Ligand Installation to Polymeric Micelles for Pediatric Brain Tumor Targeting

Medulloblastoma is a life-threatening disease with poor therapeutic outcomes. In chemotherapy, low drug accumulation has been a cause of these outcomes. Such inadequate response to treatments has been associated with low drug accumulation, particularly with a limited cellular uptake of drugs. Recently, the conjugation of drugs to ligand molecules with high affinity to tumor cells has attracted much attention for enhancing drug internalization into target cells. Moreover, combining tumor-targeting ligands with nano-scaled drug carriers can potentially improve drug loading capacity and the versatility of the delivery. Herein, we focused on the possibility of targeting CD276/B7-H3, which is highly expressed on the medulloblastoma cell membrane, as a strategy for enhancing the cellular uptake of ligand-installed nanocarriers. Thus, anti-CD276 antibodies were conjugated on the surface of model nanocarriers based on polyion complex micelles (PIC/m) via click chemistry. The results showed that the anti-CD276 antibody-installed PIC/m improved intracellular delivery into CD276-expressing medulloblastoma cells in a CD276-dependent manner. Moreover, increasing the number of antibodies on the surface of micelles improved the cellular uptake efficiency. These observations indicate the potential of anti-CD276 antibody-installed nanocarriers for promoting drug delivery in medulloblastoma.


Introduction
Medulloblastoma (MB) is the most typical malignant brain tumor in the pediatric population. It comprises 20% of all childhood brain tumors and 63% of intracranial embryonal tumors [1]. Known for its high lethality and 5-year survival rate of 40-60% [2], an effective yet safe treatment strategy is urgent [3]. State-of-the-art treatment is mostly based on surgical intervention [4], with a combination of adjuvant radiotherapy [5] and/or chemotherapy [6]. Though such treatments have shown a certain level of success, the treatment effect is unsatisfactory in a large population [7], who frequently suffer symptoms generally termed "chemobrain," caused by the side effects of the administered antitumor drugs [8]. These side effects are thought to be a product of drugs being unintendedly delivered to healthy brain tissue due to their poor tumor-targeting effect [9]. One such drug is Topotecan (TPT), which is known to accumulate poorly at a rate of approximately [8]. These side effects are thought to be a product of drugs being unintendedly delivered to healthy brain tissue due to their poor tumor-targeting effect [9]. One such drug is Topotecan (TPT), which is known to accumulate poorly at a rate of approximately 0.5% dose/g tumor [10]. Given the example of TPT, it is clear that a strategy to target and deliver drugs to the tumor at high efficiency is in high demand to improve the clinical outcomes of MB while minimally risking the patient's quality of life.
To improve the therapeutic outcomes of chemotherapy, various approaches to targeting tumor cells and delivering sufficient drug payload have been explored so far [11]. Recently, conjugating antibodies or fragmented antibodies that bind specifically to proteins highly expressed in tumor cells to drugs has provoked attention in the field of drug delivery [12]. One strategy example is conjugating an antibody with high affinity to CD276, a transmembrane protein highly expressed in pediatric tumors [13], to an antitumor drug Cu-NOTA [14]. Here, drug accumulation within breast cancer cells was significantly improved because of the endocytosis of an anti-CD276 antibody (CD276Ab) by tumor cells, followed by its subcellular localization in both early endosomes and lysosomes [15], thereby demonstrating the effectivity of utilizing antibodies to drive the drugs to the tumor. In another set of studies, nanocarriers have been used to deliver the carried drugs across the blood-brain tumor barrier (BBTB) [16][17][18]. An illustrative instance of the implementation of this approach was demonstrated using cRGD conjugated polymeric micelle for glioblastoma targeting [19]. As such, utilizing the tumor-targeting effect of antibodies and nanocarriers could offer efficient delivery of drugs to the intended cells, benefiting both the effectiveness and safety of chemotherapy.
Polyion complex micelle (PIC/m) composed of oppositely charged polymers is a nanocarrier that can sufficiently withstand the ionic strength of solvents [20,21] and environmental pH [22]. Therefore, it works as a model carrier because of its high stability. In this pilot study, influenced by the past success of antibody-drug conjugates and tumortargeted nanocarriers, fluorescently-labeled model micelles were modified with multiple anti-CD276 antibodies (CD276Ab), then tested for their efficiency in targeting tumor-derived cells (Figure 1). As a proof of concept, CD276Ab was introduced to fluorescentlylabeled azide (N3)-equipped polyion complex micelles (N3-PIC/m) through click chemistry, then added to MB patient-derived DAOY cell cultures [23]. The study results indicated that the antibodies were successfully introduced to the surface of the PIC/m, which elicited a significant improvement in the rate of internalization of the micelle by MB cells in vitro. These findings suggest that the proposed strategy can potentially improve drug delivery efficiency to tumor cells, advancing the design of effective and safe treatment for MB.

IgG Preparation as Recombinant Proteins
The amino acid sequence of the 8H9 antibody was obtained from the PDB database (PDB ID: 5CMA). The IgG antibody was expressed and purified as described previously [24]. Briefly, Expi CHO cells were co-transfected with expression vectors for heavy and light chains, and the supernatant was collected 12 days after transfection. The supernatant was purified via affinity chromatography using rProtein A Sepharose resin and subsequent size exclusion chromatography. The purity of the eluted sample was evaluated by SDS-PAGE followed by Coomassie staining.

Estimation of Number of DBCO Conjugated to CD276Ab by Fluorescamine
The number of residual primary amine groups per antibody was determined through a previously reported assay method using fluorescamine [25,26]. First, fluorescamine was dissolved in DMSO at 3.0 mg/mL and mixed thoroughly. A 2.0 mg/mL bovine serum albumin (BSA) standard sample in 0.9% saline and 0.05% sodium azide solution was adjusted to 7.8, 15.6, 31.2, 62.5, 125, 250, and 500 µg/mL through multistep dilution with D-PBS(-). Then 9 µL of each BSA solution was mixed with 3 µL fluorescamine solution and reacted for 15 min at 25 • C, and 2 µL of each solution was used to measure the fluorescence intensity to draw a standard curve. Note here it has been reported that BSA has 30 residual primary amine groups per molecule [27]. For the estimation of the primary amine groups in the CD276Ab solution, 3 µL of the CD276Ab solution (0.10 mg/mL) in D-PBS(-) was mixed with fluorescamine solution (3.0 mg/mL) in DMSO and reacted under the abovementioned conditions.
Estimation of the number of DBCO groups conjugated onto CD276Ab antibodies was conducted by measuring the remaining residual amine groups after DBCO conjugation, then comparing this to that of 'as prepared' CD276Ab antibodies. 0, 2.8, 10.5, 22.1, 88.6, and 177.1 µg/mL NHS-PEG 4 -DBCO solution (2 µL) in DMSO was mixed with CD276Ab solution (9 µL, 0.10 mg/mL) in D-PBS(-) and left still at 25 • C for 1 h. Here, the amount of each prepared NHS-PEG 4 -DBCO corresponds to 0, 0.03, 0.13, 0.25, 1.0, and 2.0, equivalent to the number of residual primary amines in the CD276Ab solution, respectively. Fluorescamine solution (3 µL, 3.0 mg/mL) was added and reacted for 15 min at 25 • C (N = 5). The number of residual primary amine and conjugated DBCO were estimated from the standard curve drawn using a BSA solution.

Fabrication of DBCO-CD276Ab and DBCO-CD276Ab-Cy5
DBCO-PEG 4 -NHS was first conjugated to the amine groups of CD276Ab. DBCO-PEG 4 -NHS in DMSO (3.3 µL, 10.0 mg/mL) was added to the antibody solution (600 µL, 1.12 mg/mL) in D-PBS(-) at concentrations equivalent to 0.25 times the number of amine groups within the solution and incubated at room temperature for 1 h.

Purification of DBCO-CD276Ab and DBCO-CD276Ab-Cy5
DBCO-CD276Ab fabricated through the aforementioned scheme was purified via a Protein G HP SpinTrap (Cytiva, Marlborough, MA, USA) [28]. The column was initially washed with phosphate buffer (20 mM, pH 7.0, 0 mM NaCl) 3 times without a lid, then the crude mixture was loaded to the column and left still for 4 min. Subsequently, the column was washed again with the same buffer, then 400 µL of 0.1 M glycine-HCl (pH 2.7) was loaded for elution. Tris-HCl (17 µL, 1 M, pH 9.0) was added to the microcentrifuge tube, which holds the column for neutralization so that the pH of the DBCO-CD276Ab solution after neutralization was 7.0. After centrifugation (2000 rpm, 20 s), DBCO-CD276Ab was collected from the microcentrifuge tube at a yield of 50.0% (N = 3). The concentration of purified antibodies was calculated by measuring the absorbance at 280 nm ( Figure S1, see Supplementary Materials). The same protocol was used to purify DyLight 488-(DBCO-CD276Ab-DyLight 488) or Cy5-labeled (DBCO-CD276Ab-Cy5) antibodies.

CD276 Detection by Flow Cytometry
DAOY cells were harvested at a confluency of 80% and were resuspended with PBS supplemented with 10% FBS (FACS Buffer). A total of 500 cells were stained in 100 µL of FACS buffer containing the CD276 antibody conjugated with phycoerythrin (PE) (Biolegend, #331605; 1:200 dilution) for 1 h at 4 • C ( Figure S2, see Supplementary Materials). FACS was performed using BD FACSCanto II (BD Biosciences) immediately after washing the cells with FACS Buffer. Data were analyzed using FlowJoTMv10 (BD Biosciences).

Immunocytochemical Evaluation
The concentration of purified Cy5-labeled DBCO-CD276Ab was determined by measuring the absorption at 280 nm by NanoDrop One (Thermo Scientific, Waltham, MA, USA) and diluted to 5.0 µg/mL with D-PBS(-). GFP-expressing DAOY cells (1.5 × 10 4 cells/well) were seeded on an 8-well chamber and then incubated in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL of streptomycin at 37 • C, 5% CO 2 , until 70% confluency. CD276Ab (200 µL) was added to the cells at 0, 12.5, 25.0, 50.0, and 100 µg/mL, followed by subsequent incubation for 1 h at 37 • C, 5% CO 2 . Each concentration was adjusted by multistep dilution using a culture medium. After washing the cells three times with D-PBS(-), DBCO-CD276Ab-Cy5 (200 µL, 30.0 µg/mL) was added to the cell cultures and incubated for 1 h at 37 • C and 5% CO 2 . The cell nuclei were counterstained with Hoechst 33258, then the cells were observed, and the images were captured using a confocal laser scanning microscope (LSM780-06, Carl Zeiss, Oberkochen, Germany). The total fluorescence intensity of Cy5 within the cell cytoplasm (determined based on GFP fluorescence) of each cell was calculated and compared amongst the different culture conditions (n = 50). All image analyses were conducted using Image J [29,30]. A significant difference in fluorescence intensity between culture conditions was detected by performing a Student's t-test followed by Bonferroni correction for multiple comparisons.

Fabrication of N 3 -PIC/m
N 3 -PIC/m was prepared based on a previous report [31]. Briefly, an aniomer solution was prepared by dissolving Methoxy-PEG-poly (α,β-aspartic acid) (PEG-PAsp, 2k-75), PEG-PAsp-Cy5 (2.2k-73), and N 3 -PEG-PAsp (2k-68) in phosphate buffer (10 mM, pH 7.4, 0 mM NaCl) at a ratio of 20:40:40, respectively, so that the final polymer concentration was 1.0 mg/mL. This mixing ratio was used to prepare N 3 groups on 20% of the product N 3 -PIC/m's surface area. A catiomer solution was prepared by dissolving Methoxy-PEGpoly[(5-aminopenthy)-α,β-aspartamide] (2k-72) in phosphate buffer (10 mM, pH 7.4, 0 mM NaCl) at 1 mg/mL. The polymer solutions were mixed at an aniomer:catiomer ratio of 5:4 to fabricate N 3 -PIC/m. The product micelles were stabilized by cross-linking the polymers with EDC/HCl, then purified with an ultracentrifugation unit, Vivaspin 6 MWCO 100,000 Da (Sartorius Stedium Biotech GmbH, Goettingen, Germany), at 2000 rpm. The final micelle solution was prepared in D-PBS(-), and its concentration was determined by measuring and comparing the Cy5-derived fluorescence with a standard curve. The characterization of N 3 -PIC/m was conducted via dynamic light scattering (DLS) measurement (Zetasizer Nano ZS90, Malvern Panalytical Ltd., Malvern, UK). The diffusion coefficient and the number of CD276Ab installed to N 3 -PIC/m were calculated through fluorescence correlation spectroscopy (FCS). As for the FCS, the diffusion time τ of Cy5 and DyLight 488 were measured. This value can be expressed using confocal ratio ω and diffusion coefficient D as in Equation (1) [33]. The diffusion coefficient of CD276Ab-PIC/m, D CD276Ab-PIC/m , was calculated using the diffusion coefficient of Cy5, (3.7 ± 0.15) × 10 −6 cm 2 /s [34], and Equation (2) at the same confocal ratio. The diameter of PIC/m and CD276Ab-PIC/m were calculated using Equation (3) [33]. In the equation, diffusion coefficient D can be expressed using the Stokes-Einstein equation, where k is the Boltzmann constant, T is the temperature, η is the viscosity, and r is the hydrodynamic radius [29].
Counts per molecule, η , reflects the fluorescence intensity of each fluorescent molecule. The number of CD276Ab installed to N 3 -PIC/m and the number of Cy5 molecules per CD276Ab-PIC/m were calculated using Equations (4) and (5) [35].

Cytotoxicity of CD276Ab-PIC/m
GFP-expressing DAOY cells (1.0 × 10 4 ) were seeded on each well of an 96-well plate and then cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL of streptomycin at 37 • C, 5% CO 2 until the chamber bottom reached 70% confluency. 50 µL of N 3 -PIC/m, CD276Ab(1)-PIC/m, and CD276Ab(2)-PIC/m (10 µg/mL) was added, then incubated in the culture medium for 6 h at 37 • C, 5% CO 2 . After washing the cells three times with D-PBS, the GFP-derived fluorescence was quantified via a multiplate reader (Spark, Tecan, Männendorf, Switzerland) at 478 nm excitation and 535 nm detection. The fluorescence of cells incubated in the growth medium was considered 100% viable and used to standardize the viability of those incubated with the PIC/m.

Cell Internalization Efficiency of CD276Ab-PIC/m
GFP-expressing DAOY cells (1.5 × 10 4 ) were seeded on each well of an 8-well chamber and then cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL of streptomycin at 37 • C, 5% CO 2 until the chamber bottom reached 70% confluency. CD276Ab (200 µL) was added to the cells at 0, 12.5, 25.0, and 50.0 µg/mL, followed by subsequent incubation for 1 h at 37 • C, 5% CO 2 . After washing the cells three times with D-PBS(-), CD276Ab-PIC/m solution (200 µL, 10 µg/mL), the cells were incubated in the culture medium for 3 h at 37 • C. Subsequently, the cultured cell nuclei were counterstained with Hoechst 33258, then the cells were observed under a confocal laser scanning microscope (LSM780-06, Carl Zeiss, Oberkochen, Germany). For quantification, the fluorescence intensity of Cy5 within the cell cytoplasm (determined based on GFP fluorescence) was calculated per cell and compared amongst the different culture conditions (n = 50 per condition). All image analyses were conducted using Image J [29,30]. A significant difference in fluorescence intensity between culture conditions was detected by performing a Student's t-test followed by Bonferroni correction for multiple comparisons.

Characterization of DBCO-CD276Ab
The fabrication scheme of CD276Ab is described in the Supplementary Materials. To introduce CD276Ab to the micelle surface, PIC/m equipped with azide (N 3 ) moiety (N 3 -PIC/m) and CD276Ab with dibenzocyclooctyne (DBCO) moiety conjugated to its primary amine groups (DBCO-CD276Ab) were prepared. Azide is known to react with DBCO at around pH 7.0 in an aqueous solution through click reaction [32,36]. The fabrication scheme of N 3 -PIC/m is described in the Supplementary Materials. To fabricate DBCO-CD276Ab at various surface DBCO densities, Dibenzocyclooctyne-poly (ethylene glycol) 4 -N-hydroxysuccinimidyl ester (NHS-PEG 4 -DBCO) was conjugated to the residual primary amine groups. DBCO-PEG 4 -NHS was added to the antibody solution at amounts equivalent to 0, 0.03, 0.125, 0.25, 1.0, and 2.0 times the amount of CD276Ab primary amine groups. The number of NHS-PEG 4 -DBCO conjugated to CD276Ab was then calculated by evaluating the number of primary amines remaining on the antibody and subtracting this from the initial amine count. Here, the number of amine groups per antibody was determined through a previously reported assay method using fluorescamine [26]. The number of NHS-PEG 4 -DBCO conjugated to CD276Ab increased with the increase in dose, reaching a maximum of 9 DBCO groups per antibody at 2.0 eq. feeding amount (Figure 2a). To accomplish the installation of CD276Ab to N 3 -PIC/m, one DBCO per CD276Ab is theoretically sufficient. To minimize the risk of CD276Ab denaturation, which can potentially cause negative impacts (e.g., a decrease in its activity, in vivo stability, and structural integrity) [37], CD276Ab conjugated with approximately five DBCOs (DBCO-PEG 4 -NHS feeding concentration of 0.25 eq. to primary amines) was chosen to be used for modifying N 3 -PIC/m. After removing the non-reacted NHS-PEG 4 -DBCO by Protein G column, the product was measured through size exclusion chromatography (SEC) (Figure 2b). The absorption of DBCO-CD276Ab was measured to check the conjugation of DBCO at 312 nm.
The absorption spectrum of DBCO-CD276Ab consisted of a unique DBCO-derived peak, indicating successful conjugation of DBCO to CD276Ab (Figure 2c). ically sufficient. To minimize the risk of CD276Ab denaturation, which can potentially cause negative impacts (e.g., a decrease in its activity, in vivo stability, and structural integrity) [37], CD276Ab conjugated with approximately five DBCOs (DBCO-PEG4-NHS feeding concentration of 0.25 eq. to primary amines) was chosen to be used for modifying N3-PIC/m. After removing the non-reacted NHS-PEG4-DBCO by Protein G column, the product was measured through size exclusion chromatography (SEC) (Figure 2b). The absorption of DBCO-CD276Ab was measured to check the conjugation of DBCO at 312 nm. The absorption spectrum of DBCO-CD276Ab consisted of a unique DBCO-derived peak, indicating successful conjugation of DBCO to CD276Ab (Figure 2c).

In Vitro Functionality Evaluation of DBCO-CD276Ab
To evaluate the functionality of the fabricated DBCO-CD276Ab, an inhibition assay using as prepared' CD276Ab and DBCO-CD276Ab was conducted. DAOY cells, derived from a pediatric brain tumor and known to express CD276 protein [13], were provided from ATCC (Manassas, VI, USA) and were gene-edited to express GFP to visualize the cell cytoplasm. DAOY cells were first incubated with abundant CD276Ab, incubated with Cy5-labeled DBCO-CD276Ab (DBCO-CD276Ab-Cy5, 2.2 Cy5 to 1 antibody), then imaged using a confocal laser scanning microscope (CLSM). The method of gene editing is described in detail in the Supplementary Materials. DAOY cells incubated without inhibition were also observed as a control. Under inhibition, DAOY cells indicated a low Cy5derived signal within the cells. In contrast, those incubated without inhibition exhibited a noticeable accumulation of fluorescence within the cells (Figure 3a). This decrease in Cy5derived fluorescence intensity under the existence of abundant inhibiting antibodies clearly indicates that DBCO-CD276Ab can selectively bind to its target, CD276. To further assess the effect of inhibition, inhibition was conducted under four conditions where the feeding concentration differed. The DBCO-CD276Ab-Cy5-derived fluorescence showed a

In Vitro Functionality Evaluation of DBCO-CD276Ab
To evaluate the functionality of the fabricated DBCO-CD276Ab, an inhibition assay using 'as prepared' CD276Ab and DBCO-CD276Ab was conducted. DAOY cells, derived from a pediatric brain tumor and known to express CD276 protein [13], were provided from ATCC (Manassas, VI, USA) and were gene-edited to express GFP to visualize the cell cytoplasm. DAOY cells were first incubated with abundant CD276Ab, incubated with Cy5-labeled DBCO-CD276Ab (DBCO-CD276Ab-Cy5, 2.2 Cy5 to 1 antibody), then imaged using a confocal laser scanning microscope (CLSM). The method of gene editing is described in detail in the Supplementary Materials. DAOY cells incubated without inhibition were also observed as a control. Under inhibition, DAOY cells indicated a low Cy5-derived signal within the cells. In contrast, those incubated without inhibition exhibited a noticeable accumulation of fluorescence within the cells (Figure 3a). This decrease in Cy5-derived fluorescence intensity under the existence of abundant inhibiting antibodies clearly indicates that DBCO-CD276Ab can selectively bind to its target, CD276. To further assess the effect of inhibition, inhibition was conducted under four conditions where the feeding concentration differed. The DBCO-CD276Ab-Cy5-derived fluorescence showed a significant decrease with an increase in inhibitor concentration (Figure 3b), which insists that the fabricated antibody competes with the inhibiting antibody when binding to CD276. These results strongly suggest that the targeting ability of the fabricated DBCO-CD276Ab was conserved throughout the fabrication process, with minimal denaturation or structural decay.

Preparation and Characterization of CD276Ab Conjugated Micelles
Model micelles, N 3 -PIC/m with cross-linked core polymers, were prepared based on a previous report [31]. Note that the comprising polymers were chemically crosslinked for stabilization so that any complication due to instability and disassembly can be excluded while characterizing the micelles. Briefly, the catiomer of Methoxy-PEG-poly[(5aminopenthy)-α,β-aspartamide] (2k-72) and aniomer of Methoxy-PEG-poly(α,β-aspartic acid) (PEG-PAsp (M n of PEG = 2000, DP of P(Asp) = 75)), PEG-PAsp-Cy5 (2.2k-73) and N 3 -PEG-PAsp (2k-68) were mixed in a phosphate buffer (10 mM, pH 7.4, 0 mM NaCl) at significant decrease with an increase in inhibitor concentration (Figure 3b), which insists that the fabricated antibody competes with the inhibiting antibody when binding to CD276. These results strongly suggest that the targeting ability of the fabricated DBCO-CD276Ab was conserved throughout the fabrication process, with minimal denaturation or structural decay.
DBCO-CD276Ab was used to modify the N3-PIC/m surface to enhance its delivery efficiency to CD276-expressing tumor cells. CD276 is a transmembrane protein highly expressed in tumor cells but absent or weakly expressed in normal tissue [38,39]. Thus, conjugation of CD276Ab to PIC/m surface was conducted to target tumor cells with minimal off-target effects. In addition, because the number of antibodies conjugated to the nanocarrier surface is known to influence the delivery efficiency [35], the number of CD276Ab that can be conjugated onto the PIC/m surface was investigated through fluorescence correlation spectroscopy (FCS). Upon FCS measurement, DBCO-CD276Ab labeled with DyLight 488 (1.2 DyLight 488 to 1 antibody) was introduced to the N3-PIC/m surface at various addition concentrations. The spherical morphology of the fabricated N3-PIC/m and DBCO-CD276Ab was used to modify the N 3 -PIC/m surface to enhance its delivery efficiency to CD276-expressing tumor cells. CD276 is a transmembrane protein highly expressed in tumor cells but absent or weakly expressed in normal tissue [38,39]. Thus, conjugation of CD276Ab to PIC/m surface was conducted to target tumor cells with minimal off-target effects. In addition, because the number of antibodies conjugated to the nanocarrier surface is known to influence the delivery efficiency [35], the number of CD276Ab that can be conjugated onto the PIC/m surface was investigated through fluorescence correlation spectroscopy (FCS). Upon FCS measurement, DBCO-CD276Ab labeled with DyLight 488 (1.2 DyLight 488 to 1 antibody) was introduced to the N 3 -PIC/m surface at various addition concentrations. The spherical morphology of the fabricated N 3 -PIC/m and CD276Ab conjugated PIC/m, which indicates successful fabrication and conjugation without causing disassembly, was confirmed via TEM imaging ( Figure S4, see Supplementary Materials).
As the initial step of characterizing the CD276Ab installed PIC/m, the number of antibodies installed was measured by counts per molecule (evaluating the fluorescence derived from DyLight 488 conjugated on DBCO-CD276Ab). Results confirmed that 1.9 antibodies could be introduced by increasing the addition concentration to 2.3 eq. (Figure 4a). Furthermore, the number of Cy5 particles retained on CD276Ab-PIC/m was similar to that of naked N 3 -PIC/m regardless of the introduction of CD276Ab (Figure 4b). Therefore, the number of CD276Ab per PIC/m did not increase due to aggregation of the carriers; instead, the increase in number solely reflects the increase in the number of CD276Ab on the PIC/m surface. This is because steric hindrance affected to lower the CD276Ab Polymers 2023, 15, 1808 9 of 14 conjugation efficiency [35,40,41]. Nevertheless, FCS measurements of the CD276Ab-PIC/m indicated successful installation of the antibody without causing disassembly of the micelle structure. However, it must be noted that the PIC/m used in this study has remarkably high stability compared to any drug-loaded micelles, which assemble through hydrophobic and/or ionic interactions. Thus, the effect of CD276Ab conjugation on the morphology of less robust micelles must be carefully considered when designing CD276Ab micelles that carry therapeutic drugs.
rived from DyLight 488 conjugated on DBCO-CD276Ab). Results confirmed that 1.9 antibodies could be introduced by increasing the addition concentration to 2.3 eq. (Figure 4a). Furthermore, the number of Cy5 particles retained on CD276Ab-PIC/m was similar to that of naked N3-PIC/m regardless of the introduction of CD276Ab (Figure 4b). Therefore, the number of CD276Ab per PIC/m did not increase due to aggregation of the carriers; instead, the increase in number solely reflects the increase in the number of CD276Ab on the PIC/m surface. This is because steric hindrance affected to lower the CD276Ab conjugation efficiency [35,40,41]. Nevertheless, FCS measurements of the CD276Ab-PIC/m indicated successful installation of the antibody without causing disassembly of the micelle structure. However, it must be noted that the PIC/m used in this study has remarkably high stability compared to any drug-loaded micelles, which assemble through hydrophobic and/or ionic interactions. Thus, the effect of CD276Ab conjugation on the morphology of less robust micelles must be carefully considered when designing CD276Ab micelles that carry therapeutic drugs. In addition to the FCS measurements described above, the particle size and polydispersity of the prepared samples were measured by dynamic light scattering (DLS) to confirm the validity of particle size and structure (Figure 4c). The measured particle size of the PIC/m modified with 1.2 CD276Ab (CD276Ab(1)-PIC/m) and 1.9 CD276Ab (CD276Ab(2)-PIC/m) were between 30-40 nm (Table 1), indicating that the change in particle size occurred due to the conjugation of the antibody. The polydispersity of the micelles was significantly increased after the introduction of CD276Ab compared to that before the introduction, presumably due to the structure changing. The polydispersity index can be represented using a derived moment [42] and decay constant Г as: Here, decay constant Г can be expressed as: In addition to the FCS measurements described above, the particle size and polydispersity of the prepared samples were measured by dynamic light scattering (DLS) to confirm the validity of particle size and structure (Figure 4c). The measured particle size of the PIC/m modified with 1.2 CD276Ab (CD276Ab(1)-PIC/m) and 1.9 CD276Ab (CD276Ab(2)-PIC/m) were between 30-40 nm (Table 1), indicating that the change in particle size occurred due to the conjugation of the antibody. The polydispersity of the micelles was significantly increased after the introduction of CD276Ab compared to that before the introduction, presumably due to the structure changing. The polydispersity index can be represented using a derived moment k 2 [42] and decay constant Γ as: Here, decay constant Γ can be expressed as: where D T is the translational diffusion coefficient and D R is the rotational diffusion coefficient. It is reported that D R decreases as the structure changes from a sphere to a prolate ellipsoid due to depolarized signals [43]. Such change in micelle morphology is suspected to be a cause of the increase in polydispersity index as the number of conjugated antibodies increased. The zeta potential of CD276Ab and CD276Ab-PIC/m was measured, as it is a highly relevant parameter affecting the stability of nanocarriers during blood circulation. The obtained results demonstrated that CD276Ab has a weakly negative charge, consistent with previous reports [44]. In addition, N 3 -PIC/m, CD276Ab(1)-PIC/m and CD276Ab(2)-PIC/m displayed a negative zeta potential. Although a positive charge typically aids in the internalization of the nanocarriers, a negative charge could positively affect the carrier circulation due to the negatively charged luminal surface of blood vessels, which causes positively charged nanoparticles to be eliminated from circulation [45]. As a result, the zeta potential of the nanocarriers may exhibit the characteristics required for extended blood circulation.

Evaluation of Cell Internalization Efficiency of CD276Ab Decorated PIC/m
CD276Ab was conjugated onto the PIC/m surface to enhance the delivery efficiency to the CD276-expressing tumor cells. The cytotoxicity of the fabricated CD276Ab-PIC/m was first evaluated, where no significant increase in cytotoxicity due to the conjugation of CD276Ab was detected ( Figure S5, see Supplementary Materials). To validate the effect of CD276Ab conjugation, CD276Ab conjugated PIC/m were incubated with DAOY cells known to express CD276, and their uptake was evaluated via the Cy5-derived fluorescence under a CLSM. While the Cy5-derived fluorescence was not visible in DAOY cells incubated with naked micelles (N 3 -PIC/m), a significant increase in internalization ratio was detected with both CD276Ab(1)-PIC/m and CD276Ab(2)-PIC/m (Figure 5a). This result suggests that installing a PIC/m surface with CD276Ab is an effective method in amplifying the delivery efficiency of micelles to DAOY cells and that the efficiency increases as the number of CD276Ab conjugated to the micelle surface increases (Figure 5b). This increase in cell uptake is presumably due to the multiple conjugated antibodies eliciting a multivalent binding effect [41,46,47]. In fact, a study showed that the degree of conjugation of antitransferrin receptor (TfR) monoclonal antibody to Cylindrical poly(ethylene glycol)-based PRINT nanoparticles greatly affects the internalization rate of nanoparticles due to the multivalent binding that occurs between TfR and anti-TfR antibodies [48]. An inhibition assay was performed to check the potency and specificity of the CD276Ab installed PIC/m. Results exhibited a significant decrease in Cy5-derived fluorescence within DAOY cells with an increase in the concentration of the inhibiting CD276Ab (Figure 5c,d). Thus, it is safe to conclude that the CD276Ab-PIC/m is selectively recognizing CD276 and using this to enter the tumor cells. Together these results suggest that modifying the micelle surface with CD276Ab is effective in homing micelles specifically to CD276-expressing cancer cells at high efficiency by utilizing the immunological recognition function of CD276Ab. Although the results obtained thus far suggest that the conjugation of CD276Ab to micelle surface holds great promise for accumulation in tumors, it is imperative for such CD276Abinstalled micelles to first penetrate the blood-brain barrier (BBB) before they can access the tumor cells. While the WNT subtype medulloblastoma has been shown to have a leaky vasculature that may facilitate the penetration of PIC/m through the BBB, other subtypes (e.g., group 3, group 4, and Sonic hedgehog subtypes) have tumor cells barricaded by tight junctions between endothelial cells [49]. Therefore, future research efforts should prioritize strategies that enhance the BBB penetrability of these micelles through additional modifications to their surface, such as the installation of transferrin receptor antibodies.
shown to have a leaky vasculature that may facilitate the penetration of PIC/m through the BBB, other subtypes (e.g., group 3, group 4, and Sonic hedgehog subtypes) have tumor cells barricaded by tight junctions between endothelial cells [49]. Therefore, future research efforts should prioritize strategies that enhance the BBB penetrability of these micelles through additional modifications to their surface, such as the installation of transferrin receptor antibodies. (red), GFP (green), and the nucleus (blue) were detected using a CLSM. The scale bar indicates 50 μm. (b) Cy5 fluorescence intensity within each cell was quantified by ImageJ (n = 50) and then tested for significant difference via a Student's t-test followed by Bonferroni correction for multiple analyses. A significant difference was detected and represented as ** p < 0.001. Error bars indicate standard deviation. An inhibition assay was conducted for (c) CD276Ab(1)-PIC/m and (d) CD276Ab(2)-PIC/m. The Cy5 fluorescence intensity within each cell was quantified by ImageJ (n = 50) and then tested for significant differences via a Student's t-test followed by Bonferroni correction for multiple comparisons. Significant difference was represented as ** p < 0.001. Error bars indicate standard deviation.

Conclusions
In this study, an anti-CD276 antibody was introduced to the N3-PIC/m surface to enhance the delivery efficiency to pediatric brain tumor cells. Successful conjugation of DBCO-modified CD276Ab to N3-PIC/m was confirmed through FCS measurement, where one or two antibodies were introduced. The immunological recognition function of (b) Cy5 fluorescence intensity within each cell was quantified by ImageJ (n = 50) and then tested for significant difference via a Student's t-test followed by Bonferroni correction for multiple analyses. A significant difference was detected and represented as ** p < 0.001. Error bars indicate standard deviation. An inhibition assay was conducted for (c) CD276Ab(1)-PIC/m and (d) CD276Ab(2)-PIC/m. The Cy5 fluorescence intensity within each cell was quantified by ImageJ (n = 50) and then tested for significant differences via a Student's t-test followed by Bonferroni correction for multiple comparisons. Significant difference was represented as ** p < 0.001. Error bars indicate standard deviation.

Conclusions
In this study, an anti-CD276 antibody was introduced to the N 3 -PIC/m surface to enhance the delivery efficiency to pediatric brain tumor cells. Successful conjugation of DBCO-modified CD276Ab to N 3 -PIC/m was confirmed through FCS measurement, where one or two antibodies were introduced. The immunological recognition function of CD276Ab-PIC/m was demonstrated via an in vitro evaluation using CD276-expressing DAOY cells, where the cell internalization efficiency was significantly enhanced. The number of CD276Ab introduced was also shown to be an important factor, as CD276Ab(2)-PIC/m showed significantly higher efficiency compared to CD276Ab(1)-PIC/m, possibly due to the multivalent bonding that occurred between CD276 and CD276Ab. Our results indicate that installing CD276Ab on the micelle surface as a ligand to target tumor cells is an effective method for improving drug delivery efficiency to pediatric brain tumors.