Vivek Gupta

Using shape effects to target antibody-coated nanoparticles to lung and brain endothelium

Vascular endothelium offers a variety of therapeutic targets for the treatment of cancer, cardiovascular diseases, inflammation, and oxidative stress. Significant research has been focused on developing agents to target the endothelium in diseased tissues. This includes identification of antibodies against adhesion molecules and neovascular expression markers or peptides discovered using phage display. Such targeting molecules also have been used to deliver nanoparticles to the endothelium of the diseased tissue. Here we report, based on in vitro and in vivo studies, that the specificity of endothelial targeting can be enhanced further by engineering the shape of ligand-displaying nanoparticles. In vitro studies performed using microfluidic systems that mimic the vasculature (synthetic microvascular networks) showed that rod-shaped nanoparticles exhibit higher specific and lower nonspecific accumulation under flow at the target compared with their spherical counterparts. Mathematical modeling of particle–surface interactions suggests that the higher avidity and specificity of nanorods originate from the balance of polyvalent interactions that favor adhesion and entropic losses as well as shear-induced detachment that reduce binding. In vivo experiments in mice confirmed that shape-induced enhancement of vascular targeting is also observed under physiological conditions in lungs and brain for nanoparticles displaying anti–intracellular adhesion molecule 1 and anti-transferrin receptor antibodies.

Delivering Nanoparticles to Lungs while Avoiding Liver and Spleen through Adsorption on Red Blood Cells

Nanoparticulate drug delivery systems are one of the most widely investigated approaches for developing novel therapies for a variety of diseases. However, rapid clearance and poor targeting limit their clinical utility. Here, we describe an approach to harness the flexibility, circulation, and vascular mobility of red blood cells (RBCs) to simultaneously overcome these limitations (cellular hitchhiking). A noncovalent attachment of nanoparticles to RBCs simultaneously increases their level in blood over a 24 h period and allows transient accumulation in the lungs, while reducing their uptake by liver and spleen. RBC-adsorbed nanoparticles exhibited ∼3-fold increase in blood persistence and ∼7-fold higher accumulation in lungs. RBC-adsorbed nanoparticles improved lung/liver and lung/spleen nanoparticle accumulation by over 15-fold and 10-fold, respectively. Accumulation in lungs is attributed to mechanical transfer of particles from the RBC surface to lung endothelium. Independent tracing of both nanoparticles and RBCs in vivo confirmed that RBCs themselves do not accumulate in lungs. Attachment of anti-ICAM-1 antibody to the exposed surface of NPs that were attached to RBCs led to further increase in lung targeting and retention over 24 h. Cellular hitchhiking onto RBCs provides a new platform for improving the blood pharmacokinetics and vascular delivery of nanoparticles while simultaneously avoiding uptake by liver and spleen, thus opening the door for new applications.

PEG-PLGA based large porous particles for pulmonary delivery of a highly soluble drug, low molecular weight heparin.

This study was designed to evaluate the feasibility of PEG-PLGA copolymers as carriers for pulmonary delivery of a highly soluble drug. We attempt to address the limitations of low entrapment efficiencies and poor release profiles that are associated with the use of conventional PLGAs. We have used low molecular weight heparin (LMWH) as a model for highly soluble and ionizable drugs and a 3 × 3 full factorial design to prepare nine formulations. We considered polymer type and percent NaCl in external phase as two independent variables at three different levels; the levels for polymer type were PLGA, PEG-PLGA and PLGA-PEG-PLGA and that for percent NaCl were 0%, 5% and 8%. Formulations were characterized for various physical properties, respirability, drug release, and evaluated for in vivo absorption, alveolar uptake, and safety. Statistical analyses suggest that both polymer type and salt concentration influenced the morphology and micromeritic properties of the particles. Compared with PLGA, PEG-PLGA copolymers produced inherently larger porous particles with high drug entrapment and a greater extent of drug release. Moreover, addition of NaCl in the external phase maximized drug entrapment but minimized burst release and produced smaller and denser particles. Fluorescent PEG-PLGA particles showed reduced uptake by alveolar macrophages, and exhibited a uniform distribution in the lungs compared with PLGA particles. Further, ~85% of the particles were cleared off the lungs within 6 days. Intratracheally administered PEG-PLGA based optimized formulation exhibited a biological half-life of 18.64 h, which was ~4.5 times longer than plain LMWH. No cytotoxic effect was observed when bronchial epithelial cells were incubated with PEG-PLGA based formulations. Similarly, no increase in the injury markers was observed in the bronchoalveolar lavage fluids collected from rats treated with PEG-PLGA particles of LMWH. Overall, this study suggests that PEG-PLGA block copolymers have the potential to be developed as efficient and biocompatible carriers for pulmonary delivery of highly water-soluble drugs.

Influence of surface charge of PLGA particles of recombinant hepatitis B surface antigen in enhancing systemic and mucosal immune responses

This study investigates the efficacy of surface-modified microspheres of hepatitis B surface antigen (HBsAg) in eliciting systemic and mucosal immune responses. Positively charged poly(D,L-lactic-co-glycolic acid) microspheres were prepared by a double-emulsion solvent-evaporation method with cationic agents--stearylamine and polyethylenimine--in the external aqueous phase. Formulations were characterized for morphology, size, density, aerodynamic diameter, entrapment efficiency and in vitro drug-release profile. Immunization was performed after pulmonary administration of the formulations to female Sprague-Dawley rats and the immune response was monitored by measuring IgG levels in serum and secretory (sIgA) levels in salivary, vaginal and bronchoalveolar lavage fluids. The cell-mediated immune response was studied by measuring cytokine levels in spleen homogenates, and a cytotoxicity study was performed with Calu-3 cell line. The aerodynamic diameter of the particles was within the respirable range, with the exception of stearylamine-modified particles. Zeta potential values moved from negative (-6.76 mV) for unmodified formulations to positive (+0.515 mV) for polyethylenimine-modified particles. Compared to unmodified formulations, polyethylenimine-based formulations showed continuous release of antigen over a period of 28-42 days and increased levels of IgG in serum and sIgA in salivary, vaginal and bronchoalveolar lavage. Further, cytokine levels-interferon gamma and interleukin-2-were increased in spleen homogenates. The viability of Calu-3 cells was not adversely affected by the microparticles. In summation, this study establishes that positive surface charges on poly(D,L-lactic-co-glycolic acid) particles containing HBsAg enhances both the systemic and mucosal immune response upon immunization via the respiratory route.

In vitro, in vivo and ex vivo models for studying particle deposition and drug absorption of inhaled pharmaceuticals

Delivery of therapeutic agents via the pulmonary route has gained significant attention over the past few decades because this route of administration offers multiple advantages over traditional routes that include localized action, non-invasive nature and favorable lung-to-plasma ratio. However, assessment of post administration behavior of inhaled pharmaceuticals-such as deposition of particles over the respiratory airways, interaction with the respiratory fluid and movement across the air-blood barrier-is challenging because the lung is a very complex organs that is composed of airways with thousands of bifurcations with variable diameters. Thus, much effort has been put forward to develop models that mimic human lungs and allow evaluation of various pharmaceutical and physiological factors that influence the deposition and absorption profiles of inhaled formulations. In this review, we sought to discuss in vitro, in vivo and ex vivo models that have been extensively used to study the behaviors of airborne particles in the lungs and determine the absorption of drugs after pulmonary administration. We have provided a summary of lung cast models, cascade impactors, noninvasive imaging, intact animals, cell culture and isolated perfused lung models as tools to evaluate the distribution and absorption of inhaled particles. We have also outlined the limitations of currently used models and proposed future studies to enhance the reproducibility of these models.

Liposomal fasudil, a rho-kinase inhibitor, for prolonged pulmonary preferential vasodilation in pulmonary arterial hypertension

Current pharmacological interventions for pulmonary arterial hypertension (PAH) require continuous infusions, multiple inhalations, or oral administration of drugs that act on various pathways involved in the pathogenesis of PAH. However, invasive methods of administration, short duration of action, and lack of pulmonary selectivity result in noncompliance and poor patient outcomes. In this study, we tested the hypothesis that encapsulation of an investigational anti-PAH molecule fasudil (HA-1077), a Rho-kinase inhibitor, into liposomal vesicles results in prolonged vasodilation in distal pulmonary arterioles. Liposomes were prepared by hydration and extrusion method and fasudil was loaded by ammonium sulfate-induced transmembrane electrochemical gradient. Liposomes were then characterized for various physicochemical properties. Optimized formulations were tested for pulmonary absorption and their pharmacological efficacy in a monocrotaline (MCT) induced rat model of PAH. The entrapment efficiency of optimized liposomal fasudil formulations was between 68.1±0.8% and 73.6±2.3%, and the cumulative release at 37°C was 98-99% over a period of 5 days. Compared to intravenous (IV) fasudil, a ~10 fold increase in the terminal plasma half-life was observed when liposomal fasudil was administered as aerosols. The t1/2 of IV fasudil was 0.39±0.12 h. and when given as liposomes via pulmonary route, the t1/2 extended to 4.71±0.72 h. One h after intratracheal instillation of liposomal fasudil, mean pulmonary arterial pressure (MPAP) was reduced by 37.6±5.7% and continued to decrease for about 3 h, suggesting that liposomal formulations produced pulmonary preferential vasodilation in MCT induced PAH rats. Overall, this study established the proof-of-principle that aerosolized liposomal fasudil is a feasible option for a non-invasive, controlled release and pulmonary preferential treatment of PAH.

Topical delivery of siRNA into skin using SPACE-peptide carriers.

Short-interfering RNAs (siRNAs) offer a potential tool for the treatment of skin disorders. However, applications of siRNA for dermatological conditions are limited by their poor permeation across the stratum corneum of the skin and low penetration into the skins viable cells. In this study, we report the use of SPACE-peptide in combination with a DOTAP-based ethosomal carrier system to enhance skin delivery of siRNA. A DOTAP-based SPACE Ethosomal System significantly enhanced siRNA penetration into porcine skin in vitro by 6.3±1.7-fold (p<0.01) with an approximately 10-fold (p<0.01) increase in epidermis accumulation of siRNA compared to that from an aqueous solution. Penetration of siRNA was also enhanced at the cellular level. Internalization of SPACE-peptide occurred in a concentration dependent manner marked by a shift in intracellular distribution from punctate spots to diffused cytoplasmic staining at a peptide concentration of 10mg/mL. In vitro delivery of GAPDH siRNA by SPACE peptide led to 83.3±3.0% knockdown relative to the control. In vivo experiments performed using female BALB/C mice also confirmed the efficacy of DOTAP-SES in delivering GAPDH-siRNA into skin. Topical application of DOTAP-SES on mice skin resulted in 63.2%±7.7% of GAPDH knockdown, which was significantly higher than that from GAPDH-siRNA PBS (p<0.05). DOTAP-SES formulation reported here may open new opportunities for cutaneous siRNA delivery.

A permeation enhancer for increasing transport of therapeutic macromolecules across the intestine

Delivery of therapeutic macromolecules is limited by the physiological limitations of the gastrointestinal tract including poor intestinal permeability, low pH and enzymatic activity. Several permeation enhancers have been proposed to enhance intestinal permeability of macromolecules; however their utility is often hindered by toxicity and limited potency. Here, we report on a novel permeation enhancer, Dimethyl palmitoyl ammonio propanesulfonate (PPS), with excellent enhancement potential and minimal toxicity. PPS was tested for dose- and time-dependent cytotoxicity, delivery of two model fluorescent molecules, sulforhodamine-B and FITC-insulin in vitro, and absorption enhancement of salmon calcitonin (sCT) in vivo. Caco-2 studies revealed that PPS is an effective enhancer of macromolecular transport while being minimally toxic. TEER measurements in Caco-2 monolayers confirmed the reversibility of the effect of PPS. Confocal microscopy studies revealed that molecules permeate via both paracellular and transcellular pathways in the presence of PPS. In vivo studies in rats showed that PPS enhanced relative bioavailability of sCT by 45-fold after intestinal administration. Histological studies showed that PPS does not induce damage to the intestine. PPS is an excellent permeation enhancer which provides new opportunities for developing efficacious oral/intestinal delivery systems for therapeutic macromolecules.

Topical delivery of hyaluronic acid into skin using SPACE-peptide carriers.

Topical penetration of macromolecules into the skin is limited by their low permeability. Here, we report the use of a skin penetrating peptide, SPACE peptide, to enhance topical delivery of a macromolecule, hyaluronic acid (HA, MW: 200-325kDa). The peptide was conjugated to phospholipids and used to prepare an ethosomal carrier system (~110nm diameter), encapsulating HA. The SPACE-ethosomal system (SES) enhanced HA penetration into porcine skin in vitro by 7.8+/-1.1-fold compared to PBS. The system also enhanced penetration of HA in human skin in vitro, penetrating deep into the epidermis and dermis in skin of both species. In vivo experiments performed using SKH1 hairless mice also confirmed increased dermal penetration of HA using the delivery system; a 5-fold enhancement in penetration was found compared to PBS control. Concentrations of HA in skin were about 1000-fold higher than those in blood; confirming the localized nature of HA delivery into skin. The SPACE-ethosomal delivery system provides a formulation for topical delivery of macromolecules that are otherwise difficult to deliver into the skin.

Mucoadhesive intestinal devices for oral delivery of salmon calcitonin.

One of the major challenges faced by therapeutic polypeptides remains their invasive route of delivery. Oral administration offers a potential alternative to injections; however, this route cannot be currently used for peptides due to their limited stability in the stomach and poor permeation across the intestine. Here, we report mucoadhesive devices for oral delivery that are inspired by the design of transdermal patches and demonstrate their capabilities in vivo for salmon calcitonin (sCT). The mucoadhesive devices were prepared by compressing a polymeric matrix containing carbopol, pectin and sodium carboxymethylcellulose (1:1:2), and were coated on all sides but one with an impermeable and flexible ethyl cellulose (EC) backing layer. Devices were tested for in vitro dissolution, mucoadhesion to intestinal mucosa, enhancement of drug absorption in vitro (Caco-2 monolayer transport) and in vivo in rats. Devices showed steady drug release with ≈75% cumulative drug released in 5h. Devices also demonstrated strong mucoadhesion to porcine small intestine to withstand forces up to 100 times their own weight. sCT-loaded mucoadhesive devices exhibited delivery of sCT across Caco-2 monolayers and across the intestinal epithelium in vivo in rats. A ≈52-fold (pharmacokinetic) and ≈44-fold (pharmacological) enhancement of oral bioavailability was observed with mucoadhesive devices when compared to direct intestinal injections. Oral delivery of devices in enteric coated capsules resulted in significant bioavailability enhancement.