Investigation Of Hydroxyethyl Starch Coated Superparamagnetic Iron Oxide Nanoparticles As Novel MRI Contrast Agents

In the past decades,the synthesis and applications of superparamagnetic iron oxide nanoparticles(SPIONs)have been active research areas in the fields of chemistry,chemical engineering,materials science,and biomedical sciences.With the unique properties such as superparamagnetism,low toxicity and biocompatibility,SPIONs have attracted increasing attentions for the biomedical applications,including magnetic resonance imaging(MRI),hyperthermia,cell labeling and imaging and targeted drug delivery.On the other hand,there are certain safety concerns of SPIONs,e.g.,oxidative stress,induced toxic cellular signaling pathways,altered gene expression profiles and overloaded iron homeostasis.In this dissertation,biocompatible SPIONs coated with hydroxyethyl starch(HES)was synthesized and modified by various surface coating materials to enhance the pharmacokinetics profiles of MRI for SPION.In addition,the toxicity of SPION was determined by the systems toxicology assessment.The main work and results are as follows:(1)The development of HES 200/0.5 coated water-soluble SPIONs for scaled up production.The synthesized SPIONs had high water solubility with the hydrodynamic size of 33.65 ± 0.92 nm and zeta potential of-6.60 ± 0.38 mV(pH 7.2).In weak acidic and alkaline solutions,the hydrodynamic size of the SPIONs remained stable over 36 months.The SPIONs had superparamagnetism at room temperature with no residual magnetism.The magnetic saturation intensity and transverse relaxivity(R2)of the SPIONs were about 62.60 emu/g and 45.73 mM-1 s-1,respectively.The SPIONs were able to enhance the MRI T2 signal of liver significantly at a low intravenous injection dose of 0.5 mg Fe/kg.(2)The assembly of the SPIONs to nanoclusters(NCs).This was achieved with the addition of polycationic poly-L-lysine(PLL)polymers through the electrostatic interaction with the negative charge of the SPIONs surface.TEM results showed that the SPIONs were spherical nanoclusters.The nanocluster size was also tightly controlled by the amount of PLL.Further study indicated that the transverse relaxivity of the nanoclusters was positively correlated with the size in the range of 30 ~ 140 nm.The in vivo MRI enhancement of orthotopic liver tumor was assessed in a mouse model after the intravenous injection of SPIO nanoclusters at 0.5 mg Fe/kg with the size about 30,80 and 120 nm,respectively.MRI revealed a significant negative contrast enhancement for tumors with diameters of 1 ~ 2 mm.In addition,the T2 signal enhancement in the liver was decreased most by the SPIO-PLL NCs with the size about 120 nm.In the normal mice,24 h after injection,there was no significant accumulation of SPIONs in various organs,suggesting a rapid tissue clearance.More importantly,most of the SPIONs had been found to be excreted through urine after 11 days.Thus,rapid bioclearance and full urine excretion of SPIONs at low injection dose had been achieved with the SPIO-PLL NCs to minimizing the potential long-term cytotoxic effects.(3)Systems toxicity assessment of SPIONs.Considering iron overload by SPIONs in the liver,especially the liver with chronic diseases may have serious toxicological effects,a systems toxicology assessment was carried out to reveal the impact of iron overload by SPIONs on a mouse cirrhosis model.A single dose injection of SPIONs at 5 mg Fe/kg in the cirrhosis mice induced a septic shock response at 24 h with significantly impacted serum biochemistry markers,such as elevated liver function markers of ALT,AST and AKP and decreased lipid metabolism markers of CHOL,HDL-C and LDL-C.Even after in 14 days,the lipid metabolism and serum iron level were still abnormal,indicating that iron overload in cirrhosis had a potential long-term toxicological effect.In contrast,the normal liver with the same injection dosage had several increased serum markers of liver function at 48 h,yet all of them returned normal within 14 days.On the other hand,low dose of 0.5 mg Fe/kg did not lead to significant toxic effects on both normal and cirrhosis liver.Analysis with RT2 PCR array of the toxicity pathways revealed that the iron overload by high injection dose of SPIONs induced significant expression changes of a distinct subset of genes in cirrhosis liver as compared to those of normal ones.All these results suggested that excess iron of the high dose of SPIONs might be a risk factor for cirrhosis because of the marked impacts of elevated lipid metabolism,disruption of iron homeostasis and possibly,aggravated loss of liver functions.Therefore,it is necessary to optimize the dosage of SPIONs to reduce the potential toxicological effects caused by iron overload of SPION.(4)The tumor targeting delivery of SPIO-PDL-pHLIP NCs.pHLIP is a low pH cell membrane insertion peptide targeting tumor acidic microenvironment.Poly-D-lysine(PDL)is not biodegradable and can maintain the integrity of nanoclusters when transported in vivo.It was demonstrated that SPIO-PDL-pHLIP NCs with size about 60 nm had the best tumor targeting efficiency and the highest tumor accumulation.In contrast,the targeting effect of SPIO-PDL-pHLIP NCs with larger size(about 80 ~ 120 nm)was significantly reduced.In addition,SPIO-PDL-pHLIP NCs with size about 60 nm had effective targeting in all Bel 7402,H22 and B16F1 subcutaneous tumors,indicating that SPIO-PDL-p HLIP NCs system may be applicable to a variety of tumors and diseases due to their unique acidic environment for diagnosis and drug delivery.In conclusion,this dissertation developed a new method for preparing water-soluble SPIONs in large scale with excellent physiochemical properties.Intravenous injection of synthesized SPIONs at low dose showed a well-enhanced T2 weighted MRI effect,rapid tissue bioclearance and full urine excretion.The potential iron overload caused by SPIONs was found to be long-term toxicity in cirrhosis liver only with high dose.What's more,the lipid and iron metabolism disorders,and even the aggravated loss of liver function was specific in cirrhosis while the impact of excess iron to the normal liver was recoverable.Those results suggested that the application of SPIONs should be minimal for potential toxicity in patients with chronic liver diseases.To enhance the safety,an active targeted SPIO nanoclusters were developed to target tumor acidic microenvironment.The nanoclusters exhibited effective tumor targeting in various tumors with high selectivity,suggesting that a targeted MRI contrast agent may be an effective solution to minimize the potential toxicity.