Construction And Evaluation Of Polydopamine-Coated Hemoglobin Particles Based On The Templated Self-Assembly Technique

Transfusion has been widely used in the therapy of chronic or acute anemia,working as a vital life-saving strategy for both civilian and military purposes.Of note,the administration of red blood cells(RBC)is important to sustain and improve the function of oxygen bind-release of the patients.The recent years have witnessed the dramatic increase of the clinical demand for blood,while its natural source is relatively limited.In addition,other factors,like the requirement for cross-match and the potential risks of blood-transmitted diseases,has made it more difficult to ensure the safety and effectiveness of transfusion.As a supplement for natural blood,RBC substitutes have gained much attention in terms of its research and development.RBC substitutes outperforms its natural counterpart in following aspects:(1)RBC substitutes can be disinfected by physical or chemical technique,lowering the risk of pathogen transmission associated with its administration.(2)Without surface antigen,there is no demand for cross-match before its use,which is helpful to save time for trauma treatment life saving.(3)RBC substitutes hold an extended storage life after its lyophilization.The RBC substitutes can be divided into two categories based on its bioactive ingredient,one is perfluorocarbons(PFCs),the other is hemoglobin-based oxygen carriers(HBOCs).A good oxygen-delivery capability of PFCs is thanks to its high solubility of oxygen,meaning that it can not continuously release oxygen as RBC do,which has hindered its applications.Generally,PFCs is used for the oxygen supply of patients suffering massive blood loss,or served as an oxygen therapeutic agent.Hemoglobin(Hb)is the fundamental ingredient in RBC to deliver oxygen,each molecule of which is composed of two?subunits and two?subunits(?₁?₁?₂?₂).Each subunit contains a heme porphyrin ring centered by Fe²⁺,which can bind an oxygen molecule.With Hb as its bioactive component,HBOCs can deliver oxygen closer to RBC.In another word,it can bind and release oxygen in a balanced manner.The research of chemically modified HBOCs has been started earlier than other varieties,and its relevant technique is more sophisticated.While,numerous toxic and side reactions induced by the transfusion of chemically modified HBOCs have became the obstacles of its further development.Stroma-free Hb will be filtrated by glomerulus following its injection,and thus lead to renal failure and hemoglobinuria,because of its structural instability and small size.The research of the first-generation HBOCs,also named as chemically modified HBOCs,is aimed at stabilizing the tetrameric structure of Hb and enlarging its size through different modification strategies.In doing so,the biosafety of Hb can be evidently improved.Relevant modification methods include intramolecular crosslinking,intermolecular polymerization,and polymer modification.Up to now,chemically modified HBOCs outperform any other type of HBOCs products in terms of the sophistication of fabrication technique,and the varieties of products undertaken clinical trials.Disappointingly,various side reactions associated with the administration of chemically modified HBOCs,like vasoconstriction,hypertension and heme-mediated oxidative toxicity,have hindered its further development and application.In fact,Hemopure,Hemolink,Polyheme and other chemically modified HBOCs products have been terminated due to the toxicity concerns in vivo.With the development of nano drug-delivery technology,the nano-and micro-sized hemoglobin particles as novel HBOCs have gained increasing attention in the HBOCs community.There two types-of nano-and micro-sized hemoglobin particles,Hb-encapsulated particles and templated self-assembled Hb particles.The synthesis principle for Hb-encapsulated particles is to enclosing Hb into nanocapsules using lipid or synthetic polymers.The capsule membrane can protect the direct contact of encapsulated Hb with the blood components,reducing its capture by reticuloendothelial system(RES)and extending its retention time in the blood.Moreover,no any chemical modification of Hb is required in its encapsulation process,meaning that its structure and function can be better maintained.One shortcoming for encapsulated Hb as HBOCs is that Hb is physically encapsulated by coating material,thus its loading capacity is relatively low.For another,lipid membrane suffers oxidation,leading to oxidative injury after injection.Templated self-assembly technique can highly concentrate Hb through the interaction of inorganic template with Hb resulting in a high Hb loading capacity can be achieved.Thanks to this,it is possible to avoid side reactions related with injection of large dose.Also,the size and morphology of templated self-assembled Hb particles can be easily adjusted by selecting templates appropriately.Templated self-assembly technique has enjoyed numerous advantages in the construction Hb-loaded particles,and shown great potential in HBOCs fields.However,several issues remain to be addressed so as to promote its further development,including its absence of surface protection layer,the instability of glutaraldehyde(GA)crosslinking and oxidative toxicity after injection.Li has fabricated Hb-loaded hollow capsules using GA as the crosslinker,based the layer-by-layer self-assembly technique.To avoid the use of GA,which has biotoxicity,Li has employed dialdehyde heparin(DHP)as the crosslinker,and fabricated Hb particles,(Hb/DHP)₆,with improved biocompatibility.To increase the Hb content in per particle,B(?)umler and his co-workers have loaded Hb through the co-precipitation of Hb and inorganic salt,followed by GA crosslinking and the templates removal with EDTA(ethylene diamine tetraacetic acid).The Hb content in each particle prepared by this method can reach up to 80%of that of RBC.Thanks to this high Hb-loading capacity,it is possible to reduce the injection dose of Hb particles,and avoid side reactions related with large-dose injection,using templated self-assembly technique.Although the templated self-assembly technique is seeing promising potential in the research of HBOCs,one of its major issues,that the loaded Hb is structurally and functionally instable,is waiting to be resolved.Firstly,this type of HBOCs is lack of surface protection barrier,meaning that Hb is exposed to the surrounding tissue and blood.Therefore,the leakage of Hb will cause side reactions.Secondly,GA is widely employed to crosslink Hb on the templates surface.The imine bond between Hb and GA is easily ruptured through hydrolysis in solvent,followed by the generation of free Hb and GA,both of which will account for toxicity in vivo.Additionally,without the reductase which is contained in natural RBC,the self-oxidation of Hb in HBOCs products will be accelerated,leading to worsened oxidative stress after administration.Polydopamine(PDA)has strong adhesion and good antioxidant capacity,providing an opportunity to solve the above problems of templated self-assembled HBOCs.As a new functional polymer,polydopamine has been widely used in environmental catalysis,anti-tumor therapy,drug delivery and water pollution treatment.Under weak alkaline conditions,dopamine can spontaneously polymerize and adhere to the surface of the substrate steadily,and its adhesive layer can serve as a protective barrier of the substrate.Therefore,the combination of polydopamine surface modification and template self-assembly technique is expected to overcome the existing problems of Hb exposure and crosslinking instability of HBOCs.In the process of polymerization,polydopamine has the loss of electrons,so it can combine with free radicals and work as an antioxidant.Previous studies have shown that 120?g polydopamine nanoparticles can scavenge 85%DPPH free radicals.The adhesive layer of polydopamine can provide antioxidant protection for hemoglobin,inhibit its autooxidation,and reduce the oxidative damage after infusion.In our previous work,polydopamine-loaded hemoglobin nanoparticles(Hb-PDA NPs)were constructed by one-step assembly method,and its ability to reduce the oxidative toxicity of hemoglobin was verified in vitro.The formation of Hb-PDA NPs is mainly based on the coating of polydopamine on the surface of a single hemoglobin molecule.The disadvantages are as follows:on the one hand,the load capacity of hemoglobin is low;On the other hand,the particle size is too small,meaning that it is easy to cause biological toxicity after infusion.Therefore,based on our previous work,this thesis combined the templated self-assembly technique with the surface modification of polydopamine.The inorganic salt co-precipitation process was used to increase the hemoglobin loading rate and increase the particle size,avoiding vascular activity caused.The adhesive layer of polydopamine served as a surface protective barrier of particles to avoid the exposure of hemoglobin and reduce its leakage.The adhesive and antioxidant properties of polydopamine can better maintain the structure and function of hemoglobin,which provides a method basis for solving the problem of insufficient stability of template self-assembled HBOCs.This study consists of the following four parts:Chapter One:the construction of hemoglobin-loaded polydopamine particles based on templated self-assembly technique.In this part,the preparation route of HBOCs by the combination of inorganic salt co-precipitation and polydopamine surface modification was developed,and the physical and chemical properties of Hb-PDA were investigated.1.Mn CO₃ presented spherical shape and dispersed uniformly in solvents.Also,it showed a high encapsulation efficiency for Hb,which can improve the load rate of Hb and optimize the oxygen supply efficiency thereby.Moreover,Mn CO₃ has an average particle size of 820.7nm,which is conducive to reducing vascular activity and avoiding the rapid clearance by phagocytes.As a consequence,Mn CO₃ was selected as the preparation template for Hb-PDA in this work.2.7.2mg/m L was screened as the optimal initial concentration of Hb for co-precipitation.Hb content in the particles as aforementioned was 23.8%-50.8%of that in red blood cells,demonstrating that a high Hb loading was achieved thanks to the co-precipitation process.3.Hb-PDA presented spherical shape,and its average size was 840 nm.The CLSM(confocal laser scanning microscopy,confocal laser microscope)results has directly confirmed that Hb was loaded into Mn CO₃.By means of spectroscopy,it was verified that the chemical structure of Hb was maintained,which was beneficial to maintain its oxygen-carrying function.Chapter Two:In vitro evaluation of hemoglobin-loaded polydopamine particles.This section aims to investigate the biocompatibility and efficacy of Hb-PDA in vitro to evaluate its potential as a novel HBOCs.1.Hb-PDA did not cause the risk of hemolysis in erythrocytes,neither interfere with coagulation function.The effect of Hb-PDA on whole blood viscosity was not more drastic than that of normal saline on whole blood,demonstrating that Hb-PDA did not cause abnormal changes in whole blood viscosity.These results indicated that Hb-PDA had good blood compatibility.2.The cell viability of HUVEC treated with Hb-PDA of 200,400,600,800 and 1000?g/m L was over 98%,indicating a low cytotoxicity of Hb-PDA.Hb-PDA was not phagocyted by RAW267.4 after co-incubation for up to three hours,which further verified a high biocompatibility of Hb-PDA.3.Hb-PDA,in the concentration range of 5.0-10.0 mg/m L,could release more oxygen than free Hb,indicating that the modification of PDA is helpful to improve the oxygen delivery efficiency of Hb,which may be related to the consumption of dissolved oxygen in the system during PDA polymerization,the reduction of Hb leakage and the antioxidant property of PDA adhesive layer.2.Hb-PDA could scavenge up to 83.1±1.8%hydroxyl radical,85%of the scavenging capacity of Trolox.The cell viability of HUVEC treated with Hb and Hb-PDA were 74.3±4.3%and 84.5±1.8%,respectively.As a result,the antioxidant property of Hb-PDA were verified in vitro.Chapter Three:stability investigation of hemoglobin-loaded polydopamine particles.The purpose of this part is to investigate the effect of polydopamine surface modification on maintaining the stability of HBOCs.In the meantime,the binding mode of PDA and Hb was discussed,to preliminarily reveal the mechanism of polydopamine surface modification enhancing the stability of the particles.1.When Hb-PDA was stored at 4?for two weeks,its average particle size and PDI did not change significantly(P>0.05),and no Hb burst release occurred.PDA as a protective layer can effectively reduce the production of free Hb,which is conducive to alleviating the toxic and side effects of free Hb.2.The flow chamber was used to simulate the blood flow environment.Under the shear rates of 200 and 400 S^-1,the particle size of Hb-PDA changed little,and there was no statistical difference with the shear time changed(P>0.05);when the shear rate was higher than 800 S^-1,the particle size corresponding to different shear time began to show statistical difference(P<0.05).It was speculated that the contact frequency between the particles and the wall of the small chamber increased under the high shear stress,thus affecting the shape and size of the particles.After treated with shear stress for different time,the Hb leakage rate was no more than 5%.It indicated that Hb-PDA can withstand shear stress,and PDA protective layer can maintain a stable micro-nano structure for the particles under flow state,avoiding substantial release of Hb.3.During the formation of the PDA adhesive layer on the particle surface,the interaction between PDA and Hb through phenolic hydroxyl-hydroxyl group and phenolic hydroxyl-amino group was the main force.Compared with cellular HBOCs based on liposomes or amphiphilic polymers,the binding of PDA to Hb through phenolic hydroxyl groups is more stable,which is helpful to avoid Hb leakage.Chapter Four:In vivo evaluation of hemoglobin-loaded polydopamine particles.In vivo investigation of Hb-PDA was conducted in this section,including efficacy,distribution,metabolism and acute phase toxicity,laying a foundation for its development and application.1.Hb-PDA could resuscitate the rats with hemorrhagic shock,and the MAP(mean arterial pressure)of the rats resuscitated by Hb-PDA was significantly higher than that of the NS(Normal Saline)group at 90 min and 120 min,indicating that Hb-PDA can maintain blood pressure more persistently.There was no abnormal increase in blood pressure after the infusion of Hb-PDA in rats,and the blood gas state of the rats were restored,possibly because the surface modification of polydopamine improved the stability of particles and decreased the vascular activity caused by free Hb.The results of the oxidative stress indicators suggested that PDA could function as an effective antioxidant in vivo.2.In vivo distribution experiments showed that particles accumulated the most in the lungs after infusion.Due to their large particle size(800 nm),the particles can easily adhere to the inner wall of the capillars on the alveolar surface.However,after the particles were desattached,they could continue to participate in the blood circulation,which can prolong their in vivo retention time.The content of Hb-Mn CO₃-PDA in the kidney,liver and lung reached its peak at 12 h after infusion,but dropped sharply from12 h to the 24 h.On the one hand,it indicated that a large number of particles were metabolized and cleared during this period;on the other hand,it suggested that Hb-Mn CO₃-PDA had a long retention.TEM results further revealed that the particles could be metabolized and removed by Kupffer cells in liver tissue and macrophages in spleen tissue.3.After Hb-PDA infusion,there was no abnormal changes in the white blood cell amount and the proportion of all kinds of white blood cells.Also,no abnormal change of the biochemical indexes was induced by Hb-PDA,and only mild lesions were observed in the kidney tissue,indicating that Hb-PDA had low toxicity in vivo.