Optimization of superfluorinated PLGA nanoparticles for enhanced cell labelling and detection by 19F-MRI

https://doi.org/10.1016/j.colsurfb.2022.112932Get rights and content

Highlights

  • High encapsulation yield of PERFECTA in PLGA NPs guarantees improved 19F-MRI sensitivity.

  • PERFECTA loaded PLGA NPs shows reduced T1 relaxation times enabling fast-imaging methods.

  • Using sodium cholate (NaC) as stabilizer on superfluorinated PLGA NPs facilitates NP cellular uptake.

Abstract

Fluorine-19 (19F) Magnetic Resonance Imaging (MRI) is an emergent imaging technique for molecular imaging and cell tracking. Lack of intrinsic 19F signals in tissues allows unambiguous in vivo detection of exogenous fluorinated probes, complementary to the anatomical and multiparametric information obtained by standard 1H-MRI. However, the intrinsic low sensitivity of MRI technique requires the need of designing increasingly effective fluorinated tracers. PERFECTA, with its 36 magnetically equivalent 19F atoms and a designed branched molecular structure, represents an excellent superfluorinated tracer. In this paper, we report the development of PERFECTA loaded PLGA NPs stabilized by different coatings as promising 19F-MRI probes. The results clearly show the optimal cellular uptake of the produced colloidally stable PERFECTA loaded PLGA NPs without impact on cells viability. Importantly, NPs stabilization with the anionic surfactant sodium cholate (NaC) clearly enhances NPs internalization within cells with respect to PVA-coated NPs. Moreover, the optimized NPs are characterized by shorter T1 relaxation times with respect to other PERFECTA formulations that would allow the increase of 19F-MRI sensitivity with fast imaging acquisitions.

Introduction

Fluorine-19 (19F) Magnetic Resonance Imaging (MRI) is a promising whole-body technique for in vivo molecular imaging and cell tracking. In fact, using the magnetic moment of 19F nucleus, which has 100% natural abundance, a gyromagnetic ratio close to that of the proton and 83% sensitivity of 1H, enables the direct detection of fluorinated exogenous products by combined 1H/19F-MRI, revealing them as “hot-spots” over anatomical images in living systems, which are naturally free of background signal [1], [2], [3]. Thanks to the insignificant endogenous amount of 19F in the body, the fluorinated product is directly localized and quantified, differently from the traditional 1H-MRI contrast agents that are detected only through pre and post image acquisitions [4], [5], [6]. These distinctive features stimulated an extensive development of 19F-MRI towards cell tracking for immunotherapy [7], [8] and inflammation imaging for various diseases, in particular multiple sclerosis, rheumatoid arthritis and myocardial infarction [9], [10], [11]. As for all MRI methods, 19F-MRI suffers for low sensitivity and requires much effort in the development of optimal 19F-probes together with suitable imaging pulse sequence and radiofrequency receiver coils.

An ideal 19F-MRI probe should have the highest number of equivalent 19F atoms resulting as a single sharp resonance signal and possess a suitable biological half-life. Some of us have proposed a promising superfluorinated probe, PERFECTA, bearing 36 magnetically equivalent 19F atoms [12], [13], [14]. Differently to other perfluorocarbons (PFC), PERFECTA is solid and possesses a hydrocarbon polar core and four ether bonds, which may promote molecular degradation in the environment through the cleavage of such bonds in physiological conditions [15]. As all PFCs, encapsulation of PERFECTA into biocompatible nanovectors is required for allowing administration both in vitro and in vivo [12], [13], [14], [16]. More recently, it has been shown by Srinivas et collaborators [17] that the local structure of poly(lactic-co-glycolic acid) co-polymer (PLGA) nanoparticles (NPs) loaded with perfluoro-15-crown ether (PFCE), a commonly used 19F-MRI probe, can strongly influence the clearance rate of PFCE, rendering it almost 15-fold faster cleared than conventional PFCE emulsions with a clear relevance for its use in clinics. Interestingly, the same authors optimized a scaled-up production of these NPs, also increasing reproducibility, for facilitating their clinical translation [18].

Thus, in the perspective of developing an effective, biocompatible PERFECTA-based formulation for 19F-MRI, which can also be easily scaled-up, PLGA seems an optimal option. PERFECTA loaded PLGA NPs (PERFECTA@PLGA NPs) have only been obtained so far in co-presence of PFCE, which seemed critical for a successful encapsulation of solid PERFECTA in the polymeric matrix [19]. In the present work, a careful experimental optimization has been performed to obtain a robust protocol for producing PERFECTA@PLGA NPs characterized by an optimal colloidal stability, high loading yields and effective MRI properties. An initial study was done to find the optimal solvent for solubilizing both PERFECTA and PLGA and guaranteeing NPs stable dispersions in aqueous media, with suitable morphology, colloidal stability and PERFECTA encapsulation yield. Different PLGA polymers and stabilizers were explored to better define the optimal composition for generating PERFECTA@PLGA NPs suitable to 19F-MRI applications. Of note, negative charged NPs, stabilized by sodium cholate (NaC) were preferentially internalized by the cells leading to a more intense 19F-MRI response. Interestingly, the optimized PERFECTA@PLGA NPs were also characterized by excellent relaxation times, which presents advantageous for increasing 19F-MRI sensitivity and using fast-imaging methods [12], [13], [16]. Overall, we report the development of effective PERFECTA@PLGA NPs as promising 19F-MRI probes.

Section snippets

Materials

PERFECTA was synthesized as previously described [12]. Poly (lactic-co-glycolic acid) (PLGA; acid terminated Resomer 502 H or ester terminated Resomer 504, Mw 7–17 kDa and 38–54 kDa respectively), polyvinyl alcohol (PVA; 30–70 kDa, 87–90% hydrolyzed), sodium cholate (NaC), and ethyl acetate (EtOAc, purity ≥ 99.5%) were supplied by Sigma Aldrich (Germany). Ultrapure type-I Milli-Q water (MilliQ water) (18.2 mΩ/cm) was obtained by a Simplicity® water purification system (MERK, Germany). For

Formulation of PERFECTA@PLGA nanoparticles

NPs were formulated by the emulsification solvent evaporation method. Briefly, 20 mg of PERFECTA were dissolved in 1.5 mL of EtOAc and heated to 65 °C to ensure its complete dissolution. PLGA (20 mg) was dissolved in the same volume of the organic solvent and was brought to the temperature of PERFECTA solution. Dissolved PLGA was then mixed with the organic solution of the fluorinated probe. The resulting organic solution was rapidly mixed pipetting it up and down and added dropwise to an

PERFECTA solubility optimization for loading in PLGA NPs

From previous studies, the superfluorinated molecule PERFECTA demonstrated to be an optimal probe for 19F-MRI [12], [13]. In addition, PERFECTA has shown to have ideal chemical configuration for functioning as bimodal imaging probe thanks to its exceptional Raman fingerprint [20]. Thus, considering the extensive use of PLGA in biomedical applications a huge effort has been dedicated to the development of novel biocompatible formulations of PERFECTA based on PLGA NPs [12], [13], [16].

The first

Conclusions

In this study we have developed for the first time PLGA NPs loaded with the superfluorinated probe PERFECTA. The preparation protocol was optimized using different polymer derivatives (with different terminal group) stabilizers in order to obtain colloidally stable NPs with high encapsulation efficiency. Moreover, the obtained NPs were also tested on a phagocytic cell line as model system of neuroinflammation process for determining their viability and cell labelling efficiency. Interestingly,

CRediT authorship contribution statement

Cristina Chirizzi: Data curation, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing. Lodovico Gatti: Data curation, Investigation, Methodology, Writing – original draft, Writing – review & editing. Maria Sancho-Albero: Data curation, Investigation, Methodology, Writing – review & editing. Victor Sebastian: Conceptualization, Data curation, Investigation, Methodology, Visualization, Funding acquisition, Supervision, Writing – review & editing. Manuel

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

F.B.B. and P.M. are thankful to the project NiFTy funded by MUR (PRIN2017, no. 2017MYBTXC) and to the NEWMED project, ID: 1175999 (funded by Regione Lombardia POR FESR 2014 2020). F.B.B. and C.C. also acknowledge financial support from P2RY12 project, ID: GR-2016- 02361325 (funded by the Italian Ministry of Health). We would like to thank Dr Marina Cretich from “Istituto di Scienze e Tecnologie Chimiche” (SCITEC-CNR) in Milan for the use of NTA Instrument. Experimental Imaging Center at IRCCS

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  • Cited by (2)

    1

    These authors have equally contributed

    2

    Present address: Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milano 20139, Italy

    3

    Present address: Department of Biochemistry and Molecular Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Via Mario Negri 2, 20156 Milano, Italy

    4

    Present address: Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, V.le F. Stagno d′Alcontres 31, 98166 Messina, Italy

    5

    Present address: Euro-BioImaging ERIC, Institute of Biostructures and Bioimaging (IBB), Italian National Research Council (CNR), Via Nizza 52, 10126 Torino, Italy

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