Nano LIFE

The study of nanomedicine research, to date, has been concentrating on developing nanovectors for medical imaging, drug/gene delivery, and cell targeting, particularly in cancer diagnosis and therapeutics. Although quite successful in some areas, critical challenges remain, especially in the clinical settings. Some of the critical issues include delivery inefficiency, targeting non-specificity, and low uptake of theranostic-payloads by the cancerous lesions upon intravenous injection. Therefore, an alternative approach may be possible, via nanotechnology, by simulating some of the immune cells. In this Technical Note, we propose using specifically designed nanoparticles to simulate neutrophils that are capable of effective cancer cells targeting and killing without the complications in drug loading, biomarker conjugation, and assembly of chemical and physical therapeutic means. The simulated artificial cells mimic neutrophils, namely, granulocytes, according to their biological characteristics, for instance, positively-charged cell surfaces and the ability to release perferin upon binding onto the cancer cells leading to cytolysis. This Technical Note is intended to deliver a new concept in artificially-engineered granulocytes for cancer diagnosis and therapeutics.

Regenerative therapies require availability of an abundant healthy cell source which can be achieved by efficient cryopreservation techniques. Here, we established a novel approach for improved cell cryopreservation using an mRNA-based dCas9-VP64 gene activation system for transient, yet highly efficient expression of epigenetic related genes in mammalian cells for repression of metabolic activity. Before freezing, mammalian cells were treated by dCas9-VP64-modified mRNA and guide RNAs for upregulation of histone deacetylase (HDAC), DNA methyltransferase (DNMT) and transcriptional co-repressor Sin3A genes. Cell viability, karyotype, pluripotency, and other cell specific functions were analyzed during post-thaw culture. Using conventional cryopreservation protocols, we found improvement of viability in dCas9-VP64 pretreated cells ($P<0.05$) compared to untreated cells. Combined with dCas9-VP64 system, a reduced amount of cryoprotectant (5% DMSO) did not negatively affect the post-thaw viability. Co-delivering chemically modified dCas9-VP64 mRNA with gRNAs is an efficient gene delivery method compared to DNA-based strategies, without the associated safety concerns. This approach is a simple, yet effective way to accelerate a wide array of cellular research and translational medical applications.

Conducting the bicinchoninic acid (BCA) assay directly after a coupling reaction using (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) (EDC) and $N$-hydroxysuccinimide (NHS) chemistry produces significant errors. Here we present a correction for the quantification of gelatin in the supernatant (SN) following gelatin conjugation to polymer microparticles using EDC and NHS chemistry. Following the conjugation reaction, SNs from the gelatin-microparticle formation reaction are treated with BCA assay reagents and quantified for the percentage of unbound gelatin in the solution. NHS was found to interfere with the BCA assay reagents and is dependent on incubation time. It is found that the large concentration (500$\mu$g/mL) of NHS in the conjugation reaction interferes with the sensitivity of gelatin present in SNs. The interference from NHS requires a careful analysis to distinguish the BCA background absorbance from the sample absorbance. Using an NHS control solution can correct NHS interference and thus decrease the expensive iterations in gelatin quantification and enable accurate analysis of gelatin content. The accuracy of gelatin quantification is further improved by reducing the BCA assay incubation time to approximately 20min, compared with the recommended 30min. This re-assessment of BCA assay is important to avoid misestimating biases in bioconjugation processes.