Preparation And Characterization Of Hemoglobin Loaded PEG-PLGA Nanoparticle And Its Targeting Function Based On Magnetic Systems

Although medical demands for blood are high, resources for transfusion are lacking. The donated blood used in medical practice often suffers various problems, such as blood-type mismatching, short storage time, virus transmission and availability. The blood substitutes were designed to overcome those problems above, and it is a persistent need in biomedicine. As one kind of the blood substitutes, hemoglobin based oxygen carriers(HBOCs) have become an advanced research focus. Hemoglobin(Hb), as the major component, has the ability to deliver and release oxygen. Modified Hbs, such as cross-linked Hb, polymerized Hb and conjugated Hb, have the functions as the red blood cells to deliver oxygen safely and effectively. In 1990 s, blood substitutes have been developed, and several kinds of them have advanced to clinical trials. However, preclinical and clinical side effects and high mortality associated with HBOC infusion have hindered the development of HBOCs. Despite the safety problem for clinic application, HBOCs can enable survival in life-threatening situations when blood is not an option. Thus, research of blood substitutes aroused great concern in this field, and many attemps have been tried to develop the novel type of the blood substitutes.Nanocarrier systems have become increasingly attractive in biomedical applications, since the material multifunction, flexibility and intelligence, inducing a crucial impulse to the development of HBOCs. Because of the function of the membrane, encapsulating hemoglobin within liposome systems or polymeric systems(Cellular HBOCs) have many advantages over the acellular HBOCs. Firstly, cellular HBOCs protect the surrounding tissues from directly contacting with Hb, and avoid vascular activity. Secondly, Hb encapsulation can prolong Hb circulation half life and not require the modification of Hb molecule. Thirdly, cellular HBOCs avoid Hb colloidal osmotic effect. Biodegradable polymeric systems have the best features of liposome systems in Hb encapsulation. Meanwhile, compared with liposomes, polymeric nanoparticles have a variety of biological properties, such as full biodegradability, mechanical stability and good permeability. The introduction of antiopsonizing moieties on nanoparticles surface, the controllability of particle size and surface charge with tailored characteristic meet the needs of the blood substitutes development.Currently, the study of polymersome encapsulated hemoglobin is still in the preliminary stage of exploration. Poly(lactic acid)(PLA) is one of the most applied polymers in the study of polymeric cellular HBOCs. Because HBOCs deliver oxygen via intravenous transfusion with high dosage, the ability of the HBOCs to remain in circulation is important for their therapeutic potentials. It is reported that the mechanical property plays an important role in avoiding phagocytosis and prolonging the circulation time. The Poly(D,L-lactide-co-gly-colide)(PLGA) was synthesized from lactic acid and glycolic acid. The mechanical flexibility of PLGA with high elastic modulus plays an important role in mimicking red blood cells and prolonging circulation time. Additionally, the biodegradable and biocompatible(PLGA) material, approved by the US FDA, is a safe-to-administrate polymer employed for in vivo applications. Because HBOCs for medical applications are given via intravenous administration, the HBOCs will contact with blood/plasma directly and induce a series of biological responses to the HBOCs, including reticulo-endothelial system and cellular internalization. Surface engineering with poly(ethylene glycol)(PEG) might create nanoparticles capable of reducing protein adsorption on the surface of the nanoparticles, evading the reticulo-endothelial system uptake, improving the biocompatibility of nanoparticles, and maintaining the bioactivity of Hb. Thus, We fabricated hemoglobin loaded nanoparticles(Hb Ps) using m PEG-PLGA(m PEG, methoxy polyethylene glycol) in our study.Owing to the frangibility of protein’s active structure, any conditions which may change protein’s character(such as the stability and solubilization) will lead to protein’s activity declined or lost. Thus, it requires an appropriate method for Hb Ps preparation. There are two differences of Hb from other hydrophilic drugs. Firstly, because of the close structure-function relationships of proteins, the changes of the multi-subunits folding will lead to changes of the oxygen carrying capacity. Secondly, the oxygen delivery carrying capacity of Hb is performed within the polymeric membrane when circulating in the blood, while the other hydrophilic drugs are functional after released from the nanosystems. Because the leaked free Hb will cause the autooxidation rate of Hb, the vascular activity and the renal toxicity, it is imperative to prevent the Hb from leaking from the Hb Ps. The preparation of nanoparticles using polymeric systems has been performed by using many techniques, including water/oil/water(w/o/w) double emulsion technique, solvent diffusion/evaporation technique and self assembly technique, etc. Because w/o/w double emulsion technique is a mild method, and avoid copolymers contacting with protein directly, it is a suitable method for Hb encapsulation. Herein, Hb Ps were fabricated through w/o/w double emulsion technique.In the first part, the applicable factors for uniform of the morphology, homogenization of the Hb Ps size and maintening of the stability of Hb Ps were screened. In the second part, the Hb Ps with good blood compatibility and effective capacity for multiple organ oxygenation were performed. In the third part, we demonstrate an intelligent switchable oxygen delivery systems for targeted oxygen supply by combining magnetic nanoparticles with the Hb Ps for controlled targeting oxygen delivery. The magnetic targeting performance of Mag-Hb Ps in vitro and in vivo was studied. Our studies will provide a strategy for noval blood substitutes development.1. The preparation and the physicochemical property of Hb PsThe effects of the emulsifier, the copolymer/drug mass ratio and the solvent type on the size, distribution and the encapsulated efficiency of Hb Ps were studied. The Soret peak at the character of hemoglobin oxidation showed that ethyl acetate was a preferable organic solvent prepared for the oil phase, because ethyl acetate has little effect on Hb oxidation. As 120 mg/m L of Hb, 20 mg/m Lof copolymer and a 7:3 ratio of PVA and F68 were selected for Hb Ps preparation, the Hb Ps of 196.3 nm with narrow size distribution were successfully fabricated. According to UV–vis spectrophotometry, Fourier transform infrared(FTIR) spectroscopy and circular dichroism(CD) spectral analysis, the Hb Ps preserve the biological and structure features of hemoglobin. According to the oxygen dissociation curve of the native Hb and Hb Ps suspensions, the Hb Ps exhibited oxygen-carrying capacity. The size of the Hb Ps showed no obvious changes from approximately 200 nm for 7 days after being dispersed in saline solution at 37 oC. The 7-day cumulative Hb release was less than 2%, when the Hb Ps were incubated at 37?C. The 42-day cumulative Hb release was about 0.5% after washing the Hb Ps by fresh normal saline, when the Hb Ps were incubated at 4?C. The investigations revealed the good stability of Hb Ps.2. The blood compatibility and effectiveness of Hb PsThe effects of Hb Ps on the blood viscosity, the red blood cells aggregation, the blood coagulation and the hemolysis were investigated. The Hb Ps showed a viscosity comparable to that of blood with no obvious effects on red blood cell aggregation. It was shown that the Hb Ps had no effects on coagulation function according to the thrombelastography result. The Hb Ps induced a percent hemolysis of less than 2%, and therefore, we assume that the Hb Ps had nohemolytic effect on the red cell suspension. The results above showed the good rheological property and blood compatibility of the Hb Ps in vitro. The male Bal B/c mice were divided into three groups(NS group, RBC group and Hb Ps group). The controlled hemorrhage model was established, and the MAP results and at baseline, after hemorrhage and after transfusion was measured. After treatment with NS solution, RBC suspension and Hb Ps suspension, the blood gas results were measured. In this controlled hemorrhage model, the Hb Ps showed blood flow recovery and systemic oxygenation for resuscitation, which indicated the potentials in blood substitutes field.3. Study on preparation of magnetic Hb Ps and its magnetic targeting activitiesAs for regional hypoxia, targeted oxygen delivery was an attractive method in biomedical therapy since it can both improve oxygen deliver efficiency and reduce the side effects of HBOCs on normal tissues. Here we demonstrate an intelligent switchable oxygen delivery systems for targeted oxygen supply by combining magnetic nanoparticles(iron oxide nanoparticles) with Hb Ps for controlled targeting oxygen delivery. After examined by trans-mission electron microscopy, the Mag-Hb Ps(Magnetic hemoglobin loaded nanoparticles) showed the typical spheres with good dispersion. According to UV–vis spectrophotometry, circular dichroism spectral analysis and oxygen dissociation curve, iron oxide nanoparticles showed negligible effects on the structure and function of Hb. The hysteresis loop and the targeting experiments in vitro indicated that the Mag-Hb Ps were superparamagnetic nanoparticles, with magnetic response property. Under the flow rate of 3.876 cm/s、5.814 cm/s and 7.752 cm/s, the magnetic response of Mag-Hb Ps was evaluated by a flow device. The slower flow speed causes the magnetic targeting gathered of Mag-Hb Ps. After the magnet removed, no aggregation was observed because of the shear stress. Good blood compatibility of Mag-Hb Ps was showed according to the hemolytic rate and blood coagulation evaluations. The magnetic targeting experiments in vivo was studied using the fluorescence imaging technique system. We encapsulated the fluorescent probe-Di R into the Mag-Hb Ps, and the distribution of Di R-Mag-Hb Ps was observed, with the magnet focusing on the specific area, which showed the magnetic targeting property of the Mag-Hb Ps in vivo.In conclusion, the Hb Ps were successfully fabricated with m PEG-PLGA via the double emulsion technique. The Hb Ps, with a narrow size distribution in the nanometer range and a nearly uniform morphology, exhibited an applicable oxygen-carrying capacity. The Hb Ps provided a more stable system and were also blood compatible. In a controlled hemorrhage model, this novel type of HBOC showed blood flow recovery and systemic oxygenation for resuscitation. Consequently, the Hb Ps showed potentials in blood substitutes field. Additionally, Mag-Hb Ps were fabricated by loading the iron oxide nanoparticles into hemoglobin loaded m PEG-PLGA nanoparticles for controlled targeting oxygen delivery, displaying oxygen-carrying capacity, good biocompatibility and magnetic targeting property. The Mag-Hb Ps create the opportunity to deliver oxygen, and externally applied magnetic forces actively increase the local Mag-Hb P concentration to improve oxygen supply efficiency. The smart Mag-Hb Ps provide a noval strategy to bridge the gap between targeted drug-delivering system and blood substitutes area.