An oral hypoglycemic agent for T2DM with the function of alleviating multi-complications

An oral hypoglycemic agent for T2DM with the function of alleviating multi-complications | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article An oral hypoglycemic agent for T2DM with the function of alleviating multi-complications Shiyong Zhang, Xiao Xiao, Xiaoluan Lu, Yi Zhang, Xingwu Ran, Yangyang Cheng, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4577178/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Although hypoglycemic drugs with the function of alleviating complications such as GLP-1RA and SGLT2i have been used in clinic, these drugs are still far from meeting the treatment needs. Herein, we report an oral hypoglycemic agent for T2DM with the function of alleviating multi-complications including neuropathy by way of self-polymerizing dietary antioxidant lipoic acid (LA) into a nanodrug called poly-lipoic acid particles (pLAPs). The self-polymerization made the oral T 1/2 and AUC 0 ~ 72 h of LA up to 23.2 h and 3761.8 ± 55.9 h*µg/mL, ~ 46 times and ~ 23 times higher than that of LA monomer, respectively. As entering the cells, pLAPs were slowly degraded to LA in response to glutathione to prolong the intracellular retention time of LA from ~ 10 min to > 6 h. This prolongation achieved a continuous activation of the insulin signaling pathway, making a long-lasting and near-normal blood glucose level hypoglycemic effect come true. Thanks to the significant improvement of pharmacokinetics and intracellular retention time, pLAPs restored the oxidative stress and inflammation-related indicators to the normal control levels in the T2DM models with neuropathy and angiopathy, leading to the outstanding therapeutic effect on these complications. Importantly, the promising efficacy of pLAPs was confirmed in the model of spontaneous diabetic rhesus monkeys with neuropathy. Considering its excellent biosafety, the oral hypoglycemic drug with multi-complication alleviation holds clinical potential. Physical sciences/Nanoscience and technology/Nanomedicine Biological sciences/Biotechnology/Biomaterials Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Type 2 diabetes (T2DM) is a metabolic syndrome characterized by insulin resistance and accompanies the high risk of multi-complications 1,2 . According to the American Diabetes Association (ADA), for patients with T2DM more than 10 years, the complications happened as high as 98%. Unfortunately, the conventional hypoglycemic drugs such as metformin, sulfonylureas, thiazolidinediones, and dipeptidylpeptidase-4 inhibitors showed no therapeutic effect on complications 3,4 . Consequently, the development of hypoglycemic drugs with the function of alleviating complications is urgently needed. The complications of T2DM are driven by persistent hyperglycemia mediated oxidative stress, including microangiopathy, macroangiopathy and neuropathy, accounting for ~25%, ~27%, and ~50% of the population with T2DM, respectively 5,6,7 . For angiopathy, there are some drugs in clinic, such as glucagon-like peptide-1 receptor agonist (GLP-1RA) and sodium-glucose cotransporter 2 inhibitor (SGLT2i), which are the first-line drugs for T2DM combined with chronic kidney disease (a type of microangiopathy) and cardiovascular disease (a type of macroangiopathy) 8,9 . In 2023, the global annual sales of these two drugs reached up to $23.6 and $12.4 billion, respectively. However, for T2DM combined with neuropathy, the complication with the highest morbidity, there is no drug in clinic and even no report in basic research. In addition, GLP-1RA needs usually to be injected subcutaneously and the long-term use will cause the deterioration of retinopathy 10,11 . The SGLT2i is prone to causing the urinary and reproductive system infections and its hypoglycemic effect will decrease with the decline of renal function 12,13 . Obviously, hypoglycemic drugs with the function of alleviating complications are far from meeting the clinical needs. As a dietary supplement with the antioxidant capacity 400 times higher than that of vitamin C, lipoic acid (LA) holds the functions of hepatic protection and anti-aging etc 14 . Besides the healthcare function, LA also is a clinical drug for diabetic peripheral neuropathy (DPN, a main type of neuropathy) by etiological treatment 15 . Interestingly, it was reported that LA can block hemoglobin glycosylation in the blood, temporarily activate the insulin receptor substrate 1 (IRS-1)/protein kinase B (Akt) and adenosine monophosphate-activated protein kinase (AMPK) signaling pathways to promote small amounts of glucose uptake after entry into the cells 16,17 . Unfortunately, the plasma half-life of LA is only ~30 minutes 18 . The quick body clearance leads to extremely limited inhibition of hemoglobin glycosylation. Moreover, LA held a very short intracellular retention time of ~10 minutes, far below the 1 h persistent activation of the IRS-1/Akt signaling pathway required for LA to produce a significant glucose uptake effect in cells 19,20 . In short, LA has no hypoglycemic effect 21,22,23 . Herein, we report an oral hypoglycemic agent for T2DM with the function of alleviating multi-complications including neuropathy by way of self-polymerizing LA to a nanodrug poly-lipoic acid particles (pLAPs). As shown in Fig. 1, pLAPs were well absorbed into the blood through the polydisulfide backbone mediated mucus penetration, epithelial cellular uptake and epithelial penetration. Due to the negative surface charge and high molecular weight, the plasma half-life of pLAPs was greatly extended from 0.5 h of LA to 23.2 h. As entering the cells, pLAPs were slowly degraded into LA by glutathione (GSH) mediated depolymerization. This unique degradation behavior prolonged the intracellular retention time of LA from ~10 min to > 6 h, making the persistent activation of the IRS-1/Akt signaling pathway become true. Thanksto the significant improvement of pharmacokinetics and intracellular retention time, pLAPs restored the oxidative stress and inflammation-related indicators to the normal control levels in the T2DM models with neuropathy and angiopathy so that the superb therapeutic effect on complications was achieved. Results and Discussion 1. pLAPs continuously activate insulin signaling pathway and promotes glucose uptake by sustained-release of LA in cells The pLAPs were prepared by a nanoprecipitation-mediated polymerization and characterized by morphology, size, zeta potential, molecular weight and the proton nuclear magnetic resonance (Supplementary Figs. 1 and 2). Benefiting from the polymerization, the pLAPs exhibited robust dilution and serum stability, and dissociated only in PBS with glutathione (GSH, 2 ~ 10 mM) that mimicking the redox level in the insulin target cells (Supplementary Fig. 3). To investigate whether the self-polymerization prolonged the intracellular retention time of LA, three insulin target cells (3T3-L1, L6, and L02) were incubated with the conditioned medium containing 100 µg/mL pLAPs. After 1 h incubation, the medium containing pLAPs was removed and the pLAPs-free medium was added. At predetermined time points, the cells were lysed and the lysate supernatant was collected to detect the content of intracellular LA by high-performance liquid chromatography (HPLC). LA was set as a control. As plotted in Fig. 2 a-c, in LA-treated cells, the LA content maintained a high level for the first 10 min, then rapidly decreased and was almost undetectable after 360 min. By comparison, in pLAPs treated cells, the LA content gave a time-dependent increase. After reaching the peak at 60 min, it remained at the high level over 360 min. We rationalized that the maintenance of the high LA level would be attributed to the balance of its dissociation and metabolism. In other words, the self-polymerization indeed significantly prolonged the intracellular retention time of LA. It should be pointed out that the self-polymerization mediated prolongation of LA intracellular time cannot be achieved by loading LA in any nanocarriers due to the unavoidance of the rapid diffusion release. Before investigating the effect of prolonged intracellular retention of LA on the activation of insulin signaling pathway, the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed to evaluate the nontoxic concentration range of pLAPs on target cells (0 ~ 250 µg/mL, Supplementary Fig. 4). The activation of insulin signaling pathway was achieved by incubating cells with palmitic acid (PA) and LA or pLAPs for 1 h before the insulin stimulation (See Supporting Information for details). PA is a classic insulin resistance inducer, which can effectively inactivate the insulin signaling pathway by inhibiting the expression of p-IRS1 and p-Akt, two key insulin signaling molecules. As shown in Fig. 2 d and Supplementary Fig. 5, the LA treatment increased only the expression of p-IRS1 and p-Akt in the first 10 min. By contrast, pLAPs caused no change of the expression of p-IRS1 and p-Akt within the first 30 min, while gave a significant increase at 60 min and maintained the high level within 60 ~ 360 min, consistent with the trend in intracellular LA content variation of the pLAPs group. As a result of the persistent activation of insulin signaling pathway, 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG), a deoxyglucose analogue used for direct monitoring of glucose uptake by living cells, showed also a remarkable uptake level within 60 ~ 360 min (Fig. 2 e-g). To confirm the achievement of pLAPs’ above effects by dissociating into LA in the cells, the GSH depletion agent diethyl maleate (DEM) was employed to inhibit the intracellular dissociation of pLAPs (Fig. 2 a-c, pink line). As shown in Fig. 2 h-n, the block of the pLAPs dissociation led to the inactivation of insulin signaling pathway and the disappearance of 2-NBDG uptake, suggesting that pLAPs activates insulin signaling pathway and promotes glucose uptake by dissociating into LA rather than in the form of nanoparticles. 2. pLAPs hold good absorption, long in vivo half-life and efficient accumulation inT2DM target organs The absorption effect of pLAPs was first investigated in vitro via the mucus penetration, epithelial cellular uptake and epithelial penetration, the major barriers of oral drug delivery. Rifampicin, a drug with oral bioavailability ~ 95%, was set as a positive control (See Supporting Information for details). As shown in Fig. 3 a-f, the mucus penetration rate of pLAPs was ~ 61.0%, over 7/10 of rifampicin (~ 84.0%). Moreover, the cellular uptake efficiency and apparent permeability coefficient (P app ) of pLAPs reached ~ 71.8% and 2.6×10 − 6 cm s − 1 , respectively, data comparable to rifampicin (~ 77.4% and 2.8×10 − 6 cm s − 1 ), indicating the good intestinal absorption of pLAPs. The good absorption was further evaluated by an ex vivo intestinal permeability model (Fig. 3 g). Briefly, the pLAPs or rifampicin was injected into the lumen (donor chamber) before ligation, followed by an incubation in the Krebs-Ringer (K-R) buffer (acceptor chamber) at 37°C. As shown in Fig. 3 h, as the drug content stabilized in the acceptor chamber, the small intestine absorption ratio of pLAPs was ~ 58.0%, up to 2/3 of rifampicin (~ 86.0%). We hypothesize that the good absorption is correlated to the polydisulfide backbone of pLAPs, which enables dynamic covalent exchange with the thiols of mucin, epithelial cell membrane protein and lysosomal membrane so as to mediate the nanoparticles to penetrate the absorption barriers 24 , 25 . To check the hypothesis, 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB), a popular thiol-reactive agent, was employed to block the thiol groups. As shown in Fig. 3 i,j, just a 30 min blocking, the mucus penetration and cellular uptake efficiency of pLAPs decreased respectively by ~ 68.8% and ~ 31.1%. The lysosomal escape behavior of pLAPs was investigated by fluorescence colocalization of Coumarin 6-labeled pLAPs (Cou@pLAPs) and LysoTracker red in Caco-2 cells via confocal laser scanning microscope (CLSM). As shown in Supplementary Fig. 6, the Cou@pLAPs were colocalized with LysoTracker at 1 h, indicating the lysosomal entrapment. As the incubation time was prolonged from 1 to 8 h, the Cou@pLAPs and LysoTracker fluorescence gave less colocalization and the colocalization Pearson’s correlation coefficient ( R r) decreased dramatically from 0.72 to 0.31, indicating the escape of pLAPs from the lysosomes. In contrast, after the blocking of thiols on the lysosomal membrane, the Cou@pLAPs were colocalized with LysoTracker over 8 h, and the R r showed no significant change. The oral terminal half-life ( T 1/2 ) of pLAPs was studied on Sprague Dawley (SD) rats via oral gavage. At predetermined time points, the blood samples were collected and treated with NaOH, then the dissociated LA was extracted and measured to reflect the plasma concentration of pLAPs. As shown in Fig. 3 k, in LA monomer-treated SD rats, the T 1/2 was 0.5 ± 0.1 h, data comparable to the previous reports. To our delight, in the pLAPs-treated SD rats, the T 1/2 was up to 23.2 ± 1.6 h, ~ 46 times higher than that of LA. We rationalized that the long T 1/2 of pLAPs would be ascribed to its negative surface charge and ~ 30,000 molecular weight, which can decrease plasma protein binding and retard the glomerular filtration. As a result of the good absorption and prolonged T 1/2 , the area under the 72 h of plasma concentration curve (AUC 0 ~ 72 h ) of pLAPs was up to 3761.8 ± 55.9 h*µg/mL, ~ 23 times higher than that of LA, indicating that the self-polymerization does remarkably improve the oral pharmacokinetics of LA. The biodistribution of pLAPs was evaluated on C57BL/6 mice through oral administration of 1,19-dioctadecyl-3,3,39,39-tetramethylindodica-rbocyanine perchlorate (Did)-labeled pLAPs (Did@pLAPs). At predetermined time points, mice were euthanized to harvest the major and insulin target organs, and the ex vivo fluorescence images were detected using an in vivo imaging system (IVIS). As shown in Fig. 3 l,m, the ex vivo IVIS images revealed that the fluorescence signals were mainly observed in the liver, heart and kidney. Besides the distribution in the above main organs, one can find that the pancreas (insulin secretion organ) and fat and skeletal muscle (insulin effector organs) gave also strong fluorescence signals, indicating the T2DM target organs-efficient accumulation of pLAPs. 3. pLAPs exert strong efficacy in the murine model of T2DM The oral hypoglycemic effect was assessed on the T2DM model of the 8-week-old male db/db mice by single and long-term administrations of pLAPs at the doses of 25, 50 and 100 mg/kg or LA at 100 mg/kg (Fig. 4 a,b). The db/db mice treated with saline and metformin (120 mg/kg, equivalent therapeutic dose for T2DM patients) were set as the negative control and positive control, respectively, and db/m mice treated with saline was set as the normal control. As shown in Fig. 4 c, LA caused no change of the blood glucose level as expected. Metformin gave a glucose decrease during 2 ~ 24 h, with a lowest level of 10.7 mmol/L at ~ 6 h. To our delight, the pLAPs showed hypoglycemic effects at all three doses, and the best one occurred in the 100 mg/kg group, where the hypoglycemic effect was maintained in a period of 2 ~ 72 h and the blood glucose level was as low as 8.4 mmol/L, 21.5% lower than that in the metformin group. Due to the nearly 3-day hypoglycemic effect of a single administration, the pLAPs were administered every three days in the long-term evaluation, while the LA and metformin were administered daily. As shown in Fig. 4 d, LA showed still no hypoglycemic effect. Metformin stabilized the blood glucose at ~ 12.7 mmol/L. By contrast, pLAPs administrated even every three days showed an excellent hypoglycemic effect, with the blood glucose stable at a low level of ~ 9.4 mmol/L (100 mg/kg), much lower than that in the metformin group and even close to the normal control. To confirm the hypoglycemic effect of pLAPs, all mice were orally administrated with glucose (2 g/kg) on day 27 and intraperitoneally injected with recombinant human insulin (1 U/kg) on day 29, and the AUC 0 ~ 120 min of oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were detected to evaluate glucose tolerance and insulin resistance, respectively. As shown in Fig. 4 e, the db/db mice showed a significant AUC 0 ~ 120 min increase of OGTT and ITT compared to the db/m mice, suggesting a seriously impaired glucose tolerance and insulin resistance. LA caused no improvement of the AUC 0 ~ 120 min of OGTT and ITT, consistent with the hypoglycemic result. Metformin showed a decrease of 21.4% for OGTT and 27.1% for ITT, comparable to the other reports 26 . By comparison, pLAPs decreased the AUC 0 ~ 120 min of OGTT by up to 39.5% and ITT by up to 48.3%, a comparable level to the normal control, implying the excellent improvement in glucose tolerance and insulin resistance. In addition, the improvement of pLAPs on insulin resistance was further evidenced by serum insulin and insulin resistance index (HOMA-IR) assays, in which the serum insulin level and HOMA-IR were decreased by 39.4% and 72.5%, respectively (Fig. 4 f,g). Notably, one can find that the pLAPs reduced the weight of epididymal and body by 34.6% and 25.6%, respectively, while metformin caused no any weight loss (Fig. 4 h,i), indicating the potential of pLAPs to reduce the risk factor of T2DM through weight loss. One can also find the weight loss of pLAPs was a little bit weaker than that of LA, possibly due to the fact that pLAPs were mainly distributed in insulin target organs and failed to block AMPK pathway in the hypothalamus to inhibit food intake 27 , 28 . 4. Besides the activation of classical IRS1/Akt signaling pathway, pLAPs exert hypoglycemic effect by promoting the normalization of pancreatic structure and secretion function In order to confirm the activation of pLAPs on the classical IRS1/Akt signaling pathway in vivo , all mice were euthanized after 30-day treatment, and the expression of p-IRS1 and p-Akt in liver and skeletal muscle were detected by Western blotting. Consistent with the in vitro results, pLAPs showed a significant increase in the expression of p-IRS1 and p-Akt, and, as a result, the levels of glucose transporter 4 (GLUT4, a key protein that promotes glucose transmembrane mobility) and glycogen deposits were also remarkably increased (Supplementary Fig. 7). In addition to the inhibition of classical IRS1/Akt signaling pathway, pancreatic damage caused by inflammation and oxidative stress is the other major factor in the progression of T2DM 29 , 30 . Considering the strong antioxidant and anti-inflammatory properties of pLAPs, we evaluated further its impact on pancreas lesions by hematoxylin and eosin (H&E), TUNEL and insulin immunohistochemical staining. As shown in Supplementary Fig. 8a-d, metformin had no improvement on pancreatic damage. To our delight, pLAPs, at the dose of 50 mg/kg, showed a significant improvement in the related indicators. When the dose was increased to 100 mg/kg, a much stronger improvement was achieved, where the size of islets was reduced by 70.9%, the number of apoptotic β-cells was reduced by 70.1%, and the expression of insulin was increased by 159.3%. Notably, one can find that all three levels were close to the normal control, indicating a very good improvement of pLAPs on pancreatic damage. To confirm the improvement of pLAPs on the pancreas injury by antioxidation and anti-inflammatory, the oxidative stress marker malondialdehyde (MDA), antioxidant markers catalase (CAT), superoxide dismutase (SOD), and glutathione peroxide (GSH-Px), and pro-inflammatory cytokines tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were assessed. As shown in Supplementary Fig. 8e-h, pLAPs gave a dose-dependent decrease on the level of oxidative stress marker and an increase on the activities of antioxidant markers, respectively, with the high-dose group (100 mg/kg) approaching to the normal control. Consistent with antioxidation results, pLAPs exhibited also a strong inhibitory effect on inflammation (Supplementary Fig. 8i-k). We note in passing that LA had no therapeutic effect on pancreatic injury, and its inhibition on oxidative stress and inflammation was also limited. This would be because LA metabolizes too fast and cannot be accumulated effectively in pancreatic tissue. 5. pLAPs show a much stronger therapeutic efficacy for neuropathy than LA and reduces blood glucose in the advanced stage of diabetes The treatment of pLAPs on neuropathy was evaluated by using the 20-week-old male db/db mice as a DPN model. Consistent with the frequency of administration in the long-term hypoglycemic evaluation, pLAPs were orally administrated every three days for 30 consecutive days at a dose of 100 mg/kg, and once-daily oral administration of LA (100 mg/kg, equivalent therapeutic dose for clinical patients with DPN) was set as the positive control. Considering the oxidative stress pathogenesis of neuropathy, we first investigated the changes of oxidative stress and inflammation-related indicators at the end of treatment. As shown in Fig. 5 a,b, pLAPs gave a significant decrease on the content of MDA and an increase on the activities of CAT, SOD and GSH-Px in serum and sciatic nerve (SN). To our delight, all these indicators showed no difference compared to the normal control ( P > 0.05), implying the complete improvement of redox imbalance. Consistent with the improvement effect of oxidative stress indicators, the pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) and inflammatory signaling molecules (p-IκBα and p-NF-κB) restored also to the normal control levels ( P > 0.05, Fig. 5 c-i). By contrast, LA, even with administration daily, had still an enormous gap in the levels of the above indicators compared to the normal control. These results indicated that by transforming LA to the stabilized nanoparticles (pLAPs), a superstrong antioxidant and anti-inflammatory capacity was achieved. As a result of the performance in antioxidant and anti-inflammatory, the mice treated by pLAPs showed an excellent improvement in behavior and nerve conduction velocity, where the hot-plate and tail-flick latencies were shortened by 40.4% and 31.1%, respectively (Fig. 5 j,k), and motor nerve conduction velocity (MCV) and sensory nerve conduction velocity (SCV) were increased by 35.2% and 36.7%, respectively (Fig. 5 l,m). At the same time, the myelin sheath of SN was significantly regenerated (Fig. 5 n). By contrast, the latencies of hot-plate and tail-flick in LA treated mice were reduced by only 13.5% and 14.2%, respectively, and the MCV and SCV were increased by only 12.9% and 19.7%, respectively. As known, the 20-week-old db/db mice, equivalent to patients with T2DM more than 10 years, were generally used as a model for advanced diabetes. In the advanced stage of diabetes, the blood glucose lowering is difficult to achieve owing to the proliferation cessation and loss of secretory function of β-cells, as well as the aggravation of insulin resistance caused by long-term hyperglycemia. To our delight, pLAPs showed also a good and stable hypoglycemic effect in the rodent model of advanced diabetes, where a stable blood glucose occurred on 6th day, with a low level of ~ 16.3 mmol/L (Fig. 5 o). 6. pLAPs exhibit excellent therapeutic effect on diabetes microvascular and macrovascular disease As known, the lesion site of DPN locates in the peripheral nerves with a single structure (composed only of neurons and glial cells) 31 , 32 . LA easily spreads to nerves and is directly taken up by cells to exert systemic antioxidant effects. Therefore, LA is approved in clinic for the DPN treatment in despite of its fast metabolism. On the contrary, the diabetic angiopathy locates mainly in the complex organs, such as heart, kidney and retina. LA hardly accumulates in the effector cells of these organs. Although driven also by high glucose-mediated oxidative stress, the angiopathy cannot be treated with LA. Encouraged by its much stronger antioxidant and anti-inflammatory capacity over LA and effective enrichment in T2DM target tissues, we tried further the potential of pLAPs in the treatment of angiopathy. The diabetic nephropathy (DN) and diabetic cardiomyopathy (DC) were employed as models for microvascular disease and macrovascular disease, respectively. Consistent with the results in the DPN treatment, pLAPs restored the oxidative stress and inflammation-related indicators to normal control levels in both models (Supplementary Fig. 9) and showed also a good and stable hypoglycemic effect (Supplementary Fig. 10). The therapeutic effect of DN was evaluated by measuring the renal function indexes [urine albumin-to-creatinine ratio (UACR), serum creatinine (Cre), and blood urea nitrogen (BUN)] and glomerular pathological changes [glomerular sizes, glomerular basement membrane (GBM) thickness, mesangial matrix expression, and width and number of foot processes]. As shown in Fig. 6 , compared to mice treated with saline, the pLAPs treated mice showed a decrease on all three renal function indexes, with the levels of UACR, Scr and BUN reduced by 71.3%, 52.6% and 50.9%, respectively (Fig. 6 a-c). The glomerular pathological results showed that pLAPs reduced the glomerular size by 64.4%, GBM thickness by 18.3%, mesangial matrix expression by 48.4%, the width of foot processes by 28.1%, and increased the number of foot processes by 64.2%, respectively (Fig. 6 d-i). The therapeutic effect of DC was evaluated by measuring the cardiac function indexes [left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-systolic diameter (LVIDs), and left ventricular end-diastolic diameter (LVIDd)], pathological changes (ventricular myocytes size and fibrotic area), and myocardial injury markers [natriuretic peptide type A ( ANP ), collagen type I alpha 1 ( Col1α1 ), matrix metallopeptidase 2 ( MMP2 ) and transforming growth factor beta 1 ( TGF-β1 )]. As shown in Fig. 6 j-n, compared to mice treated with saline, the pLAPs treated mice showed an increase of 82.9% for LVEF and 46.9% for LVFS, and a decrease of 53.6% for LVIDs and 32.4% for LVIDd. At the same time, the ventricular myocytes size, fibrotic area and myocardial injury markers levels were significantly decreased (Fig. 6 o-t). In particular, one can find that the above indicators of DC and DN showed no significant difference compared to the normal control, suggesting that the mice returned to normal after the pLAPs treatment. As a contrary, the LA treatment only provided a very limited therapeutic effect from the slight improvement of the indicators. Taken together, pLAPs hold a good clinical potential in the treatment of both angiopathy and neuropathy. 7. pLAPs improve spontaneous T2DM with DPN in rhesus monkey Encouraged by the outstanding outcomes in rodents, we challenged further the treatment of pLAPs for the spontaneous T2DM rhesus monkey with the complication of DPN since it very closely mimicked human T2DM pathological properties. Concretely, 5 spontaneous T2DM rhesus monkeys, which conform to either the slowing down of any SCV or prolonged latency by detecting SCV of the sural nerve, median nerve, and other nerves over 6 months using neuroelectromyography, were randomly divided into 2 groups. Three monkeys were given pLAPs (10 mg/kg/d) by nasogastric gavage for 8 weeks, and the other two were given placebo as the negative control. It should be pointed out that, for meeting the requirement of T2DM combined with DPN, the selected five animals are elderly diabetes monkeys aged 18–21 years (analogous to approximately 70-year-old humans) 33 , 34 . At this stage, even the classic insulin-sensitizing drugs such as metformin, sulfonylureas, or GLP-1RA would lose their hypoglycemic effect caused by islet function failure 35 , 36 , 37 , 38 , 39 . Such situation occurred also in our case. Although the fasting plasma glucose (FPG) of the pLAPs treated monkeys gave a downtrend compared to monkeys treated with placebo, there was no significant difference compared to baseline (Table 1 ). Considering that the FPG detection in this stage is no longer suitable for evaluating the true effectiveness of drugs on T2DM, the HOMA-IR, a unique indicator reflecting the insulin sensitivity without affection by the disease-developing stage, was employed to evaluate the therapeutic effect of T2DM. The lower the HOMA-IR is, the stronger the hypoglycemic effect of drugs is. As shown in Table 1 , the placebo treated monkeys showed no change of the HOMA-IR during the treatment. To our delight, the HOMA-IR of monkeys treated by pLAPs gave a continuous decrease from 7.41 to 2.02, a value located in the range of normal human (< 2.69) 40 , verifying undoubtedly the excellent therapeutic efficacy of pLAPs for T2DM. Table 1 General glucose metabolism indicators of the T2DM rhesus monkey with DPN following once daily oral administration of pLAPs (10 mg/kg) or placebo for 8 weeks. Indicators Group Baseline Day 14 Day 28 Day 42 Day 56 FPG (mmol/L) Placebo 4.93 ± 0.08 5.18 ± 0.60 4.91 ± 0.08 4.79 ± 0.25 4.80 ± 0.16 a pLAPs 6.97 ± 2.58 6.88 ± 2.81 6.84 ± 1.95 6.83 ± 2.39 6.43 ± 2.48 a FPI (µU/mL) Placebo 14.81 ± 6.16 22.76 ± 16.59 12.74 ± 2.36 18.53 ± 5.83 17.30 ± 10.22 a pLAPs 22.77 ± 7.83 13.03 ± 10.22 11.56 ± 7.17 9.46 ± 11.55 7.86 ± 7.61 b HOMA-IR Placebo 3.23 ± 1.30 5.01 ± 3.21 2.78 ± 0.47 3.91 ± 1.04 3.65 ± 2.05 a pLAPs 7.41 ± 4.51 3.75 ± 2.43 3.25 ± 1.67 2.51 ± 2.72 2.02 ± 1.58 b Note: HOMA-IR = FPG × FPI/22.5. Statistical differences were analyzed by two-tailed Student’s t-test. a P > 0.05, b P < 0.05, compared with baseline. FPG: fasting plasma glucose; FPI: fasting plasma insulin; HOMA-IR: insulin resistance index. The therapeutic effect for DPN was assessed by measuring the nerve conduction velocity (NCV) every 4 weeks, and the data were shown in Table 2. During the treatment, the placebo caused no change of the NCV of abnormal nerves compared to that at baseline ( P > 0.05), while the NCV of pLAPs treated group showed a continuous increase from 37.6 ± 3.4 m/s to 41.2 ± 3.2 m/s. By directly comparing the velocity change on day 28 and day 56, one can find that the placebo’s velocity changes were − 0.3 ± 0.8 m/s and − 0.2 ± 1.1 m/s, respectively, while the corresponding values of pLAPs gave a significant improvement of 2.4 ± 2.8 m/s and 3.6 ± 2.5 m/s, suggesting the significant improvement of pLAPs on DPN. To our knowledge, this is the first hypoglycemic drug with the function of alleviating neuropathy. Table 2. NCV and velocity change of the T2DM rhesus monkey with DPN following once daily oral administration of pLAPs (10 mg/kg) or placebo for 8 weeks. Group NCV (m/s) Velocity change (m/s) Baseline Day 28 Day 56 Day 28 Day 56 Placebo 38.1 ± 4.9 37.8 ± 4.8 a 37.9 ± 4.8 a -0.3 ± 0.8 -0.2 ± 1.1 pLAPs 37.6 ± 3.4 40.0 ± 3.6 a 41.2 ± 3.2 b 2.4 ± 2.8 c 3.6 ± 2.5 c Note: Statistical differences were analyzed by two-tailed Student’s t-test. a P > 0.05, b P < 0.05, compared with baseline. c P < 0.05, compared with placebo group. NCV: nerve conduction velocity. 8. pLAPs hold good biosafety As a chronic metabolic disease, T2DM requires long-term treatment with hypoglycemic drugs. Therefore, the drug safety is particularly important. Here, we evaluated the biosafety of pLAPs by acute and subacute toxicity tests. The acute toxicity was performed by single oral administration of 2000 mg/kg pLAPs into C57BL/6 mice, saline was used as a control. The results showed that the mice maintained healthy as confirmed by the unchanged behavior and nonweight loss over 2 weeks (Supplementary Fig. 11a). At the end of the observation, mice were euthanized and the main organs and blood were collected for the organs index, hematology profile and serum biochemistry analyses. It was found that no obvious changes in the organs index (Supplementary Fig. 11b), hematology profile (Supplementary Fig. 11c) and serum biochemical parameters (Supplementary Fig. 11d) were observed in mice treated with pLAPs. The subacute toxicity was performed by oral administration of pLAPs (100 mg/kg/d) to C57BL/6 mice for 30 days. Saline was used as a control. The levels of hematology profile, plasma biochemical parameters and the pathological changes of main organs were checked after treatment. The results showed that the mice treated with pLAPs did not show significant differences in the hematology (Supplementary Fig. 12a) and plasma biochemical parameters (Supplementary Fig. 12b) compared with the control group. In addition, the H&E staining of the main organs, including the heart, liver, spleen, lung and kidney showed no abnormalities after pLAPs oral administration (Supplementary Fig. 13). Notably, thanks to the hypoglycemic mechanism of the insulin signaling pathway activation and pancreatic injury improvement, the mice treated with pLAPs showed no hypoglycemic reactions under the doses of both acute and subacute toxicity (Supplementary Fig. 14). By the way, during the 8 weeks of administering pLAPs (10 mg/kg/d) via nasogastric gavage to treat spontaneous T2DM rhesus monkey with DPN, we did not observe any abnormalities, including body weight, food intake (Supplementary Table 1), plasma biochemical parameters (Supplementary Table 2) and hematology profile after treatment (Supplementary Table 3). Overall, the above results suggested the good biosafety of pLAPs. Conclusion In conclusion, an oral hypoglycemic agent by self-polymerizing LA to a nanodrug pLAPs has been developed for the treatment of T2DM with multi-complications. The self-polymerization made the oral T 1/2 , AUC 0 ~ 72 h and intracellular retention time of LA up to 23.2 h, 3761.8 ± 55.9 h*µg/mL and > 6 h, respectively, ~ 46, ~23 and over ~ 36 times higher than that of LA monomer. Accordingly, a single administration of pLAPs (100 mg/kg) in db/db mice gave a nearly 3-day hypoglycemic period with the blood glucose level as low as 8.4 mmol/L, a value close to the normal control, while LA showed no hypoglycemic effect. The long-term oral hypoglycemic evaluation revealed that pLAPs (100 mg/kg) administrated even every three days stabilized the blood glucose level at ~ 9.4 mmol/L. By contrast, metformin administrated daily at the equivalent therapeutic dose for patients (120 mg/kg) stabilized the blood glucose level only at ~ 12.7 mmol/L. The improvement of pharmacokinetics and intracellular retention time led also to the strong inhibition on oxidative stress and inflammation. In the mice with DPN, pLAPs restored the oxidative stress and inflammation-related indicators to the normal control. As a result, the hot-plate and tail-flick latencies of mice were shortened by 40.4% and 31.1%, respectively, and MCV and SCV were increased by 35.2% and 36.7%, respectively. All these levels were consistent with the normal control. By contrast, the latencies of hot-plate and tail-flick in LA treated mice were reduced by only 13.5% and 14.2%, respectively, and the MCV and SCV were increased by only 12.9% and 19.7%, respectively, far away from the levels of normal control. In particular, for LA’s non-indication angiopathy (e.g., DN and DC), pLAPs showed also excellent therapeutic effect, with the recovery of related indicators to the levels of normal control. Notably, the improvement of pLAPs on T2DM and its complications has been verified in the treatment of spontaneous T2DM rhesus monkey with DPN, where the HOMA-IR was decreased to 2.02 after 8 weeks of treatment, a value located in the range of normal human, and the NCV was increased by 3.8 m/s compared to the placebo. Notably, pLAPs showed no acute and subacute toxicity administered at doses of 2000 mg/kg and 100 mg/kg respectively in mice, and no adverse drug reactions were observed in the spontaneous T2DM rhesus monkey during treatment. This strategy of self-polymerizing lipoic acid into a nanodrug provides a brand-new solution for the treatment of T2DM. Methods Materials LA was purchased from Tokyo chemical industry Co., Ltd. (Japan). Metformin hydrochloride sustained release tablet was purchased from Sino-American Shanghai Squibb Pharmaceuticals Ltd. (Shanghai, China). D-(+)-Glucose was purchased from aladdin (USA). DEM was purchased from McLean Biochemical Technology Co., Ltd. (Shanghai, China). MTT and non frozen tissue RNA preservation solution (RNAwait) were obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). 2-NBDG was obtained from GlpBio Technology (Montclair, California, USA). PA was obtained from Sigma Chemical Company (Saint Louis, MO, USA). Coumarin 6 and DTNB were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). Mouse insulin, IL-1β, IL-6 and TNF-α ELISA kits were obtained from Jingmei Biotechnology (Jiangsu, China). Mouse urine albumin (ALB) ELISA kit was obtained from Shanghai Tongwei Industrial Co., Ltd. (Shanghai, China). Did, colorimetric TUNEL apoptosis assay kit, RIPA lysis buffer, protein inhibitor cocktail (100 ×), bicinchoninic acid (BCA) protein assay kit, MDA assay kit, recombinant human insulin, enhanced ECL western blotting detection kit and anti-insulin antibody were obtained from Beyotime Institute of Biotechnology (Jiangsu, China). Polyvinylidene fluoride (PVDF) and HRP-conjugated goat anti-rabbit IgG were purchased from Servicebio (Wuhan, China). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (T-BIL), BUN, Cre, CAT, GSH-Px, and SOD assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). Anti-Akt, anti-phospho-(Ser473)-Akt, and anti-β-actin antibodies were obtained from Wuhan Sanying Biotechnology Co., Ltd. (Wuhan, China). Anti-IRS-1, anti-phospho-(Ser307)-IRS1, anti-NF-κB p65, anti-IκBα, anti-phospho-(Ser32/Ser36)-IκBα antibodies were purchased from Affinity Biosciences (Jiangsu, China). Anti-GLUT4, anti-phospho-NF-κB p65 and HRP-conjugated goat anti-mouse IgG antibodies were obtained from ABclonal Biotechnology Co., Ltd. (Wuhan, China). Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, and pancreatin were purchased from Gibco BRL Life Technology (California, USA). Animals Sprague Dawley (SD) rats were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. (Chengdu, China). C57BL/6 mice, db/m mice, and db/db mice were purchased from Gempharmatech Co., Ltd. (Jiangsu, China). All rodent animal experiments were approved by the Animal Ethics Committee of Sichuan University (Chengdu, China) and carried out according to the approved guidelines. Rhesus monkeys with spontaneous type 2 diabetes mellitus were provided and housed in Sichuan Primed Shines Bio-tech Co., Ltd. (SPSB) (Chengdu, China), animal care procedures were approved by the Institutional Animal Care and Utilization Committee (IACUC) of SPSB accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). Synthesis of pLAPs The pLAPs were prepared by a nanoprecipitation mediated polymerization. Specifically, to 19 mL of ultrapure water under stirring, 1.0 mL DMSO solution of LA (10% w/v ) was added dropwise to get a milky white solution. After ultrasound treatment with 25 kHz bath for 15 min, the above solution was transferred to a photoreactor and irradiated with 365 nm ultraviolet light for 4 h. After that, the light source was removed and 3.0 mL of hydrogen peroxide (H 2 O 2 ) was added to stir for another 2 h. The resulting solution was purified by dialysis against deionized water using a 1.0 kDa MWCO tubing for 48 h followed by lyophilization overnight to get the pLAPs as a white powder (yield: 70% wt ). The pharmacokinetics study and biodistribution of pLAPs The pharmacokinetics of pLAPs were examined in male rats (~ 200 g). Briefly, SD rats were deprived of food overnight. The rats were administered intragastrically with LA (100 mg/kg) and pLAPs (100 mg/kg), respectively. The blood samples were collected from the eye ground vein into the heparinized tubes at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 60, 72 and 96 h, and followed by immediate centrifugation at 3000 × g for 10 min. After protein precipitation with acetonitrile, blood samples were added with 1 M NaOH, followed by heating 60 ℃ for 20 min to fully break the disulfide bonds. Finally, the resulting solution was adjusted the pH to 3 ~ 4 using HCl, and the dissociated LA was extracted with ethylacetate and tested by HPLC. The biodistribution of pLAPs was examined by gavage of Did-labeled pLAPs (Did@pLAPs) to the male C57BL/6 mice. Briefly, to a solvent of 4 mL of pLAPs (10 mg/mL) under stirring, 40 µL DMSO solution of Did (10 mg/mL) was added dropwise and followed by 4 h of ultrasound treatment. After that, the above solution was centrifuged for 10 min and purified by dialysis against deionized water using a 1.0 kDa MWCO tubing for 48 h to get the Did@pLAPs. The mice were sacrificed after 2, 6, 12, 24 and 36 h of oral gavage, and the spleen, lung, liver, adipose, skeletal muscle, pancreas, heart and kidney were isolated for imaging and quantitative analysis using an IVIS spectrum system with an excitation wavelength of 644 nm and an emission wavelength of 665 nm. Intracelluar LA content assay 3T3-L1, L6 and L02 cells were seeded in 60 mm dishes for 12 h at a density of 2.0 × 10 6 cells per dish, respectively. The cells were then incubated with LA (100 µg/mL), pLAPs (100 µg/mL) or [pLAPs + DEM] (pLAPs = 100 µg/mL, DEM = 1.0 mM). For the [pLAPs + DEM] group, DEM was used to deplete GSH for 24 h before the pLAPs treatment. After 1 h incubation, the medium containing LA or pLAPs was removed and the LA- or pLAPs-free medium was added. At predetermined time points, the cells were washed by phosphate buffer saline (PBS) for three times. Finally, the cells were collected and lysed, and the supernatants were collected and analyzed by HPLC. In vivo anti-diabetes study After one week of acclimation, 8-week-old male db/db mice were randomly divided into 6 groups (6 mice in each group) as follows: db/db model group, db/db + pLAPs (25 mg/kg) group, db/db + pLAPs (50 mg/kg) group, db/db + pLAPs (100 mg/kg) group, db/db + LA (100 mg/kg) group, db/db + metformin (120 mg/kg, equivalent to clinical dosage) group, and age-matched heterozygotes db/m mice were set as the negative control ( n = 6). To evaluate the hypoglycemic effect of single administration, mice in the treatment groups received a single oral administration of different formulations, while mice in the negative control and model groups received an equal volume of saline. Blood glucose levels were measured over 72 h by tail clipping using a glucometer (Roche Accu-check, Mannheim, Germany). To evaluate the hypoglycemic effect of long-term administration, mice in the LA and metformin groups were administrated with different formulations once daily for 30 consecutive days, respectively. while mice in the pLAPs groups were administrated with indicated doses every 3 days. During the treatment period, fasting blood glucose levels were measured every 3 days. The oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed on the 27th and 29th day after pLAPs treatment, respectively. For OGTT test, mice were given oral glucose at a dose of 2 g/kg and blood glucose levels were monitored at 15, 30, 60 and 120 min after the initial glucose load, respectively. For ITT test, mice were fasted for 2 h, and then recombinant human insulin was injected intraperitoneally at a dose of 1 U/kg, and blood samples were collected for blood glucose measurements at 15, 30, 60 and 120 min, respectively. The area under the 120 min of glucose concentration curve (AUC 0 ~ 120 min ) was calculated. At the end of treatment, all mice were sacrificed with inhaled isoflurane at 2 h following the last dosing. The weights of whole body and epididymal fat were recorded. Blood samples were collected by eye bleeding, and centrifuged at 1500 × g for 15 min. The isolated serum was stored at -80°C for measuring the levels of insulin, pro-inflammatory cytokines and MDA, and the activities of CAT, GSH-Px and SOD. The liver, skeletal muscle and pancreas tissue were fixed by 4% polyformaldehyde, dehydrated, embedded in paraffin wax and cut serially into 5 µm sections for histopathological analysis. In vivo anti-complications study 12-, 16- and 20-week-old male db/db mice were used as models of DN, DC and DPN, respectively. The db/db mice were randomly divided into 3 groups (6 mice in each group) as follows: db/db model group, db/db + pLAPs (100 mg/kg) group, db/db + LA (100 mg/kg) group, and age-matched db/m mice were set as the negative control ( n = 6). LA group received LA (100 mg/kg, equivalent to clinical dosage for DPN) by oral administration once daily for 30 consecutive days, and the pLAPs groups were given at a dose of 100 mg/kg by gavage every 3 days. For the DPN mice, blood glucose was measured every 3 days, and behavioral tests were performed every one week during the treatment period. On the 30th day, all mice were sacrificed and serum was collected for measuring the level of MDA, and the activities of CAT, GSH-Px and SOD. Sciatic nerves were removed for measuring the levels of pro-inflammatory cytokines and MDA, the activities of CAT, GSH-Px and SOD, and the expression of inflammatory signaling molecules (p-IκBα, IκBα, p-NF-κB and NF-κB). In addition, the rest of tissue samples were fixed by 2.5% glutaraldehyde and cut serially into 70 nm sections for TEM analysis. For the DN and DC mice, blood glucose was measured every 3 days. All mice were sacrificed on day 30, the serum samples from DN mice were collected for measuring the levels of Cre and BUN, the urine samples from DN mice were collected for ALB detection. Moreover, the kidney and heart tissues were removed for measuring the levels of pro-inflammatory cytokines and MDA, the activities of CAT, GSH-Px and SOD, the expression of target gene and protein, and evaluating histopathological changes. Measurement of nerve conduction velocity Sciatic nerve conduction velocity was detected by using orthodromic recording techniques 41 . Briefly, mice were anesthetized by intraperitoneal injection of ketamine/methylthiazide at dose of 10 mg/kg. An isolated pulse stimulator (Model 2100, A-M Systems, Everett, WA) was used to deliver the triggered square wave current pulses. The simultaneous electromyographies were recorded by two sterilized electrodes placed into the intrinsic foot muscles with a Grass Amplifier (Model P5, Grass Instruments, Quincy, MA). During the measurement process, the temperature of rectum was maintained at 37 ± 1.0°C using a feedback controlled water bath. MCV and SCV were calculated according to a previous study 41 . Tail-flick and hot plate tests According to the published methods, the thermal pain threshold was examined by tail-flick test (water immersion method) and hot plate test (IITC hot plate analgesia test) 42 , 43 . Briefly, for tail-flick test, mice were restrained in an open conical polypropylene tube and its tail was exposed. The tail of mice was immersed into a water bath at 52 ± 0.2°C the time until the rodent flicks or removes its tail was recorded. For hot plate test, mice were placed within a transparent glass chamber and acclimated for at least 20 min. A thermal stimulation meter (IITC Model 39 Hot Plate Analgesia Meter, IITC Life Science, CA) was used with floor temperature at 55°C to record the latency of paw withdrawal in response to the radiant heat. Cut-off times of 10 s and 15 s were performed to avoid tissue damage caused by tail bending and hot plate testing. Echocardiography Echocardiography of mice was carried out in M-mode with a Vevo 2100 echocardiography system (VisualSonics Inc., Toronto, ON, Canada) 44 . In brief, mice were anesthetized with isoflurane (2.5% for induction and 2.0% for maintenance). M-mode echocardiography was executed to record the left ventricular systolic and diastolic motion profile. LVIDs and LVIDd were acquired through measurement. Left ventricular end-diastolic volume (LVEDV) and end systolic volume (LVESV) were calculated using computer algorithms. In addition, the Vevo 2100 software (VisualSonics Inc.) was used to analyze echocardiographic parameters including LVEF and LVFS. All echocardiography detection and analysis processes were conducted by a researcher who was blind to the experimental treatments. LVEF and LVFS were calculated as LVEF (%) = (LVEDV - LDESV)/LVEDV × 100 and LVFS (%) = (LVIDd - LVIDs)/LVIDd × 100, respectively. Non-human primates study Eighteen to twenty one-year-old spontaneous T2DM rhesus monkeys with DPN were selected, and then divided into two groups according to the results of neuroelectromyography testing. The monkeys in pLAPs group ( n = 3) were administered with pLAPs (10 mg/kg) by nasogastric gavage once daily for 8 weeks, the monkeys ( n = 2) received placebo were set as the control group. During the study period, clinical signs of monkeys were observed and recorded. Food intake was measured and calculated (food intake = feed - discard - surplus) every one week, and body weight was monitored using electronic platform scale (Mettler Toledo, Switzerland) every two weeks. Electrophysiological procedures Neuromuscular examination in rhesus monkeys was carried out using Haishen NDI-092 EMG/EP every four weeks. All rhesus monkeys were fasted overnight at 18:00 the day before testing. The ketamine (15 mg/kg) hydrochloride and dexmedetomidine (0.015 mg/kg) were injected intramuscularly. After anesthesia, the sensory conduction was measured orthodromically in the median, ulnar, superficial peroneal, and peroneal nerves using surface stimulating and recording electrodes. Motor conduction was measured in the tibial nerve recording over the soleus muscle, the peroneal nerve recording over the intrinsic toe extensor muscles, the median nerve recording over the thenar muscles, and the ulnar nerve recording over the hypothenar muscles. MCV and SCV were recorded by Haishen NDI-092 EMG/EP. Hematological and plasma biochemical analysis Blood samples were collected into EDTA-2K anticoagulant vacuum tubes (13 × 75 mm, 3.0 mL BD Vacutainer® plastic whole blood tube containing 3.4 mg of spray-coated K2EDTA), and the hematological parameters, including white blood cells count (WBC), red blood cells count (RBC), hematocrit (HCT), hemoglobin concentration (HBG), and total platelets count (PLT) were measured using Siemens ADVIA 2120i Hematology Systems. For plasma biochemical analysis, blood samples were centrifuged at 3000 × g for 10 min. The levels of FPG, fasting plasma insulin (FPI), Cre, and BUN were detected by Roche Cobas 6000 analyzer series C501/E601 (Roche Diagnostics GmbH). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin (T-BIL) were measured using Roche Cobas 6000 analyzer series C501 (Roche Diagnostics GmbH). Statistical analysis All data was expressed as mean ± standard deviation (SD). For multiple comparisons, statistical significance was analyzed by one-way ANOVA. If inter-group differences were significant ( P < 0.05), the significance of the differences was determined by Tukey post hoc test. For data that showed a normal distribution and homogeneity of variance, differences between two groups were compared with a two-tailed Student’s t-test. All analyses of data were completed with SPSS version 16.0 software package (IBM, Chicago, IL, USA). P < 0.05 was considered statistically significant. Declarations Contributions S.Z. conceived the project. X.X. and X.L. developed the project, designed and performed the experiments and analysed the data. Y.Z., X.R., Y.C., Z.Y., C.L. and Y.W. provided technical input on this project. All authors analysed and interpreted the data. X.X., X.L. and S.Z. co-wrote the manuscript with the input from all other authors. X.R., C.L. and S.Z. supervised the work. Competing interests The authors are on a patent application filed by Sichuan University related to this work. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 22275129), the Sichuan Science and Technology Innovation Foundation (2021JDTD0015), and the Sichuan Science and Technology Program (2023NSFSC0319). We thank the Center of Testing and Analysis, Sichuan University for TEM measurements. References Suzuki K et al (2024) Genetic drivers of heterogeneity in type 2 diabetes pathophysiology. Nature 627:347–357 Xourafa G, Korbmacher M, Roden M (2024) Inter-organ crosstalk during development and progression of type 2 diabetes mellitus. Nat Rev Endocrinol 20:27–49 Jiao YR et al (2024) Exosomes derived from mesenchymal stem cells in diabetes and diabetic complications. Cell Death Dis 15:271 Liu P et al (2024) Ferroptosis: Mechanisms and role in diabetes mellitus and its complications. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4577178","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":316112022,"identity":"d46787bd-a84a-4a1a-b9a9-c1718b4d8e3c","order_by":0,"name":"Shiyong Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAw0lEQVRIiWNgGAWjYFACHhBhg8QmUksa6VoOk6BFvv/swc8Fv87b889IYHzwto1B3pyQFoMD55KlZ/bdTpxxI4HZcG4bg+HOBkJaGHsMpHl7bicYSCSwSfO2MSQYHCDksGYe49+8PefsgVrYfxOlheEYj5k0z48DjBuAtjATpcXgDI+ZNW9DcuKMMw+bJeeckzDcQNBh/WeMb/P8sbPnb08++OFNmY08YYeBAGMbmGwAEhLEqAeBP8QqHAWjYBSMghEJACM6OxfTIFToAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-7633-358X","institution":"Sichuan University","correspondingAuthor":true,"prefix":"","firstName":"Shiyong","middleName":"","lastName":"Zhang","suffix":""},{"id":316112023,"identity":"6bfe34ab-f2a9-44d0-8dd6-95e62e92d88d","order_by":1,"name":"Xiao Xiao","email":"","orcid":"","institution":"College of Biomedical Engineering and National Engineering Research Center for Biomaterials, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Xiao","suffix":""},{"id":316112024,"identity":"ca4f8035-8581-436e-82a0-3865ff5a7b81","order_by":2,"name":"Xiaoluan Lu","email":"","orcid":"","institution":"College of Biomedical Engineering and National Engineering Research Center for Biomaterials, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoluan","middleName":"","lastName":"Lu","suffix":""},{"id":316112025,"identity":"f4eeb14a-aeda-449a-b54a-8846a9cce868","order_by":3,"name":"Yi Zhang","email":"","orcid":"","institution":"College of Biomedical Engineering and National Engineering Research Center for Biomaterials, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Zhang","suffix":""},{"id":316112026,"identity":"c0baa32d-0852-4cd5-9d05-a750b7817ae0","order_by":4,"name":"Xingwu Ran","email":"","orcid":"","institution":"Diabetic Foot Care Center, Department of Endocrinology and Metabolism Diseases, West China Hospital, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Xingwu","middleName":"","lastName":"Ran","suffix":""},{"id":316112027,"identity":"b29d6f60-2252-44ff-802a-55f16cdfe499","order_by":5,"name":"Yangyang Cheng","email":"","orcid":"","institution":"Department of Endocrinology, The First Affiliated Hospital of Xi'an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Yangyang","middleName":"","lastName":"Cheng","suffix":""},{"id":316112028,"identity":"38d88489-8ecf-468c-9df1-db96c53a40c0","order_by":6,"name":"Zunyuan Yang","email":"","orcid":"","institution":"Primed Non-Human Primate Research Center (Sichuan Primed Shines Bio-tech Co., Ltd.)","correspondingAuthor":false,"prefix":"","firstName":"Zunyuan","middleName":"","lastName":"Yang","suffix":""},{"id":316112029,"identity":"a53434c3-9071-4cbd-be26-f7342bfd54ff","order_by":7,"name":"Chunyan Liao","email":"","orcid":"","institution":"College of Biomedical Engineering and National Engineering Research Center for Biomaterials, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Chunyan","middleName":"","lastName":"Liao","suffix":""},{"id":316112030,"identity":"0ef99ceb-c0ac-49bc-be60-664d4b4bf562","order_by":8,"name":"Yao Wu","email":"","orcid":"","institution":"College of Biomedical Engineering and National Engineering Research Center for Biomaterials, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Yao","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2024-06-13 15:26:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4577178/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4577178/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61732087,"identity":"6eb43c0c-8780-40ab-b1d3-3f7f550a0d19","added_by":"auto","created_at":"2024-08-05 01:33:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1767386,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of pLAPs as an oral hypoglycemic agent for T2DM with the function of alleviating multi-complications. \u003cstrong\u003ea\u003c/strong\u003e, Synthesis of pLAPs. \u003cstrong\u003eb\u003c/strong\u003e, The oral absorption, blood circulation and intracellular behavior of pLAPs.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4577178/v1/06a755fd09a321084c686c35.png"},{"id":61731906,"identity":"6513a766-8c81-4a91-aecb-19abb244b23c","added_by":"auto","created_at":"2024-08-05 01:25:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":535420,"visible":true,"origin":"","legend":"\u003cp\u003epLAPs continuously activate insulin signaling pathway and promotes glucose uptake by sustained-release of LA in cells. \u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ec\u003c/strong\u003e, The LA content in 3T3-L1 (\u003cstrong\u003ea\u003c/strong\u003e), L6 (\u003cstrong\u003eb\u003c/strong\u003e) and L02 (\u003cstrong\u003ec\u003c/strong\u003e) cells treated with pLAPs and LA overtime (\u003cem\u003en \u003c/em\u003e= 3 independent samples, mean ± SD). \u003cstrong\u003ed\u003c/strong\u003e, Representative immunoblots of phosphorylation of IRS1 and Akt determined by western blot analysis in 3T3-L1, L6 and L02 cells after treatment with pLAPs and LA at the indicated time. \u003cstrong\u003ee\u003c/strong\u003e-\u003cstrong\u003eg\u003c/strong\u003e, The relative 2-NBDG uptake levels of 3T3-L1 (\u003cstrong\u003ee\u003c/strong\u003e), L6 (\u003cstrong\u003ef\u003c/strong\u003e) and L02 (\u003cstrong\u003eg\u003c/strong\u003e) cells exposed to pLAPs and LA at the indicated time (\u003cem\u003en \u003c/em\u003e= 3 independent samples, mean ± SD). \u003cstrong\u003eh\u003c/strong\u003e, Representative immunoblots of phosphorylation of IRS1 and Akt in 3T3-L1, L6 and L02 cells after treatment with pLAPs and DEM. \u003cstrong\u003ei\u003c/strong\u003e-\u003cstrong\u003ek\u003c/strong\u003e, The quantitative analysis of phosphorylation of IRS1 and Akt in 3T3-L1 (\u003cstrong\u003ei\u003c/strong\u003e), L6 (\u003cstrong\u003ej\u003c/strong\u003e) and L02 (\u003cstrong\u003ek\u003c/strong\u003e) cells (\u003cem\u003en \u003c/em\u003e= 3 independent samples, mean ± SD). \u003cstrong\u003el\u003c/strong\u003e-\u003cstrong\u003en\u003c/strong\u003e, The relative 2-NBDG uptake levels of 3T3-L1 (\u003cstrong\u003el\u003c/strong\u003e), L6 (\u003cstrong\u003em\u003c/strong\u003e) and L02 (\u003cstrong\u003en\u003c/strong\u003e) cells exposed to pLAPs and DEM (\u003cem\u003en \u003c/em\u003e= 3 independent samples, mean ± SD). Statistical differences were analyzed by one-way ANOVA and post hoc Tukey’s test (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. ns, not significant).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4577178/v1/65032fa955c5fd562745defb.png"},{"id":61732408,"identity":"d7b577c9-4fef-4cb8-a484-a8423f21720b","added_by":"auto","created_at":"2024-08-05 01:41:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":512989,"visible":true,"origin":"","legend":"\u003cp\u003epLAPs hold good absorption, long \u003cem\u003ein vivo\u003c/em\u003e half-life and efficient accumulation inT2DM target organs. \u003cstrong\u003ea\u003c/strong\u003e, Schematic diagram of intestinal mucus penetration study. \u003cstrong\u003eb\u003c/strong\u003e, Quantitative analysis for the mucus penetration efficiency at predetermined time (\u003cem\u003en \u003c/em\u003e= 3 independent samples, mean ± SD). \u003cstrong\u003ec\u003c/strong\u003e, Schematic diagram of cellular uptake study. \u003cstrong\u003ed\u003c/strong\u003e, Quantitative analysis for the time-dependent cellular uptake efficiency on Caco-2 cells (\u003cem\u003en \u003c/em\u003e= 3 independent samples, mean ± SD). \u003cstrong\u003ee\u003c/strong\u003e, Schematic diagram of transcytosis study. \u003cstrong\u003ef\u003c/strong\u003e, The P\u003csub\u003eapp\u003c/sub\u003e value of transepithelial transport study on Caco-2 cell monolayers over 8 h (\u003cem\u003en \u003c/em\u003e= 3 independent samples, mean ± SD). \u003cstrong\u003eg\u003c/strong\u003e, Schematic diagram of the \u003cem\u003eex vivo\u003c/em\u003e intestinal permeability study. \u003cstrong\u003eh\u003c/strong\u003e, Quantitative analysis for the time-dependent the drug absorption (\u003cem\u003en \u003c/em\u003e= 3 independent samples, mean ± SD). \u003cstrong\u003ei\u003c/strong\u003e, Inhibition of the penetration efficiency of pLAPs to mucus layer by DTNB (1 mM) (\u003cem\u003en \u003c/em\u003e= 3 independent samples, mean ± SD). \u003cstrong\u003ej\u003c/strong\u003e, Inhibition of the cellular uptake efficiency of pLAPs to Caco-2 cells by DTNB (1 mM) (\u003cem\u003en \u003c/em\u003e= 3 independent samples, mean ± SD). \u003cstrong\u003ek\u003c/strong\u003e, Plasma concentration of LA in SD rats (\u003cem\u003en \u003c/em\u003e= 3 independent rats, mean ± SD). \u003cstrong\u003el\u003c/strong\u003e, Biodistribution of Did@pLAPs among organs 2, 6, 12, 24 and 36 h after intragastric gavage. \u003cstrong\u003em\u003c/strong\u003e, Quantitative values of Did@pLAPs fluorescence (total radiant efficiency) from biodistribution studies (\u003cem\u003en \u003c/em\u003e= 3 independent mice, mean ± SD). Statistical differences were analyzed by two-tailed Student’s t-test (\u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 and \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4577178/v1/b1c57cac970e254553d1dd5d.png"},{"id":61731718,"identity":"a24364a0-8a92-40af-a7b1-6804dc242e56","added_by":"auto","created_at":"2024-08-05 01:17:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":303812,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e anti-T2DM evaluation of pLAPs in leptin receptor-deficient diabetic (\u003cem\u003edb/db\u003c/em\u003e) model. \u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003eb\u003c/strong\u003e, Schematic representation of the experiment protocol for single (\u003cstrong\u003ea\u003c/strong\u003e) and long-term (\u003cstrong\u003eb\u003c/strong\u003e) administration. \u003cstrong\u003ec\u003c/strong\u003e, Blood glucose levels of mice after single oral administration with different formulations (\u003cem\u003en \u003c/em\u003e= 6 independent mice, mean ± SD). \u003cstrong\u003ed\u003c/strong\u003e, Blood glucose levels of mice after oral administration with different formulations for 30 days (\u003cem\u003en \u003c/em\u003e= 6 independent mice, mean ± SD). \u003cstrong\u003ee\u003c/strong\u003e, OGTT and ITT measured on day 27 and 29 after drug administration, respectively (\u003cem\u003en \u003c/em\u003e= 6 independent mice, mean ± SD). \u003cstrong\u003ef\u003c/strong\u003e-\u003cstrong\u003ei\u003c/strong\u003e, Changes of serum insulin level (\u003cstrong\u003ef\u003c/strong\u003e), HOMA-IR (\u003cstrong\u003eg\u003c/strong\u003e), fat weight (\u003cstrong\u003eh\u003c/strong\u003e) and body weight (\u003cstrong\u003ei\u003c/strong\u003e) in mice after treatment of 30 days (\u003cem\u003en \u003c/em\u003e= 6 independent mice, mean ± SD). Statistical differences were analyzed by one-way ANOVA and post hoc Tukey’s test (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. ns, not significant).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4577178/v1/3968559808f89fd6f05bd6eb.png"},{"id":61731723,"identity":"a4c506ef-3ec9-450e-8adc-35c153af3924","added_by":"auto","created_at":"2024-08-05 01:17:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1197309,"visible":true,"origin":"","legend":"\u003cp\u003epLAPs exert strong efficacy in a murine model of DPN. \u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003eb\u003c/strong\u003e, MDA, CAT, SOD and GSH-Px in serum (\u003cstrong\u003ea\u003c/strong\u003e) and SN (\u003cstrong\u003eb\u003c/strong\u003e) of mice after oral administration with saline, LA (100 mg/kg, daily), pLAPs (100 mg/kg, every three days) for 4 weeks (\u003cem\u003en \u003c/em\u003e= 6 independent mice, mean ± SD). \u003cstrong\u003ec\u003c/strong\u003e-\u003cstrong\u003ee\u003c/strong\u003e, Levels of TNF-α (\u003cstrong\u003ec\u003c/strong\u003e), IL-1β (\u003cstrong\u003ed\u003c/strong\u003e) and IL-6 (\u003cstrong\u003ee\u003c/strong\u003e) in SN of mice after 4 weeks of treatment (\u003cem\u003en \u003c/em\u003e= 6 independent mice, mean ± SD). \u003cstrong\u003ef\u003c/strong\u003e-\u003cstrong\u003ei\u003c/strong\u003e, Representative immunoblots (\u003cstrong\u003ef\u003c/strong\u003e) and quantitative analysis of SN homogenates for phosphorylation of I\u003cu\u003eκ\u003c/u\u003eBα (\u003cstrong\u003eg\u003c/strong\u003e), I\u003cu\u003eκ\u003c/u\u003eBα (\u003cstrong\u003eh\u003c/strong\u003e) and phosphorylation of NF-\u003cu\u003eκ\u003c/u\u003eB (\u003cstrong\u003ei\u003c/strong\u003e) in mice after 4 weeks of treatment (\u003cem\u003en \u003c/em\u003e= 6 independent mice, mean ± SD). \u003cstrong\u003ej\u003c/strong\u003e,\u003cstrong\u003ek\u003c/strong\u003e, Response to noxious thermal stimulation measured by hot plate test (\u003cstrong\u003ej\u003c/strong\u003e) and tail immersion test (\u003cstrong\u003ek\u003c/strong\u003e) in mice at the indicated time (\u003cem\u003en \u003c/em\u003e= 6 independent mice, mean ± SD). \u003cstrong\u003el\u003c/strong\u003e,\u003cstrong\u003em\u003c/strong\u003e, Changes of MCV (\u003cstrong\u003el\u003c/strong\u003e) and SCV (\u003cstrong\u003em\u003c/strong\u003e) after 4 weeks’ treatment (\u003cem\u003en \u003c/em\u003e= 6 independent mice, mean ± SD). \u003cstrong\u003en\u003c/strong\u003e, Representative TEM images of semi-thin toluidine blue-stained cross sections of SN from mice after treatment. Red asterisks and green arrow on TEM micrographs denote effaced axon and myelin, respectively (first row, scale bar: 5 μm; second row, scale bar: 2 μm). \u003cstrong\u003eo\u003c/strong\u003e, Blood glucose levels of mice during treatment for 30 days (\u003cem\u003en \u003c/em\u003e= 6 independent mice, mean ± SD). Statistical differences were analyzed by one-way ANOVA and post hoc Tukey’s test (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. ns, not significant).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4577178/v1/a30a185bda4ca5d96a27a089.png"},{"id":61731724,"identity":"a37decf5-cfb1-4335-89b5-fbd607be35e2","added_by":"auto","created_at":"2024-08-05 01:17:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4940536,"visible":true,"origin":"","legend":"\u003cp\u003epLAPs exert strong efficacy in a murine model of DN or DC. \u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ec\u003c/strong\u003e, UACR (\u003cstrong\u003ea\u003c/strong\u003e), Cre (\u003cstrong\u003eb\u003c/strong\u003e) and BUN (\u003cstrong\u003ec\u003c/strong\u003e) of mice after oral administration with saline and pLAPs (100 mg/kg, every three days) for 4 weeks (\u003cem\u003en \u003c/em\u003e= 6 independent mice, mean ± SD). \u003cstrong\u003ed\u003c/strong\u003e, Representative images of H\u0026amp;E staining (first row), PAS staining (second row), TEM micrographs (third and fourth rows with magnified inset), Masson’s trichrome staining (fifth row) and Sirius red staining (sixth row) of kidney sections from mice after treatment. Red asterisks on TEM micrographs denote effaced podocyte foot processes, and green arrows denote GBM. \u003cstrong\u003ee\u003c/strong\u003e-\u003cstrong\u003ei\u003c/strong\u003e, Quantification of glomerulus size (\u003cstrong\u003ee\u003c/strong\u003e), mean GBM thickness (\u003cstrong\u003ef\u003c/strong\u003e), number of foot processes (\u003cstrong\u003eg\u003c/strong\u003e), mean foot process width (\u003cstrong\u003eh\u003c/strong\u003e) and mean mesangial matrix expression (\u003cstrong\u003ei\u003c/strong\u003e) in different groups of mice (\u003cem\u003en \u003c/em\u003e= 6 independent mice, mean ± SD). \u003cstrong\u003ej\u003c/strong\u003e, Representative images of transthoracic echocardiography (first row, scale bars: horizontal 100 ms and vertical 2 mm), H\u0026amp;E staining (second row), and Masson’s trichrome staining (third row) from mice after treatment. \u003cstrong\u003ek\u003c/strong\u003e-\u003cstrong\u003en\u003c/strong\u003e, Assessments of LVEF (\u003cstrong\u003ek\u003c/strong\u003e), LVIDs (\u003cstrong\u003el\u003c/strong\u003e), LVFS (\u003cstrong\u003em\u003c/strong\u003e) and LVIDd (\u003cstrong\u003en\u003c/strong\u003e) in mice after treatment (\u003cem\u003en \u003c/em\u003e= 6 independent mice, mean ± SD). \u003cstrong\u003eo\u003c/strong\u003e,\u003cstrong\u003ep\u003c/strong\u003e, Quantification of ventricular myocytes size (\u003cstrong\u003eo\u003c/strong\u003e) and fibrotic area (\u003cstrong\u003ep\u003c/strong\u003e) in different groups of mice (\u003cem\u003en \u003c/em\u003e= 6 independent mice, mean ± SD). \u003cstrong\u003eq\u003c/strong\u003e-\u003cstrong\u003et\u003c/strong\u003e, The mRNA levels of \u003cem\u003eANP \u003c/em\u003e(\u003cstrong\u003eq\u003c/strong\u003e), \u003cem\u003eCol1α1 \u003c/em\u003e(\u003cstrong\u003er\u003c/strong\u003e), \u003cem\u003eMMP2 \u003c/em\u003e(\u003cstrong\u003es\u003c/strong\u003e) and \u003cem\u003eTGF-β1 \u003c/em\u003e(\u003cstrong\u003et\u003c/strong\u003e) in myocardial tissue normalized to \u003cem\u003eβ-actin\u003c/em\u003e (\u003cem\u003en \u003c/em\u003e= 6 independent mice, mean ± SD). Statistical differences were analyzed by one-way ANOVA and post hoc Tukey’s test (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. ns, not significant).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4577178/v1/0b06d5f5cc027b830d3462f6.png"},{"id":61732629,"identity":"78d05db2-c1b0-4dd5-8097-ee04ff73fabb","added_by":"auto","created_at":"2024-08-05 01:49:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10266561,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4577178/v1/84e7fa9b-dcff-40e5-a323-3bfcc36ec254.pdf"},{"id":61731909,"identity":"660a9de1-5128-4ecd-92a5-11238f28b813","added_by":"auto","created_at":"2024-08-05 01:25:07","extension":"doc","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15903921,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.doc","url":"https://assets-eu.researchsquare.com/files/rs-4577178/v1/e68c7d537629e2678ce5c503.doc"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nThe authors are on a patent application filed by Sichuan University related to this work.","formattedTitle":"An oral hypoglycemic agent for T2DM with the function of alleviating multi-complications","fulltext":[{"header":"Introduction","content":"\u003cp\u003eType 2 diabetes (T2DM) is a metabolic syndrome characterized by insulin resistance and accompanies the high risk of multi-complications\u003csup\u003e1,2\u003c/sup\u003e. According to the American Diabetes Association (ADA), for patients with T2DM more than 10 years, the complications happened as high as 98%. Unfortunately, the conventional hypoglycemic drugs such as metformin, sulfonylureas, thiazolidinediones, and dipeptidylpeptidase-4 inhibitors showed no therapeutic effect on complications\u003csup\u003e3,4\u003c/sup\u003e. Consequently, the development of hypoglycemic drugs with the function of alleviating complications is urgently needed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe complications of T2DM are driven by persistent hyperglycemia mediated oxidative stress, including microangiopathy, macroangiopathy and neuropathy, accounting for ~25%, ~27%, and ~50% of the population with T2DM, respectively\u003csup\u003e5,6,7\u003c/sup\u003e. For angiopathy, there are some drugs in clinic, such as glucagon-like peptide-1 receptor agonist (GLP-1RA) and sodium-glucose cotransporter 2 inhibitor (SGLT2i), which are the first-line drugs for T2DM combined with chronic kidney disease (a type of microangiopathy) and cardiovascular disease (a type of macroangiopathy)\u003csup\u003e8,9\u003c/sup\u003e. In 2023, the global annual sales of these two drugs reached up to $23.6 and $12.4 billion, respectively. However, for T2DM combined with neuropathy,\u0026nbsp;the complication with the highest morbidity, there is no drug in clinic and even no report in basic research. In addition, GLP-1RA needs usually to be injected subcutaneously and the long-term use will cause the deterioration of retinopathy\u003csup\u003e10,11\u003c/sup\u003e. The SGLT2i is prone to causing the urinary and reproductive system infections and its hypoglycemic effect will decrease with the decline of renal function\u003csup\u003e12,13\u003c/sup\u003e. Obviously, hypoglycemic drugs with the function of alleviating complications are far from meeting the clinical needs.\u003c/p\u003e\n\u003cp\u003eAs a dietary\u0026nbsp;supplement\u0026nbsp;with the antioxidant capacity 400 times higher than that of vitamin C, lipoic acid (LA) holds the functions of hepatic protection and anti-aging etc\u003csup\u003e14\u003c/sup\u003e. Besides the healthcare function, LA also is a clinical drug for diabetic peripheral neuropathy (DPN, a\u0026nbsp;main\u0026nbsp;type of neuropathy) by etiological treatment\u003csup\u003e15\u003c/sup\u003e. Interestingly, it was reported that LA can block hemoglobin glycosylation in the blood, temporarily activate the insulin receptor substrate 1 (IRS-1)/protein kinase B (Akt) and adenosine monophosphate-activated protein kinase (AMPK) signaling pathways to promote small amounts of glucose uptake after entry into the cells\u003csup\u003e16,17\u003c/sup\u003e.\u0026nbsp;Unfortunately, the plasma half-life of LA is only ~30 minutes\u003csup\u003e18\u003c/sup\u003e. The quick body clearance leads to extremely limited inhibition of hemoglobin glycosylation. Moreover, LA held a very short intracellular retention time of ~10 minutes, far below the 1 h persistent activation of the IRS-1/Akt signaling pathway required for LA to produce a significant glucose uptake effect in cells\u003csup\u003e19,20\u003c/sup\u003e.\u0026nbsp;In short, LA has no hypoglycemic effect\u003csup\u003e21,22,23\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eHerein, we report an oral hypoglycemic agent for T2DM with the function of alleviating multi-complications including neuropathy by way of self-polymerizing LA to a nanodrug poly-lipoic acid particles (pLAPs). As shown in Fig. 1, pLAPs were well absorbed into the blood through\u0026nbsp;the polydisulfide backbone mediated mucus penetration, epithelial cellular uptake and epithelial penetration.\u0026nbsp;Due to\u0026nbsp;the negative surface charge and high molecular weight,\u0026nbsp;the plasma half-life of pLAPs was greatly extended from 0.5 h of LA to 23.2 h. As entering the cells, pLAPs were slowly degraded into LA by glutathione (GSH) mediated\u0026nbsp;depolymerization.\u0026nbsp;This unique degradation behavior prolonged the intracellular retention time of LA from ~10 min to \u0026gt; 6 h, making the persistent activation of the IRS-1/Akt signaling pathway become true. Thanksto the significant improvement of pharmacokinetics and intracellular retention time, pLAPs restored the oxidative stress and inflammation-related indicators to the normal control levels in the T2DM models with neuropathy and angiopathy so that the superb therapeutic effect on complications was achieved.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e \u003cb\u003e1. pLAPs continuously activate insulin signaling pathway and promotes glucose uptake by sustained-release of LA in cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe pLAPs were prepared by a nanoprecipitation-mediated polymerization and characterized by morphology, size, zeta potential, molecular weight and the proton nuclear magnetic resonance (Supplementary Figs.\u0026nbsp;1 and 2). Benefiting from the polymerization, the pLAPs exhibited robust dilution and serum stability, and dissociated only in PBS with glutathione (GSH, 2\u0026thinsp;~\u0026thinsp;10 mM) that mimicking the redox level in the insulin target cells (Supplementary Fig.\u0026nbsp;3). To investigate whether the self-polymerization prolonged the intracellular retention time of LA, three insulin target cells (3T3-L1, L6, and L02) were incubated with the conditioned medium containing 100 \u0026micro;g/mL pLAPs. After 1 h incubation, the medium containing pLAPs was removed and the pLAPs-free medium was added. At predetermined time points, the cells were lysed and the lysate supernatant was collected to detect the content of intracellular LA by high-performance liquid chromatography (HPLC). LA was set as a control. As plotted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c, in LA-treated cells, the LA content maintained a high level for the first 10 min, then rapidly decreased and was almost undetectable after 360 min. By comparison, in pLAPs treated cells, the LA content gave a time-dependent increase. After reaching the peak at 60 min, it remained at the high level over 360 min. We rationalized that the maintenance of the high LA level would be attributed to the balance of its dissociation and metabolism. In other words, the self-polymerization indeed significantly prolonged the intracellular retention time of LA. It should be pointed out that the self-polymerization mediated prolongation of LA intracellular time cannot be achieved by loading LA in any nanocarriers due to the unavoidance of the rapid diffusion release.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBefore investigating the effect of prolonged intracellular retention of LA on the activation of insulin signaling pathway, the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed to evaluate the nontoxic concentration range of pLAPs on target cells (0\u0026thinsp;~\u0026thinsp;250 \u0026micro;g/mL, Supplementary Fig.\u0026nbsp;4). The activation of insulin signaling pathway was achieved by incubating cells with palmitic acid (PA) and LA or pLAPs for 1 h before the insulin stimulation (See Supporting Information for details). PA is a classic insulin resistance inducer, which can effectively inactivate the insulin signaling pathway by inhibiting the expression of p-IRS1 and p-Akt, two key insulin signaling molecules. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed and Supplementary Fig.\u0026nbsp;5, the LA treatment increased only the expression of p-IRS1 and p-Akt in the first 10 min. By contrast, pLAPs caused no change of the expression of p-IRS1 and p-Akt within the first 30 min, while gave a significant increase at 60 min and maintained the high level within 60\u0026thinsp;~\u0026thinsp;360 min, consistent with the trend in intracellular LA content variation of the pLAPs group. As a result of the persistent activation of insulin signaling pathway, 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG), a deoxyglucose analogue used for direct monitoring of glucose uptake by living cells, showed also a remarkable uptake level within 60\u0026thinsp;~\u0026thinsp;360 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee-g).\u003c/p\u003e \u003cp\u003eTo confirm the achievement of pLAPs\u0026rsquo; above effects by dissociating into LA in the cells, the GSH depletion agent diethyl maleate (DEM) was employed to inhibit the intracellular dissociation of pLAPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c, pink line). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh-n, the block of the pLAPs dissociation led to the inactivation of insulin signaling pathway and the disappearance of 2-NBDG uptake, suggesting that pLAPs activates insulin signaling pathway and promotes glucose uptake by dissociating into LA rather than in the form of nanoparticles.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2. pLAPs hold good absorption, long\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e \u003cb\u003ehalf-life and efficient accumulation inT2DM target organs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe absorption effect of pLAPs was first investigated \u003cem\u003ein vitro\u003c/em\u003e via the mucus penetration, epithelial cellular uptake and epithelial penetration, the major barriers of oral drug delivery. Rifampicin, a drug with oral bioavailability\u0026thinsp;~\u0026thinsp;95%, was set as a positive control (See Supporting Information for details). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-f, the mucus penetration rate of pLAPs was ~\u0026thinsp;61.0%, over 7/10 of rifampicin (~\u0026thinsp;84.0%). Moreover, the cellular uptake efficiency and apparent permeability coefficient (P\u003csub\u003eapp\u003c/sub\u003e) of pLAPs reached\u0026thinsp;~\u0026thinsp;71.8% and 2.6\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e cm s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, data comparable to rifampicin (~\u0026thinsp;77.4% and 2.8\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e cm s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), indicating the good intestinal absorption of pLAPs. The good absorption was further evaluated by an \u003cem\u003eex vivo\u003c/em\u003e intestinal permeability model (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). Briefly, the pLAPs or rifampicin was injected into the lumen (donor chamber) before ligation, followed by an incubation in the Krebs-Ringer (K-R) buffer (acceptor chamber) at 37\u0026deg;C. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh, as the drug content stabilized in the acceptor chamber, the small intestine absorption ratio of pLAPs was ~\u0026thinsp;58.0%, up to 2/3 of rifampicin (~\u0026thinsp;86.0%).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe hypothesize that the good absorption is correlated to the polydisulfide backbone of pLAPs, which enables dynamic covalent exchange with the thiols of mucin, epithelial cell membrane protein and lysosomal membrane so as to mediate the nanoparticles to penetrate the absorption barriers\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. To check the hypothesis, 5,5\u0026rsquo;-dithiobis(2-nitrobenzoic acid) (DTNB), a popular thiol-reactive agent, was employed to block the thiol groups. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei,j, just a 30 min blocking, the mucus penetration and cellular uptake efficiency of pLAPs decreased respectively by ~\u0026thinsp;68.8% and ~\u0026thinsp;31.1%. The lysosomal escape behavior of pLAPs was investigated by fluorescence colocalization of Coumarin 6-labeled pLAPs (Cou@pLAPs) and LysoTracker red in Caco-2 cells via confocal laser scanning microscope (CLSM). As shown in Supplementary Fig.\u0026nbsp;6, the Cou@pLAPs were colocalized with LysoTracker at 1 h, indicating the lysosomal entrapment. As the incubation time was prolonged from 1 to 8 h, the Cou@pLAPs and LysoTracker fluorescence gave less colocalization and the colocalization Pearson\u0026rsquo;s correlation coefficient (\u003cem\u003eR\u003c/em\u003er) decreased dramatically from 0.72 to 0.31, indicating the escape of pLAPs from the lysosomes. In contrast, after the blocking of thiols on the lysosomal membrane, the Cou@pLAPs were colocalized with LysoTracker over 8 h, and the \u003cem\u003eR\u003c/em\u003er showed no significant change.\u003c/p\u003e \u003cp\u003eThe oral terminal half-life (\u003cem\u003eT\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e) of pLAPs was studied on Sprague Dawley (SD) rats via oral gavage. At predetermined time points, the blood samples were collected and treated with NaOH, then the dissociated LA was extracted and measured to reflect the plasma concentration of pLAPs. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek, in LA monomer-treated SD rats, the \u003cem\u003eT\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e was 0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 h, data comparable to the previous reports. To our delight, in the pLAPs-treated SD rats, the \u003cem\u003eT\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e was up to 23.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 h, ~\u0026thinsp;46 times higher than that of LA. We rationalized that the long \u003cem\u003eT\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e of pLAPs would be ascribed to its negative surface charge and ~\u0026thinsp;30,000 molecular weight, which can decrease plasma protein binding and retard the glomerular filtration. As a result of the good absorption and prolonged \u003cem\u003eT\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e, the area under the 72 h of plasma concentration curve (AUC\u003csub\u003e0\u0026thinsp;~\u0026thinsp;72 h\u003c/sub\u003e) of pLAPs was up to 3761.8\u0026thinsp;\u0026plusmn;\u0026thinsp;55.9 h*\u0026micro;g/mL, ~\u0026thinsp;23 times higher than that of LA, indicating that the self-polymerization does remarkably improve the oral pharmacokinetics of LA.\u003c/p\u003e \u003cp\u003eThe biodistribution of pLAPs was evaluated on C57BL/6 mice through oral administration of 1,19-dioctadecyl-3,3,39,39-tetramethylindodica-rbocyanine perchlorate (Did)-labeled pLAPs (Did@pLAPs). At predetermined time points, mice were euthanized to harvest the major and insulin target organs, and the \u003cem\u003eex vivo\u003c/em\u003e fluorescence images were detected using an \u003cem\u003ein vivo\u003c/em\u003e imaging system (IVIS). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003el,m, the \u003cem\u003eex vivo\u003c/em\u003e IVIS images revealed that the fluorescence signals were mainly observed in the liver, heart and kidney. Besides the distribution in the above main organs, one can find that the pancreas (insulin secretion organ) and fat and skeletal muscle (insulin effector organs) gave also strong fluorescence signals, indicating the T2DM target organs-efficient accumulation of pLAPs.\u003c/p\u003e\n\u003ch3\u003e3. pLAPs exert strong efficacy in the murine model of T2DM\u003c/h3\u003e\n\u003cp\u003eThe oral hypoglycemic effect was assessed on the T2DM model of the 8-week-old male \u003cem\u003edb/db\u003c/em\u003e mice by single and long-term administrations of pLAPs at the doses of 25, 50 and 100 mg/kg or LA at 100 mg/kg (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b). The \u003cem\u003edb/db\u003c/em\u003e mice treated with saline and metformin (120 mg/kg, equivalent therapeutic dose for T2DM patients) were set as the negative control and positive control, respectively, and \u003cem\u003edb/m\u003c/em\u003e mice treated with saline was set as the normal control. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, LA caused no change of the blood glucose level as expected. Metformin gave a glucose decrease during 2\u0026thinsp;~\u0026thinsp;24 h, with a lowest level of 10.7 mmol/L at ~\u0026thinsp;6 h. To our delight, the pLAPs showed hypoglycemic effects at all three doses, and the best one occurred in the 100 mg/kg group, where the hypoglycemic effect was maintained in a period of 2\u0026thinsp;~\u0026thinsp;72 h and the blood glucose level was as low as 8.4 mmol/L, 21.5% lower than that in the metformin group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDue to the nearly 3-day hypoglycemic effect of a single administration, the pLAPs were administered every three days in the long-term evaluation, while the LA and metformin were administered daily. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, LA showed still no hypoglycemic effect. Metformin stabilized the blood glucose at ~\u0026thinsp;12.7 mmol/L. By contrast, pLAPs administrated even every three days showed an excellent hypoglycemic effect, with the blood glucose stable at a low level of ~\u0026thinsp;9.4 mmol/L (100 mg/kg), much lower than that in the metformin group and even close to the normal control.\u003c/p\u003e \u003cp\u003eTo confirm the hypoglycemic effect of pLAPs, all mice were orally administrated with glucose (2 g/kg) on day 27 and intraperitoneally injected with recombinant human insulin (1 U/kg) on day 29, and the AUC\u003csub\u003e0\u0026thinsp;~\u0026thinsp;120 min\u003c/sub\u003e of oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were detected to evaluate glucose tolerance and insulin resistance, respectively. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, the \u003cem\u003edb/db\u003c/em\u003e mice showed a significant AUC\u003csub\u003e0\u0026thinsp;~\u0026thinsp;120 min\u003c/sub\u003e increase of OGTT and ITT compared to the \u003cem\u003edb/m\u003c/em\u003e mice, suggesting a seriously impaired glucose tolerance and insulin resistance. LA caused no improvement of the AUC\u003csub\u003e0\u0026thinsp;~\u0026thinsp;120 min\u003c/sub\u003e of OGTT and ITT, consistent with the hypoglycemic result. Metformin showed a decrease of 21.4% for OGTT and 27.1% for ITT, comparable to the other reports\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. By comparison, pLAPs decreased the AUC\u003csub\u003e0\u0026thinsp;~\u0026thinsp;120 min\u003c/sub\u003e of OGTT by up to 39.5% and ITT by up to 48.3%, a comparable level to the normal control, implying the excellent improvement in glucose tolerance and insulin resistance. In addition, the improvement of pLAPs on insulin resistance was further evidenced by serum insulin and insulin resistance index (HOMA-IR) assays, in which the serum insulin level and HOMA-IR were decreased by 39.4% and 72.5%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef,g). Notably, one can find that the pLAPs reduced the weight of epididymal and body by 34.6% and 25.6%, respectively, while metformin caused no any weight loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh,i), indicating the potential of pLAPs to reduce the risk factor of T2DM through weight loss. One can also find the weight loss of pLAPs was a little bit weaker than that of LA, possibly due to the fact that pLAPs were mainly distributed in insulin target organs and failed to block AMPK pathway in the hypothalamus to inhibit food intake\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003e4. Besides the activation of classical IRS1/Akt signaling pathway, pLAPs exert hypoglycemic effect by promoting the normalization of pancreatic structure and secretion function\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn order to confirm the activation of pLAPs on the classical IRS1/Akt signaling pathway \u003cem\u003ein vivo\u003c/em\u003e, all mice were euthanized after 30-day treatment, and the expression of p-IRS1 and p-Akt in liver and skeletal muscle were detected by Western blotting. Consistent with the \u003cem\u003ein vitro\u003c/em\u003e results, pLAPs showed a significant increase in the expression of p-IRS1 and p-Akt, and, as a result, the levels of glucose transporter 4 (GLUT4, a key protein that promotes glucose transmembrane mobility) and glycogen deposits were also remarkably increased (Supplementary Fig.\u0026nbsp;7).\u003c/p\u003e \u003cp\u003eIn addition to the inhibition of classical IRS1/Akt signaling pathway, pancreatic damage caused by inflammation and oxidative stress is the other major factor in the progression of T2DM\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Considering the strong antioxidant and anti-inflammatory properties of pLAPs, we evaluated further its impact on pancreas lesions by hematoxylin and eosin (H\u0026amp;E), TUNEL and insulin immunohistochemical staining. As shown in Supplementary Fig.\u0026nbsp;8a-d, metformin had no improvement on pancreatic damage. To our delight, pLAPs, at the dose of 50 mg/kg, showed a significant improvement in the related indicators. When the dose was increased to 100 mg/kg, a much stronger improvement was achieved, where the size of islets was reduced by 70.9%, the number of apoptotic β-cells was reduced by 70.1%, and the expression of insulin was increased by 159.3%. Notably, one can find that all three levels were close to the normal control, indicating a very good improvement of pLAPs on pancreatic damage.\u003c/p\u003e \u003cp\u003eTo confirm the improvement of pLAPs on the pancreas injury by antioxidation and anti-inflammatory, the oxidative stress marker malondialdehyde (MDA), antioxidant markers catalase (CAT), superoxide dismutase (SOD), and glutathione peroxide (GSH-Px), and pro-inflammatory cytokines tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were assessed. As shown in Supplementary Fig.\u0026nbsp;8e-h, pLAPs gave a dose-dependent decrease on the level of oxidative stress marker and an increase on the activities of antioxidant markers, respectively, with the high-dose group (100 mg/kg) approaching to the normal control. Consistent with antioxidation results, pLAPs exhibited also a strong inhibitory effect on inflammation (Supplementary Fig.\u0026nbsp;8i-k). We note in passing that LA had no therapeutic effect on pancreatic injury, and its inhibition on oxidative stress and inflammation was also limited. This would be because LA metabolizes too fast and cannot be accumulated effectively in pancreatic tissue.\u003c/p\u003e \u003cp\u003e \u003cb\u003e5. pLAPs show a much stronger therapeutic efficacy for neuropathy than LA and reduces blood glucose in the advanced stage of diabetes\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe treatment of pLAPs on neuropathy was evaluated by using the 20-week-old male \u003cem\u003edb/db\u003c/em\u003e mice as a DPN model. Consistent with the frequency of administration in the long-term hypoglycemic evaluation, pLAPs were orally administrated every three days for 30 consecutive days at a dose of 100 mg/kg, and once-daily oral administration of LA (100 mg/kg, equivalent therapeutic dose for clinical patients with DPN) was set as the positive control. Considering the oxidative stress pathogenesis of neuropathy, we first investigated the changes of oxidative stress and inflammation-related indicators at the end of treatment. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea,b, pLAPs gave a significant decrease on the content of MDA and an increase on the activities of CAT, SOD and GSH-Px in serum and sciatic nerve (SN). To our delight, all these indicators showed no difference compared to the normal control (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), implying the complete improvement of redox imbalance. Consistent with the improvement effect of oxidative stress indicators, the pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) and inflammatory signaling molecules (p-IκBα and p-NF-κB) restored also to the normal control levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-i). By contrast, LA, even with administration daily, had still an enormous gap in the levels of the above indicators compared to the normal control. These results indicated that by transforming LA to the stabilized nanoparticles (pLAPs), a superstrong antioxidant and anti-inflammatory capacity was achieved.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs a result of the performance in antioxidant and anti-inflammatory, the mice treated by pLAPs showed an excellent improvement in behavior and nerve conduction velocity, where the hot-plate and tail-flick latencies were shortened by 40.4% and 31.1%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ej,k), and motor nerve conduction velocity (MCV) and sensory nerve conduction velocity (SCV) were increased by 35.2% and 36.7%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003el,m). At the same time, the myelin sheath of SN was significantly regenerated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003en). By contrast, the latencies of hot-plate and tail-flick in LA treated mice were reduced by only 13.5% and 14.2%, respectively, and the MCV and SCV were increased by only 12.9% and 19.7%, respectively.\u003c/p\u003e \u003cp\u003eAs known, the 20-week-old \u003cem\u003edb/db\u003c/em\u003e mice, equivalent to patients with T2DM more than 10 years, were generally used as a model for advanced diabetes. In the advanced stage of diabetes, the blood glucose lowering is difficult to achieve owing to the proliferation cessation and loss of secretory function of β-cells, as well as the aggravation of insulin resistance caused by long-term hyperglycemia. To our delight, pLAPs showed also a good and stable hypoglycemic effect in the rodent model of advanced diabetes, where a stable blood glucose occurred on 6th day, with a low level of ~\u0026thinsp;16.3 mmol/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eo).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e6. pLAPs exhibit excellent therapeutic effect on diabetes microvascular and macrovascular disease\u003c/h2\u003e \u003cp\u003eAs known, the lesion site of DPN locates in the peripheral nerves with a single structure (composed only of neurons and glial cells)\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. LA easily spreads to nerves and is directly taken up by cells to exert systemic antioxidant effects. Therefore, LA is approved in clinic for the DPN treatment in despite of its fast metabolism. On the contrary, the diabetic angiopathy locates mainly in the complex organs, such as heart, kidney and retina. LA hardly accumulates in the effector cells of these organs. Although driven also by high glucose-mediated oxidative stress, the angiopathy cannot be treated with LA. Encouraged by its much stronger antioxidant and anti-inflammatory capacity over LA and effective enrichment in T2DM target tissues, we tried further the potential of pLAPs in the treatment of angiopathy. The diabetic nephropathy (DN) and diabetic cardiomyopathy (DC) were employed as models for microvascular disease and macrovascular disease, respectively. Consistent with the results in the DPN treatment, pLAPs restored the oxidative stress and inflammation-related indicators to normal control levels in both models (Supplementary Fig.\u0026nbsp;9) and showed also a good and stable hypoglycemic effect (Supplementary Fig.\u0026nbsp;10).\u003c/p\u003e \u003cp\u003eThe therapeutic effect of DN was evaluated by measuring the renal function indexes [urine albumin-to-creatinine ratio (UACR), serum creatinine (Cre), and blood urea nitrogen (BUN)] and glomerular pathological changes [glomerular sizes, glomerular basement membrane (GBM) thickness, mesangial matrix expression, and width and number of foot processes]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, compared to mice treated with saline, the pLAPs treated mice showed a decrease on all three renal function indexes, with the levels of UACR, Scr and BUN reduced by 71.3%, 52.6% and 50.9%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-c). The glomerular pathological results showed that pLAPs reduced the glomerular size by 64.4%, GBM thickness by 18.3%, mesangial matrix expression by 48.4%, the width of foot processes by 28.1%, and increased the number of foot processes by 64.2%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed-i). The therapeutic effect of DC was evaluated by measuring the cardiac function indexes [left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-systolic diameter (LVIDs), and left ventricular end-diastolic diameter (LVIDd)], pathological changes (ventricular myocytes size and fibrotic area), and myocardial injury markers [natriuretic peptide type A (\u003cem\u003eANP\u003c/em\u003e), collagen type I alpha 1 (\u003cem\u003eCol1α1\u003c/em\u003e), matrix metallopeptidase 2 (\u003cem\u003eMMP2\u003c/em\u003e) and transforming growth factor beta 1 (\u003cem\u003eTGF-β1\u003c/em\u003e)]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ej-n, compared to mice treated with saline, the pLAPs treated mice showed an increase of 82.9% for LVEF and 46.9% for LVFS, and a decrease of 53.6% for LVIDs and 32.4% for LVIDd. At the same time, the ventricular myocytes size, fibrotic area and myocardial injury markers levels were significantly decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eo-t). In particular, one can find that the above indicators of DC and DN showed no significant difference compared to the normal control, suggesting that the mice returned to normal after the pLAPs treatment. As a contrary, the LA treatment only provided a very limited therapeutic effect from the slight improvement of the indicators. Taken together, pLAPs hold a good clinical potential in the treatment of both angiopathy and neuropathy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e7. pLAPs improve spontaneous T2DM with DPN in rhesus monkey\u003c/h3\u003e\n\u003cp\u003eEncouraged by the outstanding outcomes in rodents, we challenged further the treatment of pLAPs for the spontaneous T2DM rhesus monkey with the complication of DPN since it very closely mimicked human T2DM pathological properties. Concretely, 5 spontaneous T2DM rhesus monkeys, which conform to either the slowing down of any SCV or prolonged latency by detecting SCV of the sural nerve, median nerve, and other nerves over 6 months using neuroelectromyography, were randomly divided into 2 groups. Three monkeys were given pLAPs (10 mg/kg/d) by nasogastric gavage for 8 weeks, and the other two were given placebo as the negative control. It should be pointed out that, for meeting the requirement of T2DM combined with DPN, the selected five animals are elderly diabetes monkeys aged 18\u0026ndash;21 years (analogous to approximately 70-year-old humans)\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. At this stage, even the classic insulin-sensitizing drugs such as metformin, sulfonylureas, or GLP-1RA would lose their hypoglycemic effect caused by islet function failure\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Such situation occurred also in our case. Although the fasting plasma glucose (FPG) of the pLAPs treated monkeys gave a downtrend compared to monkeys treated with placebo, there was no significant difference compared to baseline (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Considering that the FPG detection in this stage is no longer suitable for evaluating the true effectiveness of drugs on T2DM, the HOMA-IR, a unique indicator reflecting the insulin sensitivity without affection by the disease-developing stage, was employed to evaluate the therapeutic effect of T2DM. The lower the HOMA-IR is, the stronger the hypoglycemic effect of drugs is. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the placebo treated monkeys showed no change of the HOMA-IR during the treatment. To our delight, the HOMA-IR of monkeys treated by pLAPs gave a continuous decrease from 7.41 to 2.02, a value located in the range of normal human (\u0026lt;\u0026thinsp;2.69)\u003csup\u003e40\u003c/sup\u003e, verifying undoubtedly the excellent therapeutic efficacy of pLAPs for T2DM.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGeneral glucose metabolism indicators of the T2DM rhesus monkey with DPN following once daily oral administration of pLAPs (10 mg/kg) or placebo for 8 weeks.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIndicators\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBaseline\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDay 14\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDay 28\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDay 42\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eDay 56\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFPG\u003c/p\u003e \u003cp\u003e(mmol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePlacebo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e5.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e4.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epLAPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.97\u0026thinsp;\u0026plusmn;\u0026thinsp;2.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e6.88\u0026thinsp;\u0026plusmn;\u0026thinsp;2.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e6.84\u0026thinsp;\u0026plusmn;\u0026thinsp;1.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e6.83\u0026thinsp;\u0026plusmn;\u0026thinsp;2.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.43\u0026thinsp;\u0026plusmn;\u0026thinsp;2.48 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFPI\u003c/p\u003e \u003cp\u003e(\u0026micro;U/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePlacebo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e14.81\u0026thinsp;\u0026plusmn;\u0026thinsp;6.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e22.76\u0026thinsp;\u0026plusmn;\u0026thinsp;16.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e12.74\u0026thinsp;\u0026plusmn;\u0026thinsp;2.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e18.53\u0026thinsp;\u0026plusmn;\u0026thinsp;5.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17.30\u0026thinsp;\u0026plusmn;\u0026thinsp;10.22 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epLAPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e22.77\u0026thinsp;\u0026plusmn;\u0026thinsp;7.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e13.03\u0026thinsp;\u0026plusmn;\u0026thinsp;10.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e11.56\u0026thinsp;\u0026plusmn;\u0026thinsp;7.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e9.46\u0026thinsp;\u0026plusmn;\u0026thinsp;11.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.86\u0026thinsp;\u0026plusmn;\u0026thinsp;7.61 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eHOMA-IR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePlacebo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e5.01\u0026thinsp;\u0026plusmn;\u0026thinsp;3.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e3.91\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.65\u0026thinsp;\u0026plusmn;\u0026thinsp;2.05 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epLAPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e7.41\u0026thinsp;\u0026plusmn;\u0026thinsp;4.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.75\u0026thinsp;\u0026plusmn;\u0026thinsp;2.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e3.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e2.51\u0026thinsp;\u0026plusmn;\u0026thinsp;2.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eNote: HOMA-IR\u0026thinsp;=\u0026thinsp;FPG \u0026times; FPI/22.5.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eStatistical differences were analyzed by two-tailed Student\u0026rsquo;s t-test. \u003csup\u003ea\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05, \u003csup\u003eb\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, compared with baseline.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eFPG: fasting plasma glucose; FPI: fasting plasma insulin; HOMA-IR: insulin resistance index.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe therapeutic effect for DPN was assessed by measuring the nerve conduction velocity (NCV) every 4 weeks, and the data were shown in Table\u0026nbsp;2. During the treatment, the placebo caused no change of the NCV of abnormal nerves compared to that at baseline (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), while the NCV of pLAPs treated group showed a continuous increase from 37.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4 m/s to 41.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2 m/s. By directly comparing the velocity change on day 28 and day 56, one can find that the placebo\u0026rsquo;s velocity changes were \u0026minus;\u0026thinsp;0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 m/s and \u0026minus;\u0026thinsp;0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 m/s, respectively, while the corresponding values of pLAPs gave a significant improvement of 2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8 m/s and 3.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 m/s, suggesting the significant improvement of pLAPs on DPN. To our knowledge, this is the first hypoglycemic drug with the function of alleviating neuropathy.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e NCV and velocity change of the T2DM rhesus monkey with DPN following once daily oral administration of pLAPs (10 mg/kg) or placebo for 8 weeks.\u003c/p\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"463\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.517241379310345%\" rowspan=\"2\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50.21551724137931%\" colspan=\"3\"\u003e\n \u003cp\u003eNCV (m/s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.663793103448276%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.603448275862068%\" colspan=\"2\"\u003e\n \u003cp\u003eVelocity change (m/s)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.3206106870229%\"\u003e\n \u003cp\u003eBaseline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.356234096692113%\"\u003e\n \u003cp\u003eDay 28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.610687022900763%\"\u003e\n \u003cp\u003eDay 56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.325699745547074%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.3206106870229%\"\u003e\n \u003cp\u003eDay 28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.06615776081425%\"\u003e\n \u003cp\u003eDay 56\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.483870967741936%\"\u003e\n \u003cp\u003ePlacebo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.483870967741936%\"\u003e\n \u003cp\u003e38.1 \u0026plusmn; 4.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.204301075268816%\"\u003e\n \u003cp\u003e37.8 \u0026plusmn; 4.8 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.419354838709676%\"\u003e\n \u003cp\u003e37.9 \u0026plusmn; 4.8 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.6559139784946235%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.483870967741936%\"\u003e\n \u003cp\u003e-0.3 \u0026plusmn; 0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.268817204301076%\"\u003e\n \u003cp\u003e-0.2 \u0026plusmn; 1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.483870967741936%\"\u003e\n \u003cp\u003epLAPs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.483870967741936%\"\u003e\n \u003cp\u003e37.6 \u0026plusmn; 3.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.204301075268816%\"\u003e\n \u003cp\u003e40.0 \u0026plusmn; 3.6 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.419354838709676%\"\u003e\n \u003cp\u003e41.2 \u0026plusmn; 3.2 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.6559139784946235%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.483870967741936%\"\u003e\n \u003cp\u003e2.4 \u0026plusmn; 2.8 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.268817204301076%\"\u003e\n \u003cp\u003e3.6 \u0026plusmn; 2.5 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eNote: Statistical differences were analyzed by two-tailed Student\u0026rsquo;s t-test. \u003csup\u003ea\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05, \u003csup\u003eb\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, compared with baseline. \u003csup\u003ec\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, compared with placebo group.\u003c/p\u003e\n\u003cp\u003eNCV: nerve conduction velocity.\u003c/p\u003e\n\u003ch3\u003e8. pLAPs hold good biosafety\u003c/h3\u003e\n\u003cp\u003eAs a chronic metabolic disease, T2DM requires long-term treatment with hypoglycemic drugs. Therefore, the drug safety is particularly important. Here, we evaluated the biosafety of pLAPs by acute and subacute toxicity tests. The acute toxicity was performed by single oral administration of 2000 mg/kg pLAPs into C57BL/6 mice, saline was used as a control. The results showed that the mice maintained healthy as confirmed by the unchanged behavior and nonweight loss over 2 weeks (Supplementary Fig.\u0026nbsp;11a). At the end of the observation, mice were euthanized and the main organs and blood were collected for the organs index, hematology profile and serum biochemistry analyses. It was found that no obvious changes in the organs index (Supplementary Fig.\u0026nbsp;11b), hematology profile (Supplementary Fig.\u0026nbsp;11c) and serum biochemical parameters (Supplementary Fig.\u0026nbsp;11d) were observed in mice treated with pLAPs.\u003c/p\u003e \u003cp\u003eThe subacute toxicity was performed by oral administration of pLAPs (100 mg/kg/d) to C57BL/6 mice for 30 days. Saline was used as a control. The levels of hematology profile, plasma biochemical parameters and the pathological changes of main organs were checked after treatment. The results showed that the mice treated with pLAPs did not show significant differences in the hematology (Supplementary Fig.\u0026nbsp;12a) and plasma biochemical parameters (Supplementary Fig.\u0026nbsp;12b) compared with the control group. In addition, the H\u0026amp;E staining of the main organs, including the heart, liver, spleen, lung and kidney showed no abnormalities after pLAPs oral administration (Supplementary Fig.\u0026nbsp;13). Notably, thanks to the hypoglycemic mechanism of the insulin signaling pathway activation and pancreatic injury improvement, the mice treated with pLAPs showed no hypoglycemic reactions under the doses of both acute and subacute toxicity (Supplementary Fig.\u0026nbsp;14). By the way, during the 8 weeks of administering pLAPs (10 mg/kg/d) via nasogastric gavage to treat spontaneous T2DM rhesus monkey with DPN, we did not observe any abnormalities, including body weight, food intake (Supplementary Table\u0026nbsp;1), plasma biochemical parameters (Supplementary Table\u0026nbsp;2) and hematology profile after treatment (Supplementary Table\u0026nbsp;3). Overall, the above results suggested the good biosafety of pLAPs.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, an oral hypoglycemic agent by self-polymerizing LA to a nanodrug pLAPs has been developed for the treatment of T2DM with multi-complications. The self-polymerization made the oral \u003cem\u003eT\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e, AUC\u003csub\u003e0\u0026thinsp;~\u0026thinsp;72 h\u003c/sub\u003e and intracellular retention time of LA up to 23.2 h, 3761.8\u0026thinsp;\u0026plusmn;\u0026thinsp;55.9 h*\u0026micro;g/mL and \u0026gt;\u0026thinsp;6 h, respectively, ~\u0026thinsp;46, ~23 and over ~\u0026thinsp;36 times higher than that of LA monomer. Accordingly, a single administration of pLAPs (100 mg/kg) in \u003cem\u003edb/db\u003c/em\u003e mice gave a nearly 3-day hypoglycemic period with the blood glucose level as low as 8.4 mmol/L, a value close to the normal control, while LA showed no hypoglycemic effect. The long-term oral hypoglycemic evaluation revealed that pLAPs (100 mg/kg) administrated even every three days stabilized the blood glucose level at ~\u0026thinsp;9.4 mmol/L. By contrast, metformin administrated daily at the equivalent therapeutic dose for patients (120 mg/kg) stabilized the blood glucose level only at ~\u0026thinsp;12.7 mmol/L.\u003c/p\u003e \u003cp\u003eThe improvement of pharmacokinetics and intracellular retention time led also to the strong inhibition on oxidative stress and inflammation. In the mice with DPN, pLAPs restored the oxidative stress and inflammation-related indicators to the normal control. As a result, the hot-plate and tail-flick latencies of mice were shortened by 40.4% and 31.1%, respectively, and MCV and SCV were increased by 35.2% and 36.7%, respectively. All these levels were consistent with the normal control. By contrast, the latencies of hot-plate and tail-flick in LA treated mice were reduced by only 13.5% and 14.2%, respectively, and the MCV and SCV were increased by only 12.9% and 19.7%, respectively, far away from the levels of normal control. In particular, for LA\u0026rsquo;s non-indication angiopathy (e.g., DN and DC), pLAPs showed also excellent therapeutic effect, with the recovery of related indicators to the levels of normal control. Notably, the improvement of pLAPs on T2DM and its complications has been verified in the treatment of spontaneous T2DM rhesus monkey with DPN, where the HOMA-IR was decreased to 2.02 after 8 weeks of treatment, a value located in the range of normal human, and the NCV was increased by 3.8 m/s compared to the placebo. Notably, pLAPs showed no acute and subacute toxicity administered at doses of 2000 mg/kg and 100 mg/kg respectively in mice, and no adverse drug reactions were observed in the spontaneous T2DM rhesus monkey during treatment. This strategy of self-polymerizing lipoic acid into a nanodrug provides a brand-new solution for the treatment of T2DM.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eLA was purchased from Tokyo chemical industry Co., Ltd. (Japan). Metformin hydrochloride sustained release tablet was purchased from Sino-American Shanghai Squibb Pharmaceuticals Ltd. (Shanghai, China). D-(+)-Glucose was purchased from aladdin (USA). DEM was purchased from McLean Biochemical Technology Co., Ltd. (Shanghai, China). MTT and non frozen tissue RNA preservation solution (RNAwait) were obtained from Beijing Solarbio Science \u0026amp; Technology Co., Ltd. (Beijing, China). 2-NBDG was obtained from GlpBio Technology (Montclair, California, USA). PA was obtained from Sigma Chemical Company (Saint Louis, MO, USA). Coumarin 6 and DTNB were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). Mouse insulin, IL-1β, IL-6 and TNF-α ELISA kits were obtained from Jingmei Biotechnology (Jiangsu, China). Mouse urine albumin (ALB) ELISA kit was obtained from Shanghai Tongwei Industrial Co., Ltd. (Shanghai, China). Did, colorimetric TUNEL apoptosis assay kit, RIPA lysis buffer, protein inhibitor cocktail (100 \u0026times;), bicinchoninic acid (BCA) protein assay kit, MDA assay kit, recombinant human insulin, enhanced ECL western blotting detection kit and anti-insulin antibody were obtained from Beyotime Institute of Biotechnology (Jiangsu, China). Polyvinylidene fluoride (PVDF) and HRP-conjugated goat anti-rabbit IgG were purchased from Servicebio (Wuhan, China). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (T-BIL), BUN, Cre, CAT, GSH-Px, and SOD assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). Anti-Akt, anti-phospho-(Ser473)-Akt, and anti-β-actin antibodies were obtained from Wuhan Sanying Biotechnology Co., Ltd. (Wuhan, China). Anti-IRS-1, anti-phospho-(Ser307)-IRS1, anti-NF-κB p65, anti-IκBα, anti-phospho-(Ser32/Ser36)-IκBα antibodies were purchased from Affinity Biosciences (Jiangsu, China). Anti-GLUT4, anti-phospho-NF-κB p65 and HRP-conjugated goat anti-mouse IgG antibodies were obtained from ABclonal Biotechnology Co., Ltd. (Wuhan, China). Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, and pancreatin were purchased from Gibco BRL Life Technology (California, USA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimals\u003c/h3\u003e\n\u003cp\u003eSprague Dawley (SD) rats were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. (Chengdu, China). C57BL/6 mice, \u003cem\u003edb/m\u003c/em\u003e mice, and \u003cem\u003edb/db\u003c/em\u003e mice were purchased from Gempharmatech Co., Ltd. (Jiangsu, China). All rodent animal experiments were approved by the Animal Ethics Committee of Sichuan University (Chengdu, China) and carried out according to the approved guidelines. Rhesus monkeys with spontaneous type 2 diabetes mellitus were provided and housed in Sichuan Primed Shines Bio-tech Co., Ltd. (SPSB) (Chengdu, China), animal care procedures were approved by the Institutional Animal Care and Utilization Committee (IACUC) of SPSB accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International).\u003c/p\u003e\n\u003ch3\u003eSynthesis of pLAPs\u003c/h3\u003e\n\u003cp\u003eThe pLAPs were prepared by a nanoprecipitation mediated polymerization. Specifically, to 19 mL of ultrapure water under stirring, 1.0 mL DMSO solution of LA (10% \u003cem\u003ew/v\u003c/em\u003e) was added dropwise to get a milky white solution. After ultrasound treatment with 25 kHz bath for 15 min, the above solution was transferred to a photoreactor and irradiated with 365 nm ultraviolet light for 4 h. After that, the light source was removed and 3.0 mL of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) was added to stir for another 2 h. The resulting solution was purified by dialysis against deionized water using a 1.0 kDa MWCO tubing for 48 h followed by lyophilization overnight to get the pLAPs as a white powder (yield: 70% \u003cem\u003ewt\u003c/em\u003e).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eThe pharmacokinetics study and biodistribution of pLAPs\u003c/h2\u003e \u003cp\u003eThe pharmacokinetics of pLAPs were examined in male rats (~\u0026thinsp;200 g). Briefly, SD rats were deprived of food overnight. The rats were administered intragastrically with LA (100 mg/kg) and pLAPs (100 mg/kg), respectively. The blood samples were collected from the eye ground vein into the heparinized tubes at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 60, 72 and 96 h, and followed by immediate centrifugation at 3000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min. After protein precipitation with acetonitrile, blood samples were added with 1 M NaOH, followed by heating 60 ℃ for 20 min to fully break the disulfide bonds. Finally, the resulting solution was adjusted the pH to 3\u0026thinsp;~\u0026thinsp;4 using HCl, and the dissociated LA was extracted with ethylacetate and tested by HPLC. The biodistribution of pLAPs was examined by gavage of Did-labeled pLAPs (Did@pLAPs) to the male C57BL/6 mice. Briefly, to a solvent of 4 mL of pLAPs (10 mg/mL) under stirring, 40 \u0026micro;L DMSO solution of Did (10 mg/mL) was added dropwise and followed by 4 h of ultrasound treatment. After that, the above solution was centrifuged for 10 min and purified by dialysis against deionized water using a 1.0 kDa MWCO tubing for 48 h to get the Did@pLAPs. The mice were sacrificed after 2, 6, 12, 24 and 36 h of oral gavage, and the spleen, lung, liver, adipose, skeletal muscle, pancreas, heart and kidney were isolated for imaging and quantitative analysis using an IVIS spectrum system with an excitation wavelength of 644 nm and an emission wavelength of 665 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIntracelluar LA content assay\u003c/h2\u003e \u003cp\u003e3T3-L1, L6 and L02 cells were seeded in 60 mm dishes for 12 h at a density of 2.0 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per dish, respectively. The cells were then incubated with LA (100 \u0026micro;g/mL), pLAPs (100 \u0026micro;g/mL) or [pLAPs\u0026thinsp;+\u0026thinsp;DEM] (pLAPs\u0026thinsp;=\u0026thinsp;100 \u0026micro;g/mL, DEM\u0026thinsp;=\u0026thinsp;1.0 mM). For the [pLAPs\u0026thinsp;+\u0026thinsp;DEM] group, DEM was used to deplete GSH for 24 h before the pLAPs treatment. After 1 h incubation, the medium containing LA or pLAPs was removed and the LA- or pLAPs-free medium was added. At predetermined time points, the cells were washed by phosphate buffer saline (PBS) for three times. Finally, the cells were collected and lysed, and the supernatants were collected and analyzed by HPLC.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eanti-diabetes study\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAfter one week of acclimation, 8-week-old male \u003cem\u003edb/db\u003c/em\u003e mice were randomly divided into 6 groups (6 mice in each group) as follows: \u003cem\u003edb/db\u003c/em\u003e model group, \u003cem\u003edb/db\u003c/em\u003e\u0026thinsp;+\u0026thinsp;pLAPs (25 mg/kg) group, \u003cem\u003edb/db\u003c/em\u003e\u0026thinsp;+\u0026thinsp;pLAPs (50 mg/kg) group, \u003cem\u003edb/db\u003c/em\u003e\u0026thinsp;+\u0026thinsp;pLAPs (100 mg/kg) group, \u003cem\u003edb/db\u003c/em\u003e\u0026thinsp;+\u0026thinsp;LA (100 mg/kg) group, \u003cem\u003edb/db\u003c/em\u003e\u0026thinsp;+\u0026thinsp;metformin (120 mg/kg, equivalent to clinical dosage) group, and age-matched heterozygotes \u003cem\u003edb/m\u003c/em\u003e mice were set as the negative control (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6). To evaluate the hypoglycemic effect of single administration, mice in the treatment groups received a single oral administration of different formulations, while mice in the negative control and model groups received an equal volume of saline. Blood glucose levels were measured over 72 h by tail clipping using a glucometer (Roche Accu-check, Mannheim, Germany). To evaluate the hypoglycemic effect of long-term administration, mice in the LA and metformin groups were administrated with different formulations once daily for 30 consecutive days, respectively. while mice in the pLAPs groups were administrated with indicated doses every 3 days. During the treatment period, fasting blood glucose levels were measured every 3 days. The oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed on the 27th and 29th day after pLAPs treatment, respectively. For OGTT test, mice were given oral glucose at a dose of 2 g/kg and blood glucose levels were monitored at 15, 30, 60 and 120 min after the initial glucose load, respectively. For ITT test, mice were fasted for 2 h, and then recombinant human insulin was injected intraperitoneally at a dose of 1 U/kg, and blood samples were collected for blood glucose measurements at 15, 30, 60 and 120 min, respectively. The area under the 120 min of glucose concentration curve (AUC\u003csub\u003e0\u0026thinsp;~\u0026thinsp;120 min\u003c/sub\u003e) was calculated. At the end of treatment, all mice were sacrificed with inhaled isoflurane at 2 h following the last dosing. The weights of whole body and epididymal fat were recorded. Blood samples were collected by eye bleeding, and centrifuged at 1500 \u0026times; \u003cem\u003eg\u003c/em\u003e for 15 min. The isolated serum was stored at -80\u0026deg;C for measuring the levels of insulin, pro-inflammatory cytokines and MDA, and the activities of CAT, GSH-Px and SOD. The liver, skeletal muscle and pancreas tissue were fixed by 4% polyformaldehyde, dehydrated, embedded in paraffin wax and cut serially into 5 \u0026micro;m sections for histopathological analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eanti-complications study\u003c/b\u003e\u003c/p\u003e \u003cp\u003e12-, 16- and 20-week-old male \u003cem\u003edb/db\u003c/em\u003e mice were used as models of DN, DC and DPN, respectively. The \u003cem\u003edb/db\u003c/em\u003e mice were randomly divided into 3 groups (6 mice in each group) as follows: \u003cem\u003edb/db\u003c/em\u003e model group, \u003cem\u003edb/db\u003c/em\u003e\u0026thinsp;+\u0026thinsp;pLAPs (100 mg/kg) group, \u003cem\u003edb/db\u003c/em\u003e\u0026thinsp;+\u0026thinsp;LA (100 mg/kg) group, and age-matched \u003cem\u003edb/m\u003c/em\u003e mice were set as the negative control (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6). LA group received LA (100 mg/kg, equivalent to clinical dosage for DPN) by oral administration once daily for 30 consecutive days, and the pLAPs groups were given at a dose of 100 mg/kg by gavage every 3 days. For the DPN mice, blood glucose was measured every 3 days, and behavioral tests were performed every one week during the treatment period. On the 30th day, all mice were sacrificed and serum was collected for measuring the level of MDA, and the activities of CAT, GSH-Px and SOD. Sciatic nerves were removed for measuring the levels of pro-inflammatory cytokines and MDA, the activities of CAT, GSH-Px and SOD, and the expression of inflammatory signaling molecules (p-IκBα, IκBα, p-NF-κB and NF-κB). In addition, the rest of tissue samples were fixed by 2.5% glutaraldehyde and cut serially into 70 nm sections for TEM analysis. For the DN and DC mice, blood glucose was measured every 3 days. All mice were sacrificed on day 30, the serum samples from DN mice were collected for measuring the levels of Cre and BUN, the urine samples from DN mice were collected for ALB detection. Moreover, the kidney and heart tissues were removed for measuring the levels of pro-inflammatory cytokines and MDA, the activities of CAT, GSH-Px and SOD, the expression of target gene and protein, and evaluating histopathological changes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of nerve conduction velocity\u003c/h2\u003e \u003cp\u003eSciatic nerve conduction velocity was detected by using orthodromic recording techniques\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Briefly, mice were anesthetized by intraperitoneal injection of ketamine/methylthiazide at dose of 10 mg/kg. An isolated pulse stimulator (Model 2100, A-M Systems, Everett, WA) was used to deliver the triggered square wave current pulses. The simultaneous electromyographies were recorded by two sterilized electrodes placed into the intrinsic foot muscles with a Grass Amplifier (Model P5, Grass Instruments, Quincy, MA). During the measurement process, the temperature of rectum was maintained at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u0026deg;C using a feedback controlled water bath. MCV and SCV were calculated according to a previous study\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTail-flick and hot plate tests\u003c/h2\u003e \u003cp\u003eAccording to the published methods, the thermal pain threshold was examined by tail-flick test (water immersion method) and hot plate test (IITC hot plate analgesia test)\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Briefly, for tail-flick test, mice were restrained in an open conical polypropylene tube and its tail was exposed. The tail of mice was immersed into a water bath at 52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u0026deg;C the time until the rodent flicks or removes its tail was recorded. For hot plate test, mice were placed within a transparent glass chamber and acclimated for at least 20 min. A thermal stimulation meter (IITC Model 39 Hot Plate Analgesia Meter, IITC Life Science, CA) was used with floor temperature at 55\u0026deg;C to record the latency of paw withdrawal in response to the radiant heat. Cut-off times of 10 s and 15 s were performed to avoid tissue damage caused by tail bending and hot plate testing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEchocardiography\u003c/h2\u003e \u003cp\u003eEchocardiography of mice was carried out in M-mode with a Vevo 2100 echocardiography system (VisualSonics Inc., Toronto, ON, Canada)\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. In brief, mice were anesthetized with isoflurane (2.5% for induction and 2.0% for maintenance). M-mode echocardiography was executed to record the left ventricular systolic and diastolic motion profile. LVIDs and LVIDd were acquired through measurement. Left ventricular end-diastolic volume (LVEDV) and end systolic volume (LVESV) were calculated using computer algorithms. In addition, the Vevo 2100 software (VisualSonics Inc.) was used to analyze echocardiographic parameters including LVEF and LVFS. All echocardiography detection and analysis processes were conducted by a researcher who was blind to the experimental treatments. LVEF and LVFS were calculated as LVEF (%) = (LVEDV - LDESV)/LVEDV \u0026times; 100 and LVFS (%) = (LVIDd - LVIDs)/LVIDd \u0026times; 100, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eNon-human primates study\u003c/h2\u003e \u003cp\u003eEighteen to twenty one-year-old spontaneous T2DM rhesus monkeys with DPN were selected, and then divided into two groups according to the results of neuroelectromyography testing. The monkeys in pLAPs group (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3) were administered with pLAPs (10 mg/kg) by nasogastric gavage once daily for 8 weeks, the monkeys (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2) received placebo were set as the control group. During the study period, clinical signs of monkeys were observed and recorded. Food intake was measured and calculated (food intake\u0026thinsp;=\u0026thinsp;feed - discard - surplus) every one week, and body weight was monitored using electronic platform scale (Mettler Toledo, Switzerland) every two weeks.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eElectrophysiological procedures\u003c/h2\u003e \u003cp\u003eNeuromuscular examination in rhesus monkeys was carried out using Haishen NDI-092 EMG/EP every four weeks. All rhesus monkeys were fasted overnight at 18:00 the day before testing. The ketamine (15 mg/kg) hydrochloride and dexmedetomidine (0.015 mg/kg) were injected intramuscularly. After anesthesia, the sensory conduction was measured orthodromically in the median, ulnar, superficial peroneal, and peroneal nerves using surface stimulating and recording electrodes. Motor conduction was measured in the tibial nerve recording over the soleus muscle, the peroneal nerve recording over the intrinsic toe extensor muscles, the median nerve recording over the thenar muscles, and the ulnar nerve recording over the hypothenar muscles. MCV and SCV were recorded by Haishen NDI-092 EMG/EP.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eHematological and plasma biochemical analysis\u003c/h2\u003e \u003cp\u003eBlood samples were collected into EDTA-2K anticoagulant vacuum tubes (13 \u0026times; 75 mm, 3.0 mL BD Vacutainer\u0026reg; plastic whole blood tube containing 3.4 mg of spray-coated K2EDTA), and the hematological parameters, including white blood cells count (WBC), red blood cells count (RBC), hematocrit (HCT), hemoglobin concentration (HBG), and total platelets count (PLT) were measured using Siemens ADVIA 2120i Hematology Systems. For plasma biochemical analysis, blood samples were centrifuged at 3000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min. The levels of FPG, fasting plasma insulin (FPI), Cre, and BUN were detected by Roche Cobas 6000 analyzer series C501/E601 (Roche Diagnostics GmbH). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin (T-BIL) were measured using Roche Cobas 6000 analyzer series C501 (Roche Diagnostics GmbH).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data was expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). For multiple comparisons, statistical significance was analyzed by one-way ANOVA. If inter-group differences were significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), the significance of the differences was determined by Tukey post hoc test. For data that showed a normal distribution and homogeneity of variance, differences between two groups were compared with a two-tailed Student\u0026rsquo;s t-test. All analyses of data were completed with SPSS version 16.0 software package (IBM, Chicago, IL, USA). \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eContributions\u003c/h2\u003e \u003cp\u003eS.Z. conceived the project. X.X. and X.L. developed the project, designed and performed the experiments and analysed the data. Y.Z., X.R., Y.C., Z.Y., C.L. and Y.W. provided technical input on this project. All authors analysed and interpreted the data. X.X., X.L. and S.Z. co-wrote the manuscript with the input from all other authors. X.R., C.L. and S.Z. supervised the work.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors are on a patent application filed by Sichuan University related to this work.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No. 22275129), the Sichuan Science and Technology Innovation Foundation (2021JDTD0015), and the Sichuan Science and Technology Program (2023NSFSC0319). We thank the Center of Testing and Analysis, Sichuan University for TEM measurements.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSuzuki K et al (2024) Genetic drivers of heterogeneity in type 2 diabetes pathophysiology. 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Diabetol 59:2435\u0026ndash;2447\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4577178/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4577178/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlthough hypoglycemic drugs with the function of alleviating complications such as GLP-1RA and SGLT2i have been used in clinic, these drugs are still far from meeting the treatment needs. Herein, we report an oral hypoglycemic agent for T2DM with the function of alleviating multi-complications including neuropathy by way of self-polymerizing dietary antioxidant lipoic acid (LA) into a nanodrug called poly-lipoic acid particles (pLAPs). The self-polymerization made the oral \u003cem\u003eT\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e and AUC\u003csub\u003e0 ~ 72 h\u003c/sub\u003e of LA up to 23.2 h and 3761.8 ± 55.9 h*µg/mL, ~ 46 times and ~ 23 times higher than that of LA monomer, respectively. As entering the cells, pLAPs were slowly degraded to LA in response to glutathione to prolong the intracellular retention time of LA from ~ 10 min to \u0026gt; 6 h. This prolongation achieved a continuous activation of the insulin signaling pathway, making a long-lasting and near-normal blood glucose level hypoglycemic effect come true. Thanks to the significant improvement of pharmacokinetics and intracellular retention time, pLAPs restored the oxidative stress and inflammation-related indicators to the normal control levels in the T2DM models with neuropathy and angiopathy, leading to the outstanding therapeutic effect on these complications. Importantly, the promising efficacy of pLAPs was confirmed in the model of spontaneous diabetic rhesus monkeys with neuropathy. Considering its excellent biosafety, the oral hypoglycemic drug with multi-complication alleviation holds clinical potential.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","manuscriptTitle":"An oral hypoglycemic agent for T2DM with the function of alleviating multi-complications","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-05 01:17:02","doi":"10.21203/rs.3.rs-4577178/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"86a3fb18-a7e7-400b-95fe-11758bad63ba","owner":[],"postedDate":"August 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":33429151,"name":"Physical sciences/Nanoscience and technology/Nanomedicine"},{"id":33429152,"name":"Biological sciences/Biotechnology/Biomaterials"}],"tags":[],"updatedAt":"2024-08-05T01:17:03+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-05 01:17:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4577178","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4577178","identity":"rs-4577178","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}