INTRODUCTION:Focused ultrasound (FUS) is a noninvasive modality for targeted delivery of therapeutic agents across the blood-brain barrier (BBB). We conducted a systematic review and meta-analysis to evaluate the efficacy of FUS-mediated gene therapy in preclinical orthotopic glioblastoma (GBM) in vivo models. METHODS:PubMed, Embase, Scopus, and Web of Science were searched according with PRISMA guidelines to identify studies reporting FUS-mediated delivery of genetic material in orthotopic GBM animal models. Eligible studies assessed gene delivery or therapeutic efficacy in vivo. Data were extracted on vector type, gene payload, microbubble characteristics, and FUS parameters. Primary outcomes included tumor volume reduction, survival, and gene expression in brain tissue. Random-effects meta-analyses were performed to pool effect sizes. RESULTS:Nine studies met inclusion criteria, including viral, non-viral, nanoparticle, and exosome-based vectors. Delivered genes included therapeutic transgenes (MDA-7/IL-24, HSV-TK, shBirc5, CRISPR/Cas9) and reporter genes (luciferase, GFP). FUS parameters varied (0.65-1 MHz; 120-700 kPa; 1-3 min), as did microbubble formulations. Meta-analysis demonstrated FUS significantly enhanced gene expression in brain tissue (pooled effect size 6.34, 95% CI 2.21-18.18), tumor volume reduction (pooled effect size 4.03, 95% CI 1.46-11.12), and survival (HR 1.33, 95% CI 1.13-1.56). Heterogeneity was high, reflecting protocol variability. No significant FUS-related adverse effects were reported. CONCLUSION:FUS-mediated gene therapy improved gene delivery, tumor control, and survival in preclinical glioma models. These findings support FUS as a safe and effective strategy to overcome barriers to central nervous system gene therapy. Further studies are needed to standardize parameters and evaluate long-term outcomes before clinical translation.

Glioma is one of the most common primary malignant brain tumors. Despite progress in therapeutic approaches, the median survival of patients with glioma remains less than 2 years, generating the need for new therapeutic approaches. Ultrasound (US) is widely used in medical fields and is used as a therapeutic tool mainly for improving the performance of therapeutic entities. In this study, we examined a novel approach using low frequency US (20 kHz) (LFUS) as an independent treatment tool for malignant glioma, since primary studies showed that cancer cells are more susceptible to LFUS than healthy cells. LFUS safety and efficacy were examined in a 9L gliosarcoma-bearing female Fischer 344 rats. Two LFUS protocols were examined: a one-time treatment (US1X), and two treatments 24 h apart (US2X). For safety evaluation, rats were monitored for weight change and pain measurements. For efficacy, tumor volume was measured as a function of time and the tumor structural chances were examined histopathologically. LFUS treatment showed rapid inhibition of tumor growth, seen as soon as 12 h after US application. In addition, LFUS was found to affect the tumor structure, which was more extensive (>60% of tumor area) in smaller tumors. In US2X, the tumor tissue was completely destroyed, and an extensive immune response was observed. Importantly, the treatment was highly selective, keeping the healthy tissue surrounding the tumor unharmed. We developed a highly efficient and selective therapeutic protocol for treating malignant glioma with minimal side effects based solely on LFUS.

RNA interference (RNAi) therapy has emerged as a promising cancer therapy platform; however, there is an ongoing need for a safe and effective delivery carrier for siRNA. The present study demonstrates for the first time the systemic in vivo delivery of nanosized complexes of quaternized starch (Q-starch) and siRNA to effectively target cancer in a murine model. Our results show that Q-starch/siRNA composite nanoparticle complexes are safe for systemic administration, with no evident toxicities. Encouragingly, the results demonstrate their cellular uptake in the tumor and 63% gene silencing post intratumor injection. Moreover, a biodistribution study reveals favorable accumulation of the composite nanoparticles in the tumor site post systemic administration. The gene-silencing results are encouraging, although not sufficient for therapeutic application, and raise the need to further investigate the potential of Q-starch/siRNA complexes in terms of gene-silencing efficiency and of tumor targeting and stability following systemic administration. Overall, our composite nanoparticles show promising potential for RNAi delivery to tumor sites, with their accumulation in the lungs suggesting that there may be particular merit in investigating the use of Q-starch/siRNA complexes to treat lung cancer.

Treating neuroinflammation‐related injuries and disorders through manipulation of neuroinflammation functions is being heralded as a new therapeutic strategy. In this study, a novel pectic galactan (PG) polysaccharide based gene therapy approach is developed for targeting reactive gliosis in neuroinflammation. Galectin‐3 (Gal‐3) is a cell protein with a high affinity to β‐galactoside sugars and is highly expressed in reactive gliosis. Since PG carries galactans, it can target reactive gliosis via specific carbohydrate interaction between galactan and Gal‐3 on the cell membrane, and therefore can be utilized as a carrier for delivering genes to these cells. The carrier is synthesized by modifying quaternary ammonium groups on the PG. The resulting quaternized PG (QPG) is found to form complexes with plasmid DNA with a mean diameter of 100 nm and have the characteristics required for targeted gene therapy. The complexes efficiently condense large amounts of plasmid per particle and successfully bind to Gal‐3. The in vivo study shows that the complexes are biocompatible and safe for administration and can selectively transfect reactive glial cells of an induced cortical lesion. The results confirm that this PG‐based delivery system is a promising platform for targeting Gal‐3 overexpressing neuroinflammation cells for treating neuroinflammation‐related injuries and neurodegenerative diseases.

Quaternized derivatives of pectic galactan (QPG) were synthesized by a reaction of pectic galactan (PG) with 3-chloro-2-hydroxypropyl trimethyl ammonium chloride (CHPTAC) in the presence of aqueous sodium hydroxide solution under mild reaction conditions. The results showed that the concentration of CHPTAC and NaOH has great impact on the quaternization reaction. QPG was found to interact electrostatically with plasmid DNA in aqueous solution to form complexes in globular condensed morphology in a nanometer scale size ranging from 60 to 160 nm. Complexes formed with QPG fluorescently labeled with 5-DTAF (QPG-5-DTAF) were introduced to the C6 rat glioma cell line, and were found to be able to enter the cell and approach the nucleus within 24 h. The results suggest that this type of modified natural polysaccharide may have an advantage as a biocompatible and biodegradable gene delivery carrier and furthermore may serve as a cell specific carrier.

Biodegradable polymeric nanoparticles have the potential to be safer alternatives to viruses for gene delivery; however, their use has been limited by poor efficacy in vivo. In this work, we synthesize and characterize polymeric gene delivery nanoparticles and evaluate their efficacy for DNA delivery of herpes simplex virus type I thymidine kinase (HSVtk) combined with the prodrug ganciclovir (GCV) in a malignant glioma model. We investigated polymer structure for gene delivery in two rat glioma cell lines, 9L and F98, to discover nanoparticle formulations more effective than the leading commercial reagent Lipofectamine 2000. The lead polymer structure, poly(1,4-butanediol diacrylate-co-4-amino-1-butanol) end-modified with 1-(3-aminopropyl)-4-methylpiperazine, is a poly(β-amino ester) (PBAE) and formed nanoparticles with HSVtk DNA that were 138 ± 4 nm in size and 13 ± 1 mV in zeta potential. These nanoparticles containing HSVtk DNA showed 100% cancer cell killing in vitro in the two glioma cell lines when combined with GCV exposure, while control nanoparticles encoding GFP maintained robust cell viability. For in vivo evaluation, tumor-bearing rats were treated with PBAE/HSVtk infusion via convection-enhanced delivery (CED) in combination with systemic administration of GCV. These treated animals showed a significant benefit in survival (p = 0.0012 vs control). Moreover, following a single CED infusion, labeled PBAE nanoparticles spread completely throughout the tumor. This study highlights a nanomedicine approach that is highly promising for the treatment of malignant glioma.

SUMMARY A novel targeted gene therapy nano-system for glioma brain tumors was developed and characterized. Since cell's membrane receptor, galectin-3, is found in high levels in glioma, we based our non-viral gene delivery on modified pectic galactan. Pectic galactan is a galactose-rich region of pectin which can serve as potential ligands for interaction with galectin-3. The pectic galactan was successfully modified with quaternary ammonium group and complexed with plasmid DNA. Complexes were found to be suitable for cellular uptake and with minimal cytotoxicity to C6 rat glioma cell line.