Effectiveness of Small Interfering RNA Delivery via Arginine-Rich Polyethylenimine-Based Polyplex in Metastatic and Doxorubicin-Resistant Breast Cancer Cells.

Poor cellular uptake, rapid degradation in the presence of serum, and inefficient transfection are some of the major barriers in achieving therapeutic efficacy of naked small interfering RNAs (siRNAs). We investigated the efficacy of the polyplex formulated using our synthesized polymer, polyethylene glycol (PEG)-modified l-arginine oligo(-alkylaminosiloxane) that is grafted with poly(ethyleneimine) (PEI) for siRNA delivery. We hypothesized that the polyplex formulated using the polymer with a balanced composition of PEI for siRNA condensation and its protection, PEG for polyplex stability and to minimize the PEI-associated toxicity, and with arginine facilitating cellular uptake would overcome the aforementioned issues with siRNA delivery. We tested our hypothesis using antiluciferase siRNA in luciferase-expressing metastatic breast cancer cells (MDA-MB-231-Luc-D3H2LN) and anti-ABCB1 siRNA against an efflux membrane protein, ABCB1, in doxorubicin (DOX)-resistant breast cancer cells (MCF-7/Adr). The results demonstrated that the polyplex at an optimal nucleotide/polymer ratio is stable in the presence of excess polyanions, has no cellular toxicity, and protects siRNA from RNase degradation. Transfection of MDA-MB-231-Luc-D3H2LN cells with antiluciferase siRNA polyplex showed almost complete knockdown of luciferase expression. In MCF-7/Adr cells, transfection with anti-ABCB1 siRNA effectively downregulated its target efflux protein, ABCB1; increased cellular uptake of DOX; and enhanced its cytotoxic effect. However, the cotreatment did not completely overcome drug resistance, suggesting that further optimization is needed and/or a mechanism(s) other than the efflux protein ABCB1 may be involved in drug resistance. In conclusion, our polyplex is effective for siRNA delivery and can be explored for different therapeutic applications.

Big bottlenecks in cardiovascular tissue engineering.

Although tissue engineering using human-induced pluripotent stem cells is a promising approach for treatment of cardiovascular diseases, some limiting factors include the survival, electrical integration, maturity, scalability, and immune response of three-dimensional (3D) engineered tissues. Here we discuss these important roadblocks facing the tissue engineering field and suggest potential approaches to overcome these challenges.

Mechanical Properties, Cytocompatibility and Manufacturability of Chitosan:PEGDA Hybrid-Gel Scaffolds by Stereolithography.

Extracellular matrix mimetic hydrogels which hybridize synthetic and natural polymers offer molecularly-tailored, bioactive properties and tunable mechanical strength. In addition, 3D bioprinting by stereolithography allows fabrication of internal pores and defined macroscopic shapes. In this study, we formulated a hybrid biocompatible resin using natural and synthetic polymers (chitosan and polyethylene glycol diacrylate (PEGDA), respectively) by controlling molecular weight of chitosan, feed-ratios, and photo-initiator concentration. Ear-shaped, hybrid scaffolds were fabricated by a stereolithographic method using a 405 nm laser. Hybrid hydrogel scaffolds of chitosan (50-190 kDa) and PEGDA (575 Da) were mixed at varying feed-ratios. Some of the cationic, amino groups of chitosan were neutralized by dialysis in acidic solution containing chitosan in excess of sodium acetate solution to inhibit quenching of newly formed photoradicals. A feed-ratio of 1:7.5 was found to be the most appropriate of the formulations considered in this study in terms of mechanical properties, cell adhesion, and printability. The biofabricated hybrid scaffold showed interconnected, homogeneous pores with a nominal pore size of 50 µm and an elastic modulus of ~400 kPa. Moreover, long-term cell viability and cell spreading was observed via actin filament staining. Printability of the biocompatible resin was confirmed by printing thresholded MR images of an ear and the feed ratio of 1:7.5 provided the most faithful reproduction of the shape. To the best of our knowledge, this is the first report of stereolithographic printing hybridizing cell-adhesive properties of chitosan with mechanical robustness of PEG in scaffolds suitable for repair of complex tissue geometries, such as those of the human ear.

Tissue plasminogen activator followed by antioxidant-loaded nanoparticle delivery promotes activation/mobilization of progenitor cells in infarcted rat brain.

Inherent neuronal and circulating progenitor cells play important roles in facilitating neuronal and functional recovery post stroke. However, this endogenous repair process is rather limited, primarily due to unfavorable conditions in the infarcted brain involving reactive oxygen species (ROS)-mediated oxidative stress and inflammation following ischemia/reperfusion injury. We hypothesized that during reperfusion, effective delivery of antioxidants to ischemic brain would create an environment without such oxidative stress and inflammation, thus promoting activation and mobilization of progenitor cells in the infarcted brain. We administered recombinant human tissue-type plasminogen activator (tPA) via carotid artery at 3 h post stroke in a thromboembolic rat model, followed by sequential administration of the antioxidants catalase (CAT) and superoxide dismutase (SOD), encapsulated in biodegradable nanoparticles (nano-CAT/SOD). Brains were harvested at 48 h post stroke for immunohistochemical analysis. Ipsilateral brain slices from animals that had received tPA + nano-CAT/SOD showed a widespread distribution of glial fibrillary acidic protein-positive cells (with morphology resembling radial glia-like neural precursor cells) and nestin-positive cells (indicating the presence of immature neurons); such cells were considerably fewer in untreated animals or those treated with tPA alone. Brain sections from animals receiving tPA + nano-CAT/SOD also showed much greater numbers of SOX2- and nestin-positive progenitor cells migrating from subventricular zone of the lateral ventricle and entering the rostral migratory stream than in t-PA alone treated group or untreated control. Further, animals treated with tPA + nano-CAT/SOD showed far fewer caspase-positive cells and fewer neutrophils than did other groups, as well as an inhibition of hippocampal swelling. These results suggest that the antioxidants mitigated the inflammatory response, protected neuronal cells from undergoing apoptosis, and inhibited edema formation by protecting the blood-brain barrier from ROS-mediated reperfusion injury. A longer-term study would enable us to determine if our approach would assist progenitor cells to undergo neurogenesis and to facilitate neurological and functional recovery following stroke and reperfusion injury.

Arginine-rich polyplexes for gene delivery to neuronal cells.

Neuronal gene therapy potentially offers an effective therapeutic intervention to cure or slow the progression of neurological diseases. However, neuronal cells are difficult to transfect with nonviral vectors, and in vivo their transport across the blood-brain barrier (BBB) is inefficient. We synthesized a series of arginine-rich oligopeptides, grafted with polyethyleneimine (PEI) and modified with a short-chain polyethylene glycol (PEG). We hypothesized that the arginine would enhance cellular uptake and transport of these polyplexes across the BBB, with PEG imparting biocompatibility and "stealth" properties and PEI facilitating DNA condensation and gene transfection. The optimized composition of the polyplexes demonstrated hemocompatibility with red blood cells, causing no lysis or aggregation, and showed significantly better cytocompatibility than PEI in vitro. Polyplexes formulated with luciferase-expressing plasmid DNA could transfect rat primary astrocytes and neurons in vitro. Confocal imaging data showed efficient cellular uptake of DNA and its sustained intracellular retention and nuclear localization with polyplexes. Intravenous administration of the optimized polyplexes in mice led to gene expression in the brain, which upon further immunohistochemical analysis demonstrated gene expression in neurons. In conclusion, we have successfully designed a nonviral vector for in vitro and in vivo neuronal gene delivery.

Codelivery of DNA and siRNA via arginine-rich PEI-based polyplexes.

In this study, we formulated polyplexes with different compositions for codelivery of DNA and small-interfering RNA (siRNA). Since DNA and siRNA have distinctive and complementary morphological characteristics (DNA is long and winding and siRNA is short and rigid), we hypothesized that their codelivery using polyplex would enhance each other's transfection. To test this hypothesis, cationic polymer branched polyethylenimine (bPEI) as a standard transfecting agent and its derivative arginine-rich oligopeptide-grafted bPEI modified with polyethylene glycol (P(SiDAAr)5P3), synthesized in our laboratory, were used as carriers for transfection. Polyplexes at different nucleic acid to polymer weight ratios were characterized for transfection in breast cancer sensitive (MCF-7) and resistant (MCF-7/Adr) cell lines. Gene silencing effect of polyplexes was determined in MDA-MB-231-luc-D3H2LN cell line. The results demonstrated that the polyplexes formed with derivative P(SiDAAr)5P3 show significantly lower toxicity compared to polyplexes formed using bPEI. Further, codelivery resulted in 20-fold higher DNA transfection and 2-fold higher siRNA transfection as compared to the respective single nucleotide delivery. DNA transfection was ∼100-fold lower in resistant MCF-7/Adr cells than in sensitive MCF-7 cells. Confocal imaging and flow cytometry data demonstrated that enhanced transfection does not solely depend on DNA's cellular uptake, suggesting that other mechanisms contribute to increased transfection. DNA-co-siRNA delivery could be a promising therapeutic approach to achieve synergistic effects because it can simultaneously target and interfere with multiple regulatory levels in a cell to halt and reverse disease progression.

Superoxide dismutase-loaded biodegradable nanoparticles targeted with a follicle-stimulating hormone peptide protect Sertoli cells from oxidative stress.

OBJECTIVE: To evaluate targeted superoxide dismutase (SOD)-loaded biodegradable nanoparticles' (NPs) ability to protect Sertoli cells from hydrogen peroxide (H2O2)-induced oxidative stress. DESIGN: Cell culture controlled experimental study. SETTING: Research laboratory. CELLS: Mouse testis Sertoli cells (TM4). INTERVENTIONS: Sertoli cells were exposed to 0-200 µg/mL plain media, unconjugated NPs, or FSH peptide-conjugated NPs for 2 or 24 hours to assess uptake. Next, Sertoli cells were exposed to 0-50 mmol H2O2 with 0-1 mg/mL unconjugated SOD-loaded NPs, FSH-conjugated SOD-loaded NPs, or equivalent units of SOD in solution as a control for 2-6 hours to assess influence on cell survival after oxidative stress. MAIN OUTCOME MEASURE(S): Cell viability, flow cytometry, and microscopy. RESULT(S): FSH peptide targeting improved uptake of NPs by Sertoli cells. FSH-conjugated SOD-NPs significantly protected Sertoli cells at 6 hours of H2O2--induced oxidative stress, with 100% survival with FSH-conjugated SOD-NPs compared with unconjugated SOD-NPs (45%) or SOD in solution (36%). CONCLUSION(S): Conjugation of NPs with FSH peptide improves cellular uptake and survival when SOD-loaded NPs are coincubated with Sertoli cells undergoing oxidative stress. This study represents a step toward developing NPs for the targeted treatment of testicular oxidative stress.

Advances in stroke therapy.

Stroke is a leading cause of death, long-term disability, and socioeconomic costs, highlighting the urgent need for more effective treatments. Intravenous administration of tissue plasminogen activator (t-PA) is the only FDA-approved therapy to re-establish cerebral blood flow. However, because of increased risk of hemorrhage beyond 3 h post stroke, few stroke patients (1-2%) benefit from t-PA; t-PA, which has neurotoxic effects, can also aggravate the extent of reperfusion injury by increasing blood-brain barrier permeability. An alternative strategy is needed to extend the window of intervention, minimize damage from reperfusion injury, and promote brain repair leading to neurological recovery. Reactive oxygen species (ROS), generated soon after ischemia and during reperfusion and thereafter, are considered the main mediators of ischemic injury. Antioxidant enzymes such as catalase, superoxide dismutase, etc. can neutralize ROS-mediated injury but their effective delivery to the brain remains a challenge. In this article, we review various therapeutic approaches including surgical interventions, and discuss the potential of nanoparticle-mediated delivery of antioxidants for stroke therapy.

Folate mediated l-arginine modified oligo (alkylaminosiloxane) graft poly (ethyleneimine) for tumor targeted gene delivery.

Folate receptor mediated gene targeting provides several advantages such as delivery of high concentration of gene at specific tumor sites including brain, lung, ovary, uterus and kidney where folate receptors are over expressed. In the present study for both systemic stability and tumor targeting ability, poly (ethylene glycol)-folic acid (PEG-FA) conjugate was coupled with an arginine modified oligo (alkylaminosiloxane) graft poly (ethyleneimine) having enhanced transfection efficiency compared to poly (ethyleneimine). The resultant polymer P(SiDAAr)5FP2 complexed pDNA effectively and showed protection against nuclease degradation. The PEG group provided improved blood compatibility and cell viability. Uniformly oriented arginine moiety helped to enhance cellular and nuclear localisation, which led to improved transfection. The polymer was capable of releasing pDNA at the nucleus and being cleared from the cell after its purpose. Transfection in presence of cellular uptake inhibitors showed multiple pathways for cellular uptake of the targeted polymer, out of which folate receptor mediated uptake was more prominent. The folate mediated cellular uptake of P(SiDAAr)5FP2 was then confirmed by flow cytometric evaluation. The high accumulation of targeted polymers in the tumor tissues of tumor bearing mice from 2nd hour onwards proved the active targeting effect of the polymer. Besides tumor accumulation, the material showed capability to diffuse through the vascular endothelium. This property is expected to be beneficial for brain targeting experiments.

Enhanced in-vitro transfection and biocompatibility of L-arginine modified oligo (-alkylaminosiloxanes)-graft-polyethylenimine.

Branched poly ethylene imine (PEI) has been considered as the most efficient non-viral gene transfection agent. However, its clinical application is confined due to cytotoxicity. In the present study, we tried to enhance transfection efficiency and reduce toxicity of PEI by conjugating it with arginine modified oligo(-alkylaminosiloxane) [P(SiDAAr)n]. These derivatives were complexed with plasmid DNA and the resulting nanoparticles were characterised by dynamic light scattering (DLS), Atomic force microscopy (AFM), transmission electron microscopy (TEM), gel retardation and DNase I interaction to determine surface charge, particle size, morphology, complex formation and protection of DNA respectively. Among the four P(SiDAAr)n derivatives, nanoparticles of the P(SiDAAr)5/pDNA was found to exhibit 98% cell viability and around 150% more gene transfection than branched PEI in KB cell lines. Studies performed on transfection mechanism, using inhibitor study, clearly stated that the enhancement in transfection is due to the multiple pathways for cellular uptake which offered by the presence of uniformly spaced arginine moiety by oligo(-alkylaminosiloxane) arms. The nuclear localisation ability of the arginine residue was also established by using FITC stained nanoparticles on Hoechst 33342 stained nucleus.

Folate mediated in vitro targeting of depolymerised trimethylated chitosan having arginine functionality.

Delivery vectors having targeting ligands provide an impending impact on cancer gene therapy. Our work focuses on folate mediated targeting induced by conjugating poly(ethylene glycol)-folate (PEG-FA) with arginine modified chitosan polymer having low molecular weight of 15 kDa and high degree of quaternization (ATFP15H). The ATFP15H derivative on condensation with plasmid DNA formed nanoparticles with core shell nanostructure. It also affirmed good buffering capacity. The derivative was found to protect DNA from DNase I degradation and also from disassembly in presence of negatively charged plasma proteins. It exhibited blood compatibility in terms of percentage hemolysis, erythrocyte aggregation and also by platelet activation. At a concentration of 10 microg, the capability of the derivative to enhance cell growth at normal cell growing conditions was observed. The transfection efficiency was also found to be comparable to PEI when transfected in KB cell line, which over expressed the folate receptor (FR) in presence of 10% fetal bovine serum (FBS). On comparison with native chitosan and trimethylated chitosan, ATFP15H derivative exhibited high cellular uptake and nuclear localization due to the superior colloidal stability attained on conjugation with polyethylene glycol. This has been ascertained by flow cytometry and YOYO labeling of plasmid DNA.

Folate mediated histidine derivative of quaternised chitosan as a gene delivery vector.

Folate targeted gene delivery vectors showed enhanced accumulation in folate receptor expressing tumor model. In the present work, the water solubility and transfection efficiency of chitosans were improved by modifying the depolymerised trimethylated chitosans with histidine moiety. Folate mediated targeting was induced by conjugating poly(ethylene glycol)-folate (PEG-FA) on histidine modified chitosan polymer having low molecular weight of 15 kDa and high degree of quaternisation (HTFP15-H). The zeta potential and size of the HTFP15-H/pDNA nanoparticles were determined using dynamic light scattering technique and the results were confirmed by transmission electron microscopy (TEM). The morphology of the nanoparticles was found spherical in shape having core-shell nanostructure. The HTFP15-H derivative found to buffer in the pH range from 10 to 4. The blood compatibility in terms of percentage hemolysis, erythrocyte aggregation and also by platelet activation was found to be significantly improved compared to the control vector PEI. At a concentration of 10 microg the derivative promote the cell growth up to 139% compared to control at normal cell growing conditions. The transfection efficiency in KB cell line, which over expresses the folate receptor (FR) in presence of 10% fetal bovine serum (FBS) was also found to be comparable to the control. Moreover the enhanced cellular and nuclear uptake due to the conjugation of both folic acid and histidine makes it a potential vector for gene delivery applications.

Studies on the condensation of depolymerized chitosans with DNA for preparing chitosan-DNA nanoparticles for gene delivery applications.

High molecular weight chitosan (CS) was depolymerized by oxidative degradation with NaNO(2) at room temperature to get 11 samples of CS derivatives of varying molecular weights with a view to assessing their effective molecular weight range for gene delivery applications. Viscosity studies indicated that the molecular weight of the depolymerized CS was proportional to the CS/NaNO(2) ratio. The condensation behavior of DNA/CS complexes at various charge ratios was studied using UV spectroscopy, FTIR, CD, SEM, and AFM. The results indicated that CSs having very low molecular weights and high charge density exhibited strong binding affinity to DNA compared to high molecular weight CSs. However, the very low molecular weight (1.9-7.7 kDa) CSs were found to form aggregates easily even at very low charge ratios. On the other hand, CSs having medium molecular weight (49-51 kDa) and high degree of deacetylation (DD) gave stable uniform-sized nanoparticles. Biological studies carried out with the spherical nano-sized polyplexes formed between CS of 50 kDa (DD of 94%) and pEGFP plasmid DNA at N/P ratio of 5.0 showed excellent gene transfection efficiency at pH 6.5 in HeLa cells without cytotoxicity indicating their potential as gene delivery carriers.