Monitoring the Fate of Orally Administered PLGA Nanoformulation for Local Delivery of Therapeutic Drugs

One of the goals of the pharmaceutical sciences is the amelioration of targeted drug delivery. In this context, nanocarrier-dependent transportation represents an ideal method for confronting a broad range of human disorders. In this study, we investigated the possibility of improving the selective release of the anti-cancer drug paclitaxel (PTX) in the gastro-intestinal tract by encapsulating it into the biodegradable nanoparticles made by FDA-approved poly(lactic-co-glycolic acid) (PLGA) and coated with polyethylene glycol to improve their stability (PLGA-PEG-NPs). Our study was performed by combining the synthesis and characterization of the nanodrug with in vivo studies of pharmacokinetics after oral administration in mice. Moreover, fluorescent PLGA-nanoparticles (NPs), were tested both in vitro and in vivo to observe their fate and biodistribution. Our study demonstrated that PLGA-NPs: (1) are stable in the gastric tract; (2) can easily penetrate inside carcinoma colon 2 (CaCo2) cells; (3) reduce the PTX absorption from the gastrointestinal tract, further limiting systemic exposure; (4) enable PTX local targeting. At present, the oral administration of biodegradable nanocarriers is limited because of stomach degradation and the sink effect played by the duodenum. Our findings, however, exhibit promising evidence towards our overcoming these limitations for a more specific and safer strategy against gastrointestinal disorders.


Introduction
The generation of therapeutic methods that can improve the organ specificity of many different types of therapeutic agents is one of the major challenges of 21st-century pharmacology [1].

PLGA-PEG-RhB Synthesis
For activation of the carboxylic groups of PLGA, the polymer was dissolved in DCM (15 mL, 40 mg/mL) and the reaction was performed in a round flask for 4 h (250 rpm, RT, under inert gas) after the addition of DCC (30 mg) and NHS (15 mg). Then, the solution was diluted with diethyl ether, and the polymer was collected after precipitation (20 min, 6200× g, 4 • C) and evaporation of the residual organic phase. Next, the conjugation with NH 2 -PEG-NH 2 (molar ratio PEG/PLGA = 2.7) was performed in 10 mL of CHCl 3 overnight (250 rpm, RT, under inert gas). To get rid of the unconjugated PEG, methanol (30 mL) was added, and the polymer was collected after precipitation twice [25]. The residual organic phase was then evaporated under reduced pressure, and the weight yield calculated (68.99 ± 14.94%). The degree of labeling was determined using the colorimetric ninhydrin assay (primary amine detection): Notably, 15 mg of the product (PLGA-PEG) was solubilized in DMSO (400 µL) then diluted with 100 µL of ninhydrin solution (3.5 mg/mL). Ethanolamine (18.75 mg/mL) and PLGA solutions were used as positive and negative controls, respectively. The samples were then incubated for 1 h at 65 • C (400 rpm), and the colored conjugate was detected by UV-Vis spectroscopy (Spectrophotometer Fluormax-Horiba Scientific, Rome, Italy) (λ = 600 nm), and the yield calculated using a calibration curve of ethanolamine standards. The obtained degree of labeling was 71.90 ± 12.70%.
Finally, RhB (molar ratio RhB/PLGA = 6.25) was added to the PLGA-PEG solution in CHCl 3 (15 mL) with an excess of DCC and DMAP. The reaction was carried out overnight (250 rpm, RT, under inert gas). At the end of the reaction, the solution was evaporated, and the product was solubilized with DCM (10 mL) and purified thrice by precipitation (20 min, 4 • C, 6200× g) after diethyl ether addition (20 mL). The reaction yield was 38.16 ± 13.50%. RhB linked was detected by fluorescence spectrometry (λ exitation 555 nm; λ emission 574 nm) and the degree of labeling calculated by the comparison with a calibration curve (1.43 ± 0.46 µg/mg).

Synthesis of PLGA-PEG-RhB-NPs and PTX-PLGA-PEG-RhB-NPs
NPs were synthesized according to the single emulsion method. PLGA-PEG-RhB solution (1 mL, 25 mg/mL in DCM) was emulsified with 8 mL of PVA 2% solution by sonicating twice (Sonifier Sound, Branson Ultrasonics, Shanghai, China; 30 s and 38% intensity) in an ice bath. The product was transferred immediately into a solution of PVA 2% (16 mL), allowing the organic solvent to evaporate (4 h, RT, 750 rpm). For PTX-PLGA-PEG-RhB-NPs synthesis, 5 mg of PTX was dissolved along with the polymer. After curing, NPs were collected by centrifugation at 19,500× g, 20 min, 4 • C (Heraeus Fresco 21; Thermo Fisher Scientific, Göteborg, Sweden), washed thrice with double distilled water and freeze-dried through an Alpha 1-2 LD freeze drier (Christ, Memmingen, Germany) at 0.500 mbar, −53 • C, 12 h, without the addition of any cryoprotectant. The process yield was calculated after the freeze-drying process, as the ratio between collected NPs and starting raw materials.
The amount of PTX loaded in the NPs was determined by HPLC analysis with UV detection (Waters Associates, Milford, MA, USA, model 2487 Variable Wavelength Detector, Wavelength: 230 nm). Briefly, 0.1 mL of the NPs solution was spiked with 5 µg of IS and extracted with 0.5 mL of CH 3 CN. After vortex for 10 s, samples were centrifuged at 13,000 rpm for 10 min. The organic phase was separated and dried under nitrogen, and the residues were dissolved with 250 µL of the mobile phase. Fifty microliters of the reconstituted samples was injected into the HPLC system. The apparatus was equipped with a Symmetry C18 column (5 µm, 4.6 × 150 mm), the mobile phase was composed of 50% ammonium acetate buffer (0.01 M, pH 5), 40% acetonitrile, and 10% methanol with a flux rate of 1.3 mL/min and 30 min run time. NPs solution without drug was used to prepare the calibration curve by the addition of PTX in the range 10-100 µg/mL.

Dynamic Light Scattering (DLS) and Zeta Potential Measurements
All NPs were characterized in terms of size, size distribution, and Zeta potential through dynamic light scattering (DLS, Zetasizer Nano ZS; Malvern Instruments, Cambridge, UK) in ultrapure water at 1 mg/mL, at 25 • C. The scattered light from the NPs in suspension was used to calculate NPs' hydrodynamic diameter considering medium viscosity. NPs size distribution was described by the polydispersity index (PDI), where PDI ≤ 0.2 corresponds to the monodisperse NPs population.

Scanning Transmission Electron Microscopy (STEM) Analysis
PLGA-PEG-RhB-NPs were observed by a Zeiss SEM-FEG Gemini 500, operating at 30 kV in scanning transmission electron microscopy (STEM) mode (Zeiss, Germany). The NPs suspension was deposited onto a formvar-coated 200-mesh copper grid (Ted Pella, CA, USA), negatively stained with 1% uranyl acetate and allowed to dry before examination.

In Vitro Release Studies
PTX-PLGA-PEG-RhB-NPs equivalent to 8.75 µg of PTX were suspended in 1 mL of Tween/PBS and incubated at 37 • C with constant agitation. At predetermined time points (0.5, 1, 2, 4, 6, and 24 h), the suspension was centrifuged at 19,500× g for 15 min to separate NPs pellets and supernatants. PTX in the collected supernatants was analyzed with HPLC equipped with a UV detector (1260 Infinity II Series, Agilent Technologies, Palo Alto, CA, USA) and an Atlantis C18 column (25 cm × 4.6 mm, particle size 5 µm) (Supelco, St. Louis, MO, USA). The mobile phase was a mixture of acetonitrile and water (50:50) run in the isocratic mode at a flow rate of 1 mL/min. PTX was detected at 227 nm. PTX quantitation was performed using a calibration curve in a range of 1.25-40 µg/mL, and the results were expressed as a percentage of cumulative release (mean ± SD; n = 4).

Cells
Carcinoma colon cells (CaCo 2 ) are a cell line derived from human colorectal adenocarcinoma and were purchased from ATCC ® HTB-37™. CaCo 2 cells were grown as a single layer in adhesion in DMEM-High Glucose (Dulbecco s Modified Eagle Medium High Glucose-Biowest, Nuaillé, France) with the addition of 10% of fetal bovine serum (FBS) and 1% l-glutamine (200 mM), 100 U/mL penicillin, 0.1 mg per mL streptomycin. Cells were maintained at 37 • C and in a 5% CO 2 humidified atmosphere. The ability of PLGA-PEG-RhB-NPs and free RhB to be internalized by CaCo 2 cells was evaluated. For each condition, the experiment was conducted in triplicate. Cells were seeded at a density of 40,000 cells/well on 13 mm diameter slides in 24-well plates. Forty-eight hours after sowing, CaCo 2 cells were incubated with PLGA-PEG-RhB-NPs (100 µg/mL) and with RhB free (0.06 µg/mL) for 1, 4, and 24 h, while the wells destined for control did not receive any treatment. After incubation, the cells were washed with phosphate-buffer saline (PBS) three times and fixed with a 4% paraformaldehyde solution dissolved in PBS (0.1 M, pH 7.4) for 40 min and the vital nuclear dye Hoechst 33258 (2 µg/mL) was added to each well for 45 min. The slides thus obtained were assembled and analyzed with the Olympus BX51 epifluorescence microscope (Olympus, Tokyo, Japan). All acquisition parameters, including laser settings, were kept constant during all scans. To evaluate a possible cytotoxic effect of PLGA-PEG-RhB-NPs and free RhB, the metabolic activity of CaCo 2 cells was evaluated by RealTime-Glo™ MT Cell Viability Assay (Promega, Madison, WI, USA). Cells were seeded at 16,000 cells/well in 96 opaque-walled tissue culture plates and maintained at 37 • C. The RealTime-Glo reagents were added at the same time as the test compounds, according to the manufacturer's protocol. At selected time points (4 and 24 h), the cell viability was monitored by a plate-reading luminometer (GloMax ® Discover Microplate Reader, Promega, Madison, WI, USA). For each condition, 6 replicates were prepared. The viability was expressed as a percentage compared to non-treated cells.

Animals
All procedures involving animals and their health were conducted so as to minimize the number of mice used and their collateral suffering, in accordance with institutional guidelines, national laws (DL n. All animals were housed in SPF (specific pathogen-free) conditions. The housing rooms had a temperature of 22 ± 1 • C, relative humidity values ± of 50 ± 10% and a 12 h light/dark cycle. Furthermore, the animals were kept in cages with free access to water and food.

Treatments
As regards pharmacokinetics, six weeks old CD1 mice were treated by oral gavage with 20 mg/kg of Cremophor PTX (n = 16) or PTX-PLGA-PEG-RhB-NPs (n = 16), three animals were treated with saline solution as control. Blood was collected in heparinized tubes, 30 min, 1, 4, and 24 h after treatment (4 animals for each group) and centrifuged to obtain plasma. After the mice were sacrificed, the stomach, duodenum, colon, and liver were collected and stored at −20 • C until analysis.
For the biodistribution studies, we recruited 5 animals for each experimental group. To reduce background fluorescence due to the food, mice were fed an AIN-76A diet without alfalfa (Mucedola s.r.l., Settimo Milanese, Italy) for two weeks before the analyzes. The dose of the different formulations was standardized on the quantity of RhB present and fixed at 0.6 mg/kg mouse based on previous experimental studies. Vehicle treated mice received the same volume of saline solution. Mice were sacrificed at 1, 4, or 24 h to follow the fate of RhB. The sacrifice was performed by cervical dislocation, and the stomach, intestine, liver, and blood were collected to perform ex vivo analyses. Plasma for fluorometric analysis was obtained from the blood collected. Finally, a piece of liver and GI tracts of three animals for each experimental group were frozen at −80 • C for cryostatic sections and histological analysis.

Molecular Imaging and Histology
The in vivo biodistribution of the different formulations was monitored over time using an optical fluorescence imaging system (IVIS Lumina XRMS, PerkinElmer, Waltham, MA, USA). Ex vivo scans of organs from mice sacrificed at 1, 4, and 24 h after the treatment were performed by the same instrument. The following acquisition parameters were used: Excitation filter: 580 nm, emission filter: 620 nm, exposure time: Auto, binning factor: Medium, f/Stop: 2, Field of View: D (for the gastro line-intestinal), C (for peripheral organs). Very importantly, the Living Image Software 4.3.1 (Perkin Elmer, Waltham, MA, USA) conjugated with the spectral unmixing system was used to separate the RhB signal from tissue autofluorescence, image processing, and fluorescence signal quantification analysis.
Longitudinal sections of 20 µm of thickness were prepared and then, after adhesion in glass slides, were incubated with a PBS solution of Hoechst 33258 (2 µg/mL, Sigma-Aldrich) for 45 min and, after three washes in PBS, observed at the Microscopy Virtual Slide (Olympus, Tokyo, Japan), to obtain rapid organ scans of the whole section with high anatomical resolution.

NPs Characterization from Homogenates and Biological Fluids
The fluorescence of PLGA-PEG-RhB-NPs was analyzed in tissues explanted from previously treated animals. The analyzed tissues were liver, stomach, intestine, and plasma. Each tissue sample was weighed, homogenized in PBS 1X according to a 1:4 weight ratio, and centrifuged at 1200 rpm for 10 min at 4 • C. For all samples, the analysis was performed using the Infinite ® M200 multimode plate reader exciting at the wavelength of 500 nm and recording the signal at a range of emission wavelength from 550 to 560 nm. For the stability studies in solutions mimicking gastrointestinal fluids, the NPs were incubated in stock solutions as previously described [26].

Pharmacokinetics
The total concentration of PTX in the different biological matrices was determined by HPLC-UV, as previously described [27]. For the determination of PTX in organs, tissues were previously homogenized in 0.2 M CH 3 COONH 4 pH 4.5. Each study sample (0.3 mL for plasma and 0.5 mL for homogenate tissues) was assayed together with five points of a standard calibration curve prepared in the corresponding control biological matrix obtained from untreated mice at concentrations ranging from 0.05 to 5 µg/sample. The limits of quantification (LOQ) were 0.16 µg/mL and 0.6 µg/g for plasma and organs, respectively.

Statistical Analysis
All data were expressed as mean ± SD, Student's t-test and p values were done using the GraphPad Prism version 6.00 for Windows (Graph-Pad Software, San Diego, CA, USA).

NPs Synthesis and Characterization
The protocol for the polymer modification was set up, and PLGA-PEG-RhB was employed to synthesize the NPs to be used as carriers for PTX. The covalently bound RhB chromophore allowed the tracking of the PLGA fate in vivo. The single emulsion method allowed us to obtain both unloaded and PTX-loaded NPs, having controlled size and homogenous size distributions ( Table 1). As measured by DLS analysis, the encapsulation of PTX did not significantly change the dimensional features of NPs. Moreover, a negative Z-potential was observed as opposed to PLGA-PEG-NPs, where the free amines of PEG determined the surface properties. However, a small difference in net negative charge values between PLGA-PEG-RhB-NPs and PTX-PLGA-PEG-RhB-NPs was attributable to the intramolecular reorganization of portions of the polymer chains due to hydrophobic interaction with PTX. PTX loaded NPs were further characterized by STEM, which showed pseudospherical shaped nanoparticles with the size around 200 nm ( Figure S1). PTX encapsulation efficiency detected by HPLC analysis was 10.87 ± 1.13%, while the calculated loading efficiency was 4.77 ± 0.15%, in line with previous studies [28]. The drug release performance was evaluated, and the test was conducted in PBS containing 0.2 v/v % Tween 80, considering both its poor solubility and the analysis detection limits [29]. As previously reported with similar experimental settings, a fast PTX dissolution was observed (80% within 4 h) ( Figure S2) [30]. Table 1. NPs characterization and process yield. Data represent mean ± SD (n = 3).

Formulations
Size

Stability in Mimicking Biological Fluids
The first study was carried out to verify the potential role of PEG on the fate of PLGA-NPs in solutions mimicking saliva, gastric, and proximal intestinal fluid, respectively ( Figure 1A,B). Longitudinal measurement of the NPs size by DLS was possible because, diversely from the serum, they are very poor in macromolecules, and the interference on the recording is almost zero. Figure 1A shows that the presence of the PEG confers to PLGA-NPs a long-lasting stability for at least 24 h after incubation. In contrast, the lack of PEG rapidly induced aggregation in NPs incubated in gastric fluid and, to a lesser extent in intestinal fluid. Since we aimed to conserve considerable stability up until the large intestine, we decided to carry on our studies using pegylated PLGA-NPs exclusively. attributable to the intramolecular reorganization of portions of the polymer chains due to hydrophobic interaction with PTX. PTX loaded NPs were further characterized by STEM, which showed pseudospherical shaped nanoparticles with the size around 200 nm ( Figure S1). PTX encapsulation efficiency detected by HPLC analysis was 10.87 ± 1.13%, while the calculated loading efficiency was 4.77 ± 0.15%, in line with previous studies [28]. The drug release performance was evaluated, and the test was conducted in PBS containing 0.2 v/v % Tween 80, considering both its poor solubility and the analysis detection limits [29]. As previously reported with similar experimental settings, a fast PTX dissolution was observed (80% within 4 h) ( Figure S2) [30].

Stability in Mimicking Biological Fluids
The first study was carried out to verify the potential role of PEG on the fate of PLGA-NPs in solutions mimicking saliva, gastric, and proximal intestinal fluid, respectively ( Figure 1A,B). Longitudinal measurement of the NPs size by DLS was possible because, diversely from the serum, they are very poor in macromolecules, and the interference on the recording is almost zero. Figure  1A shows that the presence of the PEG confers to PLGA-NPs a long-lasting stability for at least 24 h after incubation. In contrast, the lack of PEG rapidly induced aggregation in NPs incubated in gastric fluid and, to a lesser extent in intestinal fluid. Since we aimed to conserve considerable stability up until the large intestine, we decided to carry on our studies using pegylated PLGA-NPs exclusively.  Then, the stability of the RhB conjugation was performed by measuring the dye release in the same conditions (Figure 2A). After 24 h of incubation, the released RhB was below 15% in all cases, suggesting the system reliability for in vitro and in vivo NPs tracking. Additionally, as reported in Figure 2B, the fluorescence intensity of PLGA-PEG-RhB-NPs was not greatly affected by the incubation media showing that, once conjugated, the dye abolishes its pH-dependent emission properties [31]. Then, the stability of the RhB conjugation was performed by measuring the dye release in the same conditions (Figure 2A). After 24 h of incubation, the released RhB was below 15% in all cases, suggesting the system reliability for in vitro and in vivo NPs tracking. Additionally, as reported in Figure 2B, the fluorescence intensity of PLGA-PEG-RhB-NPs was not greatly affected by the incubation media showing that, once conjugated, the dye abolishes its pH-dependent emission properties [31].

Pharmacokinetics
To understand the possible influence of the nanoformulate on the biodistribution of drugs, we performed a pharmacokinetic study of the well-known anti-cancer agent, paclitaxel, administered orally as a free drug (PTX) or loaded into NPs (PTX-PLGA-PEG-RhB-NPs). PTX release Figure 3 shows the levels of PTX measured in the stomach (A), in the duodenum (B) and in the colon (C) of mice sacrificed 30 min, 1 and 4 h after treatment with PTX free (blue bars) or with PTX-PLGA-PEG-RhB-NPs (red bars). The drug concentration in the samples collected at 24 h after treatment resulted under the limit of detection. Overall, the drug concentration measured in all the gastrointestinal tissues was comparable between the two formulations and higher in the stomach and duodenum compared to the colon. Despite the comparable tissue drug level, interestingly, the nano-formulation reduced the systemic absorption of the drug. The measurement of the plasmatic levels, in fact, showed a clear reduction of systemic absorption of PTX when administered as PTX-PLGA-PEG-RhB-NPs, leading to a consequent reduction of liver accumulation, as shown in Figure 3D,E. Similarly to previous results achieved by our group [32,33], no evidence of acute toxicity was observed in mice receiving the single administration of PTX-PLGA-PEG-RhB-NPs.

Pharmacokinetics
To understand the possible influence of the nanoformulate on the biodistribution of drugs, we performed a pharmacokinetic study of the well-known anti-cancer agent, paclitaxel, administered orally as a free drug (PTX) or loaded into NPs (PTX-PLGA-PEG-RhB-NPs). PTX release Figure 3 shows the levels of PTX measured in the stomach (A), in the duodenum (B) and in the colon (C) of mice sacrificed 30 min, 1 and 4 h after treatment with PTX free (blue bars) or with PTX-PLGA-PEG-RhB-NPs (red bars). The drug concentration in the samples collected at 24 h after treatment resulted under the limit of detection. Overall, the drug concentration measured in all the gastrointestinal tissues was comparable between the two formulations and higher in the stomach and duodenum compared to the colon. Despite the comparable tissue drug level, interestingly, the nano-formulation reduced the systemic absorption of the drug. The measurement of the plasmatic levels, in fact, showed a clear reduction of systemic absorption of PTX when administered as PTX-PLGA-PEG-RhB-NPs, leading to a consequent reduction of liver accumulation, as shown in Figure 3D,E. Similarly to previous results achieved by our group [32,33], no evidence of acute toxicity was observed in mice receiving the single administration of PTX-PLGA-PEG-RhB-NPs. Pharmaceutics 2019, 11, x 9 of 17

NPs Biodistribution and Nanosafety
To better understand the mechanisms that govern the different behavior of the free and NPsencapsulated drug, we decided to monitor the NPs transit at the digestive tract level, the interactions with the gastric and intestinal structures, the possible transition to the bloodstream and penetration into filter organs by marking PLGA with a fluorescent molecule, RhB. Since the monitoring of biodegradable NPs through an indirect method, such as fluorescence analysis, presents the risk of artifacts caused by the dye release or degradation [34], all studies were carried out by comparing animals that received PLGA-PEG-RhB-NPs with those animals treated with the same dose of free RhB. As the in vitro studies suggested, the stability and reliability of this indirect approach (see Figure  2), enable us to obtain further evidence from the biodistribution study that explains the different behavior between an orally administered small molecule and our nanocarrier. Figure 4A shows the distribution of the signal associated with the GI tract in animals sacrificed 1, 4, and 24 h after treatment with the vehicle (left) or RhB (upper blue panel), and PLGA-PEG-RhB-NPs (lower red panel), respectively. The power of the excitation laser was set in vehicle-treated mice to avoid any possible overlapping between RhB and tissue autofluorescence. In each panel, it is possible to see an upper region shaped like a sack, which represents the stomach. The small intestine is the snake-like shape in the middle. The enlargement in the last part of the intestine includes cecum, colon, and rectum and is called "large intestine". The signal associated with RhB is clearly detectable in both groups receiving the same amount of dye. As expected, in both groups, it is also possible to see a progressive decay of signal and a shift from the distal part of the GI tract. However, in mice treated with NPs, the presence of the dye was more persistent, in particular in the distal part of the small intestine and in the large intestine. The quantification of the signal ( Figure 4B) confirmed this observational study: in particular, the levels of the signal in the intestine at the 4th hour after the treatment was markedly higher in animals receiving the nanoformulation.
To better understand the interaction between PLGA-PEG-RhB-NPs and the GI tract structures, histological evaluation was carried out, also exploiting the presence of RhB to track them along the anatomic path and their development in time. Even in this case, the same doses of RhB free were injected into a further group of mice to exclude any possible misinterpretation of the results due to the possible loss of NPs stability and the release of the dye.

NPs Biodistribution and Nanosafety
To better understand the mechanisms that govern the different behavior of the free and NPs-encapsulated drug, we decided to monitor the NPs transit at the digestive tract level, the interactions with the gastric and intestinal structures, the possible transition to the bloodstream and penetration into filter organs by marking PLGA with a fluorescent molecule, RhB. Since the monitoring of biodegradable NPs through an indirect method, such as fluorescence analysis, presents the risk of artifacts caused by the dye release or degradation [34], all studies were carried out by comparing animals that received PLGA-PEG-RhB-NPs with those animals treated with the same dose of free RhB. As the in vitro studies suggested, the stability and reliability of this indirect approach (see Figure 2), enable us to obtain further evidence from the biodistribution study that explains the different behavior between an orally administered small molecule and our nanocarrier. Figure 4A shows the distribution of the signal associated with the GI tract in animals sacrificed 1, 4, and 24 h after treatment with the vehicle (left) or RhB (upper blue panel), and PLGA-PEG-RhB-NPs (lower red panel), respectively. The power of the excitation laser was set in vehicle-treated mice to avoid any possible overlapping between RhB and tissue autofluorescence. In each panel, it is possible to see an upper region shaped like a sack, which represents the stomach. The small intestine is the snake-like shape in the middle. The enlargement in the last part of the intestine includes cecum, colon, and rectum and is called "large intestine". The signal associated with RhB is clearly detectable in both groups receiving the same amount of dye. As expected, in both groups, it is also possible to see a progressive decay of signal and a shift from the distal part of the GI tract. However, in mice treated with NPs, the presence of the dye was more persistent, in particular in the distal part of the small intestine and in the large intestine. The quantification of the signal ( Figure 4B) confirmed this observational study: In particular, the levels of the signal in the intestine at the 4th hour after the treatment was markedly higher in animals receiving the nanoformulation.
To better understand the interaction between PLGA-PEG-RhB-NPs and the GI tract structures, histological evaluation was carried out, also exploiting the presence of RhB to track them along the anatomic path and their development in time. Even in this case, the same doses of RhB free were injected into a further group of mice to exclude any possible misinterpretation of the results due to the possible loss of NPs stability and the release of the dye.  Figure 5A, upper panels, shows representative images taken from gastric sections of mice sacrificed 1, 4, and 24 h after PLGA-PEG-RhB-NPs administration. Although a progressive reduction of the signal can be seen, it is important to underline that the anatomical localization of the signal remains almost exclusively confined outside the gastric cells that are characterized by the intense blue staining due to the presence of the nuclear dye Hoechst 33258. Opposite, the RhB alone, Figure  5A lower panels, deeply penetrated inside the gastric parenchyma as clearly evidenced by the purple staining due to the merge between the red and the blue signal. This is more pronounced at the first hour and, interestingly, it does not involve the whole structure of the stomach, but it is almost confined to the superficial region of the gastric mucosa (left part of the picture at 1 h). At the 4th hour, the signal is lower but more penetrated and homogeneously spread in the parenchyma, whereas after one day, as already demonstrated by ex vivo quantification (Figure 4B), the fluorescent intensity strongly decreased. The different pattern of staining shown at the 4th and 24th hour strongly suggests that, in spite of the gastric activity, PLGA-PEG-RhB-NPs remain stable enough to avoid the release of the free dye. This is in line with the results reported in Figure 1A by DLS in solutions mimicking gastric juice.  Figure 5A, upper panels, shows representative images taken from gastric sections of mice sacrificed 1, 4, and 24 h after PLGA-PEG-RhB-NPs administration. Although a progressive reduction of the signal can be seen, it is important to underline that the anatomical localization of the signal remains almost exclusively confined outside the gastric cells that are characterized by the intense blue staining due to the presence of the nuclear dye Hoechst 33258. Opposite, the RhB alone, Figure 5A lower panels, deeply penetrated inside the gastric parenchyma as clearly evidenced by the purple staining due to the merge between the red and the blue signal. This is more pronounced at the first hour and, interestingly, it does not involve the whole structure of the stomach, but it is almost confined to the superficial region of the gastric mucosa (left part of the picture at 1 h). At the 4th hour, the signal is lower but more penetrated and homogeneously spread in the parenchyma, whereas after one day, as already demonstrated by ex vivo quantification ( Figure 4B), the fluorescent intensity strongly decreased. The different pattern of staining shown at the 4th and 24th hour strongly suggests that, in spite of the gastric activity, PLGA-PEG-RhB-NPs remain stable enough to avoid the release of the free dye. This is in line with the results reported in Figure 1A by DLS in solutions mimicking gastric juice.
The stability of the nanoparticles inside the stomach is essential to transport any encapsulated drug to the intestine. In Figure 4B, the signal measurement along the whole small intestine was evaluated, whereas histological analysis was focused on the more proximal part of the intestine, the duodenal region. The duodenum is one of the most critical portions of the GI for the absorption of metabolites and drugs. The active uptake by mucosae villi and Peyer's patches allows the absorption of many substances into the bloodstream and their consequent systemic distribution. Even if this process is required to provide energy and nutrients and to distribute therapeutic agents orally administered, it can be a hurdle for a localized gut delivery. In Figure 3, we have reported that the encapsulation in PLGA-PEG-RhB-NPs dramatically reduces the PTX absorption. This suggests that these kinds of NPs are able to pass through this first part of the intestine, maintaining their stability. Representative images from coronal sections of the duodenum from mice sacrificed 1 h after the treatment (lower panel on the left and higher magnification right in Figure 5B) shows that NPs are in the lumen and inside the intervilli space but are not absorbed by mucosa. A deeper interaction with villi can be seen at the 4th hour after treatment. However, even in this case, the red and blue signals are close but do not overlap. An overlapping was indeed clearly seen in distinction from mice treated with the same amount of RhB free at least up to the first 4 h after ingestion. A higher magnified picture furthermore confirms the restricted interaction between the red signal and the peripheral region of villi 1 h after treatment. As reported in the measurement of the intestinal levels by ex vivo scanning ( Figure 4B), an almost complete disappearance of the red signal can be seen 24 h after the treatment in both experimental groups.
By histology, we found that RhB-free treated mice showed deep red staining in villi, whereas the red signal remained in the lumen of the intestinal tube in animals receiving RhB with NPs. Since enterocytes in villi are tightly connected to the vessels, it is, therefore, possible to hypothesize that RhB can easily penetrate the circulatory tree. To confirm that NPs can dramatically reduce the passage from the small intestine to the bloodstream, we compared the RhB levels both in plasma and in the liver of mice treated with PLGA-PEG-RhB-NPs or RhB free. Figure 6A, where plasmatic levels of RhB-related signals were normalized to the value measured in mice treated with RhB-free during the first hour of analysis, clearly reveals that the nanoformulation (red bars) leads to an almost complete abolishment of hematic absorption of RhB. This striking difference between the two groups supports the hypothesis that these NPs are stable and almost completely eradicate the absorption of themselves and of the relevant cargo by gastric and intestinal mucosae. The fast and quite elevated half-life of RhB in the blood led to an expected accumulation of the dye in the main filter organ, the liver, Figure 6B. Similar to the results obtained from the blood, the animals treated with RhB exclusively showed a well detectable red signal in liver sections ( Figure 6C). By histology, we found that RhB-free treated mice showed deep red staining in villi, whereas the red signal remained in the lumen of the intestinal tube in animals receiving RhB with NPs. Since enterocytes in villi are tightly connected to the vessels, it is, therefore, possible to hypothesize that RhB can easily penetrate the circulatory tree. To confirm that NPs can dramatically reduce the complete abolishment of hematic absorption of RhB. This striking difference between the two groups supports the hypothesis that these NPs are stable and almost completely eradicate the absorption of themselves and of the relevant cargo by gastric and intestinal mucosae. The fast and quite elevated half-life of RhB in the blood led to an expected accumulation of the dye in the main filter organ, the liver, Figure 6B. Similar to the results obtained from the blood, the animals treated with RhB exclusively showed a well detectable red signal in liver sections ( Figure 6C). The last part of the study was carried out to investigate if these NPs were able to penetrate into CaCo2 cells and where they localize inside the cells. This experiment was aimed at exploring the possible application of our results to future local treatment of colorectal cancer. Figure 7A shows the progressive process of internalization of NPs (orange spots). The quantification of the occupied area of NPs inside the cell cytoplasm is reported in Figure 7B. Progressive penetration of NPs occurs and, at the 4th hour after incubation, they already occupied the 2% to 3% of the whole cell area. Although relatively low, this percentage in terms of a potential release of a therapeutic cargo cannot be considered negligible. The red arrow in Figure 7A and the higher magnified picture in Figure 7D  The last part of the study was carried out to investigate if these NPs were able to penetrate into CaCo 2 cells and where they localize inside the cells. This experiment was aimed at exploring the possible application of our results to future local treatment of colorectal cancer. Figure 7A shows the progressive process of internalization of NPs (orange spots). The quantification of the occupied area of NPs inside the cell cytoplasm is reported in Figure 7B. Progressive penetration of NPs occurs and, at the 4th hour after incubation, they already occupied the 2% to 3% of the whole cell area. Although relatively low, this percentage in terms of a potential release of a therapeutic cargo cannot be considered negligible. The red arrow in Figure 7A and the higher magnified picture in Figure 7D confirm that NPs are deeply penetrated (orange spots) and the deep internalization of NPs inside the cell cytoplasm starting from the 4th hour of incubation. Moreover, the cell viability assay confirmed the safety of the materials we selected for this study. Indeed, neither RhB nor PLGA-PEG-RhB-NPs alone modify the healthiness of the cells for the whole duration of the treatment ( Figure 7C). confirm that NPs are deeply penetrated (orange spots) and the deep internalization of NPs inside the cell cytoplasm starting from the 4th hour of incubation. Moreover, the cell viability assay confirmed the safety of the materials we selected for this study. Indeed, neither RhB nor PLGA-PEG-RhB-NPs alone modify the healthiness of the cells for the whole duration of the treatment ( Figure 7C).

Conclusions
The current study sought to evaluate the effect of the nanocarrier on the transport of a drug and its behavior within the gastrointestinal tract and absorption to the bloodstream. It is important to note, however, that although RhB was originally used as a tracer to visualize NPs. The results obtained by comparing the biodistribution in mice of the free fluorophore and linked to the NPs is of further relevance for future developments. It is, in fact, interesting how PLGA-PEG-NPs manage to preserve the gastroduodenal absorption by using chemically different molecules along with different loading approaches to the nanocarrier. The low systemic exposure and, at the same time, equivalent drug concentration at the intestinal level, with even a trend to increase in the colon 4 h after the treatment, could have a significant positive outcome on the safety of a wide range of drugs targeting inflammatory and neoplastic diseases.