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Number of Nanoparticles per Cell through a Spectrophotometric Method – A key parameter to Assess Nanoparticle-based Cellular Assays.

Engineered nanoparticles (eNPs) for biological and biomedical applications are produced from functionalised nanoparticles (NPs) after undergoing multiple handling steps, giving rise to an inevitable loss of NPs. Herein we present a practical method to quantify nanoparticles (NPs) number per volume in an aqueous suspension using standard spectrophotometers and minute amounts of the suspensions (up to 1 μL). This method allows, for the first time, to analyse cellular uptake by reporting NPs number added per cell, as opposed to current methods which are related to solid content (w/V) of NPs. In analogy to the parameter used in viral infective assays (multiplicity of infection), we propose to name this novel parameter as multiplicity of nanofection.

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In vitro and in vivo identification of ABCB1 as an efflux transporter of bosutinib.

Bosutinib is a recently approved ABL inhibitor. In spite of the well-documented effectiveness of BCR-ABL inhibitors in treating chronic myeloid leukemia, development of resistance is a continuous clinical challenge. Transporters that facilitate drug uptake and efflux have been proposed as one potential source of resistance to tyrosine kinase inhibitor treatment. Our aim was to determine which carriers are responsible for bosutinib transport.
K562S cells overexpressing the drug transporters ABCB1, ABCG2, and SLC22A1 were generated, characterized and used in proliferation assay and intracellular uptake and retention assay (IUR). In vivo experiments were performed in nude mice injected with K562S, K562DOX cells (overexpressing ABCB1), and K562DOX silenced for ABCB1 (K562DOX/sh P-GP).
The IUR assay using C-14 bosutinib showed that only ABCB1 was responsible for active bosutinib transport. K562DOX cells showed the lowest intracellular level of bosutinib, while K562DOX cells treated with the ABCB1 inhibitor verapamil showed intracellular bosutinib levels comparable with parental K562S. Proliferation assays demonstrated that K562DOX are resistant to bosutinib treatment while verapamil is able to restore the sensitivity to the drug. Nude mice injected with K562DOX and treated with bosutinib showed very limited response and quickly relapsed after stopping treatment while K562S as well as K562DOX/sh P-GP remained tumor-free.
Our data suggest that the analysis of ABCB1 expression levels might help determine treatment options for patients exhibiting resistance to bosutinib.

Drug outcome survey to evaluate anti-TNF treatment in rheumatoid arthritis: an Italian observational study (the DOSE study).

The aim of this paper is to analyse the use of anti-TNF drugs in current Italian practice, evaluate clinical responses to treatment, and identify possible predictors of negative response in patients with rheumatoid arthritis (RA).
DOSE is a non-interventional, prospective study of patients with active RA treated for the first time with anti-TNF agents in 21 Italian hospitals. Demographic and clinical characteristics of patients, treatments and outcome measures were assessed. Outcome measures used were EULAR response, DAS28 remission and HAQ remission at 12 months. A stepwise logistic regression model was used to study the predictors of non-response.
Of 299 RA patients (mean 53.8 ± 12.8 years, 76.1% female), DAS28 was &gt;5.1 in 60.5% of patients and HAQ was &gt;1 in 65.9%. Etanercept was the most prescribed anti-TNF. DMARDs were used in 77.6% of patients (methotrexate in 59.2%). Significant improvements in clinical and laboratory parameters were observed at 12 months. The proportion of patients classed as non-responders remained high, and varied according to assessment criteria. The main predictors independently and significantly associated with a high risk of non-response were: age and female gender for all outcome criteria; high DAS28 value for disease remission; and HAQ &gt;1 for disability remission.
In Italian anti-TNF treatment for RA, age, gender, and high values of both disease activity and disability were predictors of non-response to first-line therapy with anti-TNF drugs. Future studies should consider optimal second-line therapies for RA patients who do not achieve remission to their first anti-TNF treatment.

Nanocarrier-based co-delivery of small molecules and siRNA/miRNA for treatment of cancer.

Aberrant gene expression can trigger several vital molecular events that not only result in carcinogenesis but also cause chemoresistance, metastasis and relapse. Gene-based therapies using siRNA/miRNA have been suggested as new treatment method to improve the current regimen. Although these agents can restore the normal molecular cascade thereby resensitizing the cancer cells, delivering a standard regimen (either subsequently or simultaneously) is necessary to achieve the therapeutic benefit. However, co-delivery using a single carrier could give an additional advantage of similar biodistribution profile of the loaded agents. While much research has been carried out in this field in recent years, challenges involved in designing combination formulations including efficient coloading, stability, appropriate biodistribution and target specificity have hampered their clinical translation. This article highlights current aspects of nano-carriers used for co-delivery of small molecules and genes to treat cancer.

Genetic modification of oncolytic Newcastle disease virus for cancer therapy.

Clinical development of a mesogenic strain of Newcastle disease virus (NDV) as an oncolytic agent for cancer therapy has been hampered by its select agent status due to its pathogenicity in avian species. Using reverse genetics, we have generated a lead candidate oncolytic NDV based on the mesogenic NDV-73T strain that is no longer classified as a select agent for clinical development. This recombinant NDV has modification at the fusion protein (F) cleavage site to reduce the efficiency of F protein cleavage and an insertion of a 198 nucleotide sequence into the HN-L intergenic region resulting in reduced viral gene expression and viral replication in avian cells but not in mammalian cells. In mammalian cells, except for viral polymerase (L) gene expression, other viral gene expression is not negatively impacted or increased by the HN-L intergenic insertion. Furthermore, the virus can be engineered to express a foreign gene and still retain the ability to grow to high titers in cell culture. The recombinant NDV selectively replicates in and kills tumor cells and is able to drive potent tumor growth inhibition following intratumoral or intravenous administration in a mouse tumor model. The candidate is well positioned for clinical development as an oncolytic virus.
The avian paramyxovirus type 1, Newcastle disease virus (NDV) has been an attractive oncolytic agent for cancer virotherapy. However, this virus can cause epidemic disease in poultry, and concerns about the potential environmental and economic impact of an NDV outbreak have precluded its clinical development. Here we describe generation and characterization of a highly potent oncolytic NDV variant that is unlikely to cause Newcastle disease in its avian host, representing an essential step towards moving NDV forward as an oncolytic agent. Several attenuation mechanisms have been genetically engineered into the recombinant NDV that reduce chicken pathogenicity to a level that is acceptable worldwide, without impacting viral production in cell culture. The selective tumor replication of this recombinant NDV, bothin vitroandin vivo, along with efficient tumor cell killing makes it an attractive oncolytic virus candidate that may provide clinical benefit to patients.
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