUDR, an analog of 5-FU used in the treatment of col- orectal cancer, is a hydrophilic drug; to entrap it in SLN it is necessary to synthesize a lipophilic derivative. In pre- vious works lipophilic derivatives of different drugs were prepared in order to facilitate the drug loading. Wang et al. synthetized a lipophilic derivative of FUDR, the 3 ,5 -dioctanoyl-5-fluoro-2 -deoxyuridine (DO- FUdR), and incorporated it in SLN to enhance the access of the drug to the brain. In the present work, DO-FUDR was also synthetized, but, showing not sufficient lipophilic- ity to be incorporate into SLN (data not shown), it was abandoned. 3.1. Synthesis DS-FUDR In this work a more lipophilic derivative, the 3 ,5 - distearoyl-5-fluoro-2 -deoxyuridine (DS-FUDR) was 559 Lipophilic Prodrug of Floxuridine Loaded into Solid Lipid Nanoparticles: In Vitro Cytotoxicity Studies Chirio et al. Figure 2. MS-APCI+ spectrum. synthetized. A yellow-brown powder was obtained with a 61.6% overall yield. 3.2. Characterization of DS-FUDR The structure of DS-FUDR was confirmed by mass spec- trometry and by NMR studies. 3.6. SLN Characterization In Table I compositions of DS-FUDR-loaded SLN (DS- FUDR-SLN) are reported. SLN composition was the result of a preliminary screening carried out in a previous work. In this study only PVA 9000 and DS-FUDR amounts were varied in order to obtain the highest possible loading main- taining suitable SLN sizes. IP: 179.61.200.41 On: Wed, 01SLNAugsuspension201819:33:56appeared homogeneous at optical microscopy (Fig. 4(A)) as neither particle aggregates nor Delivered by Ingenta lipid/drug crystals were observed. SEM analysis permitted or to the Na or K adduct. An example is reported in Figure 2. Only signals relative to the monoester species are visible (M+H+, m/z = 513.8; M+Na+, m/z = 535.8). This is probably due to “in source ”hydrolisys of desired compound. 3.4. NMR 1H-NMR spectrum (Fig. 3) confirms the presence of the distearoyl derivatives, indeed, peak area ratio between sig- nal 7.70 ppm (aromatic proton in position 6) and signal at 0.78 ppm (metyl protons of stearoyl chians) is equal to 1/6; moreover, peak area ratio between signal 7.70 and signal at 2.30 ppm (methylen in position 2 of stearoyl chain) is equal to 1/4. No evidence of significant amount of mono-steraoyl derivative are revealed by 1H-NMR spectrum. 3.5. Partition Coefficient FUDR and DS-FUDR partition coefficients are −1.60 and +2.04 respectively. It should be noted that the DS-FUDR lipophilicity increases up to more than 4000 times that of FUDR; consequently, also DS-FUDR water solubility decreases compared to FUDR, so that this derivative is able to be entrapped into SLN prepared by coacervation method. 560 to investigate the shape and surface of SLN (Fig. 4(B)): particles are almost spherical and a certain aggregation phenomenon can be noted, probably due to the operative condition of SEM. In Table I DS-FUDR-SLN mean diameter and polidis- persity index are reported. Most SLN tested had sizes below 300 nm: only SLN prepared with the highest DS- FUDR amount showed sizes higher than 2400 nm. The use of 2% PVA 9000 permitted to obtain smaller SLN. In order to mime the conditions under which SLN undergo in vitro experiments, SLN mean sizes were determined also dilut- ing nanoparticle suspension with grown medium: no sig- nificant difference in mean diameters was noted (data not reported). In Table I also DS-FUDR EE% are reported. DS-FUDR was entrapped in SLN with high percentages from 70.8 to 82.8%; the use of 2% PVA 9000 instead of 1% slightly decreased EE% probably due to a greater micelles amount in the aqueous phase able to solubilize the drug. 3.7. In vitro Cytotoxicity In this work the effect of DS-FUDR-SLN and free FUDR on the cell growth of three different tumor cell lines was examined: M14, a melanoma cell line, HT-29, a colon can- cer cell line and MDA-MB231, a breast cancer cell line. J. Nanosci. Nanotechnol. 18, 556 –563 , 2018 Chirio et al. Lipophilic Prodrug of Floxuridine Loaded into Solid Lipid Nanoparticles: In Vitro Cytotoxicity Studies Figure 3. 1H-NMR spectrum. IP: 179.61.200.41 On: Wed, 01 Aug 2018 19:33:56 Results, reported in Figure 5, showedCopyright:that bothAmericansam- ScientificexertcellPublisherstoxicity (not related to FUDR), we evaluated the ples were effective in reducing M14 cell proliferation in a effects of empty SLN diluted as in drug loaded SLN (0.04– dose-dependent manner. Following 72 h treatment, FUDR decreased cell growth by 60% at 20 M and 35% at 4 M, becoming almost ineffective at 2 M. DS-FUDR SLN showed a greater efficacy than FUDR, since it induced a similar inhibition of proliferation already from the dose 0.4 M, revealing an efficiency about 100 times higher. 20 M) on cell growth using the MTT assay. Results showed that SLN did not significantly affect cell growth of all the cell types even at the highest concentrations. Then, similar analyses were conducted to test DS- FUDR-SLN and FUDR ability to inhibit the proliferation of HT-29 and MDA-MB231 cells. Similar results to those The IC50 was 1.6 ×10 M and 1.1 ×10 M, respectively. obtained with M14 cells were reproduced on HT29 cell In order to investigate the possibility that empty SLN may (B) (A) line. Figure 6 shows a dose-response relationship similar to the previous. The IC50 was 1.4 ×10 M and 1.3 ×10 M, respectively. Figure 4. (A): Optical microscopy SLN suspension; (B): SLN SEM image. Figure 5. M14 MTT test. ∗∗ p < 0 01. J. Nanosci. Nanotechnol. 18, 556 –563, 2018 561 Lipophilic Prodrug of Floxuridine Loaded into Solid Lipid Nanoparticles: In Vitro Cytotoxicity Studies Chirio et al. blue analysis (data not shown), proving a real signifi- cant inhibitory effect induced by DS-FUDR-SLN on cell proliferation. The differing effectiveness of DS-FUDR-SLN and FUDR may be partly due to the different uptake and subcellular localization of the drug formulations. 5-FU and FUDR prodrugs are taken up into the cytoplasm by a carrier mediated transport and fluxed out by the P-glycoprotein pumps. By contrast, we suggest that DS-FUDR-SLN are rapidly taken up by endocytosis and can escape from the P-glycoprotein pumps. Thus, SLN Figure 6. HT-29 MTT. p < 0 01. Acting on MDA-MB231 cell line (Fig. 7), FUDR was able to reduce cell proliferation only at the highest con- centration, resulting already almost inactive at 2 M. Once again DS-FUDR-SLN was significantly more effec- tive than FUDR, revealing a higher efficiency, in fact it was still efficient at 0.04 M. The IC50 was 1.6 ×10 M and 2.6 ×10 M, respectively. MTT data show that DS-FUDR-SLN inhibited the cell growth of different types of tumor cells in vitro; there- fore the inhibition was not specific for a single type of tumor. The effect was dose-dependent and clearly more potent than the one displayed by FUDR, which was effec- may act as intracellular drug depots slowly releasing the free drug into the cellular cytoplasm and enhancing the therapeutic efficacy. Clonogenic assay or colony formation assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony. The colony is defined to con- sist of at least 50 cells. The assay essentially tests every cell in the population for its ability to undergo “unlimited” division. After plating at very low density (800 cell for wells), cells are treated with the compounds for 72 h, then the drugs are removed by washing the cells with the cell medium and the cells are allowed to growth over extended period of time (7 days). Only a fraction of seeded cells retains the capacity to produce colonies. tive only at the highest doses. MTT, a yellow tetrazole, is reduced to purple formazan inIP:living179.61.200.41cells.TetrazoliumOn: Wed,cell01 clonogenicityAug201819:33:56which revealed that DS-FUDR-SLN as dye reduction is dependent on NAD(P)H-dependentCopyright: Americanoxi- Scientific Publishers doreductase enzymes largely in the cytosolic compartment of the cell. Therefore, reduction of MTT depends on the cellular metabolic activity due to NAD(P)H flux. Cells concentration; nevertheless DS-FUDR-SLN resulted even more active than FUDR at 0.2–0.4 M (Figs. 8, 9). The with a low metabolism such as thymocytes and spleno- cytes reduce very little MTT. In contrast, rapidly divid- ing cells exhibit high rates of MTT reduction. Sometimes, IC50 was 4.9 ×10 M14 and 1.9 ×10 HT29. M and 2.6 ×10 M and 1.5 ×10 M, respectively for M, respectively for assay conditions can alter metabolic activity and thus tetra- zolium dye reduction without affecting cell viability. In addition, the mechanism of reduction of tetrazolium dyes, i.e., intracellular (MTT) versus extracellular (WST-1), will also determine the amount of product. Our data demonstrated similar results using MTT, WST-1 or trypan Similar results were revealed with MDA-MB231 cells, but the highest efficacy increase respect to FUDR of DS-FUDR-SLN was obtained at 0.04–0.2 M (Fig. 10). The IC50 was 1.8 ×10 M and 2.6 ×10 M. Chirio et al. Lipophilic Prodrug of Floxuridine Loaded into Solid Lipid Nanoparticles: In Vitro Cytotoxicity Studies

4. CONCLUSIONS
The fatty acid coacervation technique, a solvent free method, was used to prepare DS-FUDR-loaded SLN with sizes in the 200–300 nm range and DS-FUDR EE% in the 70–82% range.
In vitro cytotoxicity studies on different human cancer cell lines (M14, HT-29 and MDA-MB231) confirmed the ability of FUDR and DS-FUDR-SLN to inhibit the can- cer cell growth. In particular, DS-FUDR SLN showed a greater efficacy than FUDR on all cancer cell lines reveal- ing an efficiency between 10 and 100 times higher.
In conclusion, the SLN ability to entry inside the cells

Figure 9.

HT-29 clonogenic assay.

∗∗

p < 0 01 p < 0 01. by endocytosis mechanism avoiding a carrier mediated transport means that they can be a promising vehicle to The lower difference in cytotoxicity between DS-FUDR-SLN and FUDR at concentrations higher than 0.4–2 M on clonogenic assay can be explained hypoth- esizing that FUDR does not alter the enzymatic systems of the cell (which then can still reduce the tetrazolium salts giving low cytotoxicity in the MTT test) but alters the cell replication systems so the cancer cell loses the ability to replicate (high cytotoxicity in clonogenic tests). increase FUDR efficacy in cancer therapy. Acknowledgments: The work was supported by Uni- versity of Turin research funds 2014 (ex 60%). References and Notes 1. D. J. Bharali, M. Khalil, M. Gurbuz, T. M. Simone, and S. A. Mousa, Int. J. Nanomed. 4, 1 (2009). 2. C. Avendano and J. C. Menendez, Medicinal Chemistry of Anti- cancer Drugs, Second Edition, Elsevier, Amsterdam (2015), p. 329. 3. G. Di Stefano, C. Busi, and L. Fiume, Dig. Liver Dis. 34, 439 (2002). Development of multidrug resistance in human tumors 5. Y. Tsume, B. B. Bermejo, and G. L. Amidon, Pharmaceuticals is one of the main obstacles to the success of can- 7, 169 (2014). cer chemotherapy. This phenomenon is often associ- ated with increased expression ofIP:the179.61.200.41mdr1gene, whichOn: Wed, 01Amidon,Aug2018Pharm.19:33:56Res.22,1510(2005). encodes P-glycoprotein (Pgp). As anCopyright:energy-dependentAmerican Scientific7.A. HuczyPublishersnˇski,M.Antoszczak,N. Kleczewska, M. Lewandowska, Delivered by IngentaE.Maj, J. Stefanˇska, J. Wietrzyk, J. Janczak, and L. Celewicz, Eur. J. Med. Chem. 93, 33 (2015). drugs from cells, leading to decreased drug concentrations within the cells and reduced efficacy of drugs. Other- Mol. Pharm. 12, 2328 (2015). wise a reduction of the expression of membrane nucleo- side transporters can also prevent the drug entering in the cells. We proposed that DS-FUDR-SLN may overcome these transport defects by entering into the cells without a specific carrier-mediated transport. This mechanism is under investigation and it will be described in a future work. 9. J. X. Wang, X. Sun, and Z. R. Zhang, Eur. J. Pharm. Biopharm. 54, 285 (2002). 10. L. Battaglia, M. Gallarate, R. Cavalli, and M. Trotta, J. Microencap- sulation 27, 78 (2010). 11. R. Cavalli, F. Leone, R. Minelli, R. Fantozzi, and C. Dianzani, Cur- rent Drug Delivery 11, 270 (2014). 12. C. Zinutti, M. Barberi-Heyob, M. Hoffman, and P. Maincent, Int. J. Pharm. 166, 231 (1998). 13. S. K. Jain, A. Chaurasiya, Y. Gupta, A. Jain, P. Dagur, B. Joshi, and V. M. Katoch, J. Microencapsul. 25, 289 (2008). 14. L. Zhu, J. Ma, N. Jia, Y. Zhao, and H. Shen, Colloids Surf. B- Biointerfaces 68, 1 (2009). 15. X. Li, Y. Xu, G. Chen, P. Wie, and Q. Ping, Drug Dev. Ind. Pharm. 34, 107 (2008). 16. L. Battaglia, M. Gallarate, E. Peira, D. Chirio, E. Muntoni, E. Biasibetti, M. T. Capucchio, A. Valazza, P. P. Panciani, M. Lanotte, D. Schiffer, L. Annovazzi, V. Caldera, M. Mellai, and C. Riganti, J. Pharm. Sci. 103, 2157 (2014). 17. M. Gallarate, D. Chirio, R. Bussano, E. Peira, L. Battaglia, F. Baratta, and M. Trotta, Int. J. Pharm. 440, 126 (2013). 18. L. Battaglia, M. Gallarate, E. Peira, D. Chirio, I. Solazzi, S. M. A. Giordano, C. L. Gigliotti, C. Riganti, and C. Dianzani, Nanotechnol- ogy 26, 255102 (2015). 19. D. Chirio, M. Gallarate, E. Peira, L. Battaglia, L. Serpe, and M. Trotta, J. Microencapsul. 28, 537 (2011). 20. U. Stein, W. Walther, and R. H. Shoemaker, J. Natl. Cancer Inst.