Bioinspired and self-restorable alginate-tyramine hydrogels with plasma reinforcement for arthritis treatment.

Long-standing administration of disease-modifying antirheumatic drugs confirms their clinical value for managing rheumatoid arthritis (RA). Nevertheless, there are emergent worries over unwanted adverse risks of systemic drug administration. Hence, a novel strategy that can be used in a drug-free manner while diminishing side effects is immediately needed, but challenges persist in the therapy for RA. To this end, herein we conjugated tyramine (TYR) with alginate (ALG) to form ALG-TYR and then treated it for 5 min with oxygen plasma (ALG-TYR + P/5 min). It was shown that the ALG-TYR + P/5 min hydrogel exhibited favorable viscoelastic, morphological, mechanical, biocompatible, and cellular heat-shock protein amplification behaviors. A thorough physical and structural analysis was conducted on the ALG-TYR + P/5 min hydrogel, revealing favorable physical characteristics and uniform porous structural features within the hydrogel. Moreover, ALG-TYR + P/5 min not only effectively inhibited inflammation of RA but also potentially regulated lesion immunity. Once ALG-TYR + P/5 min was intra-articularly administered to joints of rats with zymosan-induced arthritis, we observed that ALG-TYR + P/5 min could ameliorate syndromes of RA joint. This bioinspired and self-restorable ALG-TYR + P/5 min hydrogel can thus serve as a promising system to provide prospective outcomes to potentiate RA therapy.

Calcium peroxide aids tyramine-alginate gel to crosslink with tyrosinase for efficient cartilage repair.

The innate cartilage extracellular matrix is avascular and plays a vital role in innate chondrocytes. Recapping the crucial components of the extracellular matrix in engineered organs via polymeric gels and bioinspired approaches is promising for improving the regenerative aptitude of encapsulated cartilage/chondrocytes. Conventional gel formation techniques for polymeric materials rely on employing oxidative crosslinking, which is constrained in this avascular environment. Further, poor mechanical properties limit the practical applications of polymeric gels and reduce their therapeutic efficacy. Herein, the purpose of this study was to develop a bioadhesive gel possessing dual crosslinking for engineering cartilage. Tyramine (TYR) was first chemically conjugated to the alginate (ALG) backbone to form an ALG-TYR precursor, followed by the addition of calcium peroxide (CaO2); calcium ions of CaO2 physically crosslink with ALG, and oxygen atoms of CaO2 chemically crosslink TYR with tyrosinase, thus enabling dual/enhanced crosslinking and possessing injectability. The ALG-TYR/tyrosinase/CaO2 gel system was chemically, mechanically, cellularly, and microscopically characterized. The gel system developed herein was biocompatible and showed augmented mechanical strength. The results showed, for the first time, that CaO2 supplementation preserved cell viability and enhanced the crosslinking ability, bioadhesion, mechanical strength, chondrogenesis, and stability for cartilage regeneration.

Add-on repetitive transcranial magnetic stimulation in patients with opioid use disorder undergoing methadone maintenance therapy.

Background: Repetitive transcranial magnetic stimulation (rTMS) shows potential therapeutic effects for individuals with addiction, but few studies have examined individuals with opioid use disorder (OUD).Objectives: We conducted an add-on double-blinded, sham-controlled rTMS feasibility pilot trial to examine OUD participants undergoing methadone maintenance therapy (MMT). The current report focused on the effects of rTMS on (1) craving and heroin use behavior and (2) depression, impulsivity, and attention.Methods: Active or sham rTMS treatment was applied to the left dorsolateral prefrontal cortex (DLPFC) over a total of 11 sessions in 4 weeks (15-Hz frequency, 4 seconds per train, intertrain interval of 26 seconds, 40 trains per session) in OUD participants (ClinicalTrials.gov registration number: NCT03229642). Craving, heroin use severity, urine morphine tests, the Hamilton Depression Rating Scale (HDRS), the Barratt Impulsiveness Scale-11 (BIS-11), and the Continuous Performance Tests (CPTs) were measured.Results: Twenty-two OUD participants were enrolled, of which eleven (8 males) were undergoing active rTMS and nine (8 males) were in the sham rTMS group. After 12 weeks of follow-up, the active rTMS group did not show significantly greater improvements than the sham group with respect to craving, heroin use, or urine morphine test results. However, HDRS scores, BIS-11 attentional subscales, and CPTs commission T-scores (C-TS) were significantly lower in the active rTMS group (P = .003, 0.04, and 0.02, respectively) than in the sham group.Conclusion: Add-on rTMS did not appear to improve heroin use behavior but may have benefitted depressive symptoms, impulse control and attention in OUD participants undergoing MMT.

ALDH2 Gene: Its Effects on the Neuropsychological Functions in Patients with Opioid Use Disorder Undergoing Methadone Maintenance Treatment.

OBJECTIVE: Patients with opioid use disorder (OUD) have impaired attention, inhibition control, and memory function. The aldehyde dehydrogenase 2(ALDH2) gene has been associated with OUD and ALDH2 gene polymorphisms may affect aldehyde metabolism and cognitive function in other substance use disorder. Therefore, we aimed to investigate whether ALDH2 genotypes have significant effects on neuropsychological functions in OUD patients undergoing methadone maintenance therapy (MMT). METHODS: OUD patients undergoing MMT were investigated and followed-up for 12 weeks. ALDH2 gene polymorphisms were genotyped. Connors' Continuous Performance Test (CPT) and the Wechsler Memory Scale-Revised (WMS-R) were administered at baseline and after 12 weeks of MMT. Multivariate linear regressions and generalized estimating equations (GEEs) were used to examine the correlation between the ALDH2 genotypes and performance on the CPTs and WMS-R. RESULTS: We enrolled 86 patients at baseline; 61 patients completed the end-of-study assessments. The GEE analysis showed that, after the 12 weeks of MMT, OUD patients with the ALDH2 *1/*2＋*2/*2 (ALDH2 inactive) genotypes had significantly higher commission error T-scores (p= 0.03), significantly lower hit reaction time T-scores (p= 0.04), and significantly lower WMS-R visual memory index scores (p= 0.03) than did patients with the ALDH2 1*/*1 (ALDH2 active) genotype. CONCLUSION: OUD patients with the ALDH2 inactive genotypes performed worse in cognitive domains of attention, impulse control, and memory than did those with the ALDH2 active genotype. We conclude that the ALDH2 gene is important in OUD and is associated with neuropsychological performance after MMT.

Effects of the nanostructure of dendrimer/DNA complexes on their endocytosis and gene expression.

Cationic dendrimers constitute a potential nonviral vector for gene therapy due to their ability of forming electrostatic complexes with DNA (dendriplexes). However, the supramolecular structure of dendriplexes and its impact on the cellular uptake and gene transfection remain largely unknown. Using synchrotron small angle X-ray scattering, here we show that DNA in complexes with poly(amidoamine) (PAMAM) G4 dendrimers exhibited three distinct packaging states modulated by the degree of their protonation (dp). Our structure characterization suggests that the nanostructure of DNA in dendriplexes transformed from square-packed straightened chains (dp/0.1) to hexagonally-packed superhelices (dp/0.3) and eventually to a beads-on-string configuration (dp/0.6 and dp/0.9). The transfection efficiency in HT1080 cells significantly enhanced when the dp value was increased from 0.1 to 0.3. This enhancement was due to a higher positive surface charge of dendriplexes formed at higher dp, which facilitated adherence of test dendriplexes to the negatively charged cell membranes for the subsequent endocytosis. Although the surface charge of dendriplexes still increased accordingly, further increase of the dendrimer dp value to 0.9 reduced the transfection efficiency. This unexpected suppression of transfection may be attributed to the wrapping of DNA around dendrimers that frustrates the interaction between dendrimer and cholesterol in the membrane raft via the caveola-mediated endocytosis. These results can be used for the rational design of dendrimer-based gene delivery devices.

Multifunctional core-shell polymeric nanoparticles for transdermal DNA delivery and epidermal Langerhans cells tracking.

Skin is a highly immune-reactive tissue containing abundant antigen-presenting cells such as Langerhans cells (LCs), and thus is a favorable site for DNA immunization. This study developed a multifunctional core-shell nanoparticle system, which can be delivered transdermally into the epidermis via a gene gun, for use as a DNA carrier. The developed nanoparticles comprised a hydrophobic PLGA core and a positively-charged glycol chitosan (GC) shell. The core of the nanoparticles was used to load fluorescent quantum dots (QDs) for ultrasensitive detection of Langerhans cell migration following transdermal delivery, while a reporter gene was electrostatically adsorbed onto the GC shell layer of the nanoparticles. Results of fluorescence spectrophotometry, transmission electron microscopy, energy dispersive X-ray analysis, and X-ray diffraction measurement confirmed that the prepared nanoparticles had a core-shell structure with QDs in their core area. The surface charge of nanoparticles depended strongly on pH environment, enabling the intracellular release of the loaded DNA via a pH-mediated mechanism. Using a mouse model, this study demonstrated that bombardment of nanoparticles transfected DNA directly into LCs present in the epidermis; the transfected LCs then migrated and expressed the encoded gene products in the skin draining lymph nodes. These observation results suggest that the developed nanoparticle system is suitable for monitoring and fine-tuning important functional aspects of the immune system, in conjunction with the loaded fluorescence, and thus has potential for use in immunotherapy and vaccine development.

The characteristics, biodistribution, magnetic resonance imaging and biodegradability of superparamagnetic core-shell nanoparticles.

An efficient contrast agent for magnetic resonance imaging (MRI) is essential to enhance the detection and characterization of lesions within the body. In this study, we described the development of biodegradable nanoparticles with a core-shell structure to formulate superparamagnetic iron oxide (CSNP-SPIO) for MRI. The developed nanoparticles were composed of a hydrophobic PLGA core and a positively-charged glycol chitosan shell. The results obtained by transmission electron microscopy, energy dispersive X-ray analysis, electron energy loss spectroscopy, and X-ray diffraction measurement confirmed that the prepared nanoparticles had a core-shell structure with SPIO in their core area. No aggregation of nanoparticles was observed during storage in water, as a result of the electrostatic repulsion between the positively-charged nanoparticles. The magnetic properties of nanoparticles were examined by a vibrating sample magnetometer and a superconducting quantum interference device; the results showed that the superparamagnetism of SPIO was preserved after the CSNP-SPIO formulation. In tracking their cellular internalization pathway, we found that CSNP-SPIO accumulated in lysosomes. In the biodistribution study, a high level of radioactivity was observed in the liver shortly after administration of the (99m)Tc-labeled CSNP-SPIO intravenously. Once taken up by the liver cells, the liver turned dark on T(2)* images. Following cellular internalization, CSNP-SPIO were broken down gradually; therefore, with time increasing, a significant decrease in the darkness of the liver on T(2)* images was found. The aforementioned results indicate that the developed CSNP-SPIO can serve as an efficient MRI contrast agent and could be degraded after serving their imaging function.

Effects of incorporation of poly(gamma-glutamic acid) in chitosan/DNA complex nanoparticles on cellular uptake and transfection efficiency.

Chitosan (CS)/DNA complex nanoparticles (NPs) have been considered as a vector for gene delivery. Although advantageous for DNA packing and protection, CS-based complexes may lead to difficulties in DNA release once arriving at the site of action. In this study, an approach through modifying their internal structure by incorporating a negatively charged poly(gamma-glutamic acid) (gamma-PGA) in CS/DNA complexes (CS/DNA/gamma-PGA NPs) is reported. The analysis of small angle X-ray scattering results revealed that DNA and gamma-PGA formed complexes with CS separately to yield two types of domains, leading to the formation of "compounded NPs". With this internal structure, the compounded NPs might disintegrate into a number of even smaller sub-particles after cellular internalization, thus improving the dissociation capacity of CS and DNA. Consequently, after incorporating gamma-PGA in CS/DNA complexes, a significant increase in their transfection efficiency was found. Interestingly, in addition to improving the release of DNA intracellularly, the incorporation of gamma-PGA in CS/DNA complexes significantly enhanced their cellular uptake. We further demonstrated that besides a non-specific charged-mediated binding to cell membranes, there were specific trypsin-cleavable proteins involved in the internalization of CS/DNA/gamma-PGA NPs. The aforementioned results indicated that gamma-PGA played multiple important roles in enhancing the cellular uptake and transfection efficiency of CS/DNA/gamma-PGA NPs.

The use of biodegradable polymeric nanoparticles in combination with a low-pressure gene gun for transdermal DNA delivery.

Gold particles have been used as a carrier for transdermal gene delivery, which may cause adverse side effects when accumulated. In this study, biodegradable nanoparticles, composed of chitosan (CS) and poly-gamma-glutamic acid (gamma-PGA), were prepared by an ionic-gelation method for transdermal DNA delivery (CS/gamma-PGA/DNA) using a low-pressure gene gun. The conventional CS/DNA without the incorporation of gamma-PGA was used as a control. Small-angle X-ray scattering (SAXS) was used to examine the internal structures of test nanoparticles, while identification of their constituents was conducted by Fourier transformed infrared (FT-IR) spectroscopy. The CS/gamma-PGA/DNA were spherical in shape with a relatively homogeneous size distribution. In contrast, CS/DNA had a heterogeneous size distribution with a donut, rod or pretzel shape. Both test nanoparticles were able to effectively retain the encapsulated DNA and protect it from nuclease degradation. As compared with CS/DNA, CS/gamma-PGA/DNA improved their penetration depth into the mouse skin and enhanced gene expression. These observations may be attributed to the fact that CS/gamma-PGA/DNA were more compact in their internal structures and had a greater density than their CS/DNA counterparts, thus having a larger momentum to penetrate into the skin barrier. The results revealed that CS/gamma-PGA/DNA may substitute gold particles as a DNA carrier for transdermal gene delivery.

Increased oxidative damage and mitochondrial abnormalities in the peripheral blood of Huntington's disease patients.

Increased oxidative stress and mitochondrial abnormalities contribute to neuronal dysfunction in Huntington's disease (HD). We investigated whether these pathological changes in HD brains may also be present in peripheral tissues. Leukocyte 8-hydroxydeoxyguanosine (8-OHdG) and plasma malondialdehyde (MDA) were elevated, and activities of erythrocyte Cu/Zn-superoxide dismutase (Cu/Zn-SOD) and glutathione peroxidase (GPx) reduced in 16 HD patients when compared to 36 age- and gender-matched controls. Deleted and total mitochondrial DNA (mtDNA) copy numbers were increased, whereas the mRNA expression levels of mtDNA-encoded mitochondrial enzymes are not elevated in HD leukocytes compared to the normal controls. Plasma MDA levels also significantly correlated with HD disease severity. These results indicate means to suppress oxidative damage or to restore mitochondrial functions may be beneficial to HD patients. Plasma MDA may be used as a potential biomarker to test treatment efficacy in the future, if confirmed in a larger, longitudinal study.

Paclitaxel-loaded poly(gamma-glutamic acid)-poly(lactide) nanoparticles as a targeted drug delivery system against cultured HepG2 cells.

The study was to develop paclitaxel-loaded formulations using a novel type of self-assembled nanoparticles that was composed of block copolymers synthesized from poly(gamma-glutamic acid) and poly(lactide) via a simple coupling reaction. The nanoparticles (the NPs) were prepared with various feed weight ratios of paclitaxel to block copolymer (the P/BC ratio). The morphology of all prepared nanoparticles was spherical and the surfaces were smooth. Increasing the P/BC ratio significantly increased the drug loading content of the prepared nanoparticles, but remarkably reduced the drug loading efficiency. The release rate of paclitaxel from the NPs decreased significantly as the P/BC ratio increased. For the potential of targeting liver cancer cells, galactosamine was further conjugated on the prepared nanoparticles (the Gal-NPs) as a targeting moiety. It was found that the activity in inhibiting the growth of HepG2 cells (a liver cancer cell line) by the Gal-NPs was comparable to that of a clinically available paclitaxel formulation, while the NPs displayed a significantly less activity. This may be attributed to the fact that the Gal-NPs had a specific interaction with HepG2 cells via ligand-receptor recognition. Cells treated with distinct paclitaxel formulations resulted in arrest in the G2/M phase. The arrest of cells in the G2/M phase was highly suggestive of interference by paclitaxel with spindle formation and was consistent with the morphological findings presented herein. In conclusion, the active targeting nature of the Gal-NPs prepared in the study may be used as a potential drug delivery system for the targeted delivery to liver cancers.

Differential expression of mu-opioid receptor gene in CXBK and B6 mice by Sp1.

It is well known that there are individual differences in the sensitivity to analgesics. The CXBK mice are characterized by reduced sensitivity to morphine and by partial deficiency in mu-opioid receptor (MOR) expression. The sequences of MOR genes in CXBK and B6 mice are identical in their coding regions but differ at 5'-untranslated region (UTR) nucleotide -202 (C nucleotide in CXBK, but A nucleotide in B6). In this report, we identified an Sp1 element (-211 to -204) immediately before the polymorphic nucleotide. In electrophoretic mobility shift assay, nuclear protein binding to the B6-Sp1 sequence was more efficient than to the CXBK-Sp1 sequence, and anti-Sp1 but not anti-CREB antibody interfered with the formation of the DNA-protein complex. In MOR-expressing cell lines SH-SY5Y, P19, and PC12, B6 MOR promoter possessed high transcription activity than the CXBK promoter, and Sp1 inhibitor PDTC reduced the promoter activities. In SL2 cells that lack endogenous Sp1 expression, B6 and CXBK MOR promoters demonstrated equal activity, whereas overexpression of Sp1 in SL2 cells enhanced B6 MOR promoter activity better than the CXBK promoter. Together, the A-to-C change at MOR 5'-UTR decreases Sp1 binding and MOR gene transcription, which could underlie the reduced morphine expression in CXBK mice.

Transcriptional regulation of mu opioid receptor gene by cAMP pathway.

The utility of morphine for the treatment of chronic pain is hindered by the development of tolerance. Fentanyl has been shown to be a potent analgesic with a lower propensity to produce tolerance and physical dependence in the clinical setting. Previous finding has shown that fentanyl induces mu opioid receptor gene expression in PC-12 cells (Brain Res 859:217-223, 2000). In this report, we aim to identify the molecular mechanism of mu-opioid receptor (MOR) gene regulation by fentanyl. We demonstrated that the 4.7-kilobase MOR promoter could be induced by fentanyl in PC-12 cells, and we defined a partial cAMP response element (CRE) located at -106/-111 in 5'-untranslated region of the MOR gene. In electrophoretic mobility shift assay, cAMP response element-binding protein (CREB) was found in the protein-DNA complex formed on the CRE box. CREB was phosphorylated after forskolin induction, and both CREB and CREB-binding protein (CBP) binding to the endogenous MOR promoter was increased by forskolin in chromatin immunoprecipitation assay. The functional role of CREB in the induction of MOR gene was further elucidated by an experiment in which a dominant-negative mutant CREB, CREB-S133A, abolished the forskolin-mediated MOR induction. Moreover, we found that this CRE box is conserved in mouse, rat, and human MOR gene, implying physiological relevance in different species. Collectively, this study demonstrated that fentanyl-triggered MOR gene induction was mediated by the sequential activation of CREB and the binding of CREB and CBP to MOR promoter, thus provides direct evidence for lower propensity of fentanyl to produce tolerance.