Controllable Engineering and Functionalizing of Nanoparticles for Targeting Specific Proteins towards Biomedical Applications

Abstract Nanoparticles have been widely used in important biomedical applications such as imaging, drug delivery, and disease therapy, in which targeting toward specific proteins is often essential. However, current targeting strategies mainly rely on surface modification with bioligands, which not only often fail to provide desired properties but also remain challenging. Here an unprecedented approach is reported, called reverse microemulsion‐confined epitope‐oriented surface imprinting and cladding (ROSIC), for facile, versatile, and controllable engineering coreless and core/shell nanoparticles with tunable monodispersed size as well as specific targeting capability toward proteins and peptides. Via engineering coreless imprinted and cladded silica nanoparticles, the effectiveness and superiority over conventional imprinting of the proposed approach are first verified. The prepared nanoparticles exhibit both high specificity and high affinity. Using quantum dots, superparamagnetic nanoparticles, silver nanoparticles, and upconverting nanoparticles as a representative set of core substrates, a variety of imprinted and cladded single‐core/shell nanoparticles are then successfully prepared. Finally, using imprinted and cladded fluorescent nanoparticles as probes, in vitro targeted imaging of triple‐negative breast cancer (TNBC) cells and in vivo targeted imaging of TNBC‐bearing mice are achieved. This approach opens a new avenue to engineering of nanoparticles for targeting specific proteins, holding great prospects in biomedical applications.

S5 was repeated three times. For control experiments, all the procedures were the same as described above except the absence of epitope in the test samples. For optimization of conventional epitope-imprinted polymers, all the procedures were the same as described above except that cMIPs and cNIPs were replaced by MIPs and NIPs prepared under otherwise identical conditions, respectively.
Selectivity test of B2M-specific cMIP. The selectivity of B2M C-terminal epitope-imprinted cMIP at the peptide level was evaluated using the C-terminal epitopes of B2M (KIVKWDRDM), TRF (LEACTFRRP) and TfR (LEACTFRRP), and the N-terminal epitopes of alpha fetoprotein (AFP) (RTLHRNEYG) and carcinoembryonic antigen (CEA) (KLTIESTPF) as test peptides. First, each epitope standard solution (0.1 mg/mL) was separately prepared with phosphate buffer (10 mM, pH 7.4). Then equivalent corresponding cMIP and cNIP (2 mg each) were added to 200 μL of the epitope solutions in 250-μL microcentrifugal tubes. The tubes were shaken on a rotator at room temperature for 30 min. The nanoparticles were collected by centrifugation at 4,000 rpm for 30 min and rinsed with 200 μL of phosphate buffer (10 mM, pH 7.4) three times. Second, the nanoparticles were re-suspended and eluted in 20 μL of ACN:H2O:HAc = 50:49:1 (v/v) at room temperature for 10 min on a rotator. Finally, the nanoparticles were precipitated via centrifugation and the supernatant were collected. The amount of epitope bound by the cMIP was determined by measuring the UV absorbance of the supernatant at 214 nm. The measurement was repeated three times. For control experiments, all the procedure was the same as described above except the absence of epitope in the test samples. For the selectivity test of B2M C-terminal epitope-imprinted cMIP in the protein level, all the procedure was the same as described above except that the test peptides used were changed to the proteins B2M, RNase A, BSA, RNase B and OVA.
For the selectivity test of B2M C-terminal epitope-imprinted MIP at the peptide and protein level, all the procedures were the same as described above except that the B2M C-S6 terminal epitope-imprinted cMIP and cNIP were replaced by B2M C-terminal epitopeimprinted MIPs and NIPs, respectively.

Measurement of adsorption isotherm.
A series of standard solutions of fluorescently labeled B2M C-terminal epitope (FITC-KIVKWDRDM) of known concentrations were prepared with phosphate buffer (10 mM, pH 7.4). A volume of 200 μL of the above standard solutions was added to a 96-well plate, and the fluorescence intensity was measured by a microplate reader.
Then 2 mg of the B2M C-terminal epitope-imprinted cMIP were separately added to 1 mL of the above standard solutions and shaken at room temperature for 30 min. After the nanoparticles were centrifuged, 200 μL of the supernatant was added to a 96-well plate and their fluorescence intensity was measured by the microplate reader. An adsorption isotherm was established by plotting the difference between the fluorescence intensity of the standard solution before extraction and of the supernatant after extraction using B2M C-terminal epitope-imprinted cMIP against the logarithmic concentration of FITC-KIVKWDRDM. To estimate the binding affinity of B2M C-terminal epitope-cMIP, the amount of FITC-KIVKWDRDM bound by the B2M C-terminal epitope-cMIP was plotted according to the Scatchard equation as given below: For the adsorption isotherm of B2M C-terminal epitope-imprinted MIP, all the procedure was the same as described above except that the B2M C-terminal epitope-imprinted cMIP were changed to B2M C-terminal epitope-imprinted MIP. The response data and affinity parameter were obtained using the ForteBio Data analysis (ver.

Dissociation constants (Kd) determination by bio-layer interferometry (BLI
12). All the response data obtained were subtracted from the signal for blank controls, which only contained buffer. For the binding assay of anti-B2M antibody, the antibodies were immobilized onto protein A biosensors while the other conditions were the same as above.
Preparation of GPNMB-specific QD520@cMIP. The solution S3 described in microemulsion formation procedure was used for the imprinting, while slightly different peptide sequences were used as the epitope depending on the targets to test. For selectivity test and the optimization of imprinting conditions, the decapeptide AKRFHDVLGNK, which has an additional alanine at its N-terminal as compared with the native nonapeptide epitope, was used as the epitope. This was due to the fact that the protein GPNMB used in this study remained an additional alanine as its N-terminal. However, the use of additional amino acid in the epitope will not the recognition of the prepared cMIP towards its alanine-deleted analog (KRFHDVLGNK) in selectivity test (Figure S 13). While for the imaging of GPNMBoverexpressed cell lines, the native nonapeptide KRFHDVLGNK was used as the epitope (see Figure S 2 for the structure). For the imprinting, the microemulsion was first stirred at 700 rpm for 10 min at 25 ºC, and 700 μL of QD520 solution (3 mg/mL in cyclohexane) was then added S8 and stirred for 30 min. 1 mg of C13-grafted epitope was added to the above solution and continued stirring for another 30 min. After that, 1 mL of S3 was added dropwise carefully, and the mixture was allowed to stirred at 700 rpm at 25 ºC for 24 h. Subsequently, 100 μL of TEOS was added dropwise to the mixture, and the mixture was stirred at 700 rpm for another 24 h at 25 ºC. The obtained materials were released from the microemulsion by adding acetone, followed by centrifugation at 4,000 rpm for 30 min and washed with anhydrous ethanol and water five times, respectively.
To remove the C13-grafted epitope, the obtained QD520@cMIP was dispersed into 5 mL of ACN:H2O:HAc = 50:49:1 (v/v) and shaken for 20 min at room temperature. The above elution process was repeated three times. After removing the C13-grafted epitope template, the prepared GPNMB N-terminal epitope-imprinted QD520@cMIP was collected by centrifugation at 4,000 rpm for 30 min. The collected QD520@cMIP was washed with water and anhydrous ethanol three times each and then freeze-dried in a vacuum overnight.
For the preparation of QD520@cNIP, the process was the same except that no templates were added.
Preparation of HER2-specific QD620@cMIP. The N-terminal epitope of HER2 was used, the preparation process was the same as above except the C13-grafted GPNMB N-terminal epitope and QD520 were changed to C13-grafted HER2 N-terminal epitope and QD620, respectively.
Optimization of imprinting conditions of GPNMB-specific QD520@cMIP. The specific monomer ratios and the total monomers/TEOS ratios used for the preparation of GPNMB Nterminal epitope-imprinted QD520@cMIP were optimized in terms of the obtained IF value.
The optimization procedure was the same as describe above, except the materials were changed to GPNMB N-terminal epitope-imprinted QD520@cMIP.

S9
Optimization of imprinting conditions of HER2-specific QD620@cMIP. The specific monomer ratio used for the preparation of HER2 N-terminal epitope-imprinted QD620@cMIP was optimized in terms of the obtained IF value. The optimization procedure was the same as describe above, except the materials were changed to HER2 N-terminal epitope-imprinted QD620@cMIP.
Selectivity test of GPNMB-specific QD520@cMIP and HER2-specific QD620@cMIP. The selectivity test of QD520@cMIP and QD620@cMIP at the peptide and protein levels was carried out using the same procedure described above except that the B2M C-terminal epitopeimprinted cMIP and cNIP were replaced by QD520@cMIP and QD520@cNIP or QD620@cMIP and QD620@cNIP.
In vitro cytotoxicity of GPNMB-specific QD520@MIP and QD520@cMIP. Cell viability was determined by the MTT assay. Briefly, MCF-7 or MCF-10A cells were seeded on 96-well microplates with a density around 10,000 cells per well and allowed to adhere for 24 h prior to the assay. The cells were incubated with different concentrations of GPNMB-specific QD520@MIP or GPNMB-specific QD520@cMIP at 37 °C for 24 h. The zero concentration group was used as control and others were used as test group. And wells without cells were used as background group. Then 50 μL of MTT indicator dye (1 mg/mL in PBS) was added.
After incubating for another 4 h at 37°C in the dark, the supernatant was discarded, and 150 μL of DMSO was added to each well. After shaking for 10 min on a shaking table, the optical density of the solution was monitored on the microplate reader. Absorbance was measured at a wavelength of 550 nm. The cell viability was expressed as a percentage of the absorbance of test cells (added with GPNMB-specific QD520@MIP or GPNMB-specific QD520@cMIP) over that of control experiment (without the addition of GPNMB-specific QD520@MIP or S10 GPNMB-specific QD520@cMIP) (both were deducted by the background absorbance), which can be calculated by the following equation: Western blot. MCF-7 cells were cultured in the RPMI-1640 medium with 10% FBS for 2-3 days (37 ºC, 5% CO2), while MDA-MB-157 and MDA-MB-361 cells were cultured in the DMEM medium with 10% FBS for 2-3 days (37 ºC, 5% CO2). After that, cell lysis buffer containing protease inhibitor was added on the cells and the cells were lysed on ice for 30 min.
Cells were scraped with clean cell scrapes and transferred into centrifugal tubes with a pipette.
The lysate mixture was centrifuged in -4 °C at 12,000 rpm for 5 min and the supernatant was cellected for further use. Protein concentration was quantified using BCA protein quantitation assay, and 50 μg of protein from cell lysate was loaded on sodiumdodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE). Polyvinylidene fluoride (PVDF) membrane was used to transfer the gels. After blocking with 5% BSA in TBST (Tris-buffered saline with 0.1% Tween 20), the membrane was incubated with primary antibodied at 4 °C overnight and then secondary antibodies at room temperature for 1 h. Primary antibodies against GPNMB or HER2 were used at 1:1,000 dilution and secondary antibodies were used at 1: 5,000 dilution.
Unbound antibodies were washed away with the TBST, then the membrane was incubated with ECL chemiluminescence solution. The Bio-Rad GelDoc-XRTM gel imaging system was employed to expose the membrane and obtain images. β-actin was used to ensure equal loading. For immunofluorescence, the cells were fixed with 4% formaldehyde for 15 min, followed by incubation with 5% bovine serum albumin in PBS for 60 min to block the nonspecific binding sites. Then the cells were stained with anti-GPNMB antibody or anti-HER2 antibody for 60 min, followed by fluorescent goat anti-rabbit IgG H&L secondary antibody for 60 min.
Primary antibodies against GPNMB or HER2 and secondary antibodies were used at 1:1,000 dilution. After that 1×PBS and free antibodies were removed and the remaining cells were After removing the supernatant, the cells were washed with 1×PBS twice and filtrated with 200 S12 mesh sieves. The obtained cell suspensions were injected into cytoanalyzer and the count of cells was set to 10,000.
For flow cytometry assay of antibodies, the cells were harvested and washed first and the total cell number were determined. Then the cells were resuspended to approximately 1-5×10 6 cells/mL in ice cold PBS. And 100 μL of cell suspension was added to each tube, followed by incubation with ice cold 5% bovine serum albumin in PBS for 60 min to block the nonspecific binding sites. After that, the cells were stained with anti-GPNMB antibody or anti-HER2 antibody at 1:500 dilution for 60 min. The cells were washed three times by centrifugation at 400 g for 5 min and resuspended in ice cold PBS. Then the cells were stainied with fluorescent goat anti-rabbit IgG H&L secondary antibody in 5% bovine serum albumin in PBS at 1:1,000 dilution for 60 min in the dark, followed by centrifugation at 400 g for 5 min. After removing the supernatant, the cells were washed with 1×PBS three times and filtrated with 200 mesh sieves. The obtained cell suspensions were injected into cytoanalyzer and the count of cells was set to 10,000.

Preparation of GPNMB-specific NIR797-doped cMIP or cNIP. The amino group of APTES
can react with the isothiocyanate of NIR797 to yield a thiourea bridge. Typically, a volume of 5 µL of APTES and 2 mg NIR797 were dissolved in 1 mL of ethanol. After reaction in darkness with vigorous stirring for 12 h, NIR797-derivatized APTES was formed in the solution. The final products were obtained by removing ethanol by rotary evaporation. For the imprinting, the solution S3 described in microemulsion formation procedure was used for the imprinting, while slightly different peptide sequences were used as the epitope depending on the targets to test. The microemulsion was first stirred at 700 rpm at 25 ºC for 30 min. And above prepared NIR797-derivatized APTES mixed with 10 μL of TEOS was added and stirred for 30 min. Then 1 mg of C13-grafted epitope was added to the above solution and continued stirring for another S13 stirred at 700 rpm at 25 ºC for 24 h in the dark. Subsequently, 100 μL of TEOS was added dropwise to the mixture, and the mixture was stirred at 700 rpm at 25 ºC for another 24 h in the dark. The obtained materials were released from the microemulsion by adding acetone, followed by centrifugation at 4,000 rpm for 30 min and washed with anhydrous ethanol and water five times, respectively.
To remove the C13-grafted epitope, the obtained NIR797-doped cMIP was dispersed into 5 mL of ACN:H2O:HAc = 50:49:1 (v/v) and shaken for 20 min at room temperature. The above elution process was repeated three times. After removing the C13-grafted epitope template, the prepared GPNMB N-terminal epitope-imprinted NIR797-doped cMIP was collected by centrifugation at 4,000 rpm for 30 min. The collected NIR797-doped cMIP was washed with water and anhydrous ethanol three times each and then freeze-dried in a vacuum overnight.
For the preparation of NIR797-doped cNIP, the process was the same except that no templates were added.