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Nov 28, 2016 - Department of Molecular Biochemistry and Pharmacology, IRCCS - Istituto di Ricerche Farmacologiche Mario Negri, Via La Masa. 19, 20156 ...
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Biocompatible Polymer Nanoformulation To Improve the Release and Safety of a Drug Mimic Molecule Detectable via ICP-MS Raffaele Ferrari,*,† Laura Talamini,‡ Martina Bruna Violatto,‡ Paola Giangregorio,‡ Mattia Sponchioni,§ Massimo Morbidelli,† Mario Salmona,‡ Paolo Bigini,‡ and Davide Moscatelli§ †

Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zurich, Vladimir-Prelog-Weg 1, 8093 Zurich, Switzerland ‡ Department of Molecular Biochemistry and Pharmacology, IRCCS - Istituto di Ricerche Farmacologiche Mario Negri, Via La Masa 19, 20156 Milan, Italy § Department of Chemistry, Materials and Chemical Engineering, Politecnico di Milano, Via Mancinelli 7, 20131 Milan, Italy S Supporting Information *

ABSTRACT: Fluorescent poly(ε-caprolactone)-based nanoparticles (NPs) have been synthesized and successfully loaded with a titanium organometallic compound as a mimic of a water-insoluble drug. The nature of this nanovector enabled us to combine the quantification of the metal in tissues after systemic administration in healthy immunocompetent mice by inductively coupled plasma mass spectroscopy (ICP-MS) followed by the visualization of NPs in organ sections by confocal microscopy. This innovative method of nanodrug screening has enabled us to elucidate the crucial parameters of their kinetics. The organometallic compound is a good mimic of most anticancer drugs, and this approach is an interesting starting point to design the relevance of a broad range of nanoformulations in terms of safety and targeted delivery of the cargoes. KEYWORDS: biodistribution, drug delivery, ICP-MS, nanoparticle, titanium, drug development biodistribution.7−9 To achieve robust and reliable evidence of the fate of these two components, a longitudinal determination of the localization of NPs and quantification of therapeutic compounds are strongly required. This approach would be crucial to better understand the relationship between the carrier and cargo and, therefore, to build a nanovector with the best properties in terms of active or passive targeting to specific tissue.10−12 Several methods have been progressively developed to evaluate the content of the therapeutic agent inside organs and blood. Usually, the therapeutic agent can be detected ex vivo through high-performance liquid chromatography (HPLC) analyses, although certain issues are present for a specific drug. Next, innovative strategies have been introduced to facilitate understanding of the potential kinetics of a cargo, linking it with a molecule that can be followed after the administration by in vivo or ex vivo screenings. Among these strategies, a radiolabeled compound is a good opportunity unless it raises a safety concern,13 and the addition of a fluorophore is also commonly adopted to guarantee an easy detection through confocal microscopy.14 Similar strategies can be exploited when the active compound is encapsulated within nanocarriers. However, in this case, the

1. INTRODUCTION The administration of therapeutic agents has been historically limited by multiple factors, such as their low solubility, stability, and rapid clearance. Indeed, if a localized treatment cannot be applied, the therapeutic agent has to freely flow through the organs. Intravenous injection is often preferred over subcutaneous, intramuscular, and oral administration for many lifesaving drugs. Unfortunately, this method of administration often leads to a short circulation half-life of therapeutic compounds due to hepatic and splenic filtration and renal excretion. Moreover, the poor penetration through biological barriers represents strong hurdles for the specific delivery to pathological areas. Additionally, the systemic injection of drugs is nonspecific, leading to the adsorption of the therapeutic agent even by the nondiseased tissues as a result of the lack of a selective tropism, with the consequent occurrence of significant side effects. All of these issues led to the development of formulations based on nanoparticles (NPs) to prolong the circulation time, slow down the immune response, and accumulate preferentially in the target organ.1−3 With this aim, in the past decade, a wide range of nanomaterials based on organic and inorganic compounds, lipids, and proteins have been developed and translated to clinical trials.4−6 In nanotechnology, a critical step is represented by the investigation of the biodistribution of both carriers and cargoes that should modify the bioavailability of the payload, ensuring a sustained release over time together with a more governed © XXXX American Chemical Society

Received: Revised: Accepted: Published: A

August 16, 2016 October 28, 2016 November 28, 2016 November 28, 2016 DOI: 10.1021/acs.molpharmaceut.6b00753 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

our previous works, poly(methyl methacrylate) (PMMA), poly caprolactone (PCL3)-based, and poly(lactic acid) (PLA8)-based (analogous to PCL3 but with an average number of eight lactic acid repeating units) were evaluated in terms of interaction with biological fluids, cells, and tumor-bearing mice to assess their potential impact in nanomedicine.19,26 Briefly, our results showed that (1) PMMA NPs are too stable and poorly biodegradable to be proposed as a pharmacological vector, but they can be used to univocally trace the fate of the NPs; (2) PLA8-based NPs underwent rapid degradation without penetrating both the tumor cell lines and tumor mass. On the other hand, PCL3 NPs showed a longer bioavailability, reached the tumor parenchyma, and efficiently penetrated a breast cancer cell line. On the basis of this evidence, the present study was aimed at evaluating the loading and release of a cargo in physiological situations. The aims can be then summarized as follows: (i) to demonstrate that NPs can incorporate insoluble compounds; (ii) to deliver the therapeutic agent loaded with NPs to the target organ with a higher concentration than the free-based formulation; (iii) to demonstrate the possibility to positively modify the distribution of an active compound by encapsulating it into a polymer matrix; and (iv) to establish a precise and well-defined method to quantify the two components of the delivery system.

biodistribution of the carrier itself is usually evaluated instead of that of the therapeutic agent.14−17 The double tracking of both cargoes and carriers with two different and complementary techniques has been recently taken into consideration. In this context, our group extensively investigated the fate of polymeric NPs in different preclinical models of human disorders by covalently linking rhodamine B (RhB) to the carriers.18,19 In this framework, the group of Tsourkas adopted the use of inductively coupled plasma mass spectroscopy (ICP-MS) to detect different metals.20,21 This technique is very powerful because it allows the recognition of metal traces even at very low concentration (ppb). These authors’ approach is very interesting to detect the amount of carrier, lanthanum-doped NPs, but it cannot be applied to recognize small molecules, which can desorb from the particles due to diffusion and interaction with the external environment, thus having a different fate to those of the NPs. With the aim to simulate the behavior of small molecules, titanium diisopropoxide bis(acetylacetonate) (hereinafter Ti, when referred to the free molecule) has been selected as an organometallic compound that can mimic a broad spectrum of antineoplastic drugs or therapeutic agents in terms of solubility and hindrance (molecular weight); additionally, literature reports active compounds containing titanium that can be used as antitumor agents.22,23 In this way, it was possible to analyze the behavior of both the carrier and the payload with high precision for the payload quantification due to the ICPMS technique. As a hydrophobic molecule, it presents a low solubility in water; thus, it needs a formulation to be administered properly. The titanium atom is also very sensitive to be analyzed by ICP-MS; because it is not present in the body, the analysis is not affected by a background signal. Thus, the impact in the NP adoption to formulate this type of mimetic drug has been evaluated, and as a comparison, the biodistribution of the free compound is evaluated also without the presence of NP, as graphically summarized in Scheme 1.

2. EXPERIMENTAL SECTION 2.1. Materials. ε-Caprolactone (CL, 99%), 2-hydroxyethyl methacrylate (HEMA, ≥ 99%), 2-ethylhexanoic acid tin(II) salt (Sn(Oct)2, ∼ 95%), sodium sulfate, dichloromethane, potassium persulfate (KPS, ≥ 99%), poly(ethylene glycol) methyl ether methacrylate (EG45MA, molecular weight: ca. 2080 Da) and ethanol (≥99.7%), sodium sulfate (≥99%), N,N′dicyclohexylcarbodiimide (≥99%), 2-propanol (≥99.7%), cremophor, RhB, and titanium diisopropoxide bis(acetylacetonate) (Ti) were purchased from Sigma-Aldrich and were used without further treatment. 2.2. Macromonomer and Nanoparticle Production. The hydrophobic macromonomer necessary to synthesize the NPs comprises three units of ε-caprolactone functionalized with a HEMA double bond (CL3MA) and was produced following a literature recipe.27 HEMA was used as an initiator in the ROP of CL as reported in the literature. Accordingly, 3.8 g of HEMA and 60 mg of Sn(Oct)2 (i.e., Sn(Oct)2/HEMA molar ratio of 1:200) were stirred at room temperature until tin octoate dissolution. Additionally, 10 g of CL and 5 mg of sodium sulfate were heated at 130 °C, and the HEMA solution was added to initiate the reaction, which was run for 2 h. Similarly, fluorescent HEMA-RhB macromonomer was synthesized as already described to obtain a polymerizable dye that can be covalently linked to the polymer matrix.28 NP synthesis was carried out in a 50 mL three-necked glass flask. The procedure consists of loading all of the hydrophilic macromonomer (EG45MA) initially in the reactor as in a normal batch polymerization, while the more hydrophobic monomer is fed into the reactor during the process.24 In detail, 0.5 g of EG45MA was added in 50 mL of distilled water, and the solution was heated to 80 °C under a nitrogen stream for 10 min. Next, 25 mg of KPS was added to the purged solution. Thereafter, 2 g of CL3MA mixed with 2.5 mg of HEMA-RhB were injected at a rate of 2 mL/h using a syringe pump (Model NE-300, New Era Pump System, US); the reaction was run for 3 h. The solid content of the final latexes is equal to 2.5 g (5 wt %/wt). The size and particle size distribution (PSD) of the NP

Scheme 1. Diagram Representing the Consecutive Steps Composing the Present Work

Ti behaves as a lipophilic drug, and it has been formulated with the excipients used in clinical practice (e.g., paclitaxel with cremophor and ethanol). Next, the organometallic compound has been encapsulated into polymeric NPs. In detail, degradable polymer NPs based on poly(caprolactone) (PCL) and stabilized with poly(ethylene glycol) chains, which have already shown promising applications, have been selected.18,24 To univocally detect the NPs, rhodamine B (RhB) was covalently linked to the polymer backbone, thus avoiding its possible leakage with biodegradable moieties.25 The mimic compound has been loaded inside the particles (hereinafter Ti-NPs), and the biodistribution of both Ti-NPs and Ti were evaluated. In B

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environment (i.e., serum). The samples were placed in an incubator at 37 °C; at selected times, small aliquots of the PBS−albumin solution were analyzed with a plate reader (PerkinElmer Enspire 2300) to evaluate the presence of RhB outside the dialysis membrane. The samples were excited at 540 nm, while the fluorescence spectra were recorded from 560 to 650 nm. 2.5. Animals. For in vivo experiments, 12-week-old male NFR mice were used and maintained under specific pathogenfree conditions in the Institute’s Animal Care Facilities; they received food and water ad libitum and were regularly checked by a certified veterinarian who is responsible for animal welfare supervision and experimental protocol revision. Twelve mice per experimental group were enrolled and intravenously treated with a solution of (1) Ti-NPs at the dose of 12.5 mg/kgbw, (2) free Ti at different doses (12.5, 7.5, 5.0, and 2.5 mg/kgbw). Three animals were treated with PBS and were used as controls. The procedures involving animals and their care were conducted in conformity with the institutional guidelines at the IRCCS-Institute for Pharmacological Research “Mario Negri” in compliance with national (Decreto Legge nr 116/92, Gazzetta Ufficiale, supplement 40, February 18, 1992; Circolare nr 8, Gazzetta Ufficiale, July 14, 1994) and international laws and policies (EEC Council Directive 86/ 609, OJL 358, 1, Dec. 12, 1987; Guide for the Care and Use of Laboratory Animals, U.S. National Research Council, eighth edition, 2011). This research project has been reviewed by IRCCS-IRFMN Animal Care and Use Committee (IACUC) and was approved by the Italian “Istituto Superiore di Sanità” (code: 17/01 D Appl 3). 2.6. Ex Vivo Analysis. Three mice for each time point were sacrificed at 15 min, 1, 4, and 24 h after injection (Ti free or PCL3-Ti) by an overdose of ketamine (150 mg/kg) and medetotimine (2 mg/kg). To ensure optimal quality of tissues, the liver, kidneys, spleen, and lung were immediately frozen in liquid nitrogen and were equally divided into three portions devoted to histology, fluorimetry and ICP-MS analysis. Histological analysis was performed by incubating cryostat serial sections (30-μm thickness) with the vital nuclear dye Hoechst-33258 (2 μg/mL in PBS) followed by visualization in an Olympus Fluoview microscope BX61 (Tokyo, Japan) with an FV500 confocal system equipped with specific lasers λexc = 405 nm for Hoechst-33258 and λexc = 546 nm to visualize the specific signal associated with polymeric NPs. Regarding fluorimetric measurements, the tissues were weighed and homogenized in RIPA (25 mM Tris-HCl pH 7.4, 137 Mm NaCl, 1% Triton X-100, 1% dexycholate, 0,1% SDS, 1 mM EDTA) 1:4 (v/w) buffer, centrifuged at 1200 rpm for 10 min at 4 °C, and the supernatant was recovered. Spectrophotofluorimetric analyses were conducted to correlate the fluorescence intensity (FI), measured into the collected samples, with the presence of NPs. FI was determined at λexc = 546 nm for RhB using a 2300 MultilabelPlate Reader spectrophotofluorimeter (PerkinElmer, Boston, MA, U.S.A.) with a standard 96-well black plate with a transparent bottom (ProxiPlate-96, Perking Elmer). The fluorescence intensity of all of the samples was compared to that measured from blank (untreated) animals. Elemental analysis of organs and blood was carried out by ICP-MS as described previously to evaluate the Ti content. To measure its concentration, each organ was weighed before the analysis; meanwhile, for the coupled analysis of fluorescence

dispersions have been determined through dynamic light scattering (DLS) measurements (Malvern, Zetanano ZS). The PSD of the samples was estimated by adopting the cumulant method, or z-average diffusion coefficient, as defined by ISO (standard document 13321:1996E) and reported in the literature.29 All of the reported data were the average value of three measurements of the same sample. Transmission electron microscopy (TEM) images of the particles were taken using a FEI Morgagni 268, operated at 120 kV, equipped with an Orius SC1000 CCD camera. 2.3. Titanium Loading and Release. Ti was loaded using a postpolymerization process and was entrapped in the partially swollen NPs using a mixing device represented by PTFE cylinder, which was 1 cm in diameter and 1 cm in length with an axial perforation of 1 mm diameter and a radial perforation of 500 μm.24 The NP latex (50 mg/mL) and Ti dissolved in 2propanol (75 mg/mL) were loaded in two separate syringe pumps and were injected in the device (at a flow rate of 30 mL/ min and 5 mL/min, respectively). The final mass ratio between Ti and NPs is equal to 25% w/w, and the final organometallic content is equal to 10.71 mg/mL corresponding to 1.39 mg/ mL of metallic titanium (theoretical value). After the loading process, two samples were withdrawn. One was used to evaluate the drug content in the solution, and the second one was used to recover the supernatant using Vivaspin 500 filters (Sartorius Stedim) and evaluate the loading efficiency, which was calculated as follows: %loading efficiency ⎛ titanium recovered in the supernatant (t = 0) ⎞ = ⎜1 − ⎟*100 total titanium recovered in the NP solution (t = 0) ⎠ ⎝

(1) %encapsulation efficiency ⎛ titanium recovered in the supernatant (t = 0) [mg] ⎞ = ⎜1 − ⎟*100 total titanium recovered in the NP solution (t = 0) [mg] ⎠ ⎝

(2)

To remove the unloaded organometallic and isopropanol present after the loading process, 3 mL of the NP solution was loaded in a Slide-A-Lyzer cassette (Thermo Scientific; MW cutoff: 3.5 kDa) and dialyzed against 200 mL of PBS. The samples (0.2 μL) were withdrawn from the cassette after different times of dialysis and treated to determine the Ti content in the NP solution. The buffer solution was changed every 2 h to grant the preservation of the sink conditions. Elemental analysis was carried out by inductively coupled plasma mass spectroscopy (ICP-MS analysis) using an Optima model 8300 instrument (PerkinElmer, Waltham, MA). The samples were analyzed after digestion using concentrated nitric acid (Sigma-Aldrich). 2.4. NP Incubation with Albumin. To check the stability of the polymer-RhB bond, the following solution was prepared: (a) PCL3 NPs with covalently bound RhB (the ones used in the present study); (b) RhB physically entrapped into the PCL3based NPs. These NPs were synthesized as reported in section 2.2 but without using HEMA-RhB. This sample was obtained by putting it in contact with the NPs with RhB dissolved in DMSO in a mixing device as shown for the Ti loading. (c) Free RhB. One milliliter of each of these samples was put in a dialysis membrane (Thermo Scientific; 5 kDa cutoff), which was immerged in 50 mL of PBS containing BSA (40 mg/mL) to simulate the behavior of the NPs in a protein-rich C

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Molecular Pharmaceutics and Ti, each organ was split into two and separately weighed to reconstruct the right concentration. 2.7. Statistical Analysis. All of the data were expressed as the mean ± SD, and Student’s test and p values were performed using the GraphPad Prism version 6.00 for Windows (GraphPad Software, San Diego, CA).

3. RESULTS AND DISCUSSION 3.1. NP Synthesis and Ti Loading. The CL3MA monomer was synthesized using an ROP process as described above and was employed in an emulsion polymerization with EG45MA to produce surfactant-free NPs, as depicted in Scheme 2. Scheme 2. Reaction Scheme for the Production of PEGylated PCL3-Based NPs

Figure 1. TEM image of the PCL3 NPs (A) and particle size distribution as obtained from DLS data for the NPs after synthesis (blue straight line) and after loading with Ti (red dashed line) (B).

The NPs are composed of a poly(HEMA) backbone grafted with PCL and poly(ethylene glycol) chains, which are wellrecognized in the literature to prevent the opsonisation and prolong the NP circulation time.30,31 PCL3-based NPs degrade in a reasonable time, around 1 week under in vivo conditions, due to their peculiar structure, and they represent a potentially successful drug carrier, as recently reported in the literature.24 The adopted process allows the synthesis of monodispersed and well-defined NPs as reported in Figure 1. TEM analysis, reported in Figure 1A, shows that the NPs have a spherical shape and homogeneous size, comparable with DLS measurements (122 ± 2 nm and 0.037 PDI). The Ti loading was carried out on the preformed NPs using the mixing device that ensures a fast and efficient mixing of the two phases. The solvent addition allows the NP swelling (130 ± 3 and 0.094 PDI), titanium diffusion, entrapment inside the polymer, and consequent loading. DLS measurement confirms the NP swelling after the loading process as reported in Figure 1B where the shifting of the particle size distribution to higher values is observable. In addition, the same figure shows that no aggregates are formed during the loading process, as confirmed by the TEM image recorded for the loaded NPs (see Figure S3). The average content of titanium in the NP dispersion was evaluated as equal to 1.34 mg/mL via ICP-MS. The separation of the supernatant from the NPs allowed the evaluation of the organometallic titanium in the supernatant (0.36 mg/mL), and thus, the loading in the NPs was equal to 73.1%. Such values

are in agreement with literature results where the encapsulation efficiency of hydrophobic drugs ranges from 10% to 90%, whereas the drug loading efficiency (18.2% w/w) is relatively high as it is usually difficult to reach values higher than 10%.32,33 To avoid the injection of the free Ti together with the NPs, a dialysis process was applied, allowing both to exchange the buffer solution into PBS and remove the solvent added during the loading process and the organometallic Ti that is unloaded or simply stacked to the NP surface. Finally, the Ti release from NPs was measured during the dialysis, as reported in Figure 2A. Figure 2A shows that Ti is released from the NPs (red dots) until a plateau, which corresponds almost to the loading value previously evaluated. The presence of a plateau confirms that Ti is no more desorbed from the NP surface. Four-hour dialysis was then applied to the Ti-NPs before the injection with a final organometallic titanium content equal to 950 mg/kg, also confirming the measurement of the loading reported previously from the supernatant separation. In Figure 2A, the release of Ti from NPs is compared with the release of different model drugs from the same NPs. It is possible to observe that Ti (whose solubility in water is much lower than 10 g/kg35 and evaluated via ICP-MS as equal as 29 ± 8 mg/kg) behaves similarly to paclitaxel and dexamethasone, commonly considered as insoluble drugs (water solubility of 5.56 mg/kg and 89 mg/ D

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Figure 2. Release profile of Ti organometallic and other drugs together with their water solubility24,34 from the PCL3 NPs in PBS (A). Release profile of RhB in albumin-rich solution from PCL3 NPs with chemically bonded RhB (B). PCL3 NPs with physically entrapped RhB (C) and free RhB (D).

3.2. Biodistribution of Ti. This study was carried out using the PCL3 NPs exclusively in healthy mice. The first series of in vivo experiments was mainly designed to assess whether and how the formulation of Ti internalized within the PCL3 NPs, as described in the previous paragraphs, might alter the kinetics and biodistribution of the metal. Animals were enrolled in two different experimental groups (12 mice for group): one group received Ti-NPs (12.5 mg/kgbw), and the second group was treated with the same concentration of Ti diluted in a solution of 6:1 water and cremophor/ethanol to allow its solubility (this is the same formulation of antitumor drugs as Paclitaxel). The treatment with the Ti was interrupted because of its lethality. This formulation led to a very fast decease of animals (less than half of an hour after administration). The causes of the death have not been widely investigated; however, the lungs of animals were collapsed and darker than those under the physiological condition. Therefore, it is possible to argue that a strong aggregation of Ti occurred after its injection in the blood. For ethical reasons, we stopped the experiments after the third mouse; by contrast, none of the apparent hallmarks of stress (loss of hair, shaggy, irritability, tremors, or body weight loss) was observed in animals treated with Ti-NPs for the whole duration of the experiments. The fast lethality of Ti alone further confirmed the pivotal role of the organometallic compound if injected alone. The lack of lethality and other effect confirmed the vehicle tolerability. On the contrary, the fast lethality of free Ti at 12.5, 7.5, and 5.0 mg/kgbw further confirmed the pivotal role of the organometallic compound if injected alone. Due to ethical reasons we only treated one animal for each dose and we could not investigate the reasons and the mechanisms related to this high toxicity. No evidence

kg, respectively). Conversely, the behavior is significantly different from the more water-soluble Juglone (2 g/kg) and minocycline (52 g/kg). These data, together with the ones shown before for the encapsulation efficiency and loading efficiency, further confirm that Ti is correctly assumed as a good mimic molecule for insoluble drugs. Another fundamental assumption of this work is that RhB remains covalently linked to the NPs and not to the degradable PCL-grafted chains; after degradation, it remains bonded to the water-soluble poly(HEMA) backbone, which is the final degradation product. To prove this statement, PCL3 NPs with RhB covalently bound and with RhB only physically entrapped along with free RhB were dialyzed against a PBS solution containing albumin to mimic a protein-rich environment (i.e., serum). The dialysis medium of the two solutions was sampled at different times, and the emission spectrum of RhB was analyzed and shown in Figure 2B−D. The characteristic peak of the dye at 580 nm is not detected at any time if RhB is covalently bound to the NPs (panel B). On the other hand, the intensity of the signal increases for the case of free RhB (panel D). In panel C, an intermediate slower release can be observed. It is worth noting that in panel B, no RhB is detected even after 7 days, when the NPs are almost completely degraded.34 The absence of the RhB signal is due to the final degradation product of these materials being poly(HEMA-co-EG45MA-co-RhB) micelles of approximately 15 nm in size that cannot cross the membrane.36 The experiment proves that the linkage of RhB is not labile and that it follows the fate of the NPs, thus making possible to distinguish clearly the distribution of the polymer from the one of Ti, which is only physically entrapped. E

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Figure 3. Quantitative ICP-MS measurement of the percentage of the injected dose (% ID) of titanium internalized in PCL3 (Ti-NPs, dark gray bars) or free titanium (Ti, light gray bars). The data are reported as the mean ± SD (n = 3 mice for each experimental group), and the results were compared at the same time-point by Student’s t test. P values: * ≤ 0.05, **≤ 0.005, *** ≤ 0.0005 (light gray bars significantly higher than dark gray bars); P values: ∧≤ 0.05, ∧∧≤ 0.005, ∧∧∧ ≤ 0.0005 (light gray bars levels significantly lower than dark gray bars).

Figure 4. Quantitative measurement of the percentage of polymer (PCL3) (dark gray bars) and Ti-NPs (light gray bars) evaluated by fluorimetric and ICP-MS analyses, respectively. The data are reported as the mean ± SD (n = 3 mice for each experimental group), and the results were compared at the same time-point by Student’s t test. P values: ** ≤ 0.005; *** ≤ 0.0005 (light gray bars significantly higher than dark gray bars); P values: ∧ ≤ 0.05; ∧∧∧ ≤ 0.0005 (light gray bars significantly lower than dark gray bars).

metabolism of drugs over the first hours after its administration. Figure 3 shows the longitudinal measurement of Ti (reported as percentage of the total dose injected, %ID) either in mice treated with (dark-gray bars) or without NPs (light-gray bars), in blood (A), liver (B), lungs (C), and kidneys (D). The analysis of circulating levels of titanium in both formulations (Figure 3A) revealed that a sharp reduction of Ti in the bloodstream occurs the first 15 min after administration, and this trend progressively increases and almost completely disappears 4 h after injection. Despite similar kinetics, a significantly higher amount of circulating Ti is provided by its internalization in NPs. The permanence in circulation is basically influenced by two factors: tissue migration and excretion/clearance. To evaluate how the formulation may influence tissue penetration, the Ti levels in the main organs were considered in our study. Figure 3B shows the percentage of titanium in the liver parenchyma at different time points. Our previous studies, carried out by fluorimetric analysis, have shown a peculiar tropism of empty PCL3 NPs in this organ. Accordingly, the liver of animals receiving Ti-NPs shows a progressive increase in the amount of metal that became 4-fold higher from 15 min to 4 h after administration. By contrast, animals receiving the Ti showed a significant increase in the hepatic uptake over time but a lower rate of progression. However, the overall trend did not markedly differ between the two groups. The administration of Ti greatly increases the fast migration toward the lungs (Figure 3C). However, no persistent accumulation was observed, thus suggesting that an efficient pulmonary clearance occurs for both treatments. The fast penetration of Ti to the lung may be the cause of the strong and fast lethality of this formulation, and the presence of shrunken and darker lungs in animals that died

of stress was instead observed at 2.5 mg/kgbw (1/5 to the dose well-tolerated by NP-treated mice), and consequently, all the experiments reported in this study were performed at this concentration of Ti free. The histological results (see Figure S4) confirmed that the lack of lethality is accompanied by the absence of relevant alterations on the anatomy of liver, kidneys, and, above all, lungs in animals that received the lowest dose of Ti free (2.5 mg/kgbw). This is an important finding also if considering biodistribution, accumulation, and clearance. It is, indeed, well described that morphological alteration on the structure of the main filter organs may somehow influence the bionano interaction and therefore modify the kinetics and the behavior of NPs in the host tissues.2,7,15 It is also important to highlight the protective effect provided by the embedding of Ti in polymeric NPs that significantly reduced its acute toxicity. For instance, a similar behavior is exploited to increase the therapeutic index of a chemotherapeutic agent (e.g., Paclitaxel) by its incorporation in liposomes.5,37 Ti shares a low solubility with taxols in polar solutions and potential toxicity in living organisms. These two features should be underlined because even if the aim of the in vivo study is to characterize the biodistribution of Ti after the NP loading described above it is possible to mimic some problems of toxicity encountered at the clinical practice. Our recent studies have shown that the disappearance of NPs from plasma was biphasic with the distribution and elimination of half-lives of 30 min and 15 h, respectively.38 Thus, once we established the doses of both Ti and Ti-NPs able to avoid any lethal effect in mice and that did not alter the anatomy of filter organs, sacrifices were performed at different time-points ranging from 15 min to 24 h. Such temporal window is also in agreement with pharmacological analyses, it is in fact frequent to observe a progressive F

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emphasize that the deep presence of vessels in many organs (such as the kidneys, spleen, liver, and lungs) can lead to an overestimation of the actual migration of NPs into the parenchymain particular, at the earlier phases of the study. Figure 5 reports the results obtained from mice treated with Ti-NPs to evaluate the different behaviors of the polymer and

after high doses of Ti are, in part, in agreement with this result. In contrast to the lungs, the presence of Ti-NPs greatly increased its tropism to the kidneys (Figure 3D). It is important to underline that the renal levels of Ti were 5−10 times lower than the ones found in lungs. However, the difference between the two groups is not negligible, showing an effective modification of the Ti biodistribution using NPs and an increase in the Ti concentration in the organs that represent the main filters for NPs (e.g., PCL3-degraded NPs are excreted by kidneys). Overall, the internalization of Ti in NPs leads to relevant differences in the toxicity, bloodstream permanence, and organ accumulation. This is an important point; however, to better understand the reason for the different behavior between polymer alone and Ti-NPs, a deeper investigation must be provided. Figure 4 reported the fate of Ti and polymer evaluated by ICP-MS and fluorimetry, respectively, from 15 min to 24 h after a single injection in mice. Three main parameters of interaction have been considered: permanence in the bloodstream (A), organ penetration (B), carcass accumulation and excretion (C). The longitudinal analysis of the three processes highlights a relevant dichotomy among the levels of polymer and Ti-NPs since the first time-point. A few minutes after the administration in the tail vein, a strong reduction of the emetic content of both components can be observed (Figure 4A, left columns). The Ti-NPs show a faster and stronger elimination from the bloodstream; 4 h after the administration, there was a complete wash out of the metal. By contrast, the polymer progressively decreased but still remained approximately 5% of the injected dose up to 24 h after injection. The results achieved by this study are in agreement with previous results performed by calculating the permanence of the PCL3 NPs alone.38 This indicates that the loading with titanium did not markedly influence the behavior of NPs in the body. The loss of material from the bloodstream may be due either to penetration into filter organs (spleen, liver, kidneys) or into the remaining organs (lungs, heart, brain, muscles) or a fast and efficient clearance. In Figure 4B, the progressive accumulation of both polymer and Ti-NPs in filter organs is reported. It is of interest to note that approximately 40% of the injected dose of Ti-NPs was found in excised organs 15 min after injection; moreover, the ability of Ti-NPs to penetrate into tissues is manifold higher than that of the polymer alone. This difference remains for all of time points, even if it is reduced for both components 24 h after their administration. Figure 4C reports the % ID from the remaining organs, measured as the difference between the total ID and that obtained from the sum of blood and filter organs. The graph shows a progressive increase for both polymer and Ti. However, a faster and stronger increase of the polymer can be observed compared with the metal. This trend can be considered the sink effect at different times after administration, such as the excretion by feces and urines. As previously described, the combination of fluorimetry and ICP-MS revealed that, since the first time after administration, a considerable amount of Ti does not follow the route of NPs. However, the evidence that the two components have a similar trend in the blood permanence, tissue migration and excretion, leads us to hypothesize that this release is progressive and gradual over the time and it is likely related to the diffusion of the Ti from the NPs. It is known that small molecules, such as the Ti, are able to easily penetrate in cells and diffuse in tissues in few times. However, regarding NPs, it is important to

Figure 5. Quantitative measurement of the percentage of polymer (PCL3) (dark gray bars) and Ti- NPs (light gray bars) evaluated by fluorimetric and ICP-MS analyses, respectively, after the injection of NP internalizing Ti in the liver (A), kidneys (B), lungs (C), and spleen (D). The data are reported as the mean ± SD (n = 3 mice for each experimental group), and the results were compared at the same timepoint by Student’s t test. P values: * ≤ 0.05; ** ≤ 0.005; *** ≤ 0.0005 (light gray bars significantly higher than dark gray bars).

Ti-NPs (with fluorimetry and ICP-MS, respectively) in the representative organs such as the liver, kidneys, lungs and spleen. To correlate these quantitative data with NP localization, a histological analysis was carried out as shown in Figure 6. The measurement of the fluorescence level in the liver directly collected and frozen after the sacrifice (therefore containing the remaining blood) showed that the injected dose remains rather unchanged for the whole duration of the experiment ranging from values around the 20% of the total dose (Figure 5A, dark gray bars). The histology sections pinpointed the actual localization of NP in the liver (Figure 6A). The different distribution of the red staining clearly demonstrated that progressive migration of the fluorescent NPs from the vessels to the parenchyma occurs in the main filter organ because of the fourth hour after their administration. This result was quite expected because we have recently demonstrated the same spatiotemporal profile of organ penetration.19,26,38 However, it is important to note that the loading with Ti did not markedly change this typical feature of PCL3 NPs. By contrast, the percentage of Ti in the liver was G

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Figure 6. Biodistribution of Ti-NPs observed with fluorescence microscopy in the liver (A), kidneys (B), lungs (C), and spleen (D). Histological sections were collected from mice sacrificed at 0.25, 1, 4, and 24 h after intravenous injection. NPs were visualized in red (RhB signal), and nuclei were stained in blue (Hoechst-33258).

significantly higher since the first time-point, and it reached the half of the total injected dose at the fourth hour after NP injection. Figure 4 strongly suggests that this fast and strong migration cannot be accounted for by the higher presence of Ti in the blood (the percentage of ID in the blood was lower for the Ti than that for the polymer); therefore, it was related to a very efficient penetration in this organ. This marked difference in the levels of polymers and Ti can somehow be explained by a progressive release of the metal in the first 4 h after administration. This is a very important point both in pharmacokinetics and, in the case of effective compounds, in pharmacodynamics. To monitor at the same time the fate of the vector and the cargo is crucial to improve the formulation of nanodrugs. Thus, this innovative way of tracking could be extremely interesting to optimize the physicochemical parameters of NPs to improve their performance in terms of drug delivery. In this pilot study, the analysis of biodistribution was extended to the kidneys, spleen, lungs, brain, and heart. An almost complete lack of signal was found in the brain and heart (data not shown); however, in the other processed organs, the levels greatly differed among them. A very low percentage of both polymer and Ti was found in the kidneys (Figure 5B). Both materials very rapidly reached the peak a few minutes after the administration; however, they were progressively cleared very quickly. This extremely fast kinetics is due to the excretory function of the organ. The almost selective staining in vessels and glomeruli confirms that urine successfully excrete a

small part of injected materials (Figure 6B). The levels of Ti are weakly higher than those of the polymer; however, this difference, even if statistically significant, does not justify the hypothesis of a different way of clearance between the two materials. The presence of a very high amount of material in the liver and slower pattern of excretion (see Figure 4C) suggests that the common bile duct is the main way to expel both NPs and the cargo. On the other hand, systemically injected NPs do not lead to a massive accumulation in the lungs. As expected, the percentage of NPs in the lungs was very low and progressively decreased in 24 h (Figure 5C). Moreover, the signal was exclusively confined to the airways but not migrated into the parenchyma. By contrast, Ti rapidly accumulated in the parenchyma with a percentage of the injected dose from 20 to 50 times higher than those measured for NPs. However, it is important to underline that the high tropism of Ti toward this organ is not associated with NP-loading (Figure 6C). Finally, a very low % of the injected dose of both Ti and polymer was observed in the spleen. The levels of the polymer remained stable at the different time points, while it is of interest to note a slower and more prolonged accumulation of the Ti over time. Histological analysis revealed that NPs are confined in the red pulp of the spleen but did not invade the lymphatic pulp (Figure 6D). H

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4. DISCUSSION A progressive reduction in the development of drugs in many clinical settings has been recorded in the last 30 years.26 Very interestingly, this limitation on the discovery of new drugs is not primarily due to a low capacity of understanding the cellular and molecular mechanisms associated with the disease. The main obstacles that a drug encounters after systemic administration are (1) the rapid degradation of therapeutic agents in the bloodstream, (2) the metabolic activity of filter organs and fast clearance, (3) the lack of a specific tropism and the consequent occurrence of important side effects due to an increase of dosage, and (4) the low ability to cross biological barriers. The strategy to develop new formulations for molecules already in use in chemotherapy may therefore represent a major advance in terms of the tolerability of the treatment, as well as in terms of the prognosis for patients more generally. In particular, the possibility to internalize therapeutic compounds in biodegradable, biocompatible, and physicochemically wellcharacterized nanometric systems represents a large hope in oncology.39,40 Here, the use of an organometallic compound is justified by the requirements to mimic and understand the behavior of the drugs once encapsulated into polymer-based NPs and injected into the bloodstream. The main purpose of the project is to evaluate the use of polymeric nanocarriers for the biodistribution of organometallic Ti after the systemic administration in healthy immunocompetent mice. Titanium was selected in this study for two main reasons: (1) it can mimic a broad spectrum of antineoplastic drugs (characterized by high toxicity and poor solubility), and (2) it can be easily detected in organs and biological fluids because it is, in fact, almost absent in the body. This feature allows the measurement of very small quantities of this compound inside the body, and in addition, Ti has the chemical characteristics that make it extremely sensitive to analysis by ICP-MS. Because the Ti is not soluble and precipitates rapidly in saline, we administered the metal in a solution containing chremophor and ethanol, similar to that used in clinical practice for the treatment of patients with the chemotherapy of taxols and other drugs with a similar chemical nature.41 To achieve a welltolerated dose, the concentration of Ti was decreased 5 times compared to that used in the case of internalization in NPs. This strategy is not exclusively related to our study, but it is shared by the large percentage of formulations currently used in oncology to increase the therapeutic index by reducing the tropism to the heart and other vital organs.42,43 Here, we found that the lungs can be a target of toxicity (specific for the mimic compound), and the internalization in NPs markedly reduced this effect. Many other steps of development could be carried out in the future to make these NPs more targeted to specific organs (e.g., peptide to cross the blood brain barrier or antibodies to bind receptors expressed in cancer cells) to further improve the therapeutic index by increasing the tropism to the target. However, this result confirms the importance of a system to administer unmanageable compounds for hazard and chemical characteristics (high hydrophilicity). Very importantly, this study was not devoted to investigate about toxicity; however, it was devoted to defining some critical points tightly related to the nanobio interaction such as biodistribution, accumulation, clearance, and the release kinetics of the molecule internalized inside NPs. Here, we have preferred to avoid the use of a model of human diseases

(e.g., tumor-bearing mice). The characterization of a potential nanovector in healthy mice could be considered an apparent contradiction, but the principles of pharmacokinetics should be evaluated under physiological conditions before approaching pathological situations. In this work, we also labeled NPs with a fluorescent dye to maximize the sensitivity and accuracy of our results. As expected, fluorimetric data showed that these spherical NPs of approximately 100 nm have a rapid and marked tropism for the liver where they remain for more than 24 h. However, migration in the liver parenchyma is gradual. The histological data showed that, until the first hours after administration, the signal associated with the NPs is prevalently confined inside the vessels and then migrate into the parenchyma. This innovative approach, which combines the quantification in homogenates with fluorimetry to the histological observation with confocal microscopy, enabled us to associate the amount of NP accumulation with their tissue localization. In an ideal situation, the particles should be stable enough to ensure a good association between the polymer and internalized compound until the target organ is reached. In previous studies, we showed that PCL3 NPs showed progressive metabolism and clearance in filter organs and excretory systems.26 Moreover, the presence of NPs at 3 and 5 days has been shown, even if with a very low amount, to represent less than the 5% of the total injected dose. However, the plasmatic half-life is shorter, and for this reason, we focused our attention on the fast processes occurring after the NP administration. Interestingly, a massive migration of NPs from the bloodstream to the tissue and a strong release of the titanium occurred during the first hour after the administration. This integrated analysis is crucial to define the potential of each type of nanomaterial and could be extremely useful to optimize the physicochemical features of NPs to better modulate both the release of the cargo and interval of biodegradability/ clearance after their injection. In the first part of the study, we also showed that, in addition to the lower toxicity, nanoformulation led to a different tropism to many organs. The internalization of the Ti in NPs prolonged the levels in the blood and markedly increased the amount in the lungs. On the other hand, the levels of Ti were significantly lower in the kidneys, if it is freely administered in the tail vein. This difference is probably because the NPs delayed the process of filtration from the glomeruli and ureter. The histological analyses confirmed this hypothesis. A strong amount of signal was instead observed in the vessels and structures deputed to the renal filtration since the first minutes after administration but progressively faded away.

5. CONCLUSIONS We developed a reliable procedure to load degradable polymeric NPs with a drug mimic compound, namely, organometallic titanium. The nanovector is then composed of PCL3 NPs with RhB univocally bonded to the polymer chains and Ti, physically encapsulated into NPs, and precisely detectable via ICP-MS. Such a system showed the possibility to safely deliver the mimic compound, whose mimic ability was also demonstrated at a higher concentration than that of the free-based formulation and to successfully modify its biodistribution in mice. Additionally, the method of NP-loading and the combined analysis represent innovative approaches to optimize an integrated platform aimed at defining the behavior of nanodrugs to further study in a broad range of applications. I

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Passive versus Active Tumor Targeting Using RGD- and NGRModified Polymeric Nanomedicines. Nano Lett. 2014, 14 (2), 972− 981. (11) Davis, M. E.; Chen, Z.; Shin, D. M. Nanoparticle therapeutics: an emerging treatment modality for cancer. Nat. Rev. Drug Discovery 2008, 7 (9), 771−782. (12) Prabhu, R. H.; Patravale, V. B.; Joshi, M. D. Polymeric nanoparticles for targeted treatment in oncology: current insights. Int. J. Nanomed. 2015, 10, 1001−1018. (13) Xing, Y.; Zhao, J.; Conti, P. S.; Chen, K. Radiolabeled Nanoparticles for Multimodality Tumor Imaging. Theranostics 2014, 4 (3), 290−306. (14) Wu, X.; Sun, X.; Guo, Z.; Tang, J.; Shen, Y.; James, T. D.; Tian, H.; Zhu, W. In Vivo and in Situ Tracking Cancer Chemotherapy by Highly Photostable NIR Fluorescent Theranostic Prodrug. J. Am. Chem. Soc. 2014, 136 (9), 3579−3588. (15) Barua, S.; Mitragotri, S. Challenges associated with penetration of nanoparticles across cell and tissue barriers: A review of current status and future prospects. Nano Today 2014, 9 (2), 223−243. (16) Owens, D. E., III; Peppas, N. A. Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles. Int. J. Pharm. 2006, 307 (1), 93−102. (17) Key, J.; Leary, J. F. Nanoparticles for multimodal in vivo imaging in nanomedicine. Int. J. Nanomed. 2014, 9, 711−726. (18) Papa, S.; Caron, I.; Erba, E.; Panini, N.; De Paola, M.; Mariani, A.; Colombo, C.; Ferrari, R.; Pozzer, D.; Zanier, E. R.; Pischiutta, F.; Lucchetti, J.; Bassi, A.; Valentini, G.; Simonutti, G.; Rossi, F.; Moscatelli, D.; Forloni, G.; Veglianese, P. Early modulation of proinflammatory microglia by minocycline loaded nanoparticles confers long lasting protection after spinal cord injury. Biomaterials 2016, 75, 13−24. (19) Sitia, L.; Paolella, K.; Romano, M.; Violatto, M. B.; Ferrari, R.; Fumagalli, S.; Colombo, L.; Bello, E.; De Simoni, M. G.; D’Incalci, M.; Morbidelli, M.; Erba, E.; Salmona, M.; Moscatelli, D.; Bigini, P. An integrated approach for the systematic evaluation of polymeric nanoparticles in healthy and diseased organisms. J. Nanopart. Res. 2014, 16 (7), 2481. (20) Crayton, S. H.; Elias, D. R.; Al Zaki, A.; Cheng, Z.; Tsourkas, A. ICP-MS analysis of lanthanide-doped nanoparticles as a non-radiative, multiplex approach to quantify biodistribution and blood clearance. Biomaterials 2012, 33 (5), 1509−1519. (21) Elias, A.; Crayton, S. H.; Warden-Rothman, R.; Tsourkas, A. Quantitative Comparison of Tumor Delivery for Multiple Targeted Nanoparticles Simultaneously by Multiplex ICP-MS. Sci. Rep. 2014, 4, 5840. (22) Meker, S.; Margulis-Goshen, K.; Weiss, E.; Magdassi, S.; Tshuva, E. Y. High Antitumor Activity of Highly Resistant Salan-Titanium(IV) Complexes in Nanoparticles: An Identified Active Species. Angew. Chem., Int. Ed. 2012, 51 (42), 10515−10517. (23) Melendez, E. Titanium complexes in cancer treatment. Critical Reviews in Oncology Hematology 2002, 42 (3), 309−315. (24) Colombo, C.; Morosi, L.; Bello, E.; Ferrari, R.; Licandro, S. A.; Lupi, M.; Ubezio, P.; Morbidelli, M.; Zucchetti, M.; D'Incalci, M.; Moscatelli, D.; Frapolli, R. PEGylated Nanoparticles Obtained through Emulsion Polymerization as Paclitaxel Carriers. Mol. Pharmaceutics 2016, 13 (1), 40−46. (25) Ferrari, R.; Lupi, M.; Colombo, C.; Morbidelli, M.; D’Incalci, M.; Moscatelli, D. Investigation of size, surface charge, PEGylation degree and concentration on the cellular uptake of polymer nanoparticles. Colloids Surf., B 2014, 123, 639−647. (26) Sitia, L.; Ferrari, R.; Violatto, M. B.; Talamini, L.; Dragoni, L.; Colombo, C.; Colombo, L.; Lupi, M.; Ubezio, P.; D’Incalci, M.; Morbidelli, M.; Salmona, M.; Moscatelli, D.; Bigini, P. Fate of PLA and PCL-Based Polymeric Nanocarriers in Cellular and Animal Models of Triple-Negative Breast Cancer. Biomacromolecules 2016, 17 (3), 744− 755. (27) Ferrari, R.; Yu, Y.; Morbidelli, M.; Hutchinson, R. A.; Moscatelli, D. ε-Caprolactone-Based Macromonomers Suitable for Biodegradable

This aspect is not so trivial; however, it is often neglected in nanopharmacology because of careful selection and characterization of the nanovector, and its ability to release the cargo once intravenously administered plays a key role in a drug delivery system. Therefore, obtaining reliable and robust preliminary results, as reported in this study, is the only way to validate a new formulation based on nanotechnology and to think about a real translation from the bench to the bedside.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.6b00753. Characterization of HEMA-RhB macromonomer, TEM of NPs after titanium-loading process, and histological analysis of tissues treated with free Ti (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: raff[email protected]. Phone: +41 44 633 4267. ORCID

Davide Moscatelli: 0000-0003-2759-9781 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge Claudio Colombo for the release studies and Andrea Maniscalco for the ICP-MS analyses. REFERENCES

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