br fluorescence probe was chosen owing to its excellent phot
fluorescence probe was chosen owing to its excellent photostability and dramatic change of fluorescence intensity in hydrophobic particle core and outside aqueous solution (Lv, Wang, Wang, & Tang, 2012). Speci-fically, the decrease in the NR fluorescence intensity around 630 nm with the excitation wavelength of 550 nm was recorded (Fig. 3B), which indicated HA-NB-SC nanomicelles show photolabile properties upon light activation. Besides, UV–vis spectrophotometer was also chosen to study the kinetics of the photo-cleavage, and the results was exhibited in Fig. 3C. Upon photo-irradiation, bond cleavage happens at the benzylic position resulting the formation of a nitrosobenzaldehyde derivative and a free HA (Scheme S1) (Zhao, Sterner, Coughlin, & Theato, 2012). As this process involves a rearrangement of aromatic
π -electrons, it can be detected through a change of the optical prop-erties of released product (Chandra, Subramaniam, Mallik, & Srivastava, 2006). So, Puromycin spectroscopy was chosen to monitor photo-responsive behaviors of nanomicelles. For irradiation experi-ments, a longer ultraviolet wavelength at 365 nm was selected, because it does less damage to cells than shorter UV. It can be seen the ap-pearance of a new absorption band around 330 nm with the increase of irradiation time. The new band indicates the formation a more delo-calized aromatic system, which is the nitrosobenzaldehyde photo-pro-duct (Scheme S1) in keeping with the literature (Il’ichev, Schwörer, & Wirz, 2004). Experiments about photo-responsive behaviors thus clearly verified that upon UV irradiation, HA-NB-SC went through cleavage, which should, in turn, disrupt the nanomicelles. Besides, a TEM image of HA-NB-SC nanomicelles which was irradiated by UV light for 1 h was supplied in Fig. S1. In Fig. S1, many small fragments can be easily observed implying that the nanomicelles were dis-assembled after irradiation.
An ideal drug carrier should be highly stable in order to avoid drug leaking during the circulation in the blood. Results of stability tests of
HA-NB-SC nanomicelles were in Fig. 3D, in which the fluorescence intensities were not changed with time increasing at 4 °C or 37 °C, in-dicating there was no obvious leak of encapsulated NR happened. Furtherly, stability tests of HA-NB-SC nanomicelles under diﬀerent pH conditions was also performed, and the results were showed in Fig. S2. The fluorescence intensities were not changed with time increasing under diﬀerent pH conditions, which indicated there was no evident leak of NR. The results of stability tests demonstated potential of HA-NB-SC as an ideal drug carrier.
3.3. Study on cellular cytotoxicity in vitro
Biomaterials should be non- or low-cytotoxic and should not have harmful eﬀects on cellular function. To study the toxicity of nanomi-celles, HeLa cells were employed as a model system by MTT assay, which is widely applied in evaluation of cytotoxicity, cell viability and cell proliferation (Mosmann, 1983) in vitro. First, a blank control assay was conducted by simply irradiating the cells at 365 nm for 0.5 h or 1 h without any nanomicelles; Live-dead assay was also performed to evaluate the viability/cytotoxicity using the same identical conditions as MTT assay. Results showed there was no cell death was observed (Fig. S3 and Fig. S4), indicating the nonlethality of the operating con-ditions. So, in this proof-of-concept model system, UV irradiation did not cause perceptible cytotoxicity that would otherwise jeopardize the analysis of carrier-induced toxicity. The identical conditions were ap-plied to subsequent assays but with HA-NB-SC nanomicelles. To get distinct results of the cellular cytotoxicity, four kinds of experiments were set: I) HA-NB-SC nanomicelles were incubated directly with HeLa cells without irradiation for 24 h or 48 h; II) HA-NB-SC nanomicelles residues, which were prepared from HA-NB-SC nanomicelles pre-irra-diated by UV light (365 nm) for 0.5 h or 1 h, were incubated directly