pharmaceutics

Article Formulation of -Modified Raltitrexed-Loaded Nanoparticles for Colorectal Cancer Theranostics

Justin G. Rosch 1, Allison N. DuRoss 1, Madeleine R. Landry 1 and Conroy Sun 1,2,*

1 Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Portland, OR 97201, USA; [email protected] (J.G.R.); [email protected] (A.N.D.); [email protected] (M.R.L.) 2 Department of Radiation Medicine, School of Medicine, Oregon Health & Science University, Portland, OR 97239, USA * Correspondence: [email protected]

 Received: 1 January 2020; Accepted: 3 February 2020; Published: 5 February 2020 

Abstract: Multifunctional nanoparticles (NPs) that enable the imaging of drug delivery and facilitate cancer cell uptake are potentially powerful tools in tailoring oncologic treatments. Here we report the development of a layer-by-layer (LbL) formulation of folic acid (FA) and folate that have been well-established for enhanced tumor uptake and as potent chemotherapeutics, respectively. To investigate the uptake of LbL coated NPs, we deposited raltitrexed (RTX) or combined RTX-FA on fluorescent polystyrene NPs. The performance of these NP formulations was evaluated with CT26 murine colorectal cancer (CRC) cells in vitro and in vivo to examine both uptake and cytotoxicity against CRC. Fluorescence microscopy and flow cytometry indicated an increased accumulation of the coated NP formulations versus bare NPs. Ex vivo near-infrared (NIR) fluorescence imaging of major organs suggested the majority of NPs accumulated in the liver, which is typical of a majority of NP formulations. Imaging of the CRC tumors alone showed a higher average fluorescence from NPs accumulated in animals treated with the coated NPs, with the majority of RTX NP-treated animals showing the consistently-highest mean tumoral accumulation. Overall, these results contribute to the development of LbL formulations in CRC theranostic applications.

Keywords: folic acid; raltitrexed; tangential flow filtration; nanoparticles; NIR imaging

1. Introduction Folic acid (FA) is a metabolite required for DNA synthesis in the proliferation of new cells [1]. This small molecule, comprised of a pterin, p-amino benzoic acid, and glutamic acid [2], has been investigated as a targeting agent for molecular and nanoparticle-based therapies due to the prevalence of folate receptors and carriers present on various cancer cells [3–6]. By attaching FA to a drug carrier, direct targeting can potentially result in enhanced uptake in tumors [5,7,8], especially those with over-expression of the α-folate receptor [6]. Anti-folate drugs have been a common strategy for targeting various cancer cells. was one of the first of these compounds to see wide clinical use, but suffers from low aqueous solubility. Methotrexate inhibits , a necessary component in purine synthesis [9]. Therapeutic strategies evolved to target , which is also required for cell proliferation. Raltitrexed (RTX) and , among others, were compounds that saw use as a result of drug design and development in this area [9–11]. Potency varies, but is generally higher than early anti-folate drugs, with reduced side effects (such as nephrotoxicity), and more specific binding to their targets (α-folate/β-folate receptors and the reduced folate carrier) [6]. This class of drugs has been employed in the treatment of numerous cancers, including colorectal [12], lung [13], and pancreatic [14], among others, and usually in combination with

Pharmaceutics 2020, 12, 133; doi:10.3390/pharmaceutics12020133 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2020, 12, 133 2 of 14 other . Of particular note for this work is RTX’s use as adjuvant therapy in the treatment of colorectal cancer (CRC) [15–20]. Nanoparticle-based delivery of therapeutics offers numerous benefits over parenteral administration of comparable free drugs [21]. Most important here is the size, which allows passive accumulation in the tumor, likely through enhanced permeability and retention (EPR), an observed phenomena whereby damaged or “leaky” blood vessels allow for extravasation of NPs [22]. Here, we investigate cellular/tumoral uptake of RTX-laden nanoparticles (NPs) in a murine CRC model when administered intravenously to investigate the impact of RTX or raltitrexed with FA as a targeting ligand. Improved understanding of the impact of functionalizing NP surfaces on their eventual intracellular trafficking in vivo may lead to better treatment outcomes clinically, and will likely rely on investigation of specific receptor-ligand mechanisms on an individual cell type basis [23]. Early steps in determining this lie in developing systems that can be used to track NP fate, either through fluorescence, chemical composition, or other means. The presence of a near-infrared (NIR) dye loaded entrapped in a functionalized NP allows for detection of NPs by preclinical imaging systems, both in vivo and ex vivo [24–26]. By investigating the differences in accumulation in tumors by varying the targeting ligand compositions, improved targeting can be identified for future therapies. The NP surface can be functionalized with targeting ligands [22,27]; here, FA or an anti-folate drug (or both) can be adsorbed to the surface of the particle. By adsorbing various polyelectrolytes to the surface of the NP, encapsulation and targeting can be achieved [28]. This layer-by-layer (LbL) assembly strategy has been used for encapsulating fluorophores or imaging agents, therapeutics, and/or targeting ligands. Once a layer is applied, additional layers can be added sequentially after purification of excess polyelectrolyte, through centrifugation or tangential flow filtration (TFF) [29]. To develop a theranostic NP with a NIR fluorescent core and functionalized surface, we applied LbL technology to the coating of commercially available NIR dye-containing polystyrene beads. Once functionalized with an outer layer RTX or RTX/FA, the changed uptake and viability are examined in vitro, and the biodistribution versus uncoated (bare) particles is examined in vivo. The ease of changing the terminal layer allowed us to examine the differences between RTX alone and in combination with FA to investigate differences attributed to the terminal layer composition.

2. Materials and Methods

2.1. Materials Raltitrexed, and poly-l-lysine hydrochloride (PLL, 15–30 kDa) were purchased from Sigma Aldrich. Folic acid was purchased from Fisher Scientific. All LbL methods were carried out in Milli-Q water from a Milli-Q source. KR2i TFF system was purchased from Spectrum Laboratories (Repligen, Waltham, MA, USA).

2.2. Layer-by-Layer Process Nanoparticle suspensions containing 2 wt% solids were sonicated (3 applications of 20% amplitude for 10 s on a Branson 450 digital sonifer (Branson Ultrasonics, Danbury, CT, USA)) and diluted to 0.2 mg/mL, and 3 mL was aliquoted into 15 mL conical tubes. The 3 mL sample was mixed briefly with 3 mL of 1 mg/mL PLL solution, and again sonicated 3 times at 20% amplitude for 10 s. The feed and retentate lines from the TFF setup were then placed into the conical tube, with the feed line near the bottom of the conical tube. Milli-Q water was then added to nearly fill the conical tube. The TFF system was then activated, beginning the fluid circulation through the system. As the excess PLL exited as permeate, the liquid level in the conical tube descended. Once the meniscus approached <3 mL, the tube was again filled with Milli-Q water to further wash the circulating nanoparticles. The liquid level was allowed to reach the bottom of the tube, where the flow in the system was paused, the external portion of the feed line was rinsed with water, and immersed in the water. The pump was activated, and the liquid level was returned to the original volume. The system was then flushed with water. The PLL-coated particles were added to a mixture containing either RTX or RTX and FA. Pharmaceutics 2020, 12, 133 3 of 14

Mixtures were as follows: RTX—3 mL of 0.5 mg/mL with 1 mL of 0.5 M NaOH, RTX/FA—2.5 mL of 0.5 mg/mL RTX, 2.5 mL of 0.5 mg/mL FA with 1 mL of 0.5 M NaOH. Once brief mixing occurred, the mixture was purified using the same strategy as described for PLL.

2.3. Nanoparticle Characterization Size and zeta potential measurement were acquired using a Malvern Zetasizer ZS (Malvern Panalytical, Malvern, UK). Measurements were taken in triplicate in DTS 1070 zeta cells (Malvern), with values presented as the mean standard deviation of the measurements. Transmission electron ± microscopy (TEM) was used to further observe size and morphology of nanoparticle formulations. Images were acquired sing a Technai F-20 TEM operating at 4200 eV. Grids (Ted Pella, Redding, CA, USA) with copper substrate-formavar/carbon backed were used to deposit 10 µL of NP solution dropwise. Following dropwise addition of NP solution to the grid, the grids were placed in a desiccator overnight to allow for the surface to dry. Infrared spectroscopy was carried out using a Nicolet iS5 spectrophotometer (ThermoFisher, Waltham, MA, USA). Freshly prepared NP solutions were lyophilized, and the dry powder was compressed into a diamond filament. Total internal reflectance measurements were used to acquire the spectrum for each formulation at 32 resolution between 4000 1 × and 400 cm− . Drug loading and encapsulation efficiency were determined via high performance liquid chromatography (HPLC). To determine drug content, nanoparticle solutions containing RTX and RTX/FA NPs with excess polyelectrolytes were passed through a 100 kDa spin filtration unit (Pall). The samples were spun at 5500 rpm for 5 min, which allowed the drug to exit as filtrate while retaining the NPs in the retentate. Filtrate samples were run on a Shimadzu SPD-20A HPLC instrument (Shimadzu, Torrance, CA, USA) equipped with a UV-Vis detector and an Agilent Zorbax Rapid Resolution SBC-18 column (4.6 100 mm, 3.5 µm, Santa Clara, CA, USA). Drawn aliquots were run without dilution. × A five-minute gradient method was utilized with a flow rate of 1 mL/min and a 311 nm detection wavelength. The mobile phase increased from 65:35 methanol/water to 70:30 methanol/water over the first 2 min and held there until the method was complete with elution occurring at ~2.2–2.4 min. Loaded drug was calculated from the difference between inlet mass of drug and filtrate mass of drug.

2.4. Cell Uptake CT26 murine colorectal carcinoma cells (ATCC, Manassas, VA, USA) were cultured in RPMI 1640 (Corning, NY, USA) media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. When 90% confluent, the media was aspirated, the cells were washed with PBS, and 0.25% trypsin-EDTA was used to detach the cells from the culture flask. The cells were diluted in media once and counted using a Countess II FL Automated Cell Counter (ThermoFisher). The cells were then prepared for either cell uptake or cell viability assays. To evaluate enhanced uptake of the coated NPs versus bare particles, uptake in CT26 cells was evaluated by flow cytometry and fluorescence microscopy. Utilizing the culture conditions described above, 106 cells were plated into each well of a 6-well plate, followed by 2 mL of RPMI media. The cells were allowed to settle overnight. The following day, the media was aspirated, NP solutions (using with fluorescent core of 660/680 dye) were diluted in media and added to each well. A control well was left untreated. The cells were placed in an incubator for 4 h to allow uptake of the NPs. Following uptake, the cells were prepared for either flow cytometry or fluorescence microscopy. To prepare the cells for quantitative assessment of cell uptake using flow cytometry, the cells were washed with PBS, detached using trypsin, and diluted in media. The cells were centrifuged at 500 g for 5 min to form a pellet. The media was aspirated, followed by redispersion of the cells × in 1 mL of PBS. The cells were pipetted into polystyrene tubes through cell strainer caps to remove cell aggregates. Using a MACSQuant flow cytometer (Miltenyi Biotec, Cologne, Germany), cellular fluorescence of no treatment control, bare CML NP-treated, and coated NP treated cells was determined sequentially. The NP fluorescence was determined using a 632 nm excitation laser line, with a 670/30 Pharmaceutics 2020, 12, 133 4 of 14 nm emission/bandwidth filter. Forward scattering and side scattering were used to determine single cells passing through the detector, and 105 events (or cells) were passed through the detector to establish a distribution for each sample in each treatment group. To prepare the cells for fluorescent imaging, the cells were washed with PBS twice, and 2 mL of 10% formalin solution was added to each well, and allowed to fix for 30 min. Once fixed, the cells were washed twice with PBS, followed by addition of PBS containing DAPI and AlexaFluor 488-phalloidin dyes (ThermoFisher), and allowed to stain for 30 min. After staining, the cells were washed once, and the wells were filled with 2 mL of PBS. Images could then be acquired using an EVOS FL AUTO II (ThermoFisher), with filter cube sets able to capture the nucleus (blue—DAPI), cell body myelin filaments (green—AlexaFluor 488), and the NPs (red—660/680 dye).

2.5. Cell Viability CT26 cells were cultured as described in the previous section. The cells were further diluted to 20,000 cells/mL, then plated at 2000 cells per well in a 96-well plate and allowed to attach overnight. After seeding, media containing NP solution was added to the majority of wells, with half-fold serial dilutions across the plate, in triplicate. The final wells were dosed with DMSO and no treatment (i.e., media), in triplicate, for the positive and negative controls. After 72 h, 10 µL of Alamar Blue cell viability reagent (ThermoFisher) was added to each well. After 1.5 h, the fluorescent signal was determined at 560/590 excitation/emission wavelength using a Tecan M200 Infinite plate reader (Tecan Trading AG, Männedorf, Switzerland).

2.6. In Vivo Biodistribution Twenty 6-week-old BALB/c mice (Charles River Laboratories, Wilmington, MA, USA) housed in modified barrier animal facilities prior to tumor inoculation. On the day of tumor inoculation, cell suspensions containing 6 106 CT26 cells/mL were prepared in sterile PBS for injection. Fifty-microliter × injections were made into the right hind flank of isoflurane-anesthetized mice, implanting a total of 3 × 105 cells subcutaneously. The tumors were allowed to grow for 2–3 weeks to reach a threshold size of around 100 mm3 (measured twice weekly in this 2–3 week period by calipers, using formula 0.5 long × length short length2). × After the tumors in the majority of animals had reached the desired size, animals were randomized into four groups, with average size roughly even among the groups. The saline group contained two animals, the bare group contained three animals, and the RTX and RTX/FA nanoparticle groups contained four animals. Two-hundred-microliter injections of either saline, bare NPs, RTX NPs, or RTX NPs (using 715/755 fluorescent dye cores) were injected via tail-vein once per day for 3 days, roughly 24 h apart. Twenty-four hours following the third injection, animals were sacrificed, organs (including tumors) were extracted, and immediately imaged using an IVIS XRMS III imaging system (PerkinElmer, Waltham, MA, USA). Sequences of images of arranged organs were imaged at 1 s, 3 s, and 6 s exposure time with imaging settings of medium binning, f1, and filter positions at 720/790 for excitation and emission. Following initial image acquisition, all organs were immersed and stored at 4 ◦C in 10% formalin in PBS for 48 h, followed by immersion and storage in 70% ethanol. After two days in the ethanol, all tumors were arranged by treatment group and imaged in the IVIS again, with a higher exposure of 20 s in order to determine relative fluorescence away from the other organs. All animal experiments followed guidelines set forth by Oregon Health and Science University (OHSU) Institutional Animal Care and Use Committee. Following imaging of the ex vivo tumors, the samples were placed in histology grids and labeled, and transferred to the Histopathology Shared Resource at OHSU. Samples were paraffin embedded, sliced, and prepared according to the required staining protocol, which included either no staining, hematoxylin and eosin (H & E), or caspase-3 (CC3, Promega, 1:1500 dilution). Slides produced from staining were transferred to the Advanced Light Microscopy Core at OHSU. Scanned images of each slide were taken on a Leica Axios Imaging system (Leica) using a 10 objective. All tumors were × Pharmaceutics 2020, 12, 133 5 of 14

stained and imaged; complete sets of organs were stained and imaged from one representative animal Pharmaceuticsper group. 2020 Unstained, 12, x FOR PEER slices REVIEW were scanned using the 700 nm setting on an infrared imaging scanner 5 of 13 (LI-COR, Odyssey, Lincoln, NE, USA) with low pixel resolution. 2.7. Statistical Analysis 2.7. Statistical Analysis Data are presented as mean ± standard deviation. Statistical significance was evaluated with Data are presented as mean standard deviation. Statistical significance was evaluated with student t-test using GraphPad Prism± 8 software. p < 0.05 was considered statistically significant, with furtherstudent confidencet-test using indicated GraphPad by Prismasterisks 8 software. (*, p < 0.05,p < **,0.05 p < was 0.01, considered ***, p < 0.001, statistically ****, p < 0.0001). significant, with further confidence indicated by asterisks (*, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001). 3. Results and Discussion 3. Results and Discussion

3.1.3.1. Drug Drug Encapsulation Encapsulation Process Process Layer-by-layerLayer-by-layer deposition deposition onto NPNP substratessubstrates using using alternating alternating charged charged polyelectrolytes polyelectrolytes has has been beenemployed employed previously previously for numerousfor numerous applications applications [29 –[29–31].31]. Here, Here, we we use use the the process process to attachto attach FA FA and andanti-folate anti-folate drugs drugs to the to the surface surfac ofe polystyrene of polystyrene substrates. substrates. This This process process is accelerated is accelerated through through the use the of usea TFF of a setup,TFF setup, as described as described in the in the methods methods section. section. Each Each layer layer can can be be added added within within a a few few minutes. minutes. A Aschematic schematic of of the the layering layering process process is is shown shown in in Figure Figure1A. 1A. The The completed completed NP NP contains contains a fluorescent a fluorescent dye dye(either (either 660 660/680/680 or or 715 715/755)/755) terminated terminated with with a therapeutica therapeutic and and targeting targeting ligand ligand (RTX (RTX or or RTX RTX/FA),/FA), as asdepicted depicted in in Figure Figure1B. 1B.

FigureFigure 1. 1. TangentialTangential flowflow filtrationfiltration (TFF) (TFF) allows allows for rapidfor rapid purification purification after layeringafter layering of various of various materials materialsonto the onto surface the ofsurface carboxylate of carboxylate modified modified polystyrene polystyrene latex (CML) latex nanoparticles (CML) nanoparticles (NPs). (A (NPs).) Schematic (A) Schematicrepresentation representation of TFF system of TFF used syst toem purify used CML to purify from CML poly- lfrom-lysine poly- (PLL),L-lysine followed (PLL), by followed separation by of separationthe NPs from of the raltitrexed NPs from (RTX) raltitrexed or RTX (R/folicTX) acidor RTX/folic (FA) mixture. acid (FA) (B) Illustrationmixture. (B of) Illustration the Layer-by-layer of the Layer-by-layer(LbL)-coated NP(LbL)-coated formulation. NP formulation.

3.2.3.2. Nanoparticle Nanoparticle Characterization Characterization TheThe size size and and morphology morphology of of the the NPs NPs was was evalua evaluatedted by by TEM. TEM. Images Images of of the the RTX/FA RTX/FA coated coated NPs NPs takentaken using using TEM TEM are are shown shown in in Figure Figure 2A2A (RTX (RTX NPs NPs are are shown shown in in Supplementary Supplementary Info, Info, Figure Figure S1). S1). DynamicDynamic light light scattering scattering (DLS) (DLS) meas measurementsurements yielded yielded size size distribution distributionss for for the the formed formed particles, particles, as as shownshown in in Figure Figure 2B.2B. The The highest highest populations populations were were approximately approximately 40–50 40–50 nm nm in in size, size, with with the the RTX/FA RTX /FA NPsNPs appearing appearing slightly slightly smaller smaller in in overall overall population population distribution. distribution. Z-Ave Z-Ave measurements measurements from from DLS DLS measurementsmeasurements are are shown shown in in Figure Figure 2C.2C. The The particle particle cores cores appear appear to to be be roughly roughly 20–30 20–30 nm nm in in diameter, diameter, whichwhich is is consistent consistent with with DLS DLS measurements measurements (Figure (Figure 2C).2C). The The size size and and zeta zeta potential potential of of each each layer layer additionaddition was was monitored monitored during during NP NP synthesis synthesis (after (after ea eachch purification purification step) step) to to confirm confirm that that the the layers layers were being formed as intended, also shown in Figure 2C. For the RTX NPs, the size does not increase largely across the synthesis, starting at intensity weighted mean hydrodynamic diameter, or Z-Ave, of 29.53 ± 1.81 nm, and finishing at a Z-Ave of 53.08 ± 0.33 nm (PDI = 0.265 ± 0.04). For the RTX/FA NPs, the Z-Ave of the final formulation is 51.02 ± 1.70 nm (PDI = 0.215 ± 0.004). The zeta potential, though, displays marked shifts from negative (CML surface) to positive (PLL coating) to negative

Pharmaceutics 2020, 12, x FOR PEER REVIEW 6 of 13 Pharmaceutics 2020, 12, 133 6 of 14 (drug and/or folate terminal layer). This flipping of the zeta potential has been seen in similar systems [28,30,32], and is a simple way to ensure the layer is deposited on the surface, especially for smaller werepolymer being deposition formed as where intended, the size also may shown not in change Figure sign2C.ificantly. For the RTX IR spectroscopy NPs, the size was does used not increaseto identify largelychanges across in characteristic the synthesis, wavelengths starting at intensity due to weightedthe presence mean of hydrodynamicthe molecules added diameter, to the or Z-Ave, surface of of 29.53 1.81 nm, and finishing at a Z-Ave of 53.08 0.33 nm (PDI = 0.265 0.04). For the RTX/FA NPs, the NPs.± The waveforms for the synthesized NPs± are shown in Figure 2D.± The two formulations seem the Z-Ave of the final formulation is 51.02 1.70 nm (PDI = 0.215 0.004). The zeta potential, though, to add different peaks to the measured waveforms,± with the most± distinct changes appearing at ~800– displays900 cm marked−1, 1300–1500 shifts from cm− negative1, and 1600–1700 (CML surface) cm−1 to. positiveThe 800–900 (PLL coating)nm peaks to negative could (drugbe additional and/or folatecontributions terminal layer).to alkene This bonding, flipping as ofare the most zeta of potential the early haspeaks. been The seen other in similartwo distinct systems peaks [28 are,30 ,likely32], andthe is addition a simple of way theto oxygen ensure functional the layer isgroups, deposited with on carboxylic the surface, acids especially added forthrough smaller the polymer glutamic depositionsection of where both theRTX size and may FA not likely change contributing significantly. strong IR spectroscopy in these regions. was used Nitrogenous to identify functional changes ingroups characteristic are likely wavelengths to contribu duete in tothese the regions presence as of well. the molecules added to the surface of the NPs. The waveformsRaltitrexed for concentrations the synthesized were NPs estimated are shown as 148 in Figure µg/mL2D. in Thethe RTX two formulationsformulation and seem 129 to µg/mL add diinfferent the peaksRTX/FA to theformulation measured (with waveforms, a concentration with the most of 118 distinct µg/mL changes FA present appearing in atthe ~800–900 RTX/FA 1 1 1 cmformulation).− , 1300–1500 This cm− yields, and 1600–1700nearly identical cm− . encaps The 800–900ulation nm efficiencies peaks could of be29.7% additional for the contributionsRTX NPs and to30.9% alkene for bonding, the RTX/FA as are NPs most (calculated of the early as peaks. the mass The of other RTX two present distinct on the peaks final are particle/mass likely the addition of RTX ofpresent the oxygen when functional coating the groups, PLL layer). with carboxylicThe release acids of the added RTX from through the NPs theglutamic is anticipated section to ofbe bothrapid, RTXas this and was FA likelyobserved contributing with a similar strong particle in these formulated regions. Nitrogenouspreviously [31], functional with >80% groups of the are therapeutic likely to contributereleased in in the these first regions 8 h. as well.

FigureFigure 2. 2.Characterization Characterization of of RTX RTX and and RTX RTX/FA/FA NP NP formulations. formulations. (A (A) TEM) TEM images images of of formed formed RTX RTX/FA/FA NPs.NPs. Scale Scale bar bar in in lower lower magnification magnification (larger (larger image)—100 image)—100 nm, nm, scale scale bar ba inr in higher higher magnification magnification image image (inset)—20(inset)—20 nm. nm. (B ()B Intensity) Intensity derived derived distribution distribution for for the the formed formed NPs NPs as as measured measured by by dynamic dynamic light light scatteringscattering (DLS). (DLS). (C ()C Mean) Mean size size and and zeta-potential zeta-potential measurements measurements at at each each step step in in the the layering layering process process showsshows increase increase in in size size during during synthesis, synthesis, and and accompanying accompanying alternating alternating charge. charge. (D (D) IR) IR spectra spectra for for the the formulatedformulated NPs. NPs. Raltitrexed concentrations were estimated as 148 µg/mL in the RTX formulation and 129 µg/mL in 3.3. Cell Uptake the RTX/FA formulation (with a concentration of 118 µg/mL FA present in the RTX/FA formulation). This yieldsTo investigate nearly identical the effect encapsulation of the terminal effi ciencieslayer presence of 29.7% on forthe the CML RTX substrate NPs and for 30.9% cell-mediated for the RTXuptake/FA NPspurposes, (calculated two sets as the of massexperiments of RTX presentwere run. on theIn the final first, particle a quantitative/mass of RTX assessment present when of NP coatingfluorescence the PLL was layer). acquired The releaseusing flow of the cytometry. RTX from CT26 the NPsmurine is anticipated CRC cells were to be dosed rapid, with as this 5 µg/mL was

Pharmaceutics 2020, 12, 133 7 of 14 observed with a similar particle formulated previously [31], with >80% of the therapeutic released in the first 8 h.

3.3. Cell Uptake To investigate the effect of the terminal layer presence on the CML substrate for cell-mediated Pharmaceuticsuptake purposes, 2020, 12, x two FOR setsPEERof REVIEW experiments were run. In the first, a quantitative assessment 7 of of NP 13 fluorescence was acquired using flow cytometry. CT26 murine CRC cells were dosed with 5 µg/mL (CML) of particles for 4 h, washed with PBS, and fixed in 10% formalin. After fixation, the cells were (CML) of particles for 4 h, washed with PBS, and fixed in 10% formalin. After fixation, the cells were washed again, and kept at 4 °C until their fluorescence could be determined using flow cytometry. A washed again, and kept at 4 ◦C until their fluorescence could be determined using flow cytometry. A MACSQuant (Miltenyi Biotec) cytometer was used to determine NP fluorescence of ~100,000 cells, MACSQuant (Miltenyi Biotec) cytometer was used to determine NP fluorescence of ~100,000 cells, gating for single cells. In Figure 3A, the mean fluorescence intensities from the cell populations of the gating for single cells. In Figure3A, the mean fluorescence intensities from the cell populations of the various groups are shown. The bare NPs show a markedly higher signal above background (from no various groups are shown. The bare NPs show a markedly higher signal above background (from no treatment control), while both types of layered particles showed a 2–4-fold higher cell uptake over treatment control), while both types of layered particles showed a 2–4-fold higher cell uptake over the 4 h exposure period, indicating that the addition of the terminal layer for both particles led to a the 4 h exposure period, indicating that the addition of the terminal layer for both particles led to a statistically significant increase in cell uptake. RTX/FA coating had a lower signal on average than statistically significant increase in cell uptake. RTX/FA coating had a lower signal on average than the the RTX coating alone. This difference was found to be significant. The addition of the RTX and FA RTX coating alone. This difference was found to be significant. The addition of the RTX and FA to the to the terminal layer was intended to further increase the uptake both in vitro and in vivo, but inverse terminal layer was intended to further increase the uptake both in vitro and in vivo, but inverse effect effect was observed in vitro. RTX could have a higher uptake potential in CT26 cells, or the was observed in vitro. RTX could have a higher uptake potential in CT26 cells, or the combination combination with FA may interfere with specific uptake mechanisms that reduce the overall uptake. with FA may interfere with specific uptake mechanisms that reduce the overall uptake. In general, the In general, the fate of the individual NPs once they enter the media environment en route to the cells fate of the individual NPs once they enter the media environment en route to the cells is not completely is not completely understood. Factors such as size due to aggregation, apparent surface charge, and understood. Factors such as size due to aggregation, apparent surface charge, and location of RTX location of RTX and FA on the surface of the particles could all influence the intracellular trafficking and FA on the surface of the particles could all influence the intracellular trafficking of the NPs, both of the NPs, both in vitro and in vivo [23]. Representative individual histograms for the flow cytometry in vitro and in vivo [23]. Representative individual histograms for the flow cytometry data are shown data are shown in Figure 3B. in Figure3B.

Figure 3. Cellular uptake of RTX and RTX/FA NPs in vitro via flow cytometry. (A) Mean fluorescence Figure 3. Cellular uptake of RTX and RTX/FA NPs in vitro via flow cytometry. (A) Mean fluorescence intensities of cell populations analyzed using flow cytometry show that the addition of the therapeutic intensities of cell populations analyzed using flow cytometry show that the addition of the therapeutic alone and in combination with FA lead to higher uptake. **, p < 0.01, ****, p < 0.0001. (B) Representative alone and in combination with FA lead to higher uptake. **, p < 0.01, ****, p < 0.0001. (B) Representative histograms from each treatment group show marked shift in populations with the addition of the histogramstargeting layers. from each (RTX treatment and RTX gr/FAoup are show very marked similar inshift overall in popula distribution,tions with with the mostaddition of the of RTXthe targetingdistribution layers. obscuring (RTX and the viewRTX/FA of the are RTX very/FA simila distribution.).r in overall distribution, with most of the RTX distribution obscuring the view of the RTX/FA distribution.). The populations shift from left to right with increasing fluorescent signal from the red fluorophore in theThe NPs, populations with the RTX shift and from RTX /FAleft NPsto showingright with highest increasing signal intensitiesfluorescent (with signal significant from the overlap red fluorophorein their populations, in the NPs, leading with to the the RTX RTX /andFA distributionRTX/FA NPs being showing covered highest by the signal RTXdistribution intensities (with in the significantfigure). To overlap visualize in thetheir cell populations, uptake of the leading NPsqualitatively, to the RTX/FA the distribution second assessment being covered used fluorescenceby the RTX distributionmicroscopy. in CT26 the figure). cells were To visualize plated, dosed the cell for up 4take h, and of the fixed NPs to qualitatively, the surface of the a six second well assessment plate rather usedthan fluorescence in suspension. microscopy. The cells CT26 were cells stained were with plated DAPI, dosed and for Alexa 4 h, 488-phalloidinand fixed to the (blue surface and of greena six well plate rather than in suspension. The cells were stained with DAPI and Alexa 488-phalloidin (blue and green respectively) prior to imaging on an EVOS FL Auto II (Invitrogen, Waltham, MA, USA). Images taken from wells treated with each NP are shown in Figure 4.

Pharmaceutics 2020, 12, 133 8 of 14 respectively) prior to imaging on an EVOS FL Auto II (Invitrogen, Waltham, MA, USA). Images taken Pharmaceuticsfrom wells 2020 treated, 12, x with FOR PEER each REVIEW NP are shown in Figure4. 8 of 13

FigureFigure 4. Cellular uptakeuptake of of LbL LbL NP NP formulations formulationsin vitro in vitrovia fluorescence via fluorescence microscopy microscopy show an show increase an increasein red signal in red due signal to higher due to uptake higher of uptake NPs, whichof NPs, is which highest is inhighest the RTX in andthe RTX RTX /andFA images.RTX/FA Scaleimages. bar Scaleindicates bar indicates 100 µm. 100 µm.

AA no no treatment treatment well well shows shows no no red red fluorescence fluorescence,, while while bare bare CML CML particles particles show show low low signal, signal, indicatedindicated by arrowsarrows in in the the image. image. The The wells wells treated treated with with the the coated coated particles particles show show much much brighter brighter signal signalcolocalized colocalized within within the cells, the indicative cells, indicative of uptake of quantifieduptake quantified by flow cytometry.by flow cytometry. Unlike flow Unlike cytometry, flow cytometry,though, the though, difference the in signaldifference intensity in signal between intensity the diff erentbetween coating the groupsdifferent is indiscernible.coating groups is indiscernible. 3.4. Cell Viability 3.4. CellThe Viability efficacy of drug-coated formulations was evaluated in vitro using a cell viability assay, with AlamarThe Blueefficacy (Invitrogen) of drug-coated as the formulations reagent. CT26 was cells evaluated were dosed in vitro with using NPs a cell diluted viability in media assay, of with the Alamarvarious formulationsBlue (Invitrogen) for 72 as h, the after reagent. which theCT26 reagent cells waswere added dosed to with the wells.NPs diluted After 90 in min media incubation of the variouswith the formulations reagent, the for fluorescence 72 h, after which from the reag wellsent was was read added using to the a plate wells. reader. After 90 Wells min incubation containing withviable the cells reagent, have athe proportionally fluorescence higherfrom the fluorescent wells was signal, read using and are a plate presented reader. as Wells a proportion containing of a viablenegative cells control have (untreateda proportionally cells). Cellshigher una fluorescffectedent by thesignal, treatment and are show presented close to as 100% a proportion viability, while of a negativelower viability control can (untreated be attributed cells). to Cells toxic unaffected effects of theby the NPs. treatment The results show of close this assay to 100% are shownviability, in whileFigure lower5. Bare viability CML NPscan be do attributed not contain to toxic a therapeutic, effects of andthe NPs. thus The show results higher of viabilitythis assay even are shown at the inhighest Figure concentrations 5. Bare CML NPs of dosed do not NPs. contain The a RTX therapeutic, and RTX /andFA NPs thus show show greater higher toxicity,viability with even an at ICthe50 highestvalues ofconcentrations 0.024 and 0.016 ofµ dosedg/mL, NPs. respectively The RTX (free and RTX RTX/FA was found NPs show to have greate an ICr 50toxicity,of 14.44 with nM atan 72IC h).50 valuesThese valuesof 0.024 are and roughly 0.016 µg/mL, the same, respecti and thevely curves (free RTX appear was similar found overall, to have indicatingan IC50 of 14.44 that their nM at lethality 72 h). Thesemay be values similar arein roughly vivo as well,the same, even thoughand the their curves terminal appear layer similar is diff overall,erent. The indicating disparity that between their lethalitythe free drugmay ICbe50 similarand the in NPvivo IC as50 well,(the free even drug though IC50 theirvalue terminal is roughly layer a factor is different. of 10 less The than disparity the NP betweenversion) the could free be drug due toIC50 release and the kinetics NP IC or50 (the reduced free edrugfficacy IC50 due value to the is roughly adsorption a factor to the of NP 10 substrate.less than the NP version) could be due to release kinetics or reduced efficacy due to the adsorption to the NP substrate. This is only an in vitro assessment, and the NP may have higher potential to be efficacious in vivo, where the NP will aid in accumulation at the target site.

Pharmaceutics 2020, 12, 133 9 of 14

Pharmaceutics 2020, 12, x FOR PEER REVIEW 9 of 13

ThisPharmaceutics is only an2020in, 12 vitro, x FORassessment, PEER REVIEW and the NP may have higher potential to be efficacious in vivo 9 of 13, where the NP will aid in accumulation at the target site.

Figure 5. Cell viability of CT26 cells after 72 h exposure to various NPs. Toxicity is increased greatly inFigure the formulations 5. Cell viability that ofcontain CT26 cellsRTX, after with 72 no h observ exposureed toxicity to various in cells NPs. trea Toxicityted with is increased the uncoated greatly NPs. in Figure 5. Cell viability of CT26 cells after 72 h exposure to various NPs. Toxicity is increased greatly the formulations that contain RTX, with no observed toxicity in cells treated with the uncoated NPs. in the formulations that contain RTX, with no observed toxicity in cells treated with the uncoated NPs. 3.5. Biodistribution 3.5. Biodistribution 3.5.Mice Biodistribution bearing CT26 tumors were dosed with 3 × 200 µL injections of saline, bare CML NPs, RTX Mice bearing CT26 tumors were dosed with 3 200 µL injections of saline, bare CML NPs, RTX NPs, or RTX/FA NPs. One injection was made daily× over the course of 3 days. After the last injection NPs, orMice RTX bearing/FA NPs. CT26 One tumors injection were was dosed made with daily 3 over× 200 the µL course injections of 3 of days. saline, After bare the CML last injectionNPs, RTX (24 h), the animals were sacrificed, the organs and tumors were removed, and imaged on an IVIS (24NPs, h), or the RTX/FA animals NPs. were One sacrificed, injection the was organs made anddaily tumors over the were course removed, of 3 days. and After imaged the last on aninjection IVIS XRMS III system (PerkinElmer). Total radiant flux from each set of organs was acquired using the XRMS(24 h), III the system animals (PerkinElmer). were sacrificed, Total the radiant organs flux and from tumors each were set of removed, organs was and acquired imaged usingon an theIVIS associated software (Living Image), and was used to determine the relative percentage of recovered associatedXRMS III softwaresystem (PerkinElmer). (Living Image), To andtal radiant was used flux to from determine each set the of relative organs percentage was acquired of recovered using the fluorescence from the organs. These values are shown in Figure 6A. fluorescenceassociated software from the (Living organs. Image), These values and was are used shown to indetermine Figure6A. the relative percentage of recovered fluorescence from the organs. These values are shown in Figure 6A.

Figure 6. Biodistribution of NP formulations injected into mice bearing CT26 tumors. (A) Mean relative

flux in each organ across all animals in each treatment group. Liver showed high accumulation, with Figuresmall percentages 6. Biodistribution in the lungs,of NP kidneys, formulations and spleen. injected (B )into Representative mice bearing tumors CT26 from tumors. each ( treatmentA) Mean relative flux in each organ across all animals in each treatment group. Liver showed high groupFigure show 6. Biodistribution increased signal of when NP imagedformulations at a higher injected exposure into time.mice (bearingC) Mean CT26 average tumors. radiance (A) ofMean all accumulation, with small percentages in the lungs, kidneys, and spleen. (B) Representative tumors tumorsrelative imaged flux atin highereach organ exposure across time, all with animal bare,s RTX, in each and RTXtreatment/FA-treated group. organs Liver showing showed higher high from each treatment group show increased signal when imaged at a higher exposure time. (C) Mean averageaccumulation, signal due with to thesmall NPs. percentages *, p < 0.05. in the lungs, kidneys, and spleen. (B) Representative tumors average radiance of all tumors imaged at higher exposure time, with bare, RTX, and RTX/FA-treated from each treatment group show increased signal when imaged at a higher exposure time. (C) Mean organsThe majority showing of higher the NPs average for eachsignal treatment due to the groupNPs. *, accumulatesp < 0.05. in the liver, with small amounts average radiance of all tumors imaged at higher exposure time, with bare, RTX, and RTX/FA-treated present in the lungs, spleen, and kidneys. The heart and tumors show significantly lower amounts of organs showing higher average signal due to the NPs. *, p < 0.05. the injectedThe majority NPs. of While the NPs it is commonfor each treatment to see small grou percentagesp accumulates of the in injectedthe liver, material with small in the amounts tumor, present in the lungs, spleen, and kidneys. The heart and tumors show significantly lower amounts of one ofThe the majority main aims of ofthe this NPs work for each is to treatment further investigate group accumulates the potential in the di ffliver,erence with in uptakesmall amounts in the the injected NPs. While it is common to see small percentages of the injected material in the tumor, tumors.present Toin furtherthe lungs, investigate spleen, and this kidneys. difference, The thehear tumorst and tumors were isolated show significantly from the other lower organs, amounts and of one of the main aims of this work is to further investigate the potential difference in uptake in the the injected NPs. While it is common to see small percentages of the injected material in the tumor, tumors. To further investigate this difference, the tumors were isolated from the other organs, and one of the main aims of this work is to further investigate the potential difference in uptake in the tumors. To further investigate this difference, the tumors were isolated from the other organs, and

Pharmaceutics 2020, 12, x FOR PEER REVIEW 10 of 13 Pharmaceutics 2020, 12, 133 10 of 14 imaged together with a longer exposure. Representative tumors from each treatment group are shownimaged in together Figure with6B. The a longer signal exposure. increases Representativeby at least one tumors order over from the each saline treatment group. group The difference are shown inin radiance Figure6B. from The signalthe NP increases treatment by groups at least oneis diffic orderult over to interpret the saline from group. the Theimage, di ff anderence thus in radianceaverage radiancefrom the of NP the treatment tumor tissue groups was is di examinedfficult to interpretin order fromto quantify the image, the andrelative thus number average of radiance NPs in ofeach the treatmenttumor tissue organ was (Figure examined 6C). inWhile order not to significant, quantify the th relativeere appears number to be of a NPssmall in difference each treatment in the mean organ values(Figure across6C). While the groups, not significant, with the thereRTX appearsand RTX/ toFA be NPs a small showing di fference slightly in thehigher mean average values values, across likelythe groups, due to with the presence the RTX andof the RTX terminal/FA NPs targeting showing layers. slightly Furthe higherrmore, average the values, RTX group likely appears due to theto showpresence the ofmajority the terminal of the targeting tumors layers.as having Furthermore, a higher average the RTX radiance group appears than the to showbare NPs, the majority with the of meanthe tumors value as brought having down a higher by average a single radiance tumor that than shows the bare little NPs, fluorescence. with the mean Overall, value broughtthese are down low sampleby a single sizes, tumor and thatsignificance shows little cannot fluorescence. be drawn Overall,from these these results, are low but sample they may sizes, indicate and significance that this workcannot could be drawn be repeated from these with results, larger groups but they to may fully indicate elucidate that the this usefulness work could of these be repeated targeting with ligands larger ongroups the NPs. to fully elucidate the usefulness of these targeting ligands on the NPs. OrgansOrgans and and tumors tumors from from all all treatment treatment groups groups were were prepared prepared for for histology, histology, paraffin paraffin embedded, embedded, sliced,sliced, and and stained stained with with H H & & E E and and CC3. CC3. Tumors Tumors st stainedained with with CC3, CC3, shown shown in in Figure Figure 77,, showed some toxicitytoxicity from from the the RTX RTX and and RTX/FA RTX/FA treatment groups groups,, with certain area area of of the the tumor tumor showing showing brown brown stainingstaining indicative indicative of of apoptosis. apoptosis. Adjacent Adjacent slices slices stained stained with with H H & & E E potentially potentially show show the the same same trend, trend, withwith reduced reduced dark dark purple purple color color indicating indicating reduc reduceded nuclear nuclear staining. staining. Accumulation Accumulation of NPs of NPs could could be inferredbe inferred from from higher higher fluorescent fluorescent signal signal in inaccomp accompanyinganying areas areas (from (from adjacent adjacent unstained unstained slides), slides), measuredmeasured using a LI-CORLI-COR OdysseyOdyssey fluorescentfluorescent scanner. scanner. Here Here the the heterogeneous heterogeneous uptake uptake of of NPs NPs can can be beseen seen in in all all treated treated tumor tumor samples. samples. Although Although only only qualitative qualitative duedue toto smallsmall samplesample size, the general general trendtrend ofof overalloverall tumor tumor accumulation accumulation represented represented by theseby these cross-sections cross-sections is consistent is consistent with wholewith whole tumor tumorex vivo ex imaging. vivo imaging. Importantly, Importantly, co-localization co-localizati of theon fluorescence of the fluorescence signal with signal regions with of regions apoptosis of apoptosisimply NP-mediated imply NP-mediated delivery of thedelivery RTX. Thisof the is likelyRTX. notThis the is onlylikely contributor not the only to the contributor apoptotic regions,to the apoptoticthough, as regions, the overall though, larger as size the ofoverall the tumors larger indicates size of the that tumors there could indicates be apoptotic that there regions could duebe apoptoticto deoxygenation regions due from to rapiddeoxygenation growth of from the tumor.rapid growth The sample of the sizestumor. were The small sample for sizes this work,were small with forinconsistent this work, tumor with volumesinconsistent across tumor the groups.volumes In acro futuress the work, groups. the sample In future sizes work, would the besample larger sizes with wouldsimilar be tumor larger sizes with across similar all tumor of the groups,sizes across most all likely of the starting groups, at amost smaller likely tumor starting size at to a eliminate smaller tumorpotential size tumor to eliminate core apoptosis potential due tumor to lack core of apoptosis oxygen/nutrients. due to lack of oxygen/nutrients.

FigureFigure 7. TumorTumor toxicity toxicity associated associated with with NP NP treatm treatments.ents. Cleave-caspase Cleave-caspase 3 3and and H H & & E E stained stained representativerepresentative tumors tumors show show potential potential inin vivo vivo toxitoxicitycity of of the the RTX RTX and and RTX/FA RTX/FA formulations. Scale Scale bar bar indicatesindicates 1 1 mm. mm. Brown Brown color color indicates indicates regions regions of of ap apoptosis,optosis, which which are are more more prevalent prevalent in in the the RTX RTX and and RTX/FA-treatedRTX/FA-treated organs. Unstained Unstained slides slides were were scanned scanned at at lower lower resolution resolution using using a a LI-COR LI-COR Odyssey Odyssey fluorescentfluorescent scanner scanner at at the the 700 700 nm nm se setting,tting, which which appear appear to to show show higher higher signal signal in in regions regions that that showed showed higherhigher cell cell death death in in similar similar slices. slices.

Pharmaceutics 2020, 12, 133 11 of 14 Pharmaceutics 2020, 12, x FOR PEER REVIEW 11 of 13

ToTo investigate investigate systemic systemic toxicity toxicity due due to to NP NP trea treatment,tment, clearance clearance organs organs from from a a representative representative animalanimal from from each each treatment treatment group group were were stained stained with with H H& &E. E.Images Images from from each each organ organ are areshown shown in Figurein Figure 8. The8. The accumulation accumulation in these in these organs, organs, indicat indicateded from the from IVIS the imaging, IVIS imaging, does not does seem not to seem convey to noticeableconvey noticeable systemic systemic toxicity. toxicity. Spleen Spleenuptake uptakeof the ofNPs the was NPs observed was observed (Figure (Figure 6A), 6withA), with little little to no to differenceno difference in the in red the redor white or white pulp pulp regions regions of the of ti thessue tissue samples. samples. In the In kidneys, the kidneys, another another region region that displayedthat displayed fluorescence fluorescence from fromthe NPs, the NPs, the thetissue tissue is near is near identical identical to tothe the saline saline control control samples. samples. RaltitrexedRaltitrexed was was developed developed in in part part to to reduce reduce renal renal toxicity toxicity of of previous previous generations generations of of this this class class of of drugsdrugs [9,10]. [9,10]. This, This, in incombination combination with with its itsattachment attachment to a to nanocarrier, a nanocarrier, may may help help further further reduce reduce off- targetoff-target toxicity toxicity of the of therapeutic. the therapeutic.

Figure 8. Clearance organ toxicity associated with NP treatments. Images of organs stained with H & Figure 8. Clearance organ toxicity associated with NP treatments. Images of organs stained with H E& show E show little little to tono no difference difference between between saline saline treate treatedd animals animals versus versus NP NP trea treatedted animals. animals. Scale Scale bar bar indicatesindicates 100 100 µm.µm.

4.4. Conclusions Conclusions TheThe NPsNPs describeddescribed in in this this work work highlight highlight the ease the of ease LbL assemblyof LbL assembly for attachment for attachment of therapeutics of therapeuticsand targeting and ligands. targeting By ligands. varying By the varying terminal the layer terminal of the layer NPs, of we the assessed NPs, we the assessed cell uptake the cell and uptakebiodistribution and biodistribution of coated NPs of coated versus NPs uncoated versus controls. uncoated Based controls. on flow Based cytometry on flow and cytometry fluorescence and fluorescencemicroscopy, microscopy, we observed we enhanced observed uptake enhanced due uptake to the due RTX to and theRTX RTX/ FAand terminal RTX/FA layers.terminal The layers. NPs Thewere NPs also were functional, also functional, in that they in showedthat they cytotoxic showed e ffcytotoxicects on CT26 effects CRC on cells. CT26 When CRC administeredcells. When administeredintravenously, intravenously, the different formulations the different accumulated formulations in various accumulated organs andin various the implanted organs tumorsand the ex implantedvivo. The animalstumors ex treated vivo. with The RTXanimals and treated RTX/FA wi NPsth RTX showed andslightly RTX/FA higher NPs showed tumor accumulation slightly higher on tumoraverage accumulation than uncoated on NPs. average Histology than showed uncoated no observedNPs. Histology off-target showed toxicity ofno the observed formulations, off-target with toxicitypossible of higher the formulations, accumulation ofwith the possible fluorescent high NPser accumulation in the unstained of slidesthe fluorescent of the RTX andNPs RTXin the/FA unstained slides of the RTX and RTX/FA treatment groups. With higher samples sizes and higher dosing of the NPs, these formulations may be optimized for improved uptake in tumors over- expressing α-folate receptors.

Pharmaceutics 2020, 12, 133 12 of 14 treatment groups. With higher samples sizes and higher dosing of the NPs, these formulations may be optimized for improved uptake in tumors over-expressing α-folate receptors.

Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4923/12/2/133/s1, Figure S1: Transmission electron microscopy images of formed RTX NPs. Scale bar in lower magnification (larger image)—50 nm, scale bar in higher magnification image (inset)—20 nm. Author Contributions: J.G.R. and C.S. designed the experiments. J.G.R., A.N.D. and M.R.L. ran the experiments and collected the data. J.G.R. and C.S. wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the NIH NIGMS as a Maximizing Investigators’ Research Award, 1R35GM119839-01 (C.S.), and Oregon State University College of Pharmacy Start-up Funds. Acknowledgments: The authors would like to acknowledge Hayden Winter and Andrea Goforth for assistance with TEM, the Histopathology Shared Resource and Advanced Light Microscopy Core at Oregon Health and Science University, as well as the work of Yingjun Huang and Sage Morgillo, who both contributed to this work through the CURE program at OHSU. Conflicts of Interest: The authors declare no conflict of interest.

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