Design And Evaluation Of Red Blood Cell-adhesive Nanoparticles For Lung-targeted Drug Delivery System

The lungs are the gateway of the human body for gas exchange that are constantly exposed to environmental stimuli including pathogens,chemical weapons,toxic agents,and deadly viral and bacterial infections,all of which can lead to activation of inflammatory responses,cell death,and tumorigenesis.It is of great significance to develop the lung-targeted drug delivery system.Herein,based on the nanotechnology and cell-mediated drug delivery system,the red blood cell(RBC)-hitchhiking strategy has shown outstanding advantages in prolonging the circulation time of drugs in vivo and enhancing the targeted distribution of drugs in the lungs.However,existing reports have not elucidated the synergistic relationships between the key quality characteristics of nanocarrier formulations and the efficiencies of RBC-hitchhiked nanoparticle(NPs)accumulating in the lungs,which are crucial for improving lung targeting efficiencies.In this study,ivermectin,a candidate drug against the corona virus disease 2019(COVID-19),was used as a model drug and IVM-loaded NPs were adhered to the surface of RBCs in a non-covalent manner to prepare RBCs nanoparticle complexes(RBC-NPs).The RBC-hitchhiking strategy was used to deliver IVM-loaded NPs to lungs,prolong the circulation time of IVM in vivo,enhance the distribution of IVM in the lung,increase the local IVM concentration in the lung,improve the bioavailability of IVM,improve efficacy while reducing the side effects of IVM,and provide a new option for reusing IVM to treat COVID-19.In addition,the effects of key characteristics of NPs on the adhesion efficiencies between NPs and RBCs and the lung targeting efficiencies were focused on.Based on the influence of NPs properties such as sizes,materials,and surface charge on the adhesion efficiencies between RBCs and NPs,IVM-loaded negatively charged NPs(IVM-PNPs)with different materials(75/25-carboxy-terminated,75/25-ester-terminated,50/50-carboxy-terminated,50/50-ester-terminated)and particle sizes(130 nm,150 nm,180 nm,200 nm)were prepared by solvent diffusion method.Next,IVM-PNPs were coated with chitosan to obtain positively charged chitosan-coated NPs(IVM-CNPs).The prepared NPs were spherical and relatively uniform in particle sizes.The encapsulation efficiency of NPs was greater than 90%,the drug loading was about 8%,and the IVM was continuously released from NPs within72 h,with a cumulative release rate of 65%.RBC-NPs were then prepared by co-incubating erythrocytes with NPs of different properties.75/25 NPs,carboxyl-terminated NPs,and positively charged NPs exhibited high adhesion efficiencies and drug loading,due to their strong hydrophobicity,hydrogen bonding with RBCs,and electrostatic interactions,respectively.IVM-PNPs mainly adhered to the surface of RBCs through hydrophobic interaction and hydrogen bonding,and the adhesion efficiency was 42.44 ± 2.26%;IVM-CNPs mainly adhered to the surface of RBCs through electrostatic interactions,with a strong affinity and an adhesion efficiency of 92.15 ± 2.56 %.The results of confocal laser microscopy images and scanning electron microscopy images confirmed the adhesion of nanoparticles to the surface of RBCs.The results of the in vitro shear experiment showed that the RBC-NPs were relatively stable: the NPs did not desorb from the RBC surface under static or 1 Pa shear force(simulating ordinary blood vessels),while a large number of NPs were desorbed from RBCs under 5 Pa shear force(simulating capillaries).Next,the biocompatibility of the RBC-NPs system was evaluated in vitro and in vivo in terms of cell proliferation toxicity,osmotic fragility,turbulent fragility,oxidative fragility,and in vivo survival time of RBC carriers.The results showed that the NPs had no significant cytotoxicity,and a proper amount of nanoparticle adhesion would not bring side effects to the RBC carriers,which proved that the RBC-NPs had good biocompatibility.The results of in vivo pharmacokinetic experiments showed that RBC-NPs significantly prolonged the in vivo circulation time of IVM.Compared with the negative potential RBC-IVM-PNPs group,the positive potential RBC-IVM-CNPs group exhibited longer circulation time in vivo.In vivo imaging and IVM tissue distribution experiments showed that RBC-NPs significantly enhanced the distribution of nanoparticles in the lung(up to 6-fold).And different RBC-NPs exhibited different lung targeting properties.PNPs adhered to the surface of RBCs exhibited rapid accumulation and clearance in the lung,whereas CNPs adhered to the surface of RBCs exhibited longer-term lung accumulation.Next,ICR mice were used as model animals to construct an acute lung injury(ALI)mouse disease model by exposed tracheal instillation of lipopolysaccharide.The in vivo anti-inflammatory efficacy of free IVM,IVM-PNPs,IVM-CNPs,RBC-IVM-PNPs,and RBC-IVM-CNPs was examined after tail vein injection.The results showed that both the RBC-IVM-PNPs and RBC-IVM-CNPs groups significantly inhibited the inflammatory response and alleviated the progression of lung inflammation in the ALI model mice.Compared with the RBC-IVM-PNPs group,the RBC-IVM-CNPs group exhibited higher anti-inflammatory activity,which may be attributed to longer circulation time and higher bioavailability.In conclusion,the RBC-hitchhiking drug delivery strategy designed and prepared in this study exhibited high biocompatibility,prolonged the in vivo circulation time of IVM,exhibited significant lung distribution characteristics,improved the pharmacokinetics and bioavailability of IVM,had the prospect of reducing the side effects of IVM,and provided an alternative strategy for repurposing IVM for the treatment of COVID-19.Furthermore,according to different redistribution effects of different NPs,RBC-hitchhiked NPs may achieve various accumulation efficiencies and circulation time for different requirements of drug delivery.