An investigation on nanocarrier-mediated glucose-responsive delivery of antidiabetic lipophilic molecules
Creators: name: Sarkar, Sanjib
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 321405
subject: Pharmaceutical sciences

description: Diabetes mellitus (DM) is a metabolic health burden recognized by high blood glucose levels. International Diabetes Federation reported that the number of patients with DM was approximately 537 million (20−79 years) across the world in 2021, and the predicted number of individuals with DM will be 784 million by 2045. Type 2 diabetes mellitus (T2DM) is defined by impaired glucose metabolism resulting from reduced insulin production and/or decreased insulin sensitivity. T2DM represents approximately 90% of all types of DM. Uncontrolled T2DM can lead to a range of health disorders, including nephropathy, neuropathy, and retinopathy. Diabetic retinopathy (DR) is an inflammatory disease of blood vessels of the eye, the retina in particular and can cause irreversible blindness in patients with DM. 

Approximately half of all patients with DM cannot attain the required glycaemic level by using conventional medicines. An alternative strategy is required to overcome this crucial issue. Glucose-responsive delivery of antidiabetic molecules is a promising strategy for maintaining normoglycemia and reducing the risk of hypoglycaemia associated with currently available therapeutic agents. Three key types of glucose-responsive delivery systems are used in antidiabetic drug delivery. These include glucose oxidase (GOx)-based systems, glucose-binding proteins (GBP)-based systems, and phenylboronic acid (PBA)-based systems. A significant portion of the antidiabetic drugs are lipophilic molecules such as sulphonylureas, e.g., Glimepiride, Glipizide, and Glyburide; thiazolidinediones e.g., Rosiglitazone and Pioglitazone; inhibitors of dipeptidyl peptidase-4 (DPP-4), e.g., Alogliptin and Linagliptin, and inhibitors of sodium-glucose cotransporter 2 (SGLT2), e.g., Canagliflozin and Empagliflozin. Various lipid-soluble micronutrients, like vitamin K (VK) and vitamin D (VD) have also shown studies on glucose-sensitive delivery of lipophilic antidiabetic molecules are limited.  

Herein, we examine the glucose-responsive delivery of VK and VD by using dextran-capped mesoporous silica nanoparticles (MSN) functionalized with 3-carboxyphenylboronic acid (CPBA) to alleviate hyperglycemia and diabetic retinopathy. Previous studies have found a link between VD and VK deficiencies and the aetiology of diabetes and its complications. Therefore, VD and VK are used as model antidiabetic therapies in this study.

Amine functionalized-MSN were prepared by tetraethyl orthosilicate (TEOS) and (3-aminopropyl)trimethoxysilane (APTES) by using a template N-cetyl-N,N,N-trimethylammonium bromide (CTAB). CPBA functionalization was performed on the surface of MSN (MSN-CPBA). Lipid-soluble VK and VD were loaded in channel-like pores of MSN-CPBA and the pores were capped with dextran.  The CPBA-functionalization, physical nature of nanocarriers, shape, internal and surface morphology, size, pore diameter and volume, and nature of surface charge were examined using Alizarin red S test, field emission scanning electron microscopy (FESEM), high-resolution transmission electron microscopy (HRTEM), Fourier transformed infrared (FTIR) spectroscopy, nitrogen adsorption/desorption isotherm, hydrodynamic size distribution and surface charge analyser and thermogravimetric analyser, respectively. The loading and release efficiencies of lipophilic molecules were measured by using reverse-phase (RP)-HPLC. Transmission electron microscopic image analysis revealed an appropriate particle size of 238.6 ± 30.9 nm which was consistent with their hydrodynamic size. The surface charge of VK/VD-loaded dextran-gated MSN-CPBA (MSN-CPBA-VK/VD-Dex) was negative. The VK or VD was loaded inside the pores of MSN-CPBA by physical adsorption, and the loading capacity of MSN-CPBA was 206.7 and 263.6 µg/mg (w/w) in the case of VK and VD, respectively.

The pores of the nanoparticles were capped using dextran via binding with CPBA.  Glucose is a 1,2 cis-diol molecule that has a very high affinity towards carboxyphenylboronic acid compared to dextran which triggers the untethering of dextran from MSN allowing the pores to open and release VK or VD. At 24 h, the release of VK was 10.21 and 18.62 µg/mL (∼49.1% and ∼90.2%) in a phosphate-buffered saline (PBS) in exposure to 11 mM and 25 mM of glucose concentration. In contrast, VK release was 1.5 and 0.99 µg/mL in myocytes and hepatocytes, respectively, at the same time point and 25 mM glucose concentration. In cell-free systems, VD release was found to be 10.97 and 19.81 µg/mL (∼16.79% and ∼75 %) against exposure to 11- and 25-mM glucose, respectively. The VD releases were 2.12 and 3.48 μg/mL in myocytes against 11 and 25 mM of glucose exposure, respectively. The result showed that the VD release was faster compared to the VK release.  The glucose-responsive delivery of VK was examined in high-fat diet-induced diabetic mice. The levels of Gla-proteins (VK biomarkers) were significantly increased in skeletal muscles and liver of mice with diabetes supplemented with MSN-CPBA-VK-Dex in comparison with mice in the control group, demonstrating the glucose-sensitive release of VK in mice.

VK/VD loaded MSN-CPBA were internalized in two major cell types, which are involved in glucose metabolism, myocytes (C2C12) and hepatocytes (CC1), by using a confocal microscope. The green/red fluorescence was observed inside the cells suggesting the cellular uptake of fluorescent isothiocyanate/ rhodamine B isothiocyanate (FITC/RITC)-labelled MSN-CPBA. The nanoparticle internalizations in myocytes and hepatocytes were confirmed by a Flow cytometric study. Approximately 97% of myocytes and 90 % of hepatocytes engulfed MSN-CPBA-VK-Dex at 2 h. MSN-CPBA-VK/VD-Dex did not show cytotoxic effects in mammalian myocytes, hepatocytes and human retinal epithelial cells cultured in low and high glucose-treated culture medium.

The effect of MSN-CPBA-VK/VD-Dex on glucose uptake was significantly improved in the high glucose (HG)-treated myocytes. The improvement of glucose uptake was mediated by improving the GLUT4 expression in myocytes. The GLUT4 expression was increased by 1.57-fold in HG-induced myocytes supplemented with MSN-CPBA-VK/VD-Dex compared to that in HG-induced myocytes in the control. The in vivo study demonstrated that blood glucose level is markedly decreased in high-fat diet-induced diabetic mice treated with MSN-CPBA-VK-Dex compared to the control. However, VK alone at the same dose present in the nanoparticles did not show a significant hypoglycemic effect in mice with diabetes. The blood glucose levels were 6.5 ± 0.8 and 13.5 ± 4.4 mM in mice with diabetes supplemented with MSN-CPBA-VK-Dex and VK alone, respectively. Histological studies demonstrated that muscle tissues of mice with diabetes showed several complications including muscular focal necrosis, infiltration of lymphocytes, atrophic muscle fibers, and reduced thickness of muscle fibers. The lesions of muscles were mitigated in diabetic mice treated with MSN-CPBA-VK-Dex; however, VK alone could not enable the restoration of pathophysiological alterations. Likewise, liver injuries were alleviated in mice with diabetes treated with MSN-CPBA-VK-Dex, but VK alone was not able to mitigate the pathophysiological changes associated with diabetes, indicating elevated bioavailability of VK in MSN-CPBA-VK-Dex-treated mice with diabetes.

We also investigated the glucose-responsive delivery of VD by using MSN-CPBA to alleviate diabetic retinopathy (DR) in the human retinal pigmented epithelial cell culture model. In patients with diabetes, mitochondrial ROS production is dramatically raised in the retinal epithelium resulting in the destruction of epithelial cells and the occurrence of DR.  Treatment with MSN-CPBA-VD-Dex showed remarkable efficiency in reducing oxidative stress in the high glucose (25 mM)-treated adult retinal pigmented epithelial (ARPE-19) cells. In addition, VD-loaded nanoparticles were able to reduce the secretion of proinflammatory, cytokine interleukin 6 (IL-6) in retinal cells exposed to HG. Thus, MSN-CPBA-VD-Dex showed promising results in alleviating diabetic retinopathy by managing oxidative stress and inflammation associated with cellular exposure to high glucose levels.

In conclusion, the MSN-CPBA-based delivery system was able to deliver lipid-soluble molecules, VK and VD, in a glucose-sensitive manner both in vitro and in vivo. MSN-CPBA-VK/VD-Dex demonstrated its hypoglycemic effect in HG-treated myocytes as well as in HFD-induced diabetic mice. In addition, the study revealed MSN-CPBA-VD-Dex was able to reduce oxidative stress via controlling ROS production and secretion of proinflammatory cytokine IL-6 in retinal epithelial cells exposed to HG. Thus, MSN-CPBA-VD-Dex was capable of controlling DR.