Purpose

For optimizing the local, pulmonary targeting of inhaled medications, it is important to analyze the relationship between the physicochemical properties of small molecules and their absorption, retention and distribution in the various cell types of the airways and alveoli.

Methods

A computational, multiscale, cell-based model was constructed to facilitate analysis of pulmonary drug transport and distribution. The relationship between the physicochemical properties and pharmacokinetic profile of monobasic molecules was explored. Experimental absorption data of compounds with diverse structures were used to validate this model. Simulations were performed to evaluate the effect of active transport and organelle sequestration on the absorption kinetics of compounds.

Results

Relating the physicochemical properties to the pharmacokinetic profiles of small molecules reveals how the absorption half-life and distribution of compounds are expected to vary in different cell types and anatomical regions of the lung. Based on logP, pK_a and molecular radius, the absorption rate constants (K_a) calculated with the model were consistent with experimental measurements of pulmonary drug absorption.

Conclusions

The cell-based mechanistic model developed herein is an important step towards the rational design of local, lung-targeted medications, facilitating the design and interpretation of experiments aimed at optimizing drug transport properties in lung.

Purpose

To develop an efficient biocompatible and targeted drug delivery system in which platelets, an essential blood component having a natural affinity for cancer cells, are used as carrier of anticancer drug as delivery of drug to the targeted site is crucial for cancer treatment.

Methods

Doxorubicin hydrochloride, a potent anti cancer drug, was delivered in lung adenocarcinoma cell line (A549) using platelet as a delivery agent. This delivery mode was also tested in Ehrlich ascites carcinoma (EAC) bearing mice in presence and absence of platelets.

Results

The results show that platelets can uptake the drug and release the same upon activation. The efficiency of drug loaded platelets in inducing cytotoxicity was significantly higher in both in vitro and in vivo model, as compared to the free drug.

Conclusions

The proposed drug delivery strategy may lead to clinical improvement in the management of cancer treatment as lower drug concentration can be used in a targeted mode. Additionally the method can be personalized as patient's own platelet can be used for deliver various drugs.

Purpose. Nanoparticles have advantage as CNS drug delivery vehicles given they disguise drug permeation limiting characteristics. Conflicting toxicological data, however, is published with regard to blood-brain barrier integrity and gross mortality.

Methods. To address this issue two novel nanoparticle types: “emulsifying wax/Brij 78”and Brij 72/Tween 80 nanoparticles were evaluated in vivo for effect on cerebral perfusion flow, barrier integrity, and permeability using the in situ brain perfusion technique. Additional evaluation was completed in vitro using bovine brain microvessel endothelial cells for effect on integrity, permeability, cationic transport interactions, and tight junction protein expression.

Results. In the presence of either nanoparticle formulation, no overall significant differences were observed for cerebral perfusion flow in vivo. Furthermore, observed in vitro and in vivo data showed no statistical changes in barrier integrity, membrane permeability, or facilitated choline transport. Western blot analyses of occludin and claudin-1 confirmed no protein expression changes with incubation of either nanoparticle.

Conclusions. The nanoparticle formulations appear to have no effect on primary BBB parameters in established in vitro and in vivo blood-brain barrier models.

Purpose

Virus-like particles (VLPs) have been used as drug carriers for drug delivery systems. In this study, hCC49 single chain fragment variable (scFv)-displaying Rous sarcoma virus-like particles (RSV VLPs) were produced in silkworm larvae to be a specific carrier of an anti-cancer drug.

Method

RSV VLPs displaying hCC49 scFv were created by the fusion of the transmembrane and cytoplasmic domains of hemagglutinin from influenza A (H1N1) virus and produced in silkworm larvae. The display of hCC49 scFv on the surface of RSV VLPs was confirmed by enzyme-linked immunosorbent assay using tumor-associated glycoprotein-72 (TAG-72), fluorescent microscopy, and immunoelectron microscopy. Fluorescein isothiocyanate (FITC) or doxorubicin (DOX) was incorporated into hCC49 scFv-displaying RSV VLPs by electroporation and specific targeting of these VLPs was investigated by fluorescent microscopy and cytotoxicity assay using LS174T cells.

Results

FITC was delivered to LS174T human colon adenocarcinoma cells by hCC49 scFv-displaying RSV VLPs, but not by RSV VLPs. This indicated that hCC49 scFv allowed FITC-loaded RSV VLPs to be delivered to LS174T cells. DOX, which is an anti-cancer drug with intrinsic red fluorescence, was also loaded into hCC49 scFv-displaying RSV VLPs by electroporation; the DOX-loaded hCC49 scFv-displaying RSV VLPs killed LS174T cells via the specific delivery of DOX that was mediated by hCC49 scFv. HEK293 cells were alive even though in the presence of DOX-loaded hCC49 scFv-displaying RSV VLPs.

Conclusion

These results showed that hCC49 scFv-displaying RSV VLPs from silkworm larvae offered specific drug delivery to colon carcinoma cells in vitro. This scFv-displaying enveloped VLP system could be applied to drug and gene delivery to other target cells.

Purpose. Dequalinium, a drug known for over 30 years, is a dicationic amphiphile compound resembling bolaform electrolytes. The purpose of our work was to determine the state of aggregation of dequalinium in aqueous medium and to investigate both, its ability to bind DNA and its potential to serve as a novel non-viral transfection vector.

Methods. The form of aggregation was determined employing electron microscopic techniques. The DNA binding capacity of dequalinium was assayed using SYBR™ Green I stain. For in vitro cell transfection experiments plasmid DNA encoding for firefly luciferase was used.

Results. Dequalinium forms in aqueous medium liposome-like aggregates, which we term DQAsomes. These dequalinium vesicles bind DNA and they are able to transfect cells in vitro with an efficiency comparable to Lipofectin™.

Conclusions. Based on the intrinsic properties of dequalinium such as the in vivo selectivity for carcinoma cells and selective accumulation in mitochondria we propose DQAsomes as a novel and unique drug and gene delivery system.

A growing share of the pharmaceutical development pipeline is occupied by macromolecule drugs, which are primarily administered by injection. Despite decades of attempts, non-invasive delivery of macromolecules has seen only a few success stories. Potential benefits of non-invasive administration include better patient acceptance and adherence and potentially better efficacy and safety. Greater inter-disciplinary dialogue and collaboration are integral to realizing these benefits.

Purpose

The present study is focused on the development of a model drug delivery system (DDS) based on Chitosan (CS) nanoparticles using Renin substrate I (RSI) as model agent. RSI shares the main chemical-physical features of several biologically active antimicrobial peptides (AMPs). AMPs have a great therapeutic potential that is hampered by their lability in the biological fluids and as such they are perfect candidates for DDS. The development studies of quality DDS loaded with AMPs would require highly sensitive and specific quantification assays. The use of RSI allowed for the fine-tuning and optimization of the formulation parameters to promote the hydrophobic interactions between CS and the cationic peptide, favour the loading of the active ingredient and enhance the release properties of the carrier.

Methods

RSI was encapsulated in chitosan NPs by mean of ionic gelation and a chromogenic enzymatic essay was carried out for the release kinetics evaluation.

Results

The developed formulations displayed almost 100% of encapsulation efficacy, low burst percentages, and a linear release of the model peptide. A release model was created showing a direct dependence on both the amount of RSI and NPs radius.

Conclusions

Although CS has always been formulated with negatively charged active agents (e.g. oligonucleotides or anionic proteins), the use of ionotropic gelation in presence of a small cationic active agent promoted the formation of “core-shell” NPs. The described model, with tuneable linear release rates, appears eligible for further exploitation such as the loading of therapeutically active AMPs.

Purpose. To evaluate the transport characteristics of horseradish peroxidase (HRP, a nonspecific fluid-phase endocytosis marker) across an in vitro model of tight (> 2,000 ohm-cm²) rat alveolar epithelial cell monolayers grown on tissue culture-treated polycarbonate filters.

Methods. Unidirectional HRP fluxes were estimated from the appearance rate of HRP in the receiver fluid following instillation in the donor fluid as a function of donor [HRP] and temperature. Molecular species present in either bathing fluid were determined at the end of flux experiments using fluorescein isothiocyanate (FITC)-labeled HRP by gel permeation chromatography. Cell-associated HRP activity at the end of the transport experiment was determined, as were the rates of recycling and transcellular movement of HRP. An enzymatic assay was used to quantify HRP activity in the bathing fluid and cells.

Results. Unidirectional HRP fluxes were symmetric and increased linearly with up to 50 µM donor [HRP]. The apparent permeability coefficient of HRP was reduced by 3.5 times upon lowering the temperature from 37 to 4°C. About 50% of the FITC-labeled species present in either receiver fluid was intact HRP. Cell-associated HRP estimated from apical HRP incubation was about 4 times greater than that from basolateral incubation. Recycling into apical fluid of cell-associated HRP following apical incubation occurred rapidly with a half-time (T_1/2) of ~5 min, reaching a plateau at ~67% of the initial cell-associated HRP, while transcellular movement of HRP (into basolateral fluid) took place with a T_1/2 of ~20 min, attaining a steady-state at ~13% of the initial cell-associated HRP. Basolateral recycling of HRP was also rapid (T_1/2 = ~5 min) reaching a steady-state at ~35% of the initial basolaterally-bound HRP. Transcellular movement of HRP following basolateral incubation was slower (T_1/2 = ~70 min), leveling off at 50% of the initial cell-associated HRP.

Conclusions. HRP appears to be transported relatively intact (~50%) across rat alveolar epithelial barrier via nonspecific fluid-phase endocytosis. The transepithelial pinocytotic rate of alveolar epithelial cells is estimated to be about 25 nL/cm²/h.

ABSTRACT

A major challenge in the development of central nervous system drugs is to obtain therapeutic effective drug concentrations inside the brain. Many potentially effective drugs have never reached clinical application because of poor brain penetration. Currently, devices are being developed that may improve drug delivery into the brain. One approach involves the encapsulation of drugs into nanocarriers that are targeted to the brain, where the drug is released. Alternatively, living cells have been engineered to produce the pharmaceutical of interest at the target site. It is important to follow the fate of these drug delivery devices inside the body to verify their efficiency in reaching the brain. To this end, both ex-vivo approaches and in-vivo imaging techniques are used, including ex-vivo biodistribution, autoradiography, MRI, optical imaging, PET and SPECT. All these methods have their specific advantages and limitations. Consequently, selection of the tracking method should be based on the specific aims of the experiment. Here, we will discuss the methods that are currently applied for tracking brain drug delivery devices, including the most commonly used labels and labeling procedures for living cells and nanocarriers. Subsequently, we will discuss specific applications in tracking drug delivery devices.

This review introduces the basics of fluorescence recovery after photobleaching (FRAP) from a theoretical and an instrumentational approach. The most interesting and innovative applications with a pharmaceutical point of view are briefly discussed and possible future applications are suggested. These future applications include research on the mobility of macromolecular drugs in macro- or microscopic pharmaceutical dosage forms, mobility, and binding of antitumor drugs in tumor tissue, intracellular trafficking of gene complexes and mobility of drugs in membranes prior to transmembrane penetration. The paper is also intended to be an introductory guideline to those who would like to get involved in FRAP related experimental techniques. Therefore, comprehensive details on different setups and data analysis are given, as well as a brief outline of the problems that may be encountered when performing FRAP. Overall, this review shows the great potential of FRAP in pharmaceutical research. This is complemented by our own results illustrating the possibility of performing FRAP in microscopic dosage forms (microspheres) using a high resolution variant of FRAP.