Photoswitchable Nanoparticles for Triggered Tissue Penetration and Drug Delivery

We report a novel nanoparticulate drug delivery system that undergoes reversible volume change from 150 to 40 nm upon phototriggering with UV light. The volume change of these monodisperse nanoparticles comprising spiropyran, which undergoes reversible photoisomerization, and PEGylated lipid enables repetitive dosing from a single administration and enhances tissue penetration. The photoswitching allows particles to fluoresce and release drugs inside cells when illuminated with UV light. The mechanism of the light-induced size switching and triggered-release is studied. These particles provide spatiotemporal control of drug release and enhanced tissue penetration, useful properties in many disease states including cancer.

ted into the resulting aqueous solution. Deionized water (3800 μL) was added and the resulting mixture solution was sonicated in a capped glass vial for 5 minutes using a Branson 2510 bath sonicator (Cleveland, OH) at a frequency of 40 kHz and power of 130W. The resulting NP H s were collected after ultrafiltration (5 min, 3000 × g, Ultracel membrane with 10,000 NMWL, Millipore, Billerica, MA), and washed with water to remove organic solvent. The sizes of NP H s were characterized by DLS.
Lyophilization of SP NP H s in the Presence of Albumin. An aqueous solution of bovine serum albumin (BSA) (12 mg/mL, 500 L) was added to the SP NP H solution (1 mg/mL, 400 L). The mixture was lyophilized for 16 h at 50 o C. The resulting white powder was reconstituted with deionized water (2 mL) followed by addition of a concentrated PBS solution (10X, 222 L). The solution was stirred for 5 min at room temperature. The resulting NC solution was analyzed by DLS.
Determination of Drug (or Dye) Release Kinetics. To determine the release kinetics of SP NP H s loaded with different drugs, a PBS suspension of NPs was placed into Slide-A-Lyzer dialysis tubes (300 μL each tube) with a molecular weight cutoff at 3500 Da (Pierce, Rockford, IL). These microtubes were individually dialyzed in 2 L of PBS buffer (1X) at 37 o C. PBS buffer was changed every 24 hours. At scheduled times, NPs solutions from microtubes were collected separately. DMF (300 μL) was added to dissolve the NPs, and the drug/dye content in each tube was measured by HPLC. That content was serially subtracted from the measured starting quantity of drug/dye to determine release kinetics.  (1) produces equation (2), where [MC] 0 is the initial concentration of MC after UV irradiation.

− [MC] ⁄ = [MC]
(1) The [MC] concentration is directly related to the absorbance A, which can be measured at the λ max in the UV/Vis spectrum. Equation (3) can be given according to Beer-Lambert law. 4 A ∞ is the absorbance at infinity at the same wavelength (which is the the λ max of SP NP H , i.e. the λ max after MC undergoes complete ring closure to SP-C9); Δε is the difference between molar extinction coefficient of MC and of SP at λ max ; d is the cuvette's light path length. Combining equations (2) and (3), A could be expressed as a function of t, as shown in equation (4). Fitting a plot of ln((A ∞ -A 0 )/(A-A 0 )) against t with equation (4) can provide k. t ½ is calculated using equation (2)  intensity (ICG concentration) profile (C) and the distance (x) for the NP H s before and after irradiation were fitted to the following one-dimensional diffusion model (equation (5)) 6 to obtain the diffusion coefficient D in the collagen gel: where erfc is the complementary error function and a, b are the constant for the function. The curve was fitted by Origin 8 software (Northampton, MA).
For the calculation of diffusion coefficient in gels of NP H s that were irradiated by UV twice at different times, the diffusion model was modified to: where a, b, c are the constants for the function, and t 1 , t 2 are time intervals after UV treatment ( in our case t 1 = 12h, t 2 = 9h). NP H suspension was then removed and corneas were washed in PBS for 3 times. Tissues were then immediately imaged by a near infrared laser scanner (LICOR Odyssey), then processed for histology (hematoxylin-eosin staining) using standard procedures. ---  Efficiency % = Actual loading / Initial loading × 100.

Discussion:
For SP derivatives, both O 2 -dependent and O 2 -independent mechanisms have been identified for photo-fatigue (also termed photo-degradation; detailed mechanism is discussed in Scheme S1). 7 In figure  S6, the absorption intensity of MC in NP H at 551 nm faded at a rate dependent on the duration of UV (365 nm) irradiation which is consistent with an O 2 -independent fatigue mechanism for SP. 8 Furthermore, addition of antioxidant agents (e.g. 4-methoxy-1, 2, 2, 6, 6-pentamethylpiperidine and 4-methoxy-2, 2, 6, 6-tetramethylpiperidine) 7b to SP NP H s in a 10-fold molar excess to SP could not eliminate photofatigue of NPs, suggesting that O 2 -dependent photo-fatigue is not the mechanism. These studies suggest that O 2 -independent mechanisms caused the decrease in absorption by MC at λ max and the alterations in SP NP H sizes after repetitive UV/Vis triggering. S14 Figure S7. The release profiles of (a) Doxorubicin from SP-C9 and SP-C18 NP H ; and (b) Rhodamine B from SP-C9 and SP-C18 NP H . LD: loading. Data are means ± SD, N=6 Figure S8. UV-triggered release of calcein (green fluorescence) from SP NP H (calcein loading wt% = 2.7 wt%). Cells were incubated for 4 hrs with calcein-containing NP H then washed with PBS. Cells were then irradiated with UV for 2s every 5 minutes, and an image was captured 5 minutes after each UV illumination. The fluorescence intensity in HeLa cells gradually increased from (a, before irraditaion) to (b, after 4 cycles of irradiation). (c) Flow cytometry. HeLa cells were incubated with calcein-containing SP NP H for 4 hrs as in (a). Flow cytometric analysis of green fluorescence intensity was then performed for cells without (purple line) or with UV illumination (10 s, red line). HeLa cells exposed to NPs containing no dye (green line) and cells without SP NP H or dyes (blue line) were assayed for comparison. (Wavelengths for excitation and emission were 480 nm and 510 nm, respectively.)

Discussion:
The red-shift of the λ max of MC in MC NP H from 640 nm to 672 nm ( Figure S10a) reflects the assembly state of MC in NPs. The tendency of free SP/MC to undergo aggregation in organic membranes and films is well known 9 . Three types of aromatic dye aggregates have been described: dimers, Haggregates, and J-aggregates. 10 The spectra of dimers and H-aggregates are blue-shifted relative to the molecularly dispersed monomers, whereas J-aggregates are red-shifted in fluorescence spectrum. 10b,11 Therefore, it is likely that MC-C9 forms J-aggregates inside NP H (see Scheme S2 and Figure 9).

Discussion:
The λ max of MC is sensitive to its solvent or microenvironment, a property termed solvatochromism: the λ max of MC shifts to higher energy (lower wavelength in spectrum) as the polarity of the solvent or microenvironment increases. 12 Here we studied the relationship between the dielectric constant (ε), an indicator of the polarity of the solvent, and the λ max of the MC in order to make inferences about the microenvironment of MC 10b,12-13 in the NP H.
To elucidate the relationship between microenvironment polarity ε and λ max , we plotted the λ max of MC-C9 in organic solvents with various ε. 10b,12 Extrapolation of the λ max value (551 nm) for MC NP H to the abscissa (dashed line in Figure S11) suggested an effective ε around 18, which is comparable to the λ max measured in a relatively polar solvent (for isopropanol ε = 18.3, λ max = 542 nm). These findings indicated that MC-C9 molecules are likely located in relative polar microenvironments in the MC NP H . Previous studies of MC derivatives in liposomes suggest that MC-C9 might localize near the phosphorglycerol moiety linking PEG and DSPE. 10b,13 S19 Figure S12. Representative UV/Vis absorption spectra of the isomerization of MC to SP, either in (a) acetonitrile solution or in (b) NP H . MC was generated by prior UV irradiation (30s) and the reversion of MC to SP occurred in the dark (N=4). ([MC-C9] = 0.46 mM in both acetonitrile and NP H ). (c) The firstorder kinetics of the isomerization of MC to SP at λ max of panels (a) (at 560 nm, red) and (b) (at 551 nm, black).

Discussion:
The UV-triggered conversion of SP NP H to MC NP H showed an exponential increase in the absorbance at λ max of MC (551 nm) at a rate constant (k 1 ) of 0.064 ± 0.016 s -1 (t ½ = 14.8 ± 3.7 s, N=4, [SP-C9] = 0.69 mM, primary data not shown) for the ring-opening reaction of SP to MC. Subsequently, the isomerization of MC to SP occurred slowly in the dark, which in MC NP H followed first-order decay with a rate constant (k -1 ) of 3.66 ± 0.13 × 10 -4 s -1 at λ max =551 nm (N=4, t ½ = 1732 ± 70 s). This rate of MC conversion to SP in NP H was 12.2-fold slower than that of free MC in acetonitrile (N=4, k -1 = 4.46 ± 0.07 × 10 -3 s -1 at λ max =560 nm, t ½ = 155 ± 2 s). The slowness of the isomerization of MC to SP in NP H (compared to in acetonitrile) is likely due to microenvironmental restriction of MC's conformational change to SP. A similar phenomenon has been observed in SP embedded in polymeric films. 8a S20 Scheme S1. (a) Reported photo-processes of SP derivatives. 7e,14 The ring-opening pathway is depicted by purple arrows. Side reactions are depicted by blue arrows. '*' represents a vibrationally excited state. ' 1 SP*' represents the singlet excitation state and ' 3 SP*' represents the triplet excitation state. 'MC-X' represents unknown isomers and pathways that MC-cis relaxes to, as indicated by references. 1 'MC-cis' and 'MC-trans' represent the cis and trans isomers of MC, as shown in (b). The side reaction from the triplet state ' 3 MC-Cis*' to 'MC-X' causes photo-fatigue effects and undesired side products in the photo-process.

Discussion:
The O 2 -independent mechanism has been reported to be dominant in SP, 7e,14 with photodegradation occurring primarily from the triplet manifold. After UV triggering, around 90% of UVirradiated SP derivatives will be in the excited singlet SP state and will further transform to corresponding MC isomers. Approximately 10% will proceed through a triplet state with little probability of forming MCs, leading to fatigue. 15 The probability of O 2 -independent fatigue is reported to be related to the UV irradiation time, 8b,15 which is also observed in our experiments ( Figure S6).