Spatiotemporally and Sequentially-Controlled Drug Release from Polymer Gatekeeper–Hollow Silica Nanoparticles

Combination chemotherapy has become the primary strategy against cancer multidrug resistance; however, accomplishing optimal pharmacokinetic delivery of multiple drugs is still challenging. Herein, we report a sequential combination drug delivery strategy exploiting a pH-triggerable and redox switch to release cargos from hollow silica nanoparticles in a spatiotemporal manner. This versatile system further enables a large loading efficiency for both hydrophobic and hydrophilic drugs inside the nanoparticles, followed by self-crosslinking with disulfide and diisopropylamine-functionalized polymers. In acidic tumour environments, the positive charge generated by the protonation of the diisopropylamine moiety facilitated the cellular uptake of the particles. Upon internalization, the acidic endosomal pH condition and intracellular glutathione regulated the sequential release of the drugs in a time-dependent manner, providing a promising therapeutic approach to overcoming drug resistance during cancer treatment.

Immediately, TEOS (0.05 mL) was added. After 6 h, the obtained products were collected by centrifugation and redispersed in DI water (10 mL). Under vigorous stirring, 470 mg of Na 2 CO 3 were added into the well-sonicated water suspension of the above solid SiO 2 @CTAB/ SiO 2 . After the reaction was stirred at 50 for 10 h, the products were collected by centrifugation and extensively washed with deionized water and ethanol. For the extraction process, the as prepared products were dispersed in 80 mL of acetone and refluxed at 80 for 48 h. The extraction was repeated three times to fully remove CTAB. The final products were collected and washed with DI water.
Drug release profile. The release kinetics of the drug-loaded nanoparticles in phosphatebuffered saline (pH 6.5) at room temperature were analyzed at excitation wavelengths of 480 nm and 360 nm for Dox and CPT, respectively, at several time points using a Shimadzu RFPC 5430 Spectrofluorometer. For Dox and CPT loaded PHMSN, drug release profile were analyzed using HPLC with methanol or acetonitrile and water. In order to measure triggered drug release profiles, 1 mM of GSH was added to the nanoparticle solution at 4 h and the release profile was analyzed using a fluorometer and HPLC (Agilent 1200 series).
Preparation of veramphamil and Dox coloaded PHMSN. HMSNs (5.0 mg) were dispersed well in 1 mL of hydrophobic doxorubicin drug solution, (2.5 mg in DCM) and stirred for a period of 24 h at room temperature. After the stipulated time periods, the drugloaded nanoparticles were centrifuged and the supernatant was collected. The supernatant sample was used to measure the drug loading by collecting UV-Vis absorption spectra (V250 Jasco UV-Vis/NIR spectrophotometer). The drug-loaded nanoparticles were vacuum-dried, and redispersed in 1mL aqueous suspension containing 2.5 mg of veramphamil hydrochloride (Ver) and stirred for a period of 24h. To this solution PEG-PDS-DPA copolymer was added and stir for overnight. To crosslink the surface-wrapped polymer, a partial amount of DTT (20 mol% against the PDS group) was added and the resulting solution was stirred for 3 h at room temperature. Drug-loaded PMSNs were collected by centrifugation and washed extensively with pH 7.4 phosphate buffer solution and distilled water. HPLC analysis was used to measure the amount of Ver loading in the HMSN. 2 The Dox loading capacity was 21 wt%, while Ver loading capacity ranged at 9 wt% with a proportional ratio of 1:2.
Cell culture and viability analysis. Human nasopharyngeal carcinoma cells (KB) were cultured (using RPM 1640 medium) in sterile 96-well Nunc (Thermo Fisher Scientific Inc.) microtitre plate at a seeding density of 5 x 10 3 cells/well and they were allowed to settle for 24 h under incubation at 37 °C and 5% CO2. In-order to check cell viability, the cells were Initially, the nanoparticles were incubated with acidic (pH 6.5, Dulbecco's phosphate buffered saline, DPBS) and neutral (pH 7.4, DBPS) conditions for a period of 2 h, washed replaced with fresh medium, following previous reports. 7 Cell viability were measured at 48 h using Alamar blue assay with each data point measured in triplicate following manufacturer's protocol. In detail, fluorescence measurements were recorded after treating the alamar blue dye at each well, incubated until 4h at 37ºC, viability was using the plate reader (Tecan Infinite Series, Germany) by setting the excitation wavelength at 565 nm and monitoring emission at 590 nm on the 96 well plates.
In-situ confocal microscopy for cellular internalization. KB cells were seeded in one well chmabered cover glass (Lab Tek II, Thermo Scientific) at a seeding density of 2 x 10 5 cells/well. After 24 h, cells were treated with Dox-CPT-PHMSNs nanoparticles at a final concentration of Dox/CPT at 10 µg/mL at pH 6.5 DPBS and pH 7.4 DPBS medium for a period of 2 h and then replaced with fresh medium. 7 To confirm the uptake, the fluorescent signal from Dox (approx. 559 nm emission) and CPT (approx. 410 nm emission) were observed and images were captured using the confocal microscope (Olympus FV1000 series).
To check the endosomal and lysosomal escape/colocalization, the cells were pre-stained with the early endosomes-GFP and lysotracker green DND-26 by following the manufacturer's protocol. In order to check the endocytosis mediated uptake mechanism, cells were preincubated with inhibitors by previously reported methods. 8 The cellular uptake was monitored in the cover glass (Lab Tek II glass chamber cover glass, Thermo Fisher Scientific Inc) were monitored periodically using Olympus FV1000 confocal microscope connected to CO 2 incubator.
Flow-cytometry analysis. To analyze the cellular intake PHMSN nanoparticles at pH 6.

Crosslinking density and colloidal stability analysis
In order to stably hold drugs in the HMSN pores, the coating polymers were further crosslinked by adding a partial amount of dithiothreitol (15 to 36 mol% of the PDS group), and the resulting crosslinking densities were 24 mol% and 53 mol%, respectively ( Figure S3A and S3B). A pristine MSN has a limited capacity to serve as an ideal nanocarrier for drug delivery owing to unstable colloidal properties under physiological conditions, which may lead to undesired drug leakage before reaching the target. 9 To overcome these issues, polymer gatekeepers have been used in MSNs, which was shown to result in stable colloidal properties. 1 Consistently, no meaningful change in the PHMSN size was observed upon incubation with phosphate-buffered saline (PBS), pH 7.4, RPMI medium with 10% fetal bovine serum (FBS), and sodium acetate buffer, pH 5.5, for up to 72 h, a commonly used measurement period ( Figure S3D, S3E and S3F).

Cellular uptake analysis in MDK dependent drug resistant cells.
Overexpression of midkine (MDK), a cysteine-rich heparin-binding protein, in cancer cells contributes to drug resistance against Dox, 5-fluorouracil, and cisplatin in gastric cancer. 10 To evaluate the synergistic response and to overcome the Dox resistance in MDK-over expressing gastric cancer (SNU-0620-ADR/300) cells, Dox-HCl-and CPT-co-loaded PHMSNs were used. When the combination drugs were encapsulated into PHMSNs, a much enhanced cytotoxic effect was observed ( Figure S8A). The half-maximum inhibition concentration (IC 50 ) value of the Dox•HCl and CPT-loaded PHMSNs was as low as 1.2 µg/mL after 72 h of incubation, which is much lower than the values shown by single drugloaded PHMSNs (Dox-HCl-or CPT-loaded PHMSNs), which were 4.2 and 2.1 µg/mL, respectively. The internalization was further confirmed by confocal microscopy analysis in a time-dependent manner ( Figure S8B). As the time increased (from 2 h to 6 h), the Dox fluorescence signal was hardly observed, whereas the fluorescence of CPT was similarly maintained. Scrambling studies: To ensure the sequential release, one may hypothesise that the combination of Ver-Hcl (salt form as a hydrophobic drug) and Dox (neutral form as a hydrophobic drug) inside the nanoparticles may remain as the salt form and neutral form, that the proton will not simple scramble. If so, there is a possibility of concurrent release of these two drugs and one can use PTx which is not protonated as a hydrophobic drug instead of Dox.
In addition we tried to check the scrambling between Dox & Ver.HCl in test tube (in DI water). We hypothesized that the scrambling between Dox and Ver-HCl would increase the solubility of Dox due to the protonation. Initially hydrophobic Dox (2 mg) was dispersed in 1 mL of DI water and checked the solubility of hydrophobic Dox after 12 h in static condition by analyzing the absorbance. After this experiment, we performed the scrambling experiment.
For this, Ver-HCl (2 mg) was added and allowed to stand for 12 h again. We performed this experiment in the simultaneously, to maintain the same Dox concentration. Then the supernatant solution was checked to analyze the scrambling by UV-visible spectroscopy.
During this analysis, we observed that the concentration of Dox soluble in presence and absence of Ver.Hcl is negligible as shown Figure S9. As shown in Equation (1) and (2) the pK a of Ver-HCl 11 is higher that Dox-HCl 12 because Ver contains tertiary amine while Dox contains primary amine.
Eq (1) Eq (2) Dox.HCl Dox Ver.HCl Ver Therefore, there is a very little possibility of scrambling of salt between Ver-HCl and Dox due to this pK a difference. From our mixing experiment, we noted a negligible difference in absorbance in the Dox.