In-vitro and in-vivo cytotoxicity and efficacy evaluation of novel glycyl-glycine and alanyl-alanine conjugates of chitosan and trimethyl chitosan nano-particles as carriers for oral insulin delivery

Purpose

The aim of this research work was to explore the possibility of providing multifunctional oral insulin delivery system by conjugating several types of dipeptides on chitosan and trimethyl chitosan to be used as drug carriers.

Fourth Annual Conference of the American Society for Nanomedicine

The 20th conference of the Society on NeuroImmune Pharmacology will be held March 26-29, 2014. It features the latest in research examining the intersection of neuroscience, immunology and pharmacology, relevant for human health and disease. Particular emphases are placed on HIV and other infectious diseases, and abused substances, including illicit drugs and alcohol.     

Preparation and radiolabeling of a lyophilized (kit) formulation of DOTA-rituximab with 90Y and 111In for domestic radioimmunotherapy and radioscintigraphy of Non-Hodgkin’s Lymphoma

Background

On the basis of results of our previous investigations on ⁹⁰Y-DTPA-rituximab and in order to fulfil national demands to radioimmunoconjugates for radioscintigraphy and radioimmunotherapy of Non-Hodgkin’s Lymphoma (NHL), preparation and radiolabeling of a lyophilized formulation (kit) of DOTA-rituximab with ¹¹¹In and ⁹⁰Y was investigated.

Methods

¹¹¹In and ⁹⁰Y with high radiochemical and radionuclide purity were prepared by ¹¹²Cd (p,2n)¹¹¹In nuclear reaction and a locally developed ⁹⁰Sr/⁹⁰Y generator, respectively. DOTA-rituximab immunoconjugates were prepared by the reaction of solutions of p-SCN-Bz-DOTA and rituximab in carbonate buffer (pH = 9.5) and the number of DOTA per molecule of conjugates were determined by transchelation reaction between DOTA and arsenaso yttrium(III) complex. DOTA-rituximab immunoconjugates were labeled with ¹¹¹In and ⁹⁰Y and radioimmunoconjugates were checked for radiochemical purity by chromatography methods and for immunoreactivity by cell-binding assay using Raji cell line. The stability of radiolabeled conjugate with the approximate number of 7 DOTA molecules per one rituximab molecule which was prepared in moderate yield and showed moderate immunoreactivity, compared to two other prepared radioimmunoconjugates, was determined at different time intervals and against EDTA and human serum by chromatography methods and reducing SDS-polyacrylamide gel electrophoresis, respectively. The biodistribution of the selected radioimmunoconjugate in rats was determined by measurement of the radioactivity of different organs after sacrificing the animals by ether asphyxiation.

Results

The radioimmunoconjugate with approximate DOTA/rituximab molar ratio of 7 showed stability after 24 h at room temperature, after 96 h at 4°C, as the lyophilized formulation after six months storage and against EDTA and human serum. This radioimmunoconjugate had a biodistribution profile similar to that of ⁹⁰Y-ibritumomab, which is approved by FDA for radioimmunotherapy of NHL, and showed low brain and lung uptakes and low yttrium deposition into bone.

Conclusion

Findings of this study suggest that further investigations may result in a lyophilized (kit) formulation of DOTA-rituximab which could be easily radiolabeled with ⁹⁰Y and ¹¹¹In in order to be used for radioimmunotherapy and radioscintigraphy of B-cell lymphoma in Iran.     

Probing nanoparticle translocation across the permeable endothelium in experimental atherosclerosis

Therapeutic and diagnostic nanomaterials are being intensely studied for several diseases, including cancer and atherosclerosis. However, the exact mechanism by which nanomedicines accumulate at targeted sites remains a topic of investigation, especially in the context of atherosclerotic disease. Models to accurately predict transvascular permeation of nanomedicines are needed to aid in design optimization. Here we show that an endothelialized microchip with controllable permeability can be used to probe nanoparticle translocation across an endothelial cell layer. To validate our in vitro model, we studied nanoparticle translocation in an in vivo rabbit model of atherosclerosis using a variety of preclinical and clinical imaging methods. Our results reveal that the translocation of lipid–polymer hybrid nanoparticles across the atherosclerotic endothelium is dependent on microvascular permeability. These results were mimicked with our microfluidic chip, demonstrating the potential utility of the model system.Improving the design of nanomedicines is key for their success and ultimate clinical application (1). The accumulation of such therapeutic or diagnostic nanomaterials primarily relies on enhanced endothelial permeability of the microvasculature in diseased tissue (2). This holds true for a wide range of pathological conditions, including inflammation, atherosclerosis, and most notably, oncological disease (3–6). Although attributed to the enhanced permeability and retention (EPR) effect, the exact mechanism by which nanoparticles accumulate in tumors continues to be a topic of research (7, 8). The “leaky” vasculature of tumors, which facilitates the extravasation of nanoparticles from microvessels (9), is a heterogeneous phenomenon that varies between different tumor models and even more so in patients. Moreover, the exploitation of nanomedicines in other conditions with enhanced microvessel permeability has only recently begun to be studied in detail. For example, in the last 5 y, a small but increasing number of preclinical studies that apply nanoparticle therapy in atherosclerosis models has surfaced (10). Although several targeting mechanisms have been proposed (4), the exact mechanism by which nanoparticles accumulate in atherosclerotic plaques remains to be investigated, but is likely facilitated by highly permeable neovessels that penetrate into the plaque from the vasa vasorum (Fig. 1A), a network of microvessels that supplies the wall of larger vessels (11).Open in a separate windowFig. 1.Development of an endothelialized microfluidic device to probe nanoparticle translocation over a permeable microvessel. (A) Schematics of continuous normal capillaries surrounding the vessel wall as well as permeable capillaries that penetrate into the atherosclerotic plaque from the vasa vasorum. (B) Schematic of an endothelialized microfluidic device that consists of two-layer microfluidic channels that are separated by a porous membrane (3 μm pore) on which ECs are grown. (C) TEER was dynamically measured across the endothelial layer on the membrane between the upper and lower channels. (D) A well-established monolayer of the microvascular endothelium is formed at TEER ∼400 (Ω·cm²). (E) The monolayer becomes highly permeable when stimulated with the inflammatory mediator, TNF-α, as well as with shear stress, with disruption of intercellular junctional structures (i.e., adherens junctions) between ECs, as evidenced by patchy expression of VE–cadherin (green) in the image on the right versus the left. Blue depicts nuclei stained with DAPI. (Scale bar, 20 μm.) (F) FITC–albumin translocation through the endothelial monolayer increases when the chip is treated with TNF-α. (G) The chip with endothelium cultured in different culture media [base, +FBS, +growth factors (GFs)] for 6 h shows a decrease in TEER with increased FITC–albumin translocation. No cell indicates the membrane only. TEER was normalized to the level with no cells (membrane only). (H) Schematic and TEM image of PEGylated lipid-coated nanoparticles encapsulating PLGA-conjugated AuNCs and Cy5.5. The average size was 69.7 ± 14 nm, which was measured from TEM images. (Scale bar, 100 nm.) Details on labeling, synthesis, characterization, and large-scale production procedures can be found in Materials and Methods and Fig. S2.Advances in biomedical imaging allow the study of plaque-targeting nanoparticles in a dynamic fashion with exceptional detail (12, 13). Microchip technology has the potential to monitor nanoparticle behavior at the (sub)cellular level. Microfluidic chips in which endothelial cells (ECs) are grown in the channels can serve as unique in vitro test systems to study microvascular function and associated disorders (14–18). They allow the isolation of specific biological hallmarks relevant to nanoparticle accumulation, such as the leaky endothelium. In the current study, we validate the potential utility of our microchip technology to study nanoparticle translocation over the endothelium and combine this with in vivo and ex vivo multimodality imaging studies on a rabbit model to better understand nanoparticle targeting of atherosclerotic plaques.     

Engineered nanomedicine for myeloma and bone microenvironment targeting

Bone is a favorable microenvironment for tumor growth and a frequent destination for metastatic cancer cells. Targeting cancers within the bone marrow remains a crucial oncologic challenge due to issues of drug availability and microenvironment-induced resistance. Herein, we engineered bone-homing polymeric nanoparticles (NPs) for spatiotemporally controlled delivery of therapeutics to bone, which diminish off-target effects and increase local drug concentrations. The NPs consist of poly(d,l-lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG), and bisphosphonate (or alendronate, a targeting ligand). The engineered NPs were formulated by blending varying ratios of the synthesized polymers: PLGA-b-PEG and alendronate-conjugated polymer PLGA-b-PEG-Ald, which ensured long circulation and targeting capabilities, respectively. The bone-binding ability of Ald-PEG-PLGA NPs was investigated by hydroxyapatite binding assays and ex vivo imaging of adherence to bone fragments. In vivo biodistribution of fluorescently labeled NPs showed higher retention, accumulation, and bone homing of targeted Ald-PEG-PLGA NPs, compared with nontargeted PEG-PLGA NPs. A library of bortezomib-loaded NPs (bone-targeted Ald-Bort-NPs and nontargeted Bort-NPs) were developed and screened for optimal physiochemical properties, drug loading, and release profiles. Ald-Bort-NPs were tested for efficacy in mouse models of multiple myeloma (MM). Results demonstrated significantly enhanced survival and decreased tumor burden in mice pretreated with Ald-Bort-NPs versus Ald-Empty-NPs (no drug) or the free drug. We also observed that bortezomib, as a pretreatment regimen, modified the bone microenvironment and enhanced bone strength and volume. Our findings suggest that NP-based anticancer therapies with bone-targeting specificity comprise a clinically relevant method of drug delivery that can inhibit tumor progression in MM.The incidence of bone metastasis is common in 60–80% of cancer patients (1). During bone metastasis, cancer cells induce a sequence of changes in the microenvironment such as secreting cytokines to increase the activity of osteoclasts via the parathyroid hormone-related protein (PTHrP), receptor activator of nuclear factor-κB ligand (RANKL), and interleukin-6 (IL-6), resulting in increased bone resorption and secretion of growth factors from the bone matrix (2). This creates a “vicious cycle” of bone metastasis, where bone marrow becomes packed with cancer cells that develop resistance to conventional chemotherapy, and leads to devastating consequences of bone fractures, pain, hypercalcaemia, and spinal cord and nerve compression syndromes (2, 3). Multiple myeloma (MM) is a plasma cell cancer that proliferates primarily in bone marrow and causes osteolytic lesions (1). Antiresorption agents, such as bisphosphonates, may alleviate bone pain, but they are ineffective at inducing bone healing or osteogenesis in MM patients (4).Bortezomib is a proteasome inhibitor that has shown marked antitumor effects in patients with MM. Proteasome inhibitors, such as bortezomib, are also effective at increasing bone formation, both preclinically and clinically (5–9). However, the major drawback of bortezomib use in early stages of MM development is its toxicity, specifically, peripheral neuropathy (5). Therefore, we aimed to develop a method to deliver bortezomib with decreased off-target side effects by using bone-specific, bortezomib-loaded nanoparticles (NPs). The NP system was based on biodegradable, biocompatible, and Food and Drug Administration (FDA)-approved components, which are both clinically and translationally relevant. NPs derived from poly(d,l-lactic-co-glycolic acid) (PLGA), a controlled release polymer system, are an excellent choice because their safety in the clinic is well established (10, 11). Polyethylene glycol (PEG)-functionalized PLGA NPs are especially desirable as PEGylated polymeric NPs have significantly reduced systemic clearance compared with similar particles without PEG (12, 13). A number of FDA-approved drugs in clinical practice use PEG for improved pharmaceutical properties such as enhanced circulation in vivo (12, 13). To target NPs to bone [rich in the mineral hydroxyapatite (HA)], the calcium ion-chelating molecules of bisphosphonates represent a promising class of ligands (14). Bisphosphonates, upon systemic administration, are found to deposit in bone tissue, preferentially at the high bone turnover sites, such as the metastatic bone lesions, with minimal nonspecific accumulation (14) and were used herein to deliver NPs to the bone.A few systems explored for MM treatment have been tested in vitro including the following: (i) snake venom and silica NPs (15); (ii) thymoquinone and PLGA-based particles (16); (iii) curcumin and poly(oxyethylene) cholesteryl ether (PEG-Chol) NPs (17), polyethylenimine-based NPs for RNAi in MM (18), paclitaxel-Fe₃O₄ NPs (19), and liposomes (20). However, none of the above-mentioned systems have aimed to manipulate the bone marrow microenvironment rather than the myeloma cells directly (21). To date, there are no reports of using bone-targeted, controlled release, polymeric NPs with stealth properties for MM therapy. In this study, we designed NPs bearing three main components: (i) a targeting element that can selectively bind to bone mineral; (ii) a layer of stealth (PEG) to minimize immune recognition and enhance circulation; and (iii) a biodegradable polymeric material, forming an inner core, that can deliver therapeutics and/or diagnostics in a controlled manner. In this study, the physicochemical properties of a range of NPs was investigated (including NP size, charge, targeting ligand density, drug loading, and drug release kinetics) and an optimal formulation with ideal properties and maximal drug encapsulation was used for in vivo efficacy studies. We fine-tuned the NP targeting ligand density to optimize its bone-binding ability and further investigated its application for targeting myeloma in the bone microenvironment. We believe our NP system has the potential to increase drug availability by improving pharmacokinetics and biodistribution that can provide bone microenvironment specificity, which may increase the therapeutic window and most certainly decrease the off-target effects (12, 13).     

A commonly carried genetic variant in the delta opioid receptor gene,OPRD1, is associated with smaller regional brain volumes: Replication in elderly and young populations

Delta opioid receptors are implicated in a variety of psychiatric and neurological disorders. These receptors play a key role in the reinforcing properties of drugs of abuse, and polymorphisms in OPRD1 (the gene encoding delta opioid receptors) are associated with drug addiction. Delta opioid receptors are also involved in protecting neurons against hypoxic and ischemic stress. Here, we first examined a large sample of 738 elderly participants with neuroimaging and genetic data from the Alzheimer's Disease Neuroimaging Initiative. We hypothesized that common variants in OPRD1 would be associated with differences in brain structure, particularly in regions relevant to addictive and neurodegenerative disorders. One very common variant (rs678849) predicted differences in regional brain volumes. We replicated the association of this single‐nucleotide polymorphism with regional tissue volumes in a large sample of young participants in the Queensland Twin Imaging study. Although the same allele was associated with reduced volumes in both cohorts, the brain regions affected differed between the two samples. In healthy elderly, exploratory analyses suggested that the genotype associated with reduced brain volumes in both cohorts may also predict cerebrospinal fluid levels of neurodegenerative biomarkers, but this requires confirmation. If opiate receptor genetic variants are related to individual differences in brain structure, genotyping of these variants may be helpful when designing clinical trials targeting delta opioid receptors to treat neurological disorders. Hum Brain Mapp 35:1226–1236, 2014. © 2013 Wiley Periodicals, Inc.     

Reinfection risk of novel coronavirus (COVID-19): A systematic ‎review of current evidence

BACKGROUNDThere is recently a concern regarding the reinfection and reactivation of previously reCoVered coronavirus disease 2019 (CoVID-19) patients.AIMTo summarize the recent findings and reports of CoVID-19 reinfection in patients previously reCoVered from the disease.METHODSThis study was a systematic review of current evidence conducted in August 2020. The authors studied the probable reinfection risk of novel coronavirus (CoVID-19). We performed a systematic search using the keywords in online databases. The investigation adheres to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist to ensure the reliability and validity of this study and results.RESULTSWe reviewed 31 studies. Eight studies described reCoVered patients with reinfection. Only one study reported reinfected patients who died. In 26 studies, there was no information about the status of the patients. Several studies indicated that reinfection is not probable and that post-infection immunity is at least temporary and short.CONCLUSIONBased on our review, we concluded that a positive polymerase chain reaction retest could be due to several reasons and should not always be considered as reinfection or reactivation of the disease. Most relevant studies in positive retest patients have shown relative and probably temporary immunity after the reCoVery of the disease.     

Muscle strength and its relationship with skeletal muscle mass indices as determined by segmental bio-impedance analysis

Purpose

Despite increasing interest in bio-impedance analysis (BIA) for estimation of segmental skeletal muscle mass (SMM), published results have not been entirely convincing. Furthermore, a better understanding of the relationship between muscle strength and SMM will be useful in interpreting outcomes of physical/training interventions particularly in groups with diverse body sizes (e.g. men vs women). This study aimed to measure SMM in the upper body (upper extremity and torso), to determine its correlation with muscle strength and to examine the effects of gender on muscle strength–muscle mass relationship.

Methods

Segmental (upper extremity and torso) SMM and muscle strength in five distinct shoulder planes (forward flexion, abduction in scapular plane, abduction in coronal plane, internal and external rotation) were measured in 45 healthy participants (22 males, 23 females) with mean age 30.3 years. Statistical analysis included independent t tests, Pearson correlation, and multiple regression analysis.

Results

Men and women differed significantly in body mass (BMI: 25.9 ± 4.3 vs 23 ± 3.6) and SMM (p < 0.01). A strong relationship correlation was found between the five shoulder strength measurements and upper extremity SMM (r = 0.66–0.80, p < 0.01), which was not affected by gender. There was a significant gender difference (p < 0.01) in absolute shoulder strength, but not after normalisation to the SMM.

Conclusion

BIA-estimated SMM of upper extremity and torso was highly correlated with upper extremity (shoulder) strength independent of gender. SMM may, therefore, be useful for the normalisation of muscle strength allowing size-independent comparisons of muscle strength in individuals with diverse physical characteristics.     

Antioxidant effects of oleuropein versus oxidative stress induced by ethanol in the rat intestine

Purified oleuropein from olive leaf extract indicated antioxidant properties in our previous reports. The recent study demonstrated that the lesions of absolute ethanol could be attributed to the increasing reactive oxygen species, subsequently lipid peroxidation and gastric ulcers. Therefore, the antioxidant effects of oleuropein as a natural antioxidant agent on intestine mucosal damages induced by absolute ethanol were investigated in the present study. The 42 adult male rats were divided into four (control, oleuropein, ethanol, and oleuropein plus ethanol) groups. Oleuropein at a dosage of 12 mg/kg was used for 10 consecutive days in oleuropein and oleuropein plus ethanol groups, thereafter absolute ethanol (once, 1 ml/rat) was administrated orally in ethanol and oleuropein plus ethanol groups at fasting state by gavage. The duodena of rats were removed for histopathology and antioxidant assay. Histological evidence of severe mucosal damages were confirmed by histopathology findings in ethanol group and prevented in oleuropein plus ethanol group. In this setting, glutathione peroxidase and catalase activities were significantly higher in oleuropein and oleuropein plus ethanol groups than the ethanol-treated rats. In contrast, thiobarbituric acid reactive substances concentration (as a lipid peroxidation marker) significantly increased in ethanol-treated rats when compared to the other groups. Our results suggest that olive leaf extract (containing oleuropein) exerts a potent antioxidant agent against oxidative stress. These findings allow us to exploit the advantages of oleuropein treatment in various diseases induced by oxidative stress in humans.