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Abstract: Diabetic neuropathy (DN) is the most common peripheral neuropathy and long-term complication of diabetes. In view of the pathological basis for the treatment of DN, it is important to prevent nerve degeneration. Most of the current treatment strategies are symptomatic therapies. In this study, we evaluated the effectiveness of magnesium-25, carrying porphyrin-fullerene nanoparticles, on diabetes-induced neuropathy. Previous studies have suggested that dorsal root ganglion (DRG) neurons comprise a specific target and may be responsible for the known complications of DN. Experimental DN was induced by intraperitoneal injection of streptozotocin (STZ) (45 mg/kg). Different forms of magnesium including 25Mg-PMC16, 24Mg-PMC16 and MgCl2 were administered intravenously in equal dose (0.5 LD50) at 48-hr intervals before STZ injection. Peripheral nerves were studied after 2 months of diabetes in groups using qualitative approaches, morphometric analysis of DRG neurons and motor function tests. We showed that STZ-induced DN caused morphological abnormalities in DRG neurons comprising changes in area, diameter and number of A and B cells as well as motor dysfunction in DN. Moreover, our findings indicated that administration of 25Mg-PMC16 as a magnetic form of Mg improved morphological abnormalities and motor dysfunctions significantly, whereas other forms of Mg were ineffective.
Diabetic neuropathy (DN) is the most common late complication of diabetes showing an increasing prevalence . Sixty per cent of patients with diabetes show evidence of peripheral nerve disease . Similar to other diabetes complications, DN has been ascribed to hyperglycaemia and subsequent metabolic abnormalities such as increased polyol pathway activity leading to the accumulation of sorbitol and fructose, imbalances in NADP/NAD+, auto-oxidation of glucose causing the formation of reactive oxygen species, advanced glycation end-products produced by non-enzymatic glycation of proteins, inappropriate activation of protein kinase C (PKC) and a deficit of neurotrophic supports . The pathology of DN includes axonal atrophy, demyelination, loss of nerve fibres and decreased regeneration of nerve fibres . Abnormalities of peripheral nerve function are typical late complications of diabetes. However, by applying streptozotocin (STZ) injections in rats, such a spectrum of disease characteristics can be seen much earlier, i.e. only several weeks after induction of diabetes .
Based on these observations, pharmacological therapies including aldose reductase inhibitors, antioxidant drugs, aminoguanidine and neurotrophic factors have been used in the treatment of DN . Notwithstanding their importance, these treatments are solely symptomatic therapies, because peripheral nerve tissue damage cannot be reversed. Thus, early diagnosis of DN followed by a stringent pharmacological therapy may be effective to prevent the progression of DN, i.e. arrest the degenerative changes of nerve fibre pathology . Oxidative stress mechanisms by induction of mitochondrial dysfunction, decrease in adenosine triphosphate (ATP) and neural death play important roles in peripheral neuropathy [6–8]. Our recent study also indicated the usefulness of PMC16 in DN in terms of improvement of oxidative stress and mitochondrial ATP production .
According to growing advances in the field of nanomedicine, we evaluated in this study the effects of magnesium-25 carrying porphyrin-fullerene nanoparticles that have the potential to prevent dorsal root ganglion (DRG) neuron degeneration and motor dysfunction symptomatically for DN. These nanoparticles are a novel pharmaceutical nano-tool based on the porphyrin-attached fullerene-C60 ‘ball’ (porphylleren-MC16 or PMC16) . Regarding accumulation of this nanoparticle in neural mitochondria [6,10], we aimed to analyse the efficacy of PMC16 on morphological changes of DRG neurons and motor function recovery of DN rats in the present work. Therefore, the benefits of magnetic (25Mg-PMC16), non-magnetic (24Mg-PMC16) and ordinary (MgCl2) forms of Mg were compared in a model of experimental DN.
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The major findings of this work were as follows: (i) 25Mg-PMC16 prevented degeneration of DRG neurons in DN rats; (ii) This type of Mg nanoparticle improved motor function deficiencies in the DN group; and (iii) Other types of Mg did not compensate or ameliorate DN effects.
In the present study, we used amphiphilic and membranotropic nanoparticles that contain ferroporphyrin and fullerene in their structure to analyse their potency to diminish cell destructive effects seen under DN-like conditions in rats. These nanoparticles serve as nanocation particles both in vitro and in vivo and are putatively beneficial in terms of regulating ‘smart release’ of magnesium in hypoxia-induced acidosis in DN. They activate oxidative phosphorylation pathways and increase ATP level. They are safe to use because non-allergic and anti-inflammation properties of most fullerene derivatives ever tested as well as their generally high level of ‘biocompatibility’ make PMC16 suitable for in vivo safety and pharmacological activity studies . Some data also showed that porphyrin-modified negatively charged fullerenes were completely safe products with no tendency to interact with immunological responses of any sort . In addition, the hepatic metabolic attack occurs mainly on the porphyrin domain of PMC16, which causes the formation of haem precursor with neutral biological properties . These effects, along with high rate of renal elimination, lead to consider PMC16 particles perfectly safe for chronic administration as well. These are the reasons why we observed no notable adverse effects and confirm additional support for safety potential of the PMC16 nanoparticles.
As generally accepted, 2,3-DPG (2,3-diphosphoglycerate) resembles a biological indicator of tissue hypoxia and DN . A decrease in the red blood cell 2,3-DPG concentration, which is one of the factors contributing to tissue hypoxia induced by DN, has been reported previously . The decrease in erythrocyte 2,3-DPG concentration in DN rats might be due to hyperglycaemia-induced metabolic deficits that would lead to erythrocyte dysfunction. Erythrocytes with normal functions can increase 2,3-DPG in the hypoxic condition, but abnormal erythrocytes in DN show an increased oxygen affinity and decreased oxygen release . Recently, we have shown that 25Mg-PMC16 nanoparticles improve 2,3-DPG in DN .
Progressive loss of sensory fibres in peripheral nerves is a characteristic phenomenon of DN and is accompanied by degeneration and loss of parent DRG neurons. Hyperglycaemia and subsequent metabolic abnormalities such as increased polyol activity, nerve hypoxaemia and oxidative stress have been suggested as the main causes of DN [2,7,8]. Motor and sensory conduction abnormalities are present in chronic DN. Because motor conduction defects emerge early during DN and progress in a period of time , we evaluated motor functions on days 0, 7 and 60 after STZ administration.
In general, neurons from diabetic rats tend to be smaller, reveal a stronger basophilic staining attitude and have more and larger vacuoles . There is a selective atrophy of the largest DRG neurons in DN. Total count of A and B cells are almost equal in control and under DN conditions, but the ratio of large to small neurons is reduced in diabetic rats. Nerve conduction studies showed reduced conduction velocities in DN . These findings were confirmed by our results. It is conceivable that an impaired synthesis of neuroskeletal proteins may play a role. The vacuolar changes are likely to be caused by an impaired neuronal energy metabolism and perturbed sodium handling possibly via modified Na+/K+-ATPase activity and/or decreased DRG blood flow in DN .
Conduction velocity is directly related to the size of DRG neurons. In DN models, however, atrophy or loss of DRG neurons appears much later than do changes in conduction velocity . It is believed that a slowing of conduction may be the cause of metabolic changes. These changes are associated with impaired mitochondrial generation of high-energy metabolites such as ATP .
A decreased mitochondrial membrane potential induced by diabetes can also cause a decrease in ATP synthesis. Impairment of the bioenergetic system of sensory neurons alters cellular functions  that results in atrophy of neurons and consequently in a deficiency of motor function. Motor abnormality which is because of the mitochondrial dysfunction and accompanied by an accumulation of oxygen radicals indirectly supports this ‘oxidative hypothesis’ .
As we have shown in previous studies, the decrease in 2,3-DPG, hypoxia and a subsequent acidosis in DN are conditions that activate nanoparticles . Under such conditions, 25Mg-PMC16 can increase ATP and thereby affect phosphorylation and oxidation of substrates. This can ameliorate neuronal atrophy and motor function deficiency. A neuroprotective strategy involves mitigating oxidative stress, which is believed to be a neuropathological key process that contributes to degenerative disorders . Fullerenes and metalloporphyrins are also known as neuroprotective agents [21,23]. The neuroprotective activity of fullerenes is based on their capability to react with oxygen radical species such as superoxide (O2˙−) and hydroxyl (˙OH) radicals . It was shown that metalloporphyrins such as ferroporphyrins possess cell protective effects which are related to their ability to detoxify superoxide, H2O2 and peroxynitrite .
Our findings indicate that 25Mg-PMC16, by affecting stress oxidative biomarkers such as total antioxidant capacity, sulfhydryl molecules and lipid peroxidation, induce antioxidant effects (data not shown). These data confirm the results of our previous work . Considering the antioxidant properties of both magnesium and fullerene derivatives, it is thus reasonable to deduce that 25Mg-PMC16, by significant elevation of intra-neuronal Mg and positive role of fullerene structure, improves DN-mediated damage of DRG neurons and motor dysfunction in DN.
The porphyrin receptors on the mitochondrial membrane are tissue selective sites for interaction with the porphyrin domain of PMC16. The water-soluble activity of C60-fullerene derivatives provides selective ability for accumulation of PMC16 inside mammalian mitochondria . Regarding the reports indicating that DRG neurons have large mitochondria with a highly oxidative metabolism which makes these much susceptible to oxidative injury , these nanoparticles can be navigated towards the DRG neurons in a suitably targeted delivery manner. However, the mitochondrial intake, PMC16 pharmacokinetics, particle size and the porphyrin domain–receptor recognition are involved in penetration and accumulation of nanoparticles in DRG neurons.
Another finding of this study is the days long-lasting protective effects of 25Mg-PMC16. The most plausible explanation for this effect is a presence of the PMC16 high-affinity receptors in mitochondrial membrane and activation of a signalling pathway by selective drug–receptor interaction. A protein signalling cascade is known for a number of porphyrin derivatives [6,24–26]. Moreover, the resistance of PMC16 to hepatic metabolism and increase of its half-life in hypoxia conditions  are other possibilities for accumulation and retaining of PMC16 in DN-DRG neurons for a longer period of time.
It should be mentioned that we observed beneficial effects only with 25Mg-PMC16 and not with other forms such as 24Mg-PMC16 and MgCl2. Some reports demonstrated that the separate and targeted PMC16 delivery of magnetic and non-magnetic isotopes provide a high level of ATP production once the magnetic isotope has been applied . On the other hand 25Mg-PMC16 is unable to release Mg efficiently in plasma with normal pH of 7.4 but when intracellular pH will alter to acidic level by cellular damage or hypoxic condition, the magnetic form of nanoparticle can release Mg as much as causing a significant increase in Mg inside DRG cells. Because we used equal doses of three forms of Mg in our study, the first reason to be concluded is that magnetic magnesium by better reaching and accumulation in neuronal cells shows a protective effect much better than other forms of Mg. However, the positive effects of metaloporphyrin and fullerene should not be excluded.
Taken together, our study demonstrates that multiple cellular and molecular pathways are responsible for DN-mediated damages in DRG neurons which are associated with neuronal degeneration, atrophy and impaired motor function. The potency of 25Mg PMC16 to abolish the observed toxic effects may open novel therapeutic aspects for the treatment of diabetic symptoms associated with neuronal destruction.