Pathogenic Hydrogel? A Novel-Entrapment Neuropathy Model Induced by Ultrasound-Guided Perineural Injections.

Entrapment neuropathy (EN) is a prevalent and debilitative condition caused by a complex pathogenesis that involves a chronic compression-edema-ischemia cascade and perineural adhesion that results in excessive shear stress during motion. Despite decades of research, an easily accessible and surgery-free animal model mimicking the mixed etiology is currently lacking, thus limiting our understanding of the disease and the development of effective therapies. In this proof-of-concept study, we used ultrasound-guided perineural injection of a methoxy poly(ethylene glycol)-b-Poly(lactide-co-glycoilide) carboxylic acid (mPEG-PLGA-BOX) hydrogel near the rat's sciatic nerve to induce EN, as confirmed sonographically, electrophysiologically, and histologically. The nerve that was injected with hydrogel appeared unevenly contoured and swollen proximally with slowed nerve conduction velocities across the injected segments, thus showing the compressive features of EN. Histology showed perineural cellular infiltration, deposition of irregular collagen fibers, and a possible early demyelination process, thus indicating the existence of adhesions. The novel method provides a surgery-free and cost-effective way to establish a small-animal model of EN that has mixed compression and adhesion features, thus facilitating the additional elucidation of the pathophysiology of EN and the search for promising treatments.

Synergistic Effect of PEI and PDMAEMA on Transgene Expression in Vitro.

Polyethylenimine (PEI) and poly(2-(dimethylamino) ethyl methacrylate) (PDMAEMA) have both been used for DNA delivery. PDMAEMA has been shown to exhibit better gene transfection efficiency but lower expression ability than PEI. We mixed the two polymers at different ratios to investigate whether the resulting "dual" polyplex (PEI/PDMAEMA/DNA) could enhance both gene transfection efficiency and DNA expression ability. Experimental results showed a significant increase in DNA internalization and DNA expression for the PDMAEMA/PEI/DNA polyplexes at a ratio of 1:3 or 1:9 (PDMAEMA: PEI), depending on cell type, in comparison with PEI/DNA, PDMAEMA/DNA, and PDMAEMA/PEI/DNA at other ratios. PDMAEMA/PEI/DNA polyplexes did not reduce cell viability. In contrast to with the conventional approach using covalently modified PEI, the proposed "combination" approach provided a more convenient and effective way to improve transgene expression efficiency.

Ultrasound enhanced PEI-mediated gene delivery through increasing the intracellular calcium level and PKC-δ protein expression.

PURPOSE: Polyethylenimine (PEI), a cationic polymer, has been shown to aggregate plasmid DNA and facilitate its internalization. It has also been shown that combining ultrasound (US) with PEI could enhance and prolong in vitro and in vivo transgene expression. However, the role US in the enhancement of PEI uptake is poorly understood. This study investigates the impact of US on PEI-mediated gene transfection. METHODS: Specific endocytosis pathway siRNA, including clathrin HC siRNA, caveolin-1 siRNA and protein kinase C-delta (PKC-δ) siRNA, are used to block the corresponding endocytosis pathways prior to the transfection of luciferase DNA/PEI polyplexes to cultured cells by 1-MHz pulsed US with ultrasound contrast agent SonoVue®. RESULTS: Transgene expression was found not to be enhanced by US treatment in the presence of the PKC-δ siRNA. We further demonstrated that PKC-δ protein could be enhanced at 6 h after US exposure. Moreover, intracellular calcium levels were found to be significantly increased at 3 h after US exposure, while transgene expressions were significantly reduced in the presence of calcium channel blockers both in vitro and in vivo. CONCLUSIONS: Our results suggest that US enhanced PEI-mediated gene transfection specifically by increasing PKC-δ related fluid phase endocytosis, which was induced by increasing the intracellular calcium levels.

One injection for one-week controlled release: In vitro and in vivo assessment of ultrasound-triggered drug release from injectable thermoresponsive biocompatible hydrogels.

Episodic release of bioactive compounds is often necessary for appropriate biological effects under specific physiological conditions. Here, we aimed to develop an injectable, biocompatible, and thermosensitive hydrogel system for ultrasound (US)-triggered drug release. An mPEG-PLGA-BOX block copolymer hydrogel was synthesized. The viscosity of 15 wt% hydrogel is 0.03 Pa*s at 25 °C (liquid form) and 34.37 Pa*s at 37 °C (gel form). Baseline and US-responsive in vitro release profile of a small molecule (doxorubicin) and that of a large molecule (FITC-dextran), from the hydrogel, was tested. A constant baseline release was observed in vitro for 7 d. When triggered by US (1 MHz, continuous, 0.4 W/cm2), the release rate increased by approximately 70 times. Without US, the release rate returned to baseline. Baseline and US-responsive in vivo release profile of doxorubicin was tested by subcutaneous injection in the back of mice and rats. Following injection into the subcutaneous layer, in vivo results also suggested that the hydrogels remained in situ and provided a steady release for at least 7 d; in the presence of the US-trigger, in vivo release from the hydrogel increased by approximately 10 times. Therefore, the mPEG-PLGA-BOX block copolymer hydrogel may serve as an injectable, biocompatible, and thermosensitive hydrogel system that is applicable for US-triggered drug release.

Quantitative evaluation of the use of microbubbles with transcranial focused ultrasound on blood-brain-barrier disruption.

It has been shown that focused ultrasound (FUS) can disrupt the blood-brain barrier (BBB) noninvasively and reversibly at target locations when applied in the presence of ultrasound contrast agent (UCA). In this study, the dose-dependent effects of UCA on BBB disruption were investigated in the brains of 16 male Wistar rats sonicated by 1.0-MHz transcranial FUS, with the UCA present at four doses. The BBB disruption was evaluated quantitatively based on the extravasation of Evans blue (EB). The amount of EB extravasation in the brain increased with the quantity of UCA injected into the femoral vein prior to sonication. Moreover, the use of a suitable dose of UCA resulted in the BBB disruption being concentrated in the focal region instead of the entire brain. Our results indicate that injecting an appropriate quantity of UCA effectively increases and localizes the BBB disruption induced by transcranial FUS sonications.

Tertiary-amine functionalized polyplexes enhanced cellular uptake and prolonged gene expression.

Ultrasound (US) has been found to facilitate the transport of DNA across cell membranes. However, the transfection efficiency is generally low, and the expression duration of the transfected gene is brief. In this study, a tertiary polycation, Poly(2-(dimethylamino) ethyl methacrylate) (PDMAEMA), was used as a carrier for US-mediated gene transfection. Its in-vitro and in-vivo effects on the transfection efficiency and the expression duration were evaluated. A mixture of pCI-neo-luc and PDMAEMA was transfected to cultured cells or mouse muscle by exposure to 1-MHz pulse US. A strong expression of luciferase was found 10 days after the transfection in vitro regardless of US exposure. However, effective transfection only occurred in the US groups in vivo. The transfection ability depended on the weight ratio of PDMAEMA to DNA, and was different for the in-vitro and in-vivo conditions. Lower weight ratios, e.g., 0.25, exhibited better in-vivo expression for at least 45 days.

Prolonging the expression duration of ultrasound-mediated gene transfection using PEI nanoparticles.

Ultrasound (US) irradiation has been found to facilitate the inward transport of genetic materials across cell membranes (sonoporation). However, its transfection efficiency is generally low, and the expression duration of transfected gene is short. Polyethylenimine (PEI), a cationic polymer, has been shown to aggregate plasmid DNA and facilitate its internalization. The purpose of this study is to determine whether PEI can also prolong the expression duration after US-mediated transfection. A mixture of pCMViLUC and 22-kDa linear PEI was transfected to cultured cells or mouse muscle by exposure to 1-MHz pulsed US. The duration of expression was assessed periodically following US treatment. As expected, strong expression of luciferase could be found 30days after the treatment of DNA-PEI complex with US exposure, both in vitro and in vivo. However, without US, only very low transfection level could be obtained in vivo. The DNA/PEI complex showed protective effect against digestion of DNase I enzymes as compared with groups without PEI or to which PEI was added following the mixing of plasmid DNA with DNase I. PEI enhanced the US transfection efficiency by increasing both the intracellular uptake of plasmid DNA and the percentage of transfected cells. Most of the DNA uptake occurred at 3h after US exposure, suggesting that endocytosis took place. Moreover, the PEI-facilitated US gene transfection depended on the ratio of PEI and DNA (N/P ratio), which was different for in-vitro and in-vivo conditions. This system could be applied in future human gene therapies.

Thermal effect of sonophoresis for accelerating the analgesic effect of local anesthetics on rat tail nerve.

Sonophoresis is an ultrasound transdermal drugs delivery system. The eutectic mixture of local anesthetics (EMLA) has been used clinically for anesthesia but requires at least one hour to take effect and lacks of analgesia's objective assessment. We proposed that sonophoresis could reduce the duration of EMLA analgesia effect onset and be assessed by sensory conduction studies. Thirty Wistar adult rats were randomized into normal, control, ultrasound-, and heat-treatment groups. Normal group was received no EMLA cream or ultrasound and heat treatment. The control group received the EMLA cream on the rat tail at 3.5 cm distal to the rat tail base for local anesthesia of tail nerve. Ultrasound- and heat-treatment groups were received ultrasound with different parameters and heat treatment, respectively, before EMLA cream applied. Sensory conduction studies of tail nerve were made before and after treatment every 5 min at least for 60 min in all rats. There was no significant difference between the EMLA control group and heat treatment group. All rats in ultrasound-treatment group exhibit significant difference with EMLA control group and heat-treatment group in time for decreased 20% SNCV except for the 2 W/cm(2), 25 min, 20% in ultrasound-treatment group having no significant difference with heat-treatment group. There was no significant difference between ultrasound-treatment subgroups. In the decrease of amplitude, only the 2 W/cm(2), 5 min, 100% and the 2 W/cm(2), 10 min, 50% in ultrasound-treatment group had significant difference between EMLA control and heat-treatment groups. We have objectively examined the sonophoresis effect of ultrasound by investigating the effects of EMLA. Applying ultrasound for 5 min reduces the onset time of EMLA analgesia from 60 min to less than 20 min. Ultrasound sonophoresis of analgesic drugs is potentially useful in the treatment of carpal tunnel syndrome, tooth extraction, and other applications of analgesia.