Spinal cord tissue engineering via covalent interaction between biomaterials and cells.

Noncovalent interactions between cells and environmental cues have been recognized as fundamental physiological interactions that regulate cell behavior. However, the effects of the covalent interactions between cells and biomaterials on cell behavior have not been examined. Here, we demonstrate a combined strategy based on covalent conjugation between biomaterials (collagen fibers/lipid nanoparticles) and various cells (exogenous neural progenitor cells/astrocytes/endogenous tissue-resident cells) to promote neural regeneration after spinal cord injury (SCI). We found that metabolic azido-labeled human neural progenitor cells conjugated on dibenzocyclooctyne-modified collagen fibers significantly promoted cell adhesion, spreading, and differentiation compared with noncovalent adhesion. In addition, dibenzocyclooctyne-modified lipid nanoparticles containing edaravone, a well-known ROS scavenger, could target azide-labeled spinal cord tissues or transplanted azide-modified astrocytes to improve the SCI microenvironment. The combined application of these covalent conjugation strategies in a rat SCI model boosted neural regeneration, suggesting that the covalent interactions between cells and biomaterials have great potential for tissue regeneration.

Enhanced bioavailability of L-carnitine after painless intradermal delivery vs. oral administration in rats.

PURPOSE: In vitro and in vivo permeation studies were conducted to evaluate the characteristic of percutaneous administration of high hydrophilic drug L-carnitine (LC) by Functional MicroArray (FMA) painless intradermal delivery system. METHODS: In vitro study was designed to assess the effects of various skins, donor concentration and hydrogels from different carbomer derivatives on the release of LC in a Franz-type diffusion cell. The LC gel patches with carbomer 980 P were prepared and successfully applied to pharmacokinetic study of SD rats with and without FMA. Intravenous injection and oral administration were performed to support pharmacokinetic calculations and comparison of bioavailability. RESULTS: Enhanced delivery of LC using FMA was achieved in skin of different species in vitro studies. The 750 mg LC gel patches were applied to rats over 6 h, and approximately 27% of loaded dose was transported into rat. A 2.8-fold enhancement of absolute bioavailability for LC with FMA intradermal delivery system was observed compared with oral LC administration in vivo study. CONCLUSIONS: Both in vitro and in vivo studies demonstrated that the FMA intradermal delivery system can enhance the delivery and bioavailability of LC.

A modified collagen scaffold facilitates endogenous neurogenesis for acute spinal cord injury repair.

Due to irreversible neuronal loss and glial scar deposition, spinal cord injury (SCI) ultimately results in permanent neurological dysfunction. Neuronal regeneration of neural stem cells (NSCs) residing in the spinal cord could be an ideal strategy for replenishing the lost neurons and restore function. However, many myelin-associated inhibitors in the SCI microenvironment limit the ability of spinal cord NSCs to regenerate into neurons. Here, a linearly ordered collagen scaffold was used to prevent scar deposition, guide nerve regeneration and carry drugs to neutralize the inhibitory molecules. A collagen-binding EGFR antibody Fab fragment, CBD-Fab, was constructed to neutralize the myelin inhibitory molecules, which was demonstrated to promote neuronal differentiation and neurite outgrowth under myelin in vitro. This fragment could also specifically bind to the collagen and undergo sustained release from collagen scaffold. Then, the scaffolds modified with CBD-Fab were transplanted into an acute rat SCI model. The robust neurogenesis of endogenous injury-activated NSCs was observed, and these NSCs could not only differentiate into neurons but further mature into functional neurons to reconnect the injured gap. The results indicated that the modified collagen scaffold could be an ideal candidate for spinal cord regeneration after acute SCI. STATEMENTS OF SIGNIFICANCE: A linearly ordered collagen scaffold was specifically modified with collagen-binding EGFR antibody, allowed for sustained release of this EGFR neutralizing factor, to block the myelin associated inhibitory molecules and guide spinal cord regeneration along its linear fibers. Dorsal root ganglion neurons and neural stem cells induced by CBD-Fab exhibited enhanced neurite outgrowth and neuronal differentiation rate under myelin in vitro. Transplantation of the modified collagen scaffold with moderate EGFR neutralizing proteins showed greatest advantage on endogenous neurogenesis of injury-activated neural stem cells for acute spinal cord injury repair.

DNA-based vaccination against hepatitis B virus using dissolving microneedle arrays adjuvanted by cationic liposomes and CpG ODN.

DNA vaccines are simple to produce and can generate strong cellular and humoral immune response, making them attractive vaccine candidates. However, a major shortcoming of DNA vaccines is their poor immunogenicity when administered intramuscularly. Transcutaneous immunization (TCI) via microneedles is a promising alternative delivery route to enhance the vaccination efficacy. A novel dissolving microneedle array (DMA)-based TCI system loaded with cationic liposomes encapsulated with hepatitis B DNA vaccine and adjuvant CpG ODN was developed and characterized. The pGFP expression in mouse skin using DMA was imaged over time. In vivo immunity tests in mice were performed to observe the capability of DMA to induce immune response after delivery of DNA. The results showed that pGFP could be delivered into skin by DMA and expressed in skin. Further, the amount of expressed GFP was likely to peak at day 4. The immunity tests showed that the DMA-based DNA vaccination could induce effective immune response. CpG ODN significantly improved the immune response and achieved the shift of immune type from predominate Th2 type to a balance Th1/Th2 type. The cationic liposomes could further improve the immunogenicity of DNA vaccine. In conclusion, the novel DMA-based TCI system can effectively deliver hepatitis B DNA vaccine into skin, inducing effective immune response and change the immune type by adjuvant CpG ODN.

Identification of a novel mutation in a Chinese family with Nance-Horan syndrome by whole exome sequencing.

OBJECTIVE: Nance-Horan syndrome (NHS) is a rare X-linked disorder characterized by congenital nuclear cataracts, dental anomalies, and craniofacial dysmorphisms. Mental retardation was present in about 30% of the reported cases. The purpose of this study was to investigate the genetic and clinical features of NHS in a Chinese family. METHODS: Whole exome sequencing analysis was performed on DNA from an affected male to scan for candidate mutations on the X-chromosome. Sanger sequencing was used to verify these candidate mutations in the whole family. Clinical and ophthalmological examinations were performed on all members of the family. RESULTS: A combination of exome sequencing and Sanger sequencing revealed a nonsense mutation c.322G>T (E108X) in exon 1 of NHS gene, co-segregating with the disease in the family. The nonsense mutation led to the conversion of glutamic acid to a stop codon (E108X), resulting in truncation of the NHS protein. Multiple sequence alignments showed that codon 108, where the mutation (c.322G>T) occurred, was located within a phylogenetically conserved region. The clinical features in all affected males and female carriers are described in detail. CONCLUSIONS: We report a nonsense mutation c.322G>T (E108X) in a Chinese family with NHS. Our findings broaden the spectrum of NHS mutations and provide molecular insight into future NHS clinical genetic diagnosis.

Enhanced transcutaneous immunization via dissolving microneedle array loaded with liposome encapsulated antigen and adjuvant.

Transcutaneous immunization (TCI) with dissolving microneedle arrays (DMAs) is a promising vaccine administration method. In this work, we developed a TCI device consisting of dissolving polyvinylpyrrolidone (PVP) microneedles array, where in the tips are loaded with antigen and adjuvant encapsulated in liposomes. The microneedles could effectively be inserted into the skin and completely dissolve within 3 min. As a test-case, we selected ovalbumin (OVA) as a model antigen, CpG OND as adjuvant and cationic liposome (Lip) as a microparticulate vehicle for co-deliver antigens and adjuvant. Mice were immunized transcutaneously with DMAs containing OVA, OVA-CpG OND, OVA encapsulated in Lip, OVA-CpG OND encapsulated in Lip and conventional intramuscular injection (IM) with OVA solution, respectively. The results show that the anti-OVA IgG antibody level in the group immunized with the DMA containing OVA-CpG OND encapsulated in Lip was significantly higher than that of the other groups. Furthermore, it significantly increased the level of IgG2a (P<0.05) and achieved the shift of immune type from predominate Th2 type to a balance Th1/Th2 type. In conclusion, the DMA TCI device can effectively deliver the Lip encapsulating CpG OND-OVA into skin, enhancing the immune response and change the immune type.

Biodistribution in mice and severity of damage in rat lungs following pulmonary delivery of 9-nitrocamptothecin liposomes.

This study was designed to investigate in vitro release, in vivo tissue distribution and the damage to the lungs of 9-nitrocamptothecin (9-NC) liposomes. In vitro release of 9-NC from liposomes was carried out in phosphate buffer saline solution (PBS) pH 7.4. The tissue distribution of 9-NC liposomes and 9-NC solution was determined after pulmonary delivery to mice. The tissue distribution of 9-NC liposomes after intravenous administration was also studied. The changes of pulmonary edema index and histology of lungs in rats were investigated to evaluate the severity of the damage after pulmonary delivery. The results showed that 9-NC was continuously released from liposomes in PBS pH 7.4 for 24h at 37 degrees C. After pulmonary delivery, the mean residence time (MRT) of 9-NC liposomes in the lungs was 3.4 times as long as that of 9-NC solution and the total AUC0-t of all tissues in mice of the liposomes was 2.2-fold higher than that of the solution, indicating that the liposomes had sustained-release characteristics. Following intravenous administration and pulmonary delivery, the targeting efficiency (Te) to the lung of 9-NC liposomes was 0.14 and 2.02, respectively, which showed that intratracheal instillation can deliver the drug mainly to the lung and decrease the accumulation of the drug in other tissues at different concentrations. The pulmonary edema index and the histological changes of the lungs in 9-NC liposome group were significantly different from those in 9-NC solution group. The lung damage by liposomes was less severe than that by solution. Pulmonary delivery of 9-NC liposomes could directly deliver the drug to the lung and make the drug accumulate in the lung with sustained-release characteristics, changing the disposition behavior in vivo, decreasing the toxic and side effects on other tissues and reduce the severity of damage to lungs following intratracheal instillation.