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Theranostics 2020, Vol. 10, Issue 20 Although 50 nm, 70 nm and 80 nm nanoparticles can effectively penetrate both, 100 nm nanoparticles cannot penetrate normal fenestrations, suggesting that there might be an optimal size for effective leakage of nanoparticles from the microvasculature into the tissue. Rona et al. [41] demonstrated that size of AgNPs could influence cellular uptake and toxicity. Smaller particles (10 nm, 20 nm) easily penetrate LoVo cells and then significantly increase intracellular ROS levels, while larger particles (100 nm) appeared mainly on the cell surface. Alicia et al. [220] also found that smaller AgNPs were more cytotoxic than larger AgNPs when studying the therapeutic effects of AgNPs on human hepatoma and leukemia. Our team [21] used an evaporation–condensation system to obtain silver particles approaching the Ångstrom dimension. By comparing AgNPs with larger size, we found Ångstrom-scale silver particles had greater cytotoxicity to tumor cells, but did not induce notable toxicity on normal tissues. The applications of AgNPs can be extended by tailoring the shape of nanoparticle, which may optimize the physicochemical and biological properties of AgNPs [26, 221]. The shape-controlled AgNPs can be obtained by changing the parameters in different synthesis methods. Though AgNPs with various shapes are prepared, such as sphere, triangle, cuboid, rod, tube, disk and wire, only a few among these are chosen for anticancer therapy. The cellular uptakes of AgNPs, as well as particle-to-cell or particle-to-protein interactions, are partly dependent on the shape of nanoparticles [216, 222]. In general, spherical AgNPs may display stronger endocytosis and more active anticancer effect than other shapes. Because it is more efficient for spherical AgNPs than non-spherical nanoparticles to pass through vascular endothelium, and their higher specific surface area is more beneficial for them to enter cancer cells [216, 222]. In addition, the active or weak endocytosis may be related to the different membrane bending energies of various shaped AgNPs. Ying Li et al. [223] compared the internalization rates of spherical-, cubic-, disk- and rod-shaped nanoparticles to find out the shape effect on endocytosis. They realized that the spherical nanoparticles exhibited the fastest internalization rate, followed by the cubic nanoparticles, while the disk- and rod-shaped nanoparticles exhibited the slowest internalization rate. After analyzing the free energies of four shaped nanoparticles, they speculated that the membrane bending energy of nanoparticles during endocytosis might be the main factor inducing the shape effect of the nanoparticles. Among these four shaped nanoparticles, compared with the non-spherical, the spherical nanoparticles only needed to overcome a 9010 minimal membrane bending energy barrier, while the disk shaped nanoparticles faced a larger free energy barrier caused by stronger membrane deformation. In order to understand the effect of more complex shaped particles on cellular uptake, Yuanzu He et al. [224] treated LnCAP cells with particles of different keyboard character shapes and compared the cell endocytosis. Compared with shapes without sharp features, like number 0, letter O and pound key, the rod-like microparticles, such as number 1, letter I, and arrow key, were more likely to adhere, penetrate and enter the cancer cells. The results explained that the shapes of microparticles with sharper angular features and higher aspect ratio might have a higher chance to contact and be internalized by cancer cells. Dose and Exposure Time The AgNPs exhibit dose- and time-dependent cytotoxicity against cancer [21, 225-227]. In general, increased dose and prolonged exposure time can cause more tumor cell apoptosis [228, 229]. Increasing dosage and prolonged exposure time can provide more opportunities for AgNPs to enter cells and trigger multiple anticancer mechanisms. Muthu et al. [226] studied the anticancer effect of AgNPs on Dalton’s lymphoma ascites (DLA) cell lines and found that AgNPs showed dose-dependent cytotoxicity to DLA cells through activation of caspase 3 enzyme, ultimately inducing apoptosis. Bita Mousavi et al. [230] found that AgNPs synthesized by Artemisia turcomanica leaf extract showed both dose- and time-dependent anticancer effect on gastric cancer cell line. Although increased dose of AgNPs and prolonged exposure time can result in better anticancer effects, the potential toxicity to normal tissues needs to be carefully considered. Surface Charge and Protein Corona Surface charges participate in the formation of AgNPs surface chemistry, which play an important role in cytotoxicity [231-233]. The surface charges of AgNPs determine the binding with serum albumin, as well as the adhesion and uptake of cells [25]. Negatively charged and neutrally charged AgNPs can adhere to cell membranes but internalize in small amounts, while positively charged AgNPs exhibit more efficient cell membrane penetration and internalization [25]. Besides, the positively charged AgNPs tend to stagnate on the surface of the tissue and the lumen of the blood vessels for a long time, which may be beneficial for the targeted delivery of anticancer agents [234]. AgNPs with opposite surface charges exhibit different cytotoxicity in tumor cells. The greater cytotoxicity and more ROS production are observed in tumor cells exposed to high positive charged AgNPs [234]. 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