In vitro and in vivo mRNA delivery using lipid-enveloped pHresponsive polymer nanoparticles

Biodegradable core−shell structured nanoparticles with a poly(β-amino ester) (PBAE) core enveloped by a phospholipid bilayer shell were developed for in vivo mRNA delivery with a view toward delivery of mRNA-based vaccines. The pH-responsive PBAE component was chosen to promote endosome disruption, while the lipid surface layer was selected to minimize toxicity of the polycation core. Messenger RNA was efficiently adsorbed via electrostatic interactions onto the surface of these net positively charged nanoparticles. In vitro, mRNA-loaded particle uptake by dendritic cells led to mRNA delivery into the cytosol with low cytotoxicity, followed by translation of the encoded protein in these difficult-to-transfect cells at a frequency of 30%. Particles loaded with mRNA administered intranasally (i.n.) in mice led to the expression of the reporter protein luciferase in vivo as soon as 6 h after administration, a time point when naked mRNA given i.n. showed no expression. At later time points, luciferase expression was detected in naked mRNA-treated mice, but this group showed a wide variation in levels of transfection, compared to particle-treated mice. This system may thus be promising for noninvasive delivery of mRNA-based vaccines.United States. Dept. of Defense (Institute for Soldier Nanotechnology, contract W911NF-07-D-0004)Ragon Institute of MGH, MIT and HarvardSingapore. Agency for Science, Technology and ResearchHoward Hughes Medical Institute (Investigator

Permeability enhancers dramatically increase zanamivir absolute bioavailability in rats: implications for an orally bioavailable influenza treatment.

We have demonstrated that simple formulations composed of the parent drug in combination with generally regarded as safe (GRAS) permeability enhancers are capable of dramatically increasing the absolute bioavailability of zanamivir. This has the advantage of not requiring modification of the drug structure to promote absorption, thus reducing the regulatory challenges involved in conversion of an inhaled to oral route of administration of an approved drug. Absolute bioavailability increases of up to 24-fold were observed when Capmul MCM L8 (composed of mono- and diglycerides of caprylic/capric acids in glycerol) was mixed with 1.5 mg of zanamivir and administered intraduodenally to rats. Rapid uptake (t(max) of 5 min) and a C(max) of over 7200 ng/mL was achieved. Variation of the drug load or amount of enhancer demonstrated a generally linear variation in absorption, indicating an ability to optimize a formulation for a desired outcome such as a targeted C(max) for enzyme saturation. No absorption enhancement was observed when the enhancer was given 2 hr prior to drug administration, indicating, in combination with the observed tmax, that absorption enhancement is temporary. This property is significant and aligns well with therapeutic applications to limit undesirable drug-drug interactions, potentially due to the presence of other poorly absorbed polar drugs. These results suggest that optimal human oral dosage forms of zanamivir should be enteric-coated gelcaps or softgels for intraduodenal release. There continues to be a strong need and market for multiple neuraminidase inhibitors for influenza treatment. Creation of orally available formulations of inhibitor drugs that are currently administered intravenously or by inhalation would provide a significant improvement in treatment of influenza. The very simple GRAS formulation components and anticipated dosage forms would require low manufacturing costs and yield enhanced convenience. These results are being utilized to design prototype dosage forms for initial human pharmacokinetic studies

Augmentation of Therapeutic Efficacy in Drug-Resistant Tumor Models Using Ceramide Coadministration in Temporal-Controlled Polymer-Blend Nanoparticle Delivery Systems

The development of multidrug resistance (MDR) is a major hindrance to cancer eradication as it renders tumors unresponsive to most chemotherapeutic treatments and is associated with cancer resurgence. This study describes a novel mechanism to overcome MDR through a polymer-blend nanoparticle platform that delivers a combination therapy of C6-ceramide (CER), a synthetic analog of an endogenously occurring apoptotic modulator, together with the chemotherapeutic drug paclitaxel (PTX), in a single formulation. The PTX/CER combination therapy circumvents another cellular mechanism whereby MDR develops, by lowering the threshold for apoptotic signaling. In vivo studies in a resistant subcutaneous SKOV3 human ovarian and in an orthotopic MCF7 human breast adenocarcinoma xenograft showed that the PTX and CER nanoparticle combination therapy reduced the final tumor volume at least twofold over treatment with the standard PTX therapy alone. The study also revealed that the cotherapy accomplished this enhanced efficacy by generating an enhancement in apoptotic signaling in both tumor types. Additionally, acute evaluation of safety with the combination therapy did not show significant changes in body weight, white blood cell counts, or liver enzyme levels. The temporal-controlled nanoparticle delivery system presented in this study allows for a simultaneous delivery of PTX + CER in breast and ovarian tumor model drug, leading to a modulation of the apoptotic threshold. This strategy has tremendous potential for effective treatment of refractory disease in cancer patients

Permeability of zanamivir through Caco-2 cell monolayers in the absence and presence of absorption enhancers.

<p>The enhancers 0.25% Capmul MCM L8 and 5% glycerol resulted in 5.2- and 5.6-fold increased permeability, respectively, compared to the no-enhancer negative control. Zanamivir had significantly higher (p<0.01) permeability in the presence of the indicated enhancers compared to the PBS control. Error bars depict standard deviation.</p

Timecourse of changes in plasma zanamivir concentrations with differing intraduodenally administered formulations versus intravenous administration of zanamivir in PBS.

<p>A rapid uptake from the intraduodenally administered Capmul MCM L8 formulation was observed which was rapidly cleared in a manner corresponding to the intravenous administration route. All animals were dosed with 1.5 mg of zanamivir regardless of dosing route or formulation. Error bars depict standard deviation.</p

Effect of increasing Capmul MCM L8 on the absolute bioavailability of 1.5 mg of zanamivir after intraduodenal administration in rats.

<p>Zanamivir had significantly higher (p<0.01) absolute bioavailability when dosed in the Capmul MCM L8 formulation compared to the control and there was a significant difference (p<0.05) in the absolute bioavailability observed between 25 µL and 75 µL Capmul MCM L8 volume dosed groups. Error bars depict standard deviation.</p

Effect of increasing zanamivir in the presence of 50 µL of Capmul MCM L8 on zanamivir absolute bioavailability after intraduodenal administration in rats.

<p>Zanamivir had significantly higher (p<0.01) absolute bioavailability when dosed in the Capmul MCM L8 formulation compared to the PBS control. Error bars depict standard deviation.</p

Summary of Pharmacokinetic Parameters for Zanamivir from Different Formulations after Intraduodenal Administration in Male Sprague-Dawley Rats at 1.5 mg/animal.

<p>C_max: Maximum plasma concentration; t_max: Time to maximum plasma concentration; t_1/2: half-life; AUC_last: Area Under the Curve, calculated to the last observable time point; AUC_∞: Area Under the Curve, extrapolated to infinity;</p>1<p>Dose normalized by dividing the parameter by the nominal dose of 1.5 mg/animal.</p