Targeted anti-cancer therapy: Co-delivery of VEGF siRNA and Phenethyl isothiocyanate (PEITC) via cRGD-modified lipid nanoparticles for enhanced anti-angiogenic efficacy.

Anti-tumor angiogenesis therapy, targeting the suppression of blood vessel growth in tumors, presents a potent approach in the battle against cancer. Traditional therapies have primarily concentrated on single-target techniques, with a specific emphasis on targeting the vascular endothelial growth factor, but have not reached ideal therapeutic efficacy. In response to this issue, our study introduced a novel nanoparticle system known as CS-siRNA/PEITC&L-cRGD NPs. These chitosan-based nanoparticles have been recognized for their excellent biocompatibility and ability to deliver genes. To enhance their targeted delivery capability, they were combined with a cyclic RGD peptide (cRGD). Targeted co-delivery of gene and chemotherapeutic agents was achieved through the use of a negatively charged lipid shell and cRGD, which possesses high affinity for integrin αvß3 overexpressed in tumor cells and neovasculature. In this multifaceted approach, co-delivery of VEGF siRNA and phenethyl isothiocyanate (PEITC) was employed to target both tumor vascular endothelial cells and tumor cells simultaneously. The co-delivery of VEGF siRNA and PEITC could achieve precise silencing of VEGF, inhibit the accumulation of HIF-1α under hypoxic conditions, and induce apoptosis in tumor cells. In summary, we have successfully developed a nanoparticle delivery platform that utilizes a dual mechanism of action of anti-tumor angiogenesis and pro-tumor apoptosis, which provides a robust and potent strategy for the delivery of anti-cancer therapeutics.

Erythrocyte Membrane-Coated Invisible Acoustic-Sensitive Nanoparticle for Inducing Tumor Thrombotic Infarction by Precisely Damaging Tumor Vascular Endothelium.

Selective induction of tumor thrombus infarction is a promising antitumor strategy. Non-persistent embolism due to non-compacted thrombus and activated fibrinolytic system within the tumor large blood vessels and tumor margin recurrence are the main therapeutic bottlenecks. Herein, an erythrocyte membrane-coated invisible acoustic-sensitive nanoparticle (TXA+DOX/PFH/RBCM@cRGD) is described, which can induce tumor thrombus infarction by precisely damaging tumor vascular endothelium. It is revealed that TXA+DOX/PFH/RBCM@cRGD can effectively accumulate on the endothelial surface of tumor vessels with the help of the red blood cell membrane (RBCM) stealth coating and RGD cyclic peptide (cRGD), which can be delivered in a targeted manner as nanoparticle missiles. As a kind of phase-change material, perfluorohexane (PFH) nanodroplets possess excellent acoustic responsiveness. Acoustic-sensitive missiles can undergo an acoustic phase transition and intense cavitation with response to low-intensity focused ultrasound (LIFU), damaging the tumor vascular endothelium, rapidly initiating the coagulation cascade, and forming thromboembolism in the tumor vessels. The drugs loaded in the inner water phase are released explosively. Tranexamic acid (TXA) inhibits the fibrinolytic system, and doxorubicin (DOX) eliminates the margin survival. In summary, a stealthy and acoustically responsive multifunctional nanoparticle delivery platform is successfully developed for inducing thrombus infarction by precisely damaging tumor vascular endothelium.

The synergistic effect of Angelica sinensis (Oliv.) Diels and Rehmannia glutinosa (Gaertn.) DC. on antioxidant activity and protective ability against cell injury.

Oxidative stress-induced dysfunction of nerve cells has been implicated as a crucial cause of cell death in neurodegenerative diseases. In Asian countries, herbs, such as Angelica sinensis (Oliv.) Diels (DG) and Rehmannia glutinosa (Gaertn.) DC. (SDH), have long be considered to have antiaging abilities. The herbs act as neuro protectants that rescue nerve cells from oxidative stress damage and apoptosis. Thus, developing herbal formulas can potentially lead to new treatments for neurodegenerative diseases. In this study, we compared the effective active components and antioxidant properties of extractive of DG and SDH (DG-SDH) when formulated at different ratios. DG-SDH formulated at a ratio of 3:2 (DG-SDH [3:2]) produced the highest content of polysaccharides, polyphenols, and flavonoids. It also showed the best ability in removing DPPH and hydroxyl free radicals compared to single herb or other compounding ratio. The antioxidant activity of DG-SDH (3:2) showed best synergistic effects in scavenging activity assays of DPPH free radicals and hydroxyl free radicals. DG-SDH (3:2) could increase the cell viability of SHSY-5Y cells, PC-12 cells, and BV-2 cells. In particular, DG-SDH (3:2) protected SHSY-5Y cells from H2 O2 -induced cell injury by inhibiting excessive expression of reactive oxygen species (ROS), reducing the rate of apoptosis and restoring mitochondrial membrane potential. Actin-Tracker Green and DAPI staining and fluorescence microscope observation confirmed that DG-SDH (3:2) helped in preserving cell morphology under oxidative stress. These findings support that DG-SDH (3:2) promote the neuroprotection against hydrogen peroxide and can serve as a novel therapy for neurodegenerative diseases. PRACTICAL APPLICATIONS: This is the first study to investigate DG and SDH interaction between effective ingredients. These findings support that DG-SDH (3:2) has the best synergistic effects in antioxidant activity and promote the neuroprotection against hydrogen peroxide. Hence, DG-SDH (3:2) will be an excellent candidate to be developed as a functional food ingredients or nutraceuticals for neurodegenerative diseases.

A dual-functional buformin-mimicking poly(amido amine) for efficient and safe gene delivery.

Biguanides (i.e. metformin, phenformin and buformin) are antidiabetic drugs with potential antitumor effects. Herein, a polycationic polymer, N,N'-bis(cystamine)acrylamide-buformin (CBA-Bu), containing multiple biodegradable disulphide bonds and buformin-mimicking side chains was synthesised. CBA-Bu was equipped with high efficiency and safety profile for gene delivery, meanwhile exhibiting potential antitumor efficacy. As a gene vector, CBA-Bu was able to condense plasmid DNA (pDNA) into nano-sized (<200 nm), positively-charged (>30 mV) uniform polyplexes that were well resistant to heparin and DNase I. Due to the reduction responsiveness of the disulphide bonds, CBA-Bu/pDNA polyplexes could release the loaded pDNA in the presence of dithiothreitol, and induce extremely low cytotoxicity in NIH/3T3 and U87 MG cells. The transfection results showed that CBA-Bu had a cellular uptake efficiency comparable to 25 kDa PEI, while a significantly higher gene expression level. Additionally, CBA-Bu had a lower IC50 value than its non-biguanide counterpart in two cancer cell lines. Furthermore, CBA-Bu could activate AMPK and inhibit mTOR pathways in U87 MG cells, a mechanism involved in the antitumor effect of biguanides. Taken together, CBA-Bu represented an advanced gene vector combining desirable gene delivery capability with potential antitumor activity, which was promising to achieve enhanced therapeutic efficacy in antitumor gene therapy.

A biodegradable poly(amido amine) based on the antimicrobial polymer polyhexamethylene biguanide for efficient and safe gene delivery.

Inspired by the excellent membrane affinity of antimicrobial polymers, we synthesized a novel biodegradable poly(amino amine) polymer with pendent side chains that mimic the widely used biocide polyhexamethylene biguanide (PHMB) for gene delivery. Michael addition polymerization was utilized to form the polymer scaffold between N,N'-cystaminebisacrylamide (CBA) and N-Boc-1,6-diaminohexane (Boc-DAH) followed by N-Boc deprotection. Then the exposed primary amino groups were partly (about 75%) transformed into biguanide by an addition reaction with dicyandiamide to obtain the final product CBA-DAH-biguanide (CBA-DAH-BG). The polymer CBA-DAH-BG was able to condense plasmid DNA (pDNA) into nano-sized (<200 nm), positively-charged (>35 mV) polyplexes that were well resistant to heparin and DNase I. Rapid DNA release was observed in the presence of dithiothreitol (DTT), indicating that CBA-DAH-BG was equipped with biodegradability by the cleavage of disulfide bonds, which was helpful for unpacking DNA and decreasing cytotoxicity. CBA-DAH-BG/pDNA polyplexes were characterized by efficient cellular uptake efficacy, extremely low cytotoxicity, and high transfection efficiency in two cell lines (i.e., NIH/3T3 and U87 MG), compared to 25 kDa polyethyleneimine (PEI) and the intermediate product CBA-DAH that were both devoid of biguanide groups. Of note, clathrin-mediated endocytosis and lipid rafts played an important role in the internalization of the polyplexes. Taken together, this strategy described herein may represent an innovative avenue for the design of more advanced nonviral gene vectors with high transfection efficiency and biocompatibility.

Nuclear localization signal peptide enhances transfection efficiency and decreases cytotoxicity of poly(agmatine/N,N'-cystamine-bis-acrylamide)/pDNA complexes.

At present, nonviral gene vectors develop rapidly, especially cationic polymers. A series of bioreducible poly(amide amine) (PAA) polymers containing guanidino groups have been synthesized by our research team. These novel polymer vectors demonstrated significantly higher transfection efficiency and lower cytotoxicity than polyethylenimine (PEI)-25kDa. However, compared with viral gene vectors, relatively low transfection efficiency, and high cytotoxicity are still critical problems confronting these polymers. In this study, poly(agmatine/N,N'-cystamine-bis-acrylamide) p(AGM-CBA) was selected as a model polymer, nuclear localization signal (NLS) peptide PV7 (PKKKRKV) with good biocompatibility and nuclear localization effect was introduced to investigate its impact on transfection efficiency and cytotoxicity. NLS peptide-mediated in vitro transfection was performed in NIH 3T3 cells by directly incorporating NLS peptide with the complexes of p(AGM-CBA)/pDNA. Meanwhile, the transfection efficiency and cytotoxicity of these complexes were evaluated. The results showed that the transfection efficiency could be increased by 5.7 times under the appropriate proportion, and the cytotoxicity brought by the polymer vector could be significantly reduced.

Nuclear delivery of plasmid DNA determines the efficiency of gene expression.

As a cationic non-viral gene delivery vector, poly(agmatine/ N, N'-cystamine-bis-acrylamide) (AGM-CBA) showed significantly higher plasmid DNA (pDNA) transfection ability than polyethylenimine (PEI) in NIH/3T3 cells. The transfection expression of AGM-CBA/pDNA polyplexes was found to have a non-linear relationship with AGM-CBA/pDNA weight ratios. To further investigate the mechanism involved in the transfection process of poly(AGM-CBA), we used pGL3-control luciferase reporter gene (pLUC) as a reporter pDNA in this study. The distribution of pLUC in NIH/3T3 cells and nuclei after AGM-CBA/pLUC and PEI/pLUC transfection were determined by quantitative polymerase chain reaction (qPCR) analysis. The intracellular trafficking of the polyplexes was evaluated by cellular uptake and nuclei delivery of pLUC, and the intracellular availability was evaluated by the ratio of transfection expression to the numbers of pLUC delivered in nuclei. It was found that pLUC intracellular trafficking did not have any correlation with the transfection expression, while an excellent correlation was found between the nuclei pLUC availability and transfection expression. These results suggested that the intracellular availability of pLUC in nuclei was the rate-limiting step for pLUC transfection expression. Further optimization of the non-viral gene delivery system can be focused on the improvement of gene intracellular availability.

Verapamil and riluzole cocktail liposomes overcome pharmacoresistance by inhibiting P-glycoprotein in brain endothelial and astrocyte cells: A potent approach to treat amyotrophic lateral sclerosis.

Riluzole is currently one of two approved medications for the treatment of amyotrophic lateral sclerosis (ALS). However, brain disposition of riluzole, as a substrate of P-glycoprotein (P-gp), is limited by the efflux transporters at the blood-brain barrier (BBB). We propose to develop a liposomal co-delivery system that could effectively transport riluzole to brain cells by reducing efflux pumps with a P-gp inhibitor, verapamil. Riluzole and verapamil cocktail liposomes were prepared by lipid film hydration. The average particle size of cocktail liposomes was 194.3 ±â€¯6.0 nm and their polydispersity index (PDI) was 0.272 ±â€¯0.017. The encapsulation efficiencies of verapamil and riluzole in the cocktail liposomes were 86.0 ±â€¯1.4% and 85.6 ±â€¯1.1%, respectively. The drug release from cocktail liposomes after 8 h in PBS at 37 °C was 78.4 ±â€¯6.2% of riluzole and 76.7 ±â€¯3.8% of verapamil. The average particle size of liposomes did not show significant changes at 4 °C after three months. Verapamil cocktail liposomes inhibited P-gp levels measured by western blotting in dose and time-dependent manners in brain endothelial bEND.3 cells. Increased drug efflux transporters were detected in bEND.3 and astrocytes C8D1A cells, promoted by tumor necrosis factor (TNF-α) or hydrogen peroxide (H2O2). Restored accumulations of riluzole and fluorescent dye rhodamine 123 were observed in bEND.3 cells after treatments with cocktail liposomes. It indicated that inhibitory potential of co-delivery liposome system towards P-gp could mediate the transport of both P-gp substrates. Verapamil and riluzole co-loaded liposomes may be used to overcome pharmacoresistance of riluzole for improving ALS therapy.

Disulfide-bond-containing agamatine-cystaminebisacrylamide polymer demonstrates better transfection efficiency and lower cytotoxicity than polyethylenimine in NIH/3T3 cells.

Previously, we synthesized a non-viral vector containing disulfide bond by polymerization of agamatine (AGM) and N,N'-cystaminebisacrylamide (CBA). In this study, we investigated the transfection efficiency of disulfide bond (SS) containing AGM-CBA polymer in gene delivery into NIH/3T3 cells, and examined the factors affecting its transfection efficiency by comparing with polyethylenimine (PEI). In addition, experiments were carried out to determine the mechanisms of cell entry pathways and intracellular behavior of AGM-CBA/pDNA polyplexes. The transfection efficiency of AGM-CBA/pDNA with different weight ratios and different amounts of pDNA was measured and the pathways mediated transfection processes were studied by using various endocytosis inhibitors. To determine the intracellular behavior of AGM-CBA/pDNA polyplexes, the transfection efficiencies of AGM-CBA/pDNA and PEI/pDNA polyplexes with different combination structures were determined by using reporter gene and fake plasmid DNA. The transfection efficiency of AGM-CBA/pDNA polyplexes was correlated with its weight ratio of AGM-CBA and pDNA, and the amount of pDNA. Both AGM-CBA/pDNA and PEI/pDNA polyplexes enter into cell by clathrin- and caveolae-mediated endocytic pathways. However, AGM-CBA/pDNA showed different intracellular behavior in NIH/3T3 cells compared to PEI/pDNA polyplexes. It was hypothesized that disulfide bond in AGM-CBA could be an important factor contributing to its intracellular behavior and better transfection efficiency. Overall, AGM-CBA demonstrated better transfection efficiency and lower cytotoxicity than PEI in NIH/3T3 cells as a gene delivery vector.

Exosome-based small RNA delivery: Progress and prospects.

RNA interfering (RNAi), mediated by small interfering RNAs and microRNAs, is currently one of the most promising tools of gene therapy. Small RNAs are capable of inducing specific post-transcriptional gene silencing, providing a potentially effective platform for the treatment of a wide array of diseases. However, similar to other nucleic acid-based drugs, the major hurdle of RNAi therapy is lack of efficient and non-immunogenic delivery vehicles. Currently, viruses, synthetic polymers, and lipid-based carriers are among the most widely studied vehicles for small RNA delivery. However, many drawbacks are reported to be associated with these delivery vehicles. There is a pressing need to replace them with more efficient and better-tolerated approaches. Exosomes secreted from the endocytic compartment of live cells, are a subtype of endogenous extracellular vesicles that transfer genetic and biochemical information among different cells, thus playing an important role in cell-cell communication. Recently, accumulating attention has been focused on harnessing exosomes as nanaocarriers for small RNAs delivery. Due to their natural role in shuttling endogenous nucleic acid in our body, exosomes may exhibit higher delivery efficiency, lower immunogenicity, and better compatibility than existing foreign RNA carriers. Importantly, exosomes own intrinsic homing capacity that can guide small RNAs across natural membranous barriers. Moreover, such a capacity can be further improved by adding appropriate targeting moieties. In this manuscript, we briefly review the progress and challenges of RNAi therapy, and discuss the potential of exosomes' applications in small RNA delivery with focus on the most recent advances in exosome-based small RNA delivery for disease therapy.

Intracellular distribution and internalization pathways of guanidinylated bioresponsive poly(amido amine)s in gene delivery.

Guanidinylated bioresponsive poly(amido amine)s polymers, CAR-CBA and CHL-CBA, were synthesized by Michael-type addition reaction between guanidine hydrochloride (CAR) or chlorhexidine (CHL) and N,N'-cystaminebisacrylamide (CBA). Previous studies have shown that both polymers had high transfection efficiencies as gene delivery carriers. In this study, we investigated the nucleolus localization abilities and cellular internalization pathways of these two polymers in gene delivery. Each polymer condensed plasmid DNA (pDNA) and formed nanoparticle complexes, and then their transfection studies were performed in MCF-7 cells. Both complexes were found enriched in nucleolus after cellular transfection, and their transfection efficiencies were significantly improved when transfection was performed on MCF-7 cells arrested at M phase. The transfection efficiency of CAR-CBA-pDNA was inhibited by chlorpromazine, and cell endosomes were disrupted after being exposed to CAR-CBA-pDNA. In regards to CHL-CBA-pDNA, its transfection efficiency was not affected by three types of endocytosis inhibitors used in the study, and CHL-CBA-pDNA showed no effect on endosomes. Cellular lactate dehydrogenase release and membrane morphology were changed after cells were transfected by the two complexes. The results indicated that both CAR-CBA and CHL-CBA polymers demonstrated good nucleolus localization abilities. It was beneficial for transfection when cells were arrested at M phase. CAR-CBA-pDNA cellular internalization was involved with clathrin-mediated endocytosis pathway, and escaping from endosomal entrapment, while the cellular uptake of CHL-CBA-pDNA occurs via clathrin- and caveolae-independent mechanism.

Exploring the role of peptides in polymer-based gene delivery.

Polymers are widely studied as non-viral gene vectors because of their strong DNA binding ability, capacity to carry large payload, flexibility of chemical modifications, low immunogenicity, and facile processes for manufacturing. However, high cytotoxicity and low transfection efficiency substantially restrict their application in clinical trials. Incorporating functional peptides is a promising approach to address these issues. Peptides demonstrate various functions in polymer-based gene delivery systems, such as targeting to specific cells, breaching membrane barriers, facilitating DNA condensation and release, and lowering cytotoxicity. In this review, we systematically summarize the role of peptides in polymer-based gene delivery, and elaborate how to rationally design polymer-peptide based gene delivery vectors. STATEMENT OF SIGNIFICANCE: Polymers are widely studied as non-viral gene vectors, but suffer from high cytotoxicity and low transfection efficiency. Incorporating short, bioactive peptides into polymer-based gene delivery systems can address this issue. Peptides demonstrate various functions in polymer-based gene delivery systems, such as targeting to specific cells, breaching membrane barriers, facilitating DNA condensation and release, and lowering cytotoxicity. In this review, we highlight the peptides' roles in polymer-based gene delivery, and elaborate how to utilize various functional peptides to enhance the transfection efficiency of polymers. The optimized peptide-polymer vectors should be able to alter their structures and functions according to biological microenvironments and utilize inherent intracellular pathways of cells, and consequently overcome the barriers during gene delivery to enhance transfection efficiency.

Molecular weight determination of a newly synthesized guanidinylated disulfide-containing poly(amido amine) by gel permeation chromatography.

A cationic gene delivery vector, guanidinylated disulfide-containing poly(amido amine) (CAR-CBA), was synthesized by Michael addition reaction between N,N'-cystaminebisacrylamide (CBA) and guanidine hydrochloride (CAR). Gel permeation chromatography (GPC) was used to evaluate the molecular weight of synthesized CAR-CBA. Polyethyleneimine (PEI) with molecular weight of 25 kDa was adopted as a reference, and polyethylene glycols (PEG) with different molecular weights were used to establish a standard curve for determining the molecular weight of CAR-CBA. The effects of two critical factors, namely columns and eluents, on the molecular weight measurement of CAR-CBA were investigated to optimize the GPC quantitative method. The results showed that Ultrahydrogel columns (120, 250) and HAc-NaAc (0.5 M, pH 4.5) buffer solution were the optimal column and GPC eluent, respectively. The molecular weight of the synthesized CAR-CBA was analyzed by the optimized GPC method and determined to be 24.66 kDa.

Delivery of Small Interfering RNA to Inhibit Vascular Endothelial Growth Factor in Zebrafish Using Natural Brain Endothelia Cell-Secreted Exosome Nanovesicles for the Treatment of Brain Cancer.

Although small interfering RNA (siRNA) holds great therapeutic promise, its delivery to the disease site remains a paramount obstacle. In this study, we tested whether brain endothelial cell-derived exosomes could deliver siRNA across the blood-brain barrier (BBB) in zebrafish. Natural exosomes were isolated from brain endothelial bEND.3 cell culture media and vascular endothelial growth factor (VEGF) siRNA was loaded in exosomes with the assistance of a transfection reagent. While fluorescence-activated cell flow cytometry and immunocytochemistry staining studies indicated that wild-type exosomes significantly increased the uptake of fluorescence-labeled siRNA in the autologous brain endothelial cells, decreased fluorescence intensity was observed in the cells treated with the tetraspanin CD63 antibody-blocked exosome-delivered formulation (p < 0.05). In the transport study, exosomes also enhanced the permeability of rhodamine 123 in a co-cultured monolayer of brain endothelial bEND.3 cell and astrocyte. Inhibition at the expression of VEGF RNA and protein levels was observed in glioblastoma-astrocytoma U-87 MG cells treated with exosome-delivered siRNAs. Imaging results showed that exosome delivered more siRNAs across the BBB in Tg(fli1:GFP) zebrafish. In a xenotransplanted brain tumor model, exosome-delivered VEGF siRNAs decreased the fluorescence intensity of labeled cancer cells in the brain of zebrafish. Brain endothelial cell-derived exosomes could be potentially used as a natural carrier for the brain delivery of exogenous siRNA.

Uptake Pathways of Guandinylated Disulfide Containing Polymers as Nonviral Gene Carrier Delivering DNA to Cells.

Polymers of guanidinylated disulfide containing poly(amido amine)s (Gua-SS-PAAs), have shown high transfection efficiency and low cytotoxicity. Previously, we synthesized two Gua-SS-PAA polymers, using guanidino containing monomers (i.e., arginine and agmatine, denoted as ARG and AGM, respectively) and N,N'-cystaminebisacrylamide (CBA). In this study, these two polymers, AGM-CBA and ARG-CBA were complexed with plasmid DNA, and their uptake pathway was investigated. Complexes distribution in MCF-7 cells, and changes on cell endosomes/lysosomes and membrane after the cells were exposed to complexes were tested. In addition, how the transfection efficiency changed with the cell cycle status as well as endocytosis inhibitors were studied. The polymers of AGM-CBA and ARG-CBA can avoid endosomal/lysosomal trap, therefore, greatly delivering plasmid DNA (pDNA) to the cell nucleoli. It is the guanidine groups in the polymers that enhanced complexes' permeation through cell membrane with slight membrane damage, and targeting to the nucleoli. J. Cell. Biochem. 118: 903-913, 2017. © 2016 Wiley Periodicals, Inc.

Novel guanidinylated bioresponsive poly(amidoamine)s designed for short hairpin RNA delivery.

Two different disulfide (SS)-containing poly(amidoamine) (PAA) polymers were constructed using guanidino (Gua)-containing monomers (ie, arginine [Arg] and agmatine [Agm]) and N,N'-cystamine bisacrylamide (CBA) by Michael-addition polymerization. In order to characterize these two Gua-SS-PAA polymers and investigate their potentials as short hairpin RNA (shRNA)-delivery carriers, pSilencer 4.1-CMV FANCF shRNA was chosen as a model plasmid DNA to form complexes with these two polymers. The Gua-SS-PAAs and plasmid DNA complexes were determined with particle sizes less than 90 nm and positive ζ-potentials under 20 mV at nucleic acid:polymer weight ratios lower than 1:24. Bioresponsive release of plasmid DNA was observed from both newly constructed complexes. Significantly lower cytotoxicity was observed for both polymer complexes compared with polyethylenimine and Lipofectamine 2000, two widely used transfection reagents as reference carriers. Arg-CBA showed higher transfection efficiency and gene-silencing efficiency in MCF7 cells than Agm-CBA and the reference carriers. In addition, the cellular uptake of Arg-CBA in MCF7 cells was found to be higher and faster than Agm-CBA and the reference carriers. Similarly, plasmid DNA transport into the nucleus mediated by Arg-CBA was more than that by Agm-CBA and the reference carriers. The study suggested that guanidine and carboxyl introduced into Gua-SS-PAAs polymers resulted in a better nuclear localization effect, which played a key role in the observed enhancement of transfection efficiency and low cytotoxicity. Overall, two newly synthesized Gua-SS-PAAs polymers demonstrated great potential to be used as shRNA carriers for gene-therapy applications.

Exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in Danio rerio.

PURPOSE: The blood-brain barrier (BBB) essentially restricts therapeutic drugs from entering into the brain. This study tests the hypothesis that brain endothelial cell derived exosomes can deliver anticancer drug across the BBB for the treatment of brain cancer in a zebrafish (Danio rerio) model. MATERIALS AND METHODS: Four types of exosomes were isolated from brain cell culture media and characterized by particle size, morphology, total protein, and transmembrane protein markers. Transport mechanism, cell uptake, and cytotoxicity of optimized exosome delivery system were tested. Brain distribution of exosome delivered anticancer drugs was evaluated using transgenic zebrafish TG (fli1: GFP) embryos and efficacies of optimized formations were examined in a xenotransplanted zebrafish model of brain cancer model. RESULTS: Four exosomes in 30-100 diameters showed different morphologies and exosomes derived from brain endothelial cells expressed more CD63 tetraspanins transmembrane proteins. Optimized exosomes increased the uptake of fluorescent marker via receptor mediated endocytosis and cytotoxicity of anticancer drugs in cancer cells. Images of the zebrafish showed exosome delivered anticancer drugs crossed the BBB and entered into the brain. In the brain cancer model, exosome delivered anticancer drugs significantly decreased fluorescent intensity of xenotransplanted cancer cells and tumor growth marker. CONCLUSIONS: Brain endothelial cell derived exosomes could be potentially used as a carrier for brain delivery of anticancer drug for the treatment of brain cancer.

In vitro evaluation of optimized liposomes for delivery of small interfering RNA.

One of the biggest challenges for small interfering RNAs (siRNAs) as therapeutic agents is their insufficient cellular delivery efficiency. We developed long circulating and cationic liposomes to improve the cell uptake and inhibitory effectiveness of siRNA on the expression of vascular endothelial growth factor (VEGF) in cancer cells. SiRNA liposomes were obtained by polyelectrolyte complexation between negatively charged siRNA and positively charged liposome prepared by a hydration method. Gel electrophoresis was used to evaluate the loading efficiency of siRNA on the cationic liposome. The optimized siRNA liposomes were observed to be spherical in shape and had smooth surfaces with particle sizes of 167.7 ± 2.0 nm and zeta potentials of 4.03 ± 0.69 mV, which had no significant change when stored at 4 °C for three months. Fluorescence-activated cell sorting studies and confocal laser scanning images indicated that the cationic liposomes significantly increased the uptake of fluorescence-labeled siRNA in cancer cells. Effects of the siRNA on the inhibition of VEGF were tested by measuring concentrations of VEGF in cell culture media via an enzyme-linked immunosorbent assay and intracellular VEGF levels using a western blotting method. The liposomal siRNA was significantly effective at inhibiting the expression of VEGF in lung, liver and breast cancer cells. Optimal liposomes could effectively deliver siRNA into cancer cells and inhibit VEGF as a therapy agent.

Comparative studies on chitosan and polylactic-co-glycolic acid incorporated nanoparticles of low molecular weight heparin.

This study was performed to test the feasibility of chitosan and polylactic-co-glycolic acid (PLGA) incorporated nanoparticles as sustained-release carriers for the delivery of negatively charged low molecular weight heparin (LMWH). Fourier transform infrared (FTIR) spectrometry was used to evaluate the interactions between chitosan and LMWH. The shifts, intensity, and broadening of the characteristic peaks for the functional groups in the FTIR spectra indicated that strong interactions occur between the positively charged chitosans and the negatively charged LMWHs. Three types of LMWH nanoparticles (NP-1, NP-2, and NP-3) were prepared using chitosan with or without PLGA: NP-1 nanoparticles were formed by polyelectrolyte complexation after single mixing, NP-2 nanoparticles were prepared by polyelectrolyte complexation after single emulsion-diffusion-evaporation, and NP-3 nanoparticles were optimized by double emulsion-diffusion-evaporation. NP-3 nanoparticles of LMWH prepared by the emulsion-diffusion-evaporation method showed significant differences in particle morphology, size, zeta potential, and drug release profile compared to NP-1 nanoparticles formed by polyelectrolyte complexation. Another ionic complex of LMWH with chitosan-incorporated PLGA nanoparticles (NP-2) showed lower drug entrapment efficiency than that of NP-1 and NP-3. The drug release rate of NP-3 was slower than the release rates of NP-1 and NP-2, although particle morphology of NP-3 was similar to that of NP-2. Cell viability was not adversely affected when cells were treated with all three types of nanoparticles. The data presented in this study demonstrate that nanoparticles formulated with chitosan-PLGA could be a safe sustained-release carrier for the delivery of LMWH.

Nicotine exacerbates brain edema during in vitro and in vivo focal ischemic conditions.

We have previously shown that nicotine, the addictive component of tobacco products, alters the blood-brain barrier (BBB) Na(+),K(+),2Cl(-) cotransporter (NKCC) during in vitro hypoxia-aglycemia exposure. Attenuation of abluminal NKCC suggests that accumulation of ions in the brain extracellular fluid would result in an increase of fluid or cytotoxic edema in the brain during hypoxia-aglycemia or stroke conditions. To further investigate whether nicotine products have the potential to worsen stroke outcome by increasing edema formation, two separate models to mimic stroke conditions were utilized to decipher the effects of short-term and long-term administrations of nicotine products on brain edema following stroke. Oxygen glucose deprivation (OGD) was studied in rat hippocampal slices with short-term or long-term exposure to nicotine and cigarette smoke constituents. During short-term exposure, the presence of nicotine at a concentration mimicking heavy smokers increased water content of hippocampal slices during OGD. Furthermore, long-term 1-week administration of nicotine increased water content in hippocampal slices that could be attenuated with nicotine acetylcholine receptor (nAChR) antagonists, suggesting nicotine increase edema during OGD via nAChRs. A second model of focal ischemia, middle cerebral artery occlusion, showed an increase of infarct size during short-term exposure to nicotine and an increase of edema during both short-term and long-term administration of nicotine, compared with saline controls. These findings support the paradigm that nicotine products not only increase the incidence of stroke but also have the potential to worsen stroke outcome by increased edema formation.