Fatty acid-based strategy for efficient brain targeted gene delivery.

PURPOSE: To investigate a fatty acid-based strategy for efficient brain targeted gene delivery and to understand mechanism(s) of this small molecule-mediated brain gene delivery strategy. METHODS: A series of fatty acids (FAs) were conjugated with polyethylenimine (PEI25k). A near-infrared fluorescence probe, IR820, was used to study in vivo and ex vivo brain targeting ability of these fatty acid-PEI conjugates (FA-PEIs). Brain uptake of FA-PEI25k/rhodamine-6-isothiocyanate (RITC)-labeled DNA nanoparticles was investigated via a fluorescence imaging method. Moreover, pEGFP was used as a model gene to study in vitro and in vivo transfection effect of the ideal FA-PEI25k conjugate. RESULTS: FA modification did not have interference with the complexation between DNA and the PEI25k. The FA-PEI25k conjugates showed excellent brain targeting ability compared with unmodified PEI25k. Among these FA-PEI25k conjugates studied, myristic acid (MC)-PEI25k showed sustained brain distribution profile and higher brain DNA uptake. Furthermore, MC-PEI25k/pEGFP nanoparticles was able to achieve efficient in vitro and in vivo gene transfection. GFP expression was observed at different brain regions in vivo. CONCLUSIONS: These results demonstrated that the small molecule fatty acid, particularly myristic acid-based brain gene delivery strategy, is promising to mediate efficient gene transfection in the brain.

Ternary complexes with core-shell bilayer for double level targeted gene delivery: in vitro and in vivo evaluation.

PURPOSE: Hyaluronic acid (HA)/polyethyleneimine-dexamethasone (PEI-Dex)/DNA ternary complexes with "core-shell" bilayer were developed for double level targeted gene delivery. A PEI1800-Dex, as a core, was applied to compact DNA into a nano-sized structure and facilitate the nuclear translocation of DNA after endocytosis into tumor cells, and a polyanion HA, as the outer corona, was employed to improve targeted tumor delivery and reduce cytotoxicity. METHODS: PEI-Dex was synthesized and characterized by (1)H NMR. The characterizations of ternary complexes were investigated. Their biological properties, including transfection efficiency, cytotoxicity, cellular uptake and in vivo efficacy were evaluated systemically. RESULTS: Ternary complexes with the size of about 160 nm exhibited the lowest cytotoxicity and the highest transfection efficiency in B16F10 cells among investigated complexes. The sub-cellular localization study confirmed that ternary complexes could facilitate more efficient cell uptake and nuclear transport of DNA than binary complexes. Moreover, Cy7-labeled ternary complexes obviously accumulated in the tumor after i.v. administration, indicating that ternary complexes could assist the DNA targeting to the tumor. In in vivo studies, HA/PEI1800-Dex/DNA ternary complexes showed confirmed anti-inflammation activity, and could significantly suppress tumor growth of tumor-bearing nude mice. CONCLUSIONS: HA/PEI-Dex/DNA ternary complexes might be a promising targeted gene delivery system.

The use of myristic acid as a ligand of polyethylenimine/DNA nanoparticles for targeted gene therapy of glioblastoma.

To establish a gene delivery system for brain targeting, a low molecular weight polyethylenimine (PEI(10 K)) was modified with myristic acid (MC), and complexed with DNA, yielding MC-PEI(10 K)/DNA nanoparticles successfully. The nanoparticles were observed to be successfully taken up by the brains of mice. The transfection efficiency of the nanoparticles was then investigated, and both the in vitro and in vivo gene expression of MC-PEI(10 K)/DNA nanoparticles is significantly higher than that of unmodified PEI(10 K)/DNA nanoparticles. The anti-glioblastoma effect of MC-PEI(10 K)/pORF-hTRAIL was demonstrated by the survival time of intracranial U87 glioblastoma-bearing mice. The median survival time of the MC-PEI(10 K)/pORF-hTRAIL group (28 days) was significantly longer than that of the PEI(10 K)/pORF-hTRAIL group (24 days), the MC-PEI(10 K)/pGL(3) group (21 days) and the saline group (22 days). Therefore, our results suggested that MC-PEI(10 K) could be potentially used for brain-targeted gene delivery and in the treatment of glioblastoma.

Preparation and evaluation of an Arg-Gly-Asp-modified chitosan/hydroxyapatite scaffold for application in bone tissue engineering.

Bone tissue engineering has become a promising method for the repair of bone defects, and the production of a scaffold with high cell affinity and osseointegrative properties is crucial for successful bone substitute. Chitosan (CS)/hydroxyapatite (HA) composite was prepared by in situ compositing combined with lyophilization, and further modified by arginine­glycine­aspartic acid (RGD) via physical adsorption. In order to evaluate the cell adhesion rate, viability, morphology, and alkaline phosphatase (ALP) activity, the RGD­CS/HA scaffold was seeded with bone marrow stromal cells (BMSCs). The osseointegrative properties of the RGD­CS/HA scaffold were evaluated by in vivo heterotopic ossification and in vivo bone defect repair. After 4 h culture with the RGD­CS/HA scaffold, the adhesion rate of the BMSCs was 80.7%. After 3 days, BMSCs were fusiform in shape and evenly distributed on the RGD­CS/HA scaffold. Formation of extracellular matrix and numerous cell­cell interactions were observed after 48 h of culture, with an ALP content of 0.006 ± 0.0008 U/l/ng. Furthermore, the osseointegrative ability and biomechanical properties of the RGD­CS/HA scaffold were comparable to that of normal bone tissue. The biocompatibility, cytocompatibility, histocompatibility and osseointegrative properties of the RGD­CS/HA scaffold support its use in bone tissue engineering applications.

Cyclic RGD-polyethylene glycol-polyethylenimine for intracranial glioblastoma-targeted gene delivery.

Even though the blood-brain barrier (BBB) is compromised for angiogenesis, therapeutic agents for glioblastoma multiforme (GBM) are particularly inefficient due to the existence of a blood-tumor barrier (BTB), which hampers tumor accumulation and uptake. Integrin α(v)ß(3) is overexpressed on glioblastoma U87 cells and neovasculture, thus making its ligands such as the RGD motif target glioblastoma in vitro and in vivo. In the present work, we have designed a modified polyethylene glycol-polyethylenimine (PEG-PEI) gene carrier by conjugating it with a cyclic RGD sequence, c(RGDyK) (cyclic arginine-glycine-aspartic acid-D-tyrosine-lysine). When complexed with plasmid DNA, this gene carrier, termed RGD-PEG-PEI, formed homogenous nanoparticles with a mean diameter of 73 nm. These nanoparticles had a high binding affinity with U87 cells and facilitated targeted gene delivery against intracranial glioblastoma in vivo, thereby leading to a higher gene transfer efficiency compared to the PEG-PEI gene carrier without RGD decoration. This intracranial glioblastoma-targeted gene carrier also enhanced the therapeutic efficacy of pORF-hTRAIL, as evidenced by a significantly prolonged survival of intracranial glioblastoma-bearing nude mice. Considering the contribution of glioblastoma neovasculature to the BBB under angiogenic conditions, our results demonstrated the therapeutic feasibility of treating a brain tumor through mediation of integrin α(v)ß(3), as well as the potential of using RGD-PEG-PEI as a targeted gene carrier in the treatment of intracranial glioblastoma.

Benzamide analogue-conjugated polyethylenimine for brain-targeting and gene delivery.

In this study, a small molecule, benzamide analogue, p-hydroxybenzoic acid (p-HA), was used as a novel ligand for brain-targeting gene delivery. p-HA was conjugated to polyethylenimine and further labeled with a near infrared dye, IR820, for in vivo and ex vivo imaging study. Significant fluorescent signal was detected in brain from 0.5 to 24 h after injection compared with unmodified PEI. Then nanoparticles were prepared with p-HA-PEI to encapsulate pEGFP and pGL2 as reporter genes and characterized on the cell level. In 5 y cells green fluorescent protein expression could be observed by fluorescent microscopy and significant higher expression of firefly luciferase was detected in p-HA-PEI/pGL2 group than in PEI/pGL2 group. For in vivo gene expression study, comparable high expression of green fluorescent protein in brain sections was confirmed using both confocal fluorescent microscopy and in vivo fluorescent imaging. All these results suggested that p-HA-PEI could be potentially used for brain targeted gene delivery.

Revision hip arthroplasties with use of the modular S-ROM prosthesis.

OBJECTIVE: The aim of this study was to revision hip arthroplasties in ankylosing spondylitis. MATERIALS AND METHODS: We performed 21 revision hip arthroplasty in 16 patients with ankylosing spondylitis (AS) using S-ROM modular femoral stem. At the final follow-up, all patients were able to ambulate without any walking aids. RESULTS: The mean Harris hip score improved from 57.2 (35 to 74) pre-operatively to 90.2 points (73 to 100) post-operatively, and the outcome was classified as good or excellent in 17 hips (81%). Fixation of the femoral component was classified as stable with bone ingrowth in 15 hips (71.4%), stable with fibrous ingrowth in 2 hips (9.5%), and radiolucent loosening in 4 hips (19.1%). Five hips developed a pedestal at the tip of femoral component. Femoral osteolysis was found in 3 hips (14.2%): 3 hips in Gruen zones 1 and 7, one hip in zone 7, and two hips in zone 1. One hip underwent acetabular revision because of breakage of polyethylene liner, and the well-fixed femoral component was left in situ. Thigh pain developed in 1 patient (6.2%). Kaplan-Meier survival was 81% at 64 months, with radiographic loosening as an end-point when two hips were at risk. CONCLUSION: Satisfactory results of midterm clinical and radiographic follow-up can be achieved using S-ROM modular femoral stem for revision of femoral stem in ankylosing spondylitis.

Poly(ethylene glycol)-block-poly(D,L-lactide acid) micelles anchored with angiopep-2 for brain-targeting delivery.

In this study, angiopep with high transcytosis capacity and parenchymal accumulation was used as a novel ligand for the brain-targeting delivery of poly(ethylene glycol)-block-poly(D,L-lactide acid) (PEG-PLA) micelles. Angiopep-2 was synthesized by solid-phase peptide synthesis, and then conjugated with maleimide-PEG-PLA to form angiopep-PEG-PLA. The micelles composed of methoxy-PEG-PLA (mPEG-PLA) and angiopep-PEG-PLA was prepared by film-hydration method. Near-infrared fluorescence dye, DiR was loaded into micelles to evaluate the brain-targeting ability of micelles with or without angiopep modification by near-infrared fluorescence imaging in vivo and ex vivo. Significant near-infrared (NIR) fluorescent signal was detected in the brain after angiopep-anchored micelles administration and further confirmed by imaging the whole brain and brain slices, compared with that of the micelles without modification. ¹²5I-radiolabeled angiopep-PEG-PLA micelles after intravenous administration in mice showed high brain accumulation for up to 24 h. These results indicate that angiopep-modified PEG-PLA micelle is a promising brain-targeting nanocarrier for lipophilic drugs.

Myristic acid-conjugated polyethylenimine for brain-targeting delivery: in vivo and ex vivo imaging evaluation.

To investigate the potential of myristic acid (MC) to mediate brain delivery of polyethylenimine (PEI) as a gene delivery system, a covalent conjugate (MC-PEI) of MC, and PEI was synthesized. A near-infrared fluorescence probe, IR820 was conjugated to MC-PEI to explore its in vivo distribution after intravenous (i.v.) administration in mice. The brain targeting ability of MC-PEI was evaluated by near-infrared fluorescence imaging and analyzed semiquantitatively by fluorescence intensity, respectively. Significant NIR fluorescent signal was detected in the brain 12 h after i.v. administration and further confirmed by imaging the whole brain and brain slices. Semiquantitative results from fluorescence intensity further supported the successful brain delivery of MC-PEI which led to a very significant increase ( approximately 200%) in the brain uptake after i.v. injection in comparison with unmodified PEI. The capability of MC-PEI to condense DNA was further confirmed using agarose gel retardation assay, indicating its potential for gene delivery. The significant in vivo and ex vivo results suggest that MC-PEI is a promising brain-targeting drug delivery system, especially for gene delivery.