RAFT-PISA Controlled Preparation Of PFPE-Based ¹⁹F MRI Nanoprobe And Its Application Study

Objective:Fluorine-19 magnetic resonance imaging(¹⁹F MRI)is a non-invasive,non-radioactive,non-tissue penetration depth-limited and quantifiable imaging technique with a wide range of biomedical applications.¹⁹F MRI contrast agents（CA）have gained extensive research interest because of their unique advantages of being free from in vivo background signal interference and quantifiable detection.Linear perfluoropolyethers（PFPE）with multiple effective imaging fluorine atoms and simple ¹⁹F MRI signal peaks can provide strong fluorine imaging signals while ensuring sufficient solubility.The introduction of biocompatible hydrophilic monomers,such as polyethylene glycol,by reversible addition-rupture chain transfer（RAFT）radical polymerization method improves the inherent hydrophobicity of PFPE to obtain water-soluble ¹⁹F MRI probes with good ¹⁹F MRI signals,which can be applied to targeted imaging,drug delivery,and cell localization and tracking.In this study,perfluoropolyether methacrylate（PFPEMA）was selected as the fluorine-19 imaging signal source,and the improvement effect of the introduction of common hydrophilic monomers 2-（methylsulfinyl）ethyl acrylate（MSEA）or oligo（ethylene glycol）methacrylate（OEGA）on the hydrophobicity of PFPEMA was firstly investigated,and the polymer structure optimization was used to screen out the more water-soluble and better imaging The polymer structure optimization was used to screen for more water-soluble and better imaging polymers,and their in vitro imaging,biocompatibility,and cytotoxicity were systematically investigated.Further,PFPE-based nanoprobes of different sizes and morphologies were prepared using 2-（perfluorooctyl ethyl methacrylate）（FOEMA）as nucleating monomer by RAFT polymerization-induced self-assembly（PISA）technique,and their potential as ¹⁹F MRI CA was evaluated by ¹⁹F MRI tests and cytotoxicity studies.Methods:1.The dimeric random copolymers ploy（MSEA-co-PFPEMA）and ploy（OEGA-co-PFPEMA）with controlled molecular weight and narrow molecular weight distribution were synthesized by RAFT polymerization method using（propionic acid-based）butyl trithiocarbonate as chain transfer agent（CTA）,MSEA and OEGA as water-soluble monomers,and PFPEMA as ¹⁹F fluorine-derived monomer,respectively.2.Comparison of the differences in water solubility between the copolymer ploy（MSEA-co-PFPEMA）and ploy（OEGA-co-PFPEMA）.3.The random copolymer ploy（OEGA-co-PFPEMA）was used as a macromolecular chain transfer agent（macro-CTA）by RAFT-PISA technique,and the monomer FOEMA was used for chain expansion to prepare nanoparticles（NP）with precisely controllable size and different morphology.4.The ¹⁹F MRI nanoprobes properties of hydrophilic random copolymers and NPs were investigated by measuring the ¹⁹F spin-lattice relaxation time（T₁）,spin-spin relaxation time（T₂）and ¹⁹F MRI.5.The toxicity of hydrophilic random copolymers and NPs to human umbilical vein endothelial cells（HUVEC）was tested by the 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT）method to evaluate their biocompatibility.Results:1.Compared to ploy（MSEA-co-PFPEMA）,the random copolymer ploy（OEGA-co-PFPEMA）,which is synthesized by introducing OEGA as a hydrophilic chain segment,is more water soluble.The T₂ value of ploy（OEGA-co-PFPEMA）reached a maximum of 66ms when the OEGA to PFPEMA repeat unit ratio was close to 5:1,at which point the fluorine content was 9 wt%.The best ¹⁹F imaging was achieved at 10 mg m L^-1 with a signal-to-noise ratio（SNR）of 136.87.The decrease in SNR values for the polymer ploy（OEGA-co-PFPEMA）below and above this concentration suggests that the strength of the ¹⁹F signal is related to the fluorine atom concentration and aggregation.The polymer ploy（OEGA-co-PFPEMA）with different ratios of repeating units was not significantly toxic to HUVEC in the concentration range of 0-50μM.2.Further chain expansion of ploy（OEGA-co-PFPEMA）was performed using FOEMA and spindle,vesicle and spherical NPs of different sizes were obtained when the molar ratio of FOEMA to ploy（OEGA-co-PFPEMA）was 50,100 and 200,respectively.The NPs were dispersed in aqueous solution at a concentration of 10 mg m L^-1 and a SNR value of 85.81was measured for spindle-shaped NPs with a PFOEMA polymerization of 38 and 25.65 for spherical NPs with a polymerization of 172,this change being caused by a decrease in fluorine content.NPs with FOEMA to ploy（OEGA-co-PFPEMA）molar ratios of 50,100and 200 were incubated with HUVEC in the concentration range of 0-50μM for 24 hours and HUVEC still maintained over 80%survival,indicating that the NPs are biocompatible and are an excellent potential tracer.Conclusion:In this study,water-soluble PFPE-based random polymers with controllable molecular weight and molecular weight distribution were successfully synthesized by RAFT polymerization.A series of polymeric NPs with controlled morphology and uniform size were successfully prepared as ¹⁹F MRI nanoprobes by RAFT-PISA technique.The imaging performance study results illustrated that the SNR values of ploy（OEGA-co-PFPEMA）were up to 136.87 and the SNR values of NPs were up to 85.81 under the suitable fluorine content and solution concentration conditions The bright ¹⁹F MRI images indicated good imaging signals of ploy（OEGA-co-PFPEMA）and NPs.In addition,cytocompatibility and cytotoxicity experiments demonstrated that both ploy（OEGA-co-PFPEMA）and NPs were not biotoxic to HUVEC within the concentration of 0-50μM,and the prepared ¹⁹F MRI CA had a good basis for biological applications.