Kenneth A. Howard

Size-Dependent Accumulation of PEGylated Silane-Coated Magnetic Iron Oxide Nanoparticles in Murine Tumors.

Magnetic nanoparticles (MNP) can be used as contrast-enhancing agents to visualize tumors by magnetic resonance imaging (MRI). Here we describe an easy synthesis method of magnetic nanoparticles coated with polyethylene glycol (PEG) and demonstrate size-dependent accumulation in murine tumors following intravenous injection. Biocompatible iron oxide MNPs coated with PEG were prepared by replacing oleic acid with a biocompatible and commercially available silane-PEG to provide an easy and effective method for chemical coating. The colloidal stable PEGylated MNPs were magnetically separated into two distinct size subpopulations of 20 and 40 nm mean diameters with increased phagocytic uptake observed for the 40 nm size range in vitro. MRI detection revealed greater iron accumulation in murine tumors for 40 nm nanoparticles after intravenous injection. The enhanced MRI contrast of the larger MNPs in the tumor may be a combined result of the size-dependent extravasation and capture by macrophages in the tumor, providing important considerations for improved bioimaging approaches.

Delivery of RNA interference therapeutics using polycation-based nanoparticles

RNAi-based therapies are dependent on extracellular and intracellular delivery of RNA molecules for enabling target interaction. Polycation-based nanoparticles (or polyplexes) formed by self-assembly with RNA can be used to modulate pharmacokinetics and intracellular trafficking to improve the therapeutic efficacy of RNAi-based therapeutics. This review describes the application of polyplexes for extracellular and intracellular delivery of synthetic RNA molecules. Focus is given to routes of administration and silencing effects in animal disease models. The inclusion of functional components into the nanoparticle for controlling cellular trafficking and RNA release is discussed. This work highlights the versatile nature of polycation-based nanoparticles to fulfil the delivery requirements for RNA molecules with flexibility in design to evolve alongside an expanding repertoire of RNAi-based drugs.

The Effect of Chemical Modification and Nanoparticle Formulation on Stability and Biodistribution of siRNA in Mice

Instability and inadequate biodistribution of double-stranded RNA are major drawbacks to the clinical use of RNA interference. This work compares chemical modification and nanoparticle formulation as strategies to improve the systemic delivery of small interfering RNA (siRNA). Variable levels of chemical modified siRNA, either naked or within nanoparticle, were intravenously injected into mice to study temporal stability and biodistribution detected by direct radioactive labeling or by northern blotting. Naked siRNA showed rapid renal clearance, with circulatory half-life of <5 minutes that could be extended to >30 minutes by cholesterol conjugation. The integrity of the chemically stabilized siRNA was maintained in blood for at least 30 minutes, whereas, unmodified siRNA duplex was degraded within 1 minute. Intact chemically modified siRNA could also be detected in all analyzed organs at 30 minutes but disappeared at 24 hours, except for heavy locked nucleic acid (LNA)-modified and cholesterol-conjugated siRNA in the lungs. Chitosan, liposomal, or JetPEI formulation greatly improved the stability and biodistribution of siRNA. Interestingly, high siRNA accumulation of the chitosan/siRNA formulation within the kidney was observed 24 hours postadministration. This comparative study highlights improvements to siRNA stability and pharmacokinetics, key determinants for development of clinically relevant RNAi therapeutics.

Chitosan/siRNA Nanoparticles Encapsulated in PLGA Nanofibers for siRNA Delivery

Composite nanofibers of biodegradable poly(D,L-lactic-co-glycolic acid) (PLGA) encapsulating chitosan/siRNA nanoparticles (NPs) were prepared by electrospinning. Acidic/alkaline hydrolysis and a bulk/surface degradation mechanism were investigated in order to achieve an optimized release profile for prolonged and efficient gene silencing. Thermo-controlled AFM in situ imaging not only revealed the integrity of the encapsulated chitosan/siRNA polyplex but also shed light on the decreasing T(g) of PLGA on the fiber surfaces during release. A triphasic release profile based on bulk erosion was obtained at pH 7.4, while a triphasic release profile involving both surface erosion and bulk erosion was obtained at pH 5.5. A short alkaline pretreatment provided a homogeneous hydrolysis and consequently a nearly zero-order release profile. The interesting release profile was further investigated for siRNA transfection, where the encapsulated chitosan/siRNA NPs exhibited up to 50% EGFP gene silencing activity after 48 h post-transfection on H1299 cells.

Cellular disposal of miR23b by RAB27-dependent exosome release is linked to acquisition of metastatic properties.

Exosomes are small secreted vesicles that can transfer their content to recipient cells. In cancer, exosome secretion has been implicated in tumor growth and metastatic spread. In this study, we explored the possibility that exosomal pathways might discard tumor-suppressor miRNA that restricts metastatic progression. Secreted miRNA characterized from isogenic bladder carcinoma cell lines with differing metastatic potential were uncoupled from binding to target transcripts or the AGO2-miRISC complex. In metastatic cells, we observed a relative increase in secretion of miRNA with tumor-suppressor functions, including miR23b, miR224, and miR921. Ectopic expression of miR23b inhibited invasion, anoikis, angiogenesis, and pulmonary metastasis. Silencing of the exocytotic RAB family members RAB27A or RAB27B halted miR23b and miR921 secretion and reduced cellular invasion. Clinically, elevated levels of RAB27B expression were linked to poor prognosis in two independent cohorts of patients with bladder cancer. Moreover, highly exocytosed miRNA from metastatic cells, such as miR23b, were reduced in lymph node metastases compared with patient-matched primary tumors and were correlated with increments in miRNA-targeted RNA. Taken together, our results suggested that exosome-mediated secretion of tumor-suppressor miRNA is selected during tumor progression as a mechanism to coordinate activation of a metastatic cascade.

Polycation-based nanoparticle delivery of RNAi therapeutics: adverse effects and solutions.

Small interfering RNA (siRNA) that silence genes by the process of RNA interference offers a new therapeutic modality for disease treatment. Polycation-based nanoparticles termed polyplexes have been developed to maximise extracellular and intracellular siRNA delivery, a key requirement for enabling the clinical translation of RNAi-based drugs. Medical applications are dependent on safety; therefore, detailed investigation into potential toxicity to the cell or organism is required. This review addresses potential adverse effects arising from cellular and tissue interactions, immune stimulation and altered gene expression that can be associated with the assembled polyplex or the polycation and siRNA component parts. A greater understanding of the cellular mechanisms involved allows design-based solutions for rationale development of safe, effective and clinically relevant polyplex-based RNAi drugs.

Multifunctional Bismuth Selenide Nanocomposites for Antitumor Thermo-Chemotherapy and Imaging

To integrate real-time monitoring and therapeutic functions into a single nanoagent, we have designed and synthesized a drug-delivery platform based on a polydopamine(PDA)/human serum albumin (HSA)/doxorubicin (DOX) coated bismuth selenide (Bi2Se3) nanoparticle (NP). The resultant product exhibits high stability and biocompatibility both in vitro and in vivo. In addition to the excellent capability for both X-ray computed tomography (CT) and infrared thermal imaging, the NPs possess strong near-infrared (NIR) absorbance, and high capability and stability of photothermal conversion for efficient photothermal therapy (PTT) applications. Furthermore, a bimodal on-demand pH/photothermal-sensitive drug release has been achieved, resulting in a significant chemotherapeutic effect. Most importantly, the tumor-growth inhibition ratio achieved from thermo-chemotherapy of the Bi2Se3@PDA/DOX/HSA NPs was 92.6%, in comparison to the chemotherapy (27.8%) or PTT (73.6%) alone, showing a superior synergistic therapeutic effect. In addition, there is no noticeable toxicity induced by the NPs in vivo. This multifunctional platform is, therefore, promising for effective, safe and precise antitumor treatment and may stimulate interest in further exploration of drug loading on Bi2Se3 and other competent PTT agents combined with in situ imaging for biomedical applications.

Antimicrobial Effect of Chitosan Nanoparticles on Streptococcus mutans Biofilms

ABSTRACT Nanoparticle complexes were prepared from chitosans of various molecular weights (MW) and degrees of deacetylation (DD). The antimicrobial effect was assessed by the Live/Dead BacLight technique in conjunction with confocal scanning laser microscopy (CSLM) and image analysis. Nanocomplexes prepared from chitosans with high MW showed a low antimicrobial effect (20 to 25% of cells damaged), whereas those prepared from low-MW chitosans showed high antimicrobial effect (>95% of cells damaged).

Comparative analysis of discrete exosome fractions obtained by differential centrifugation

Background Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ in size, density and composition. The standard isolation method for exosomes is centrifugation of fluid samples, typically at 100,000×g or above. Knowledge of the effect of discrete ultracentrifugation speeds on the purification from different cell types, however, is limited. Methods We examined the effect of applying differential centrifugation g-forces ranging from 33,000×g to 200,000×g on exosome yield and purity, using 2 unrelated human cell lines, embryonic kidney HEK293 cells and bladder carcinoma FL3 cells. The fractions were evaluated by nanoparticle tracking analysis (NTA), total protein quantification and immunoblotting for CD81, TSG101, syntenin, VDAC1 and calreticulin. Results NTA revealed the lowest background particle count in Dulbeccos Modified Eagles Medium media devoid of phenol red and cleared by 200,000×g overnight centrifugation. The centrifugation tube fill level impacted the sedimentation efficacy. Comparative analysis by NTA, protein quantification, and detection of exosomal and contamination markers identified differences in vesicle size, concentration and composition of the obtained fractions. In addition, HEK293 and FL3 vesicles displayed marked differences in sedimentation characteristics. Exosomes were pelleted already at 33,000×g, a g-force which also removed most contaminating microsomes. Optimal vesicle-to-protein yield was obtained at 67,000×g for HEK293 cells but 100,000×g for FL3 cells. Relative expression of exosomal markers (TSG101, CD81, syntenin) suggested presence of exosome subpopulations with variable sedimentation characteristics. Conclusions Specific g-force/k factor usage during differential centrifugation greatly influences the purity and yield of exosomes. The vesicle sedimentation profile differed between the 2 cell lines.

Pulmonary Gene Silencing in Transgenic EGFP Mice Using Aerosolised Chitosan/siRNA Nanoparticles

ABSTRACTPurposeThis work describes the production and application of an aerosolised formulation of chitosan nanoparticles for improved pulmonary siRNA delivery and gene silencing in mice.MethodsAerosolised chitosan/siRNA nanoparticles were pneumatically formed using a nebulising catheter and sized by laser diffraction. In vitro silencing of aerosolised and non-aerosolised formulations was evaluated in an EGFP endogenous-expressing H1299 cell line by flow cytometry. Non-invasive intratracheal insertion of the catheter was used to study nanoparticle deposition by histological detection of Cy3-labeled siRNA and gene silencing in transgenic EGFP mouse lungs using a flow cytometric method.ResultsFlow cytometric analysis demonstrated minimal alteration in gene silencing efficiency before (68%) and after (62%) aerosolisation in EGFP-expressing H1299 cells. Intratracheal catheter administration in mice resulted in nanoparticle deposition throughout the entire lung in both alveoli and bronchiolar regions using low amounts of siRNA. Transgenic EGFP mice dosed with the aerosolised nanoparticle formulation showed significant EGFP gene silencing (68% reduction compared to mismatch group).ConclusionsThis work provides a technology platform for effective pulmonary delivery and gene silencing of RNAi therapeutics with potential use in preclinical studies of respiratory disease treatment.