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Wei-Jie Cheng,1,* Ling-Chun Chen,2,* Hsiu-O Ho,1 Hong-Liang Lin,3 Ming-Thau Sheu1 1School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan; 2Department of Biotechnology and Pharmaceutical Technology, Yuanpei University of Medical Technology, Hsinchu, Taiwan; 3School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan *These authors contributed equally to this work Purpose: In this study, a double emulsion method for complexing plasmids with stearyl polyethylenimine (stPEI) as the core to form human serum albumin (HSA) (plasmid/stPEI/HSA) nanoparticles (NPs) was developed for gene delivery by non-covalently binding onto plasmid/stPEI/HSA nanoparticles with CRISPR/Cas9 or siRNA, which disrupts or silences the expression of programmed cell death ligand-1 (PD-L1) for immunotherapy. Materials and methods: Chemically synthesized stearyl-polyethyenimine (stPEI)/ plasmids /HSA nanoparticles were maded by double emulsion method. They were characterized by dynamic light scattering (DLS), transmission electron microscope and also evaluated by in vitro study on CT 26 cells. Results: stPEI was synthesized by an N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC)–N-hydroxysuccinimide (NHS) reaction, and we found that the degree of substitution was ~1.0 when the ratio of PEI to stearic acid was 1:7 in the reaction. Then, two sgRNA sequences were selected and evaluated for their ability to knock out PD-L1 by decreasing its expression by about 20%. Based on the trend of particle size/zeta potential values as a function of ratio, F25P1 containing 25 µg of plasmid/stPEI/HSA NPs noncovalently bound to 1 µg plasmids via charge–charge interactions was found to be optimal. Its particle size was about 202.7±4.5 nm, and zeta potential was 12.60±0.15 mV. In an in vitro study, these NPs showed little cytotoxicity but high cellular uptake. Moreover, they revealed the potential for transfection and PD-L1 knockout in an in vitro cell model. Furthermore, F25P1S0.5 containing 25 µg of plasmid/stPEI/HSA NPs noncovalently bound to 1 µg of plasmids and 0.5 µg siRNA was prepared to simultaneously deliver plasmids and siRNA. An in vitro study demonstrated that the siRNA did not interfere with the transfection of plasmids and showed a high-transfection efficiency with a synergistic effect on inhibition of PD-L1 expression by 21.95%. Conclusion: The plasmids/stPEI/HSA NPs could be a promising tool for gene delivery and improved immunotherapy. Keywords: stearyl polyethylenimine, human serum albumin nanoparticles, PD-L1, CRISPR, Cas9, siRNA, gene delivery

Pyrazolo[3,4-d]pyrimidine derivatives 1–5, active as c-Src inhibitors, have been selected to be formulated as drug-loaded human serum albumin (HSA) nanoparticles, with the aim of improving their solubility and pharmacokinetic properties. The present study includes the optimization of a desolvation method-based procedure for preparing HSA nanoparticles. First, characterization by HPLC-MS and Dynamic Light Scattering (DLS) showed a good entrapment efficacy, a controllable particle size (between 100 and 200 nm) and an optimal stability over time, confirmed by an in vitro drug release assay. Then, 1–4 and the corresponding NPs were tested for their antiproliferative activity against neuroblastoma SH-SY5Y cell line. Notably, 3-NPs and 4-NPs were identified as the most promising formulation showing a profitable balance of stability, small size and a similar activity compared to the free drugs in cell-based assays. In addition, albumin formulations increase the solubility of pyrazolo[3,4-d]pyrimidine avoiding the use of DMSO as solubilizing agent.

Brain metastasis from triple-negative breast cancer (TNBC) has continued to lack effective clinical treatments until present. However, the feature of epidermal growth factor receptor (EGFR) frequently overexpressed in TNBC offers the opportunity to employ lapatinib, a dual-tyrosine kinase inhibitor of human epidermal growth factor receptor-2 (HER2) and EGFR, in the treatment of brain metastasis of TNBC. Unfortunately, the low oral bioavailability of lapatinib and drug efflux by blood-brain barrier have resulted in low drug delivery efficiency into the brain and limited therapeutic effects for patients with brain metastasis in clinical trials. To overcome such disadvantages, we developed lapatinib-loaded human serum albumin (HSA) nanoparticles, named LHNPs, by modified nanoparticle albumin-bound (Nab) technology. LHNPs had a core-shell structure and the new HSA/phosphatidylcholine sheath made LHNPs stable in bloodstream. Compared to free lapatinib, LHNPs could inhibit the adhesion, migration and invasion ability of high brain-metastatic 4T1 cells more effectively in vitro. Tissue distribution following intravenous administration revealed that LHNPs (i.v., 10 mg/kg) achieved increased delivery to the metastatic brain at 5.43 and 4.36 times the levels of Tykerb (p.o., 100 mg/kg) and lapatinib solution (LS, i.v., 10 mg/kg), respectively. Compared to the marketed Tykerb group, LHNPs had markedly better inhibition effects on brain micrometastasis and significantly extended the median survival time of 4T1 brain metastatic mice in consequence. The improved anti-tumor efficacy of LHNPs could be partly ascribed to down-regulating metastasis-related proteins. Therefore, these results clearly indicated that LHNPs could become a promising candidate for clinical applications against brain metastasis of TNBC.