Molecularly Imprinted Polymer‐Based Smart Prodrug Delivery System for Specific Targeting, Prolonged Retention, and Tumor Microenvironment‐Triggered Release

Abstract Prodrug and drug delivery systems are two effective strategies for improving the selectivity of chemotherapeutics. Molecularly imprinted polymers (MIPs) have emerged as promising carriers in targeted drug delivery for cancer treatment, but they have not yet been integrated with the prodrug strategy. Reported here is an MIP‐based smart prodrug delivery system for specific targeting, prolonged retention time, and tumor microenvironment‐triggered release. 5′‐Deoxy‐5‐fluorocytidine (DFCR) and sialic acid (SA) were used as a prodrug and a marker for tumor targeting, respectively. Their co‐imprinted nanoparticles were prepared as a smart carrier. Prodrug‐loaded MIP specifically and sustainably accumulated at the tumor site and then gradually released. Unlike conventional prodrug designs, which often require in‐liver bioconversion, this MIP‐based prodrug delivery is liver‐independent but tumor‐dependent. Thus, this study opens new access to the development of smart prodrug delivery nanoplatforms.

substrate and the imprinted NPs. After excessive nanoparticles were removed through washing with phosphate buffer (0.01 M, pH 7.4), the fluorescence signal was detected on the SynergyMX microreader. The boronic acid-modified 96-well microplate was prepared according to a previous method. [2][3][4] Firstly the wells were filled with a 3:1 (v/v) mixture of H2SO4 (98%) and HNO3 (63%) (250 μL/well) and kept at room temperature for 12 h. After being washed with deionized water to achieve a neutral pH, the wells were airdried. After that, the wells were filled with 5% aqueous APTES solution (pH 6.9, 250 μL/well), slightly shaken at room temperature for 2 h, and then dried by air. The wells were then filled with methanol solution containing 5 mg/mL 4-formylphenylboronic acid and 5 mg/mL sodium cyanoborohydride dissolved in anhydrous methanol (250 μL/well). The microplate was sealed and slightly shaken at room temperature for 12 h, and then the wells were washed with ethanol for 5-10 times. The obtained microplate was dried by air and then kept at 4º C for later experiments.

Characterization of boronic acid-modified 96-well microplate.
To characterize the modification of boronic acid onto the 96-well microplate, the glycoprotein HRP was used as a test compound. Three random boronic acid-modified wells and three unmodified wells were added with 250 μL of 0.5 mg/mL HRP dissolved in 0.01 M phosphate buffer (pH 7.4). The three unmodified wells filled with equal volume phosphate buffer with HRP were used as controls. After incubation for 30 min, the wells were washed with 0.01 M phosphate buffer (pH 7.4) three times. The UV absorbance at 260 nm of each well was read on the SynergyMX microreader.

Evaluation of boronic acid-functionalization of FITC-doped NPs.
The boronic acid functionalization of FITC-doped NPs was evaluated according to the boronate affinity of the prepared material using the cis-diol group containing compound adenosine as a test compound and non-cisdiol containing compound deoxyadenosine as a control. FPBA-fuctionalized NPs were dispersed in 0.01 M phosphate buffer (pH 7.4) to make the final concentration of 1 mg/mL. A volume of 1 mL of 1 mg/mL boronic acid-functionalization NPs was incubated with 1 mg adenosine or deoxyadenosine dissolved in 0.01 M phosphate buffer (pH 7.4) for 1 h. After that, the NPs were collected via centrifugation and washed with 0.01 M phosphate buffer (pH 7.4) for three times. The collected NPs were further eluted with 40 μL of 0.1 M HAc for 30 min. Finally, the supernatant was collected via centrifugation and tested by UV absorbance at 260 nm.

Calibration curves of DFCR and SA.
A series of standard mixed solution of SA and DFCR (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 mg/mL for SA as well as DFCR) were prepared with 0.01 M phosphate buffer (pH 7.4)  DFCR imprinted NPs were prepared according to the boronate affinity oriented surface imprinting approach reported previously [1] with modifications. 4 mg of DFCR was added into 4 mL of FPBA-functionalized NPs To prepare non-imprinted NPs for comparison, the processing procedure was the same except that no template was immobilized onto FPBA-functionalized NPs.
The imprinting time was optimized in terms of the imprinting factor (IF) obtained with different imprinting times.
where Qe is the amount of the target compound bound by the dt-MIP at equilibrium, Qmax is the saturated adsorption capacity, x is the concentration of free target compound, and n is and the Hill coefficient.
In vitro cytotoxicity of FITC-doped dual-templated MIP NPs.

Proliferation inhibition test in vitro and IC50 calculation.
Exponentially growing HepG-2 were harvested and placed in 96-well plates at concentration of 5,000 cells/well. After incubation at 37C for 24 h, the HepG-2 cells were respectively treated with DFCR and DFCRloaded dt-MIP dispersed in cell culture medium with different concentrations (the concentration of DFCR is from 0 μg/mL-200 μg/ mL, and the concentration of DFCR-loaded dt-MIP is from 0 μg/mL-2 mg/ mL) for 24 h.
The cells without NPs were used as control. The wells with equal volume of cell culture medium were used as background. Then 50 μL of MTT (1 mg/mL) was added to each well and the plates were incubated at 37C for another 4 h in dark. Subsequently, the supernatant was discarded and 150 μL of DMSO was added to each well. After shaking for 10 min, the absorption of the solution was monitored on a Varioskan Flash.
Absorbance at 550 nm was measured. Inhibition of cell growth was calculated according to the following equation: Data are reported as the mean of three independent experiments.
The IC50 value of tested drug was obtained by fitting the data according to the following equation: where X is the drug concentration, Y is the cell vialibity%, and h is the Hill coefficient. Min stands for the minimum Y value, while Max stands for the maximal Y value.

Proliferation inhibition test in vitro for different time.
Exponentially growing MCF-7, HepG-2 were harvested and placed in 96-well plates at concentration of 5,000 cells/well. After incubation at 37C for 24 h, the HepG-2 and MCF-7 cells were respectively treated with DFCR-

Preparation of NIR797-doped dual-templated NPs.
NIR797-doped dual-templated MIP NPs were prepared as the same as the preparation of FITC-doped NPs described above except that the NIR797 dye was reacted with APTES.

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In vivo biodistribution in the mice. All