Sunday A. Shoyele

The Effects of Excipients and Particle Engineering on the Biophysical Stability and Aerosol Performance of Parathyroid Hormone (1-34) Prepared as a Dry Powder for Inhalation

Pulmonary delivery of therapeutic peptides and proteins has many advantages including high relative bioavailability, rapid systemic absorption and onset of action and a non-invasive mode of administration which improves patient compliance. In this study, we investigated the effect of spray-drying (SD) and spray freeze-drying processes on the stability and aerosol performance of parathyroid hormone (PTH) (1-34) microparticles. In this study, the stabilisation effect of trehalose (a non-reducing sugar) and Brij 97 (a non-ionic surfactant) on spray-dried PTH particles was assessed using analytical techniques including circular dichroism (CD), fluorescence spectroscopy, modulated differential scanning calorimetry and an in vitro bioactivity assay. Physical characterisation also included electron microscopy, tap density measurement and laser light diffraction. The aerosol aerodynamic performance of the formulations was assessed using the Andersen cascade impactor. Based on these studies, a formulation for spray freeze-drying was selected and the effects of the two particle engineering techniques on the biophysical stability and aerosol performance of the resulting powders was determined. CD, fluorescence spectroscopy and bioactivity data suggest that trehalose when used alone as a stabilising excipient produces a superior stabilising effect than when used in combination with a non-ionic surfactant. This highlights the utility of CD and fluorescence spectroscopy studies for the prediction of protein bioactivity post-processing. Therefore, a method and formulation suitable for the preparation of PTH as a dry powder was developed based on spray-drying PTH with trehalose as a stabiliser with the bioactivity of SD PTH containing trehalose being equivalent to that of unprocessed PTH.

Quercetin regulates β-catenin signaling and reduces the migration of triple negative breast cancer.

Triple negative breast cancer (TNBC) is characterized by a lack in estrogen, progesterone, and epidermal growth factor 2 receptors. TNBC exhibits most of the characteristics of basal‐like and claudin‐low breast cancer subtypes. The main contributor in the mortality of TNBC is due to the higher invasive and migratory ability of these tumor cells. Some plant flavonoids inhibit the epithelial mesenchymal transition (EMT) of tumor cells and suppress cancer metastasis. In this study, we aimed to determine whether the flavonoid quercetin is effective in modulating the molecular signaling associated with EMT in TNBC. Our data indicated that quercetin can induce the expression of E‐cadherin and also downregulate vimentin levels in TNBC. The ability of quercetin to modulate these EMT markers resulted in a mesenchymal‐to‐epithelial transition (MET). Quercetin‐induced MET was linked with the alteration of nuclear localization of β‐catenin and modulation of β‐catenin target genes such as cyclin D1 and c‐Myc. Furthermore, we observed that quercetin induced the anti‐tumor activity of doxorubicin by inhibiting the migratory ability of TNBC cells. These results suggested that quercetin may inhibit TNBC metastasis and also improve the therapeutic efficacy of existing chemotherapeutic drugs.

Controlling the release of proteins/peptides via the pulmonary route.

The inhalation route is seen as the most promising non-invasive alternative for the delivery of proteins; however, the short duration of activity of drugs delivered via this route brought about by the activities of alveolar macrophages and mucociliary clearance means there is a need to develop controlled release system to prolong the activities of proteins delivered to the lung. Polymeric materials such as (D,L)-poly(lactic glycolic acid) (PLGA), chitosan and poly(ethylene glycol) (PEGs) have been used for controlled release of proteins. Other systems such as liposomes and microcrystallization have also proved effective. This chapter gives a more detailed understanding of these techniques and the manufacture of the delivery systems.

Investigation of the stability and cellular uptake of self-associated monoclonal antibody (MAb) nanoparticles by non-small lung cancer cells.

The inability to deliver MAbs to intracellular targets still remains a limitation to their application in cancer therapy and diagnosis. Selective targeting of MAbs to oncoproteins in cancer cells while avoiding their accumulation in normal cells may reduce some of the well-documented adverse effects accompanying antibody therapy. One of the remarkable characteristics of malignant cells is the alteration in the biological properties of the cellular plasma membrane. Taking advantage of this alteration, we hope to selectively deliver self-associated MAb nanoparticles to cancer cells while reducing their accumulation in normal cells. We hypothesized that self-associated MAb nanoparticles can be preferentially taken up by non-small lung cancer cells in comparison to normal cells due to the absence or dysfunction of tight junctions (TJ) in confluent cancer cells and increased permeability of the cancer cell membrane. Self-associated bevacizumab nanoparticles were prepared and characterized for particle size and biochemical stability. Fluorescence microscopy, TEM, and flow cytometry revealed that these bevacizumab nanoparticles were internalized by A549 cells three times more than MRC-5 cells. Macropinocytosis and energy-dependent pathways were elucidated to be involved in their uptake by A549 cells. Further, uptake was by nonspecific interaction with cell membrane. Results obtained from this study suggest that self-associated MAb nanoparticles can be selectively delivered to cancer cells.

Engineering Protein Particles for Pulmonary Drug Delivery

Pulmonary delivery of proteins requires particles for delivery to be in the aerodynamic size range 1-5 microm for deep lung deposition. However, the traditional particle size reduction technique of jet-milling normally used for inhalation is not suitable for processing these protein particles because of their lability brought about by the weak physical interactions making up their higher order structures. Advanced techniques such as spray drying, spray freeze drying and the use of supercritical fluid technology have been developed to produce particles in the suitable size range and morphology for deep long deposition without altering the native conformation of these biomolecules. Judicious use of excipients and operating conditions are some of the factors needed for a successful particle design.

Novel targeted siRNA-loaded hybrid nanoparticles: preparation, characterization and in vitro evaluation

BackgroundsiRNAs have a high potential for silencing critical molecular pathways that are pathogenic. Nevertheless, their clinical application has been limited by a lack of effective and safe nanotechnology-based delivery system that allows a controlled and safe transfection to cytosol of targeted cells without the associated adverse effects. Our group recently reported a very effective and safe hybrid nanoparticle delivery system composing human IgG and poloxamer-188 for siRNA delivery to cancer cells. However, these nanoparticles need to be optimized in terms of particle size, loading capacity and encapsulation efficiency. In the present study, we explored the effects of certain production parameters on particle size, loading capacity and encapsulation efficiency. Further, to make these nanoparticles more specific in their delivery of siRNA, we conjugated anti-NTSR1-mAb to the surface of these nanoparticles to target NTSR1-overexpressing cancer cells. The mechanism of siRNA release from these antiNTSR1-mAb functionalized nanoparticles was also elucidated.ResultsIt was demonstrated that the concentration of human IgG in the starting nanoprecipitation medium and the rotation speed of the magnetic stirrer influenced the encapsulation efficiency, loading capacity and the size of the nanoparticles produced. We also successfully transformed these nanoparticles into actively targeted nanoparticles by functionalizing with anti-NTSR1-mAb to specifically target NTSR1-overexpressing cancer cells, hence able to avoid undesired accumulation in normal cells. The mechanism of siRNA release from these nanoparticles was elucidated to be by Fickian diffusion. Using flow cytometry and fluorescence microscopy, we were able to confirm the active involvement of NTSR1 in the uptake of these anti-NTSR1-mAb functionalized hybrid nanoparticles by lung adenocarcinoma cells.ConclusionsThis hybrid nanoparticle delivery system can be used as a platform technology for intracellular delivery of siRNAs to NTSR1-overexpressing tumor cells.

Biodistribution and Pharmacokinetics Study of siRNA-loaded Anti-NTSR1-mAb-functionalized Novel Hybrid Nanoparticles in a Metastatic Orthotopic Murine Lung Cancer Model

Small interfering RNA (siRNA) is effective in silencing critical molecular pathways in cancer. The use of this tool as a treatment modality is limited by lack of an intelligent carrier system to enhance the preferential delivery of this molecule to specific targets in vivo. In the present study, the in vivo behavior of novel anti-NTSR1-mAb-functionalized antimutant K-ras siRNA-loaded hybrid nanoparticles, delivered by i.p. injection to non-small-cell lung cancer in mice models, was investigated and compared to that of a naked siRNA formulation. The siRNA in anti-NTSR1-mAb-functionalized hybrid nanoparticles was preferentially accumulated in tumor-bearing lungs and metastasized tumor for at least 48 hours while the naked siRNA formulation showed lack of preferential accumulation in all of the organs monitored. The plasma terminal half-life of nanoparticle-delivered siRNA was 11 times higher (17–1.5 hours) than that of the naked siRNA formulation. The mean residence time and AUClast were 3.4 and 33 times higher than the corresponding naked siRNA formulation, respectively. High-performance liquid chromatography analysis showed that the hybrid nanoparticle carrier system protected the encapsulated siRNA against degradation in vivo. Our novel anti-NTSR1-mAb-functionalized hybrid nanoparticles provide a useful platform for in vivo targeting of siRNA for both experimental and clinical purposes.

Aptamer-hybrid nanoparticle bioconjugate efficiently delivers miRNA-29b to non-small-cell lung cancer cells and inhibits growth by downregulating essential oncoproteins

MicroRNAs (miRNAs) are potentially attractive candidates for cancer therapy. However, their therapeutic application is limited by lack of availability of an efficient delivery system to stably deliver these potent molecules intracellularly to cancer cells while avoiding healthy cells. We developed a novel aptamer-hybrid nanoparticle bioconjugate delivery system to selectively deliver miRNA-29b to MUC1-expressing cancer cells. Significant downregulation of oncoproteins DNMT3b and MCL1 was demonstrated by these MUC1 aptamer-functionalized hybrid nanoparticles in A549 cells. Furthermore, downregulation of these oncoproteins led to antiproliferative effect and induction of apoptosis in a superior version when compared with Lipofectamine 2000. This novel aptamer-hybrid nanoparticle bioconjugate delivery system could potentially serve as a platform for intracellular delivery of miRNAs to cancer cells, hence improving the therapeutic outcome of lung cancer.

Recent advancements in the field of nanotechnology for the delivery of anti-Alzheimer drug in the brain region

ABSTRACT Introduction: Brain is supposed to be the most complicated part of the body which is very far from the reach of drug moieties. The drug entry in to the brain region depends upon various factors, and among those, the blood-brain-barrier remains the most prominent one. This barrier restricts the entry of almost all the drug and most of the essential biological components like proteins, peptides, etc. and hinders treatment of the CNS disorders. Alzheimer Disease (AD) is one such brain disorder, more specifically a neurodegenerative disorder which primarily affects the older adults. Areas covered: From solubility enhancement to targeted delivery, the nanoparticulate system became the answer for almost all the criticality related to drug delivery. Hence, nanoparticulate drug carrier system has been widely utilizing to remove the hurdles of brain drug delivery. Keeping this in mind, we have underlined the proficiencies of the nanocarrier systems which claim to improve the drug efficacy for the treatment of the AD. Expert opinion: The nanotechnological approaches are highly exploited by the researchers to enhance the drug permeation across the BBB to improve its bioavailability and efficacy by protecting the drug from peripheral degradation. However, still in this area of drug targeting provides vast scope for discoveries towards the enhancement of drug efficacy through surface modifications, site specification, reduced toxicity of the nanocarrier system and so on.

siRNA-Encapsulated Hybrid Nanoparticles Target Mutant K-ras and Inhibit Metastatic Tumor Burden in a Mouse Model of Lung Cancer

There is an unmet need in the development of an effective therapy for mutant K-ras-expressing non-small-cell lung cancer (NSCLC). Although various small molecules have been evaluated, an effective therapy remains a dream. siRNAs have the potential to downregulate mutant K-ras both at the protein and mRNA levels. However, a safe and effective delivery of siRNAs to tumors remains a limitation to their translational application in the treatment of this highly debilitating disease. Here we developed a novel hybrid nanoparticle carrier for effective delivery of anti-mutant K-ras to NSCLC (AKSLHN). The ability of this treatment modality to regress lung tumors in mouse models was evaluated as a monotherapy or as a combination treatment with erlotinib. Further, the toxicity of this treatment modality to healthy tissues was evaluated, along with its ability to elicit immune/inflammatory reactions. The results suggest that this treatment modality is a promising prospect for the treatment of mutant K-ras-expressing NSCLC without any accompanying toxicity. However, further understanding of the cellular-level interaction between AHSLHN and erlotinib needs to be attained before this promising treatment modality can be brought to the bedside.