As a first step toward synthesizing MCNP-ATAP constructs (Figure 2a), the magnetic cores were synthesized by making slight modifications to a previously reported protocol.³⁷ Then, citrate-capped MCNPs were synthesized according to previously reported methods with slight modifications.³² The as-synthesized citrate-capped MCNPs were found to have a hydrodynamic diameter of 15.4 nm (Figure 2b). Thereafter these MCNPs were coated with branched polyethylenimine (PEI, MW = 10 kDa) via electrostatic interactions to afford an overall positive charge to the complex. Additionally, PEI is known to act as a proton sponge and induce endosomolysis within the cytoplasm,³⁸ such that the MCNP-ATAP constructs will not be subjected to the highly acidic endosomal environment and be released within the cytoplasm in their most potent form. Subsequently, these amine-terminated MCNPs were coupled to the thiol-terminated ATAP moieties via a heterobifunctional cross-linker, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), to form redox-responsive MCNP-ATAP constructs. Additionally, thiol-PEG moieties bearing targeting moieties (iRGD)⁶ were also conjugated to the MCNPs using the SPDP linker to improve the overall aqueous solubility of the MCNP-ATAP constructs and to allow for selective delivery of MCNP-ATAP constructs to U87 glioblastoma cells, which contain upregulated levels of integrins.³⁹

The particle diameter and zeta potential of the MCNP-ATAP constructs was measured using dynamic light scattering after each step of conjugation (Figure 2c). The final MCNP constructs had a hydrodynamic diameter of 46.8 ± 2.3 nm and were positively charged (ζ = +15.78 mV) to allow for increased cellular uptake. Additionally, these MCNP-ATAPs showed a slight red shift in the characteristic surface plasmon resonance band as compared to unconjugated MCNPs, thus confirming the presence of ATAP moieties on the surface (Figure 2d).⁴⁰ The amount of ATAP present on the MCNPs was quantified by measuring the concentration of the unconjugated ATAP moieties present in the supernatant using UV–vis spectroscopy (Experimental Section). Approximately, 50% of the loading amount of ATAP was conjugated on the nanoparticles and it was found that 10 nM ATAP was present per 1 μg/mL of MCNPs (∼500 ATAP molecules per NP). The synthesized MCNPs were also found to be biocompatible within a wide range of concentrations (5–50 μg/mL) and did not affect the cell viability in either U87vIII or MDA-MB-231 cells (Supporting Information, Figures S1 and S2). Additionally, we tested the biocompatibility of MCNPs coated with PEI (to mimic the conditions found after the cleavage of disulfide bond and subsequent release of ATAP within the cytoplasm) and found that these MCNP-PEI constructs also did not induce any noticeable cytotoxicity at the concentrations present in U87vIII cells (Supporting Information, Figure S3).

Once the MCNP-ATAP constructs were generated and characterized, we then went on to test their targeted delivery and the subsequent apoptotic efficacy in brain cancer cells (U87vIII) overexpressing the mutant epidermal growth factor receptor vIII (EGFRvIII).⁴¹ The overexpression of EGFRvIII has been implicated in enhancing the tumorigenicity and resistance to radiation and chemotherapy in GBM.⁴² As mentioned earlier, ATAP lacks a tumor targeting moiety, thereby restricting its widespread clinical application. Hence, we modified the carboxyl end-groups of the PEG chains, with a targeting ligand, iRGD, which has been reported to home to the αvβ3 integrin surface receptors present in glioblastoma and other cancer cells (Figure 3a).⁴³ These iRGD-grafted PEG chains were then conjugated on the surface of MCNPs as described in the Experimental Section. Additionally, we conjugated a fluorophore (Alexa Fluor 594) on the surface of the MCNPs to make them amenable for visualization using fluorescence microscopy. The iRGD-conjugated MCNPs were then incubated with U87vIII and MCF-7 (breast cancer) cells, which have low integrin expression;⁴⁴ the cells were then washed thrice with PBS to remove any excess MCNPs and thereafter imaged using fluorescence microscopy. As seen in Figure 3b, U87vIII cells, having higher integrin levels show significantly higher uptake of the iRGD conjugated MCNPs as compared to MCF-7 cells, which have low levels of integrins. Taken together, these results indicate that by simply conjugating targeting ligands on the surface of the MCNPs, we can adopt a facile approach for conferring tumor targeting capability to the ATAP moieties without making any structural modifications to ATAP, which can compromise its activity.

We next tested the apoptotic capability of these MCNP-ATAP constructs in both glioblastoma (U87vIII) and breast cancer (MDA-MB-231) cells. Prior to testing MCNP-ATAP constructs, we first tested the effect of varying concentrations of unconjugated ATAP (in DMSO) on the viability of U87vIII and MDA-MB-231 cells using the MTS assay (Supporting Information, Figure S4). Consistent with our previous study,¹⁸ the unconjugated ATAP (in DMSO) had negligible effect on the viability of both cell lines, even at significantly higher ATAP concentrations ([ATAP] = 200 nM); Figure 3c). Next, we tested the chemotherapeutic efficacy of MCNP-ATAP constructs delivered to brain and breast cancer cells using magnetically facilitated delivery.^35,45 We first optimized the duration of magnetic field exposure (Supporting Information, Figure S5) and found that 30 min was the optimal exposure time, resulting in significantly higher uptake of MCNP-ATAP constructs. We then tested the apoptosis-inducing ability of MCNP-ATAP constructs in U87vIII and MDA-MB-231 cells by delivering varying concentrations of MCNP-ATAP constructs (Figure 3c and d) to cells using magnetically facilitated delivery (30 min magnetic field exposure). The viability of the treated cells was then determined using the MTS assay 48 h post-transfection. As can be seen from Figure 3c,d, MCNP-ATAP constructs led to a significant increase in cell death, as compared to unconjugated ATAP at all concentrations in both U87vIII and MDA-MB-231 cells. Taken together, these results indicate that the potency of ATAP is greatly enhanced when conjugated to MCNPs, possibly due to an increase in its aqueous solubility in the presence of PEG molecules. Additionally, we tested whether release of ATAP from the MCNPs was required to achieve a maximal effect on cell viability. For this purpose, we compared the effect of cell viability of the above-mentioned MCNP-ATAP constructs (with a cleavable disulfide bond) with MCNP-ATAP constructs (with a noncleavable amide bond) in brain cancer cells. From this, it is clearly evident that MCNP-ATAP constructs with the noncleavable amide bond show a modest decrease in the cell viability as compared to the cleavable constructs (Supporting Information, Figure S6). Taken together, these results suggest that while the conjugation of ATAP to the MCNPs increases its overall chemotherapeutic efficacy, release of ATAP from the MCNPs is essential to achieve the maximal apoptotic effect.

As mentioned earlier, our MCNPs could induce localized hyperthermia in the presence of an AMF, which can act in a synergistic manner with the ATAP moieties to increase the permeabilization of the mitochondrial membrane and thus enhance apoptosis (Figure 1b).^28,29 Hence, we wanted to evaluate whether the combination of MCNP-mediated hyperthermia and ATAP delivery would lead to a synergistic enhancement of cancer cell apoptosis. However, prior to testing the MCNP-mediated magnetic hyperthermia, we tested the combination therapy of MCNP-ATAP and water-bath hyperthermia. The MCNP-ATAP-treated cells were exposed to 45 min of water-bath hyperthermia (43 °C), and the percent cell viability was quantified as before. Combined treatment of water-bath hyperthermia with MCNP-ATAP constructs did not lead to significant increase in cancer cell apoptosis (Supporting Information, Figure S7). Hence, in the next step, we tested the combination therapy of MCNP-mediated magnetic hyperthermia and ATAP delivery. We first optimized the duration of hyperthermia by exposing the cells transfected with MCNPs to either 15, 30, 45, or 60 min of hyperthermia (Supporting Information, Figure S8). From this, it was found that 45 min of hyperthermia was optimal to induce further cell death (an additional ∼20% based on MTS), and further increases in the exposure time did not lead to a corresponding increase in cell death. Thereafter, we tested the combined therapy by incubating the U87vIII and MDA-MB-231 cells with MCNP-ATAP constructs and inducing hyperthermia 24 h post-transfection. The cell viability following the combined therapy was determined 48 h post-transfection using an MTS cell viability assay. From Figure 3c, it can be seen that the combined therapy of MCNP-ATAP ([ATAP] = 200 nM) and hyperthermia (45 min) caused significant cell death, as compared to either treatment alone. Collectively, these results support our hypothesis that hyperthermia can significantly enhance ATAP-mediated cell death. In addition, these results also clearly suggest that such an enhancement in cell death can only be seen in the case of MCNP-mediated hyperthermia and ATAP delivery, as compared to water-bath hyperthermia.

In order to confirm that the apoptotic effect of MCNP-ATAP constructs was caused as a result of mitochondrial dysfunction, we investigated the loss of mitochondrial membrane potential (ΔΨm), using a flow-cytometry-based JC-1 assay.¹¹ Mitochondrial depolarization occurs as a result of mitochondrial dysfunction and is regarded as a hallmark of apoptosis (Figure 4a). JC-1 is a lipophilic, cationic dye, which selectively translocates to the mitochondria and undergoes a color change as a function of the mitochondrial membrane potential (ΔΨm).⁴⁶ In healthy cells with high ΔΨm, JC-1 spontaneously forms intense red colored complexes known as J-aggregates, whereas in apoptotic cells with low ΔΨm, JC-1 remains in the green monomeric form.⁴⁷ From Figure 4b, it can be seen that there is no obvious change in mitochondrial membrane potential in U87vIII cells treated with MCNPs alone, as is evident from the formation of red J-aggregates (96.0%) with minimal green JC-1 monomers (2.6%). When the U87 cells are treated with MCNP-ATAP, there is a dramatic increase in the green fluorescence (JC-1 monomer in the cytoplasm, 25.7%), suggesting increased mitochondrial depolarization and hence apoptosis. This is also coupled with a decrease in red fluorescence (normal mitochondria, 67.0%). Taken together, these results suggest that the MCNP-mediated delivery of ATAP resulted in significantly higher mitochondrial dysfunction and eventually increased apoptosis, as compared to unconjugated ATAP (Supporting Information, Figure S9). Furthermore, when hyperthermia is induced in cells treated with MCNP-ATAP, it causes a further increase in the green fluorescence (38.1%, as compared to 25.7% seen with MCNP-ATAP), thus suggesting the enhanced effect of hyperthermia (HT) on the mitochondrial-regulated apoptosis caused by ATAP, because of their cooperative mechanism of action. To further quantify the ATAP-induced mitochondria-dependent apoptosis of U87 cells, we conducted a flow-cytometry-based annexin V-FITC/PI assay (Supporting Information, Figure S10). Combined treatment of MCNP-mediated ATAP and hyperthermia (MCNP-ATAP+HT) showed the highest percentage of apoptotic cells (38.3%), as compared to the individual MCNP-ATAP only and MCNP-induced hyperthermia treatments.

Once we established that the potency of ATAP increases exponentially upon conjugation to MCNPs, we then went on to test the potency of MCNP-ATAP constructs in vivo using a mouse xenograft model. Since such peptide–nanoparticle constructs are administered systemically in repeated dosages in a clinical setting, it is therefore essential to identify the potential of these constructs to induce an immune response in the mice. We therefore systemically injected MCNP-ATAP-IRGD constructs in mice and found that the constructs show no obvious immunogenicity when tested over a period of 3 weeks. In addition, we tested the tumor-targeting capability of ATAP-iRGD constructs in an esophageal cancer (KYSE)⁴⁸ xenograft model. These studies showed that ATAP-iRGD had similar tumor suppression effects to those of BH3-iRGD peptide (equal molar concentration), thus indicating ATAP-iRGD suppresses esophageal tumor growth with limited off-target toxicity in the mouse model (Supporting Information, Figure S11). While the IC₅₀ for ATAP-iRGD was found to be 4–5 μM, after MCNP packaging of ATAP, we found that MCNP-ATAP remarkably improved the IC₅₀ from the μM range to the 50 nM range in the MTT assay. Although these in vivo experiments are still in the preliminary stage, these in vivo results strongly suggest the potential of MCNPs to enhance the delivery and efficacy of ATAP in tumors.

To obtain PEI-coated MNPs, the water-soluble MNPs from above were first diluted with DPBS to reach a final concentration of 0.1 mg/mL. Afterward, excess 10 kDa branched PEI (Sigma-Aldrich) was added dropwise (1 mg/mL). After spinning overnight, the PEI-coated MNPs were filtered using a centrifugal filter unit (EMD Millipore, 10 000 MW). Thereafter, the PEI-coated MNPs were mixed with heterobifunctional linker and SPDP (0.1 mM) and incubated at room temperature for 4–6 h with continuous shaking. Simultaneously, SH-PEG-COOH moieties were linked to iRGD-NH₂ moieties using EDC coupling. Thereafter, varying ratios of thiolated ATAP and thiolated PEG-iRGD constructs were added to the thiol-reactive SPDP-linked MCNP-PEI complexes. The resulting mixture was allowed to react overnight, followed by purification using centrifugation. The resulting MCNP-ATAP complexes were then dispersed in sterile DPBS (Dulbecco’s Phosphate Buffered Saline) for further use. For targeted delivery, the MCNP-PEI complexes were also conjugated with Alexa-Fluor594-succinimide to allow for monitoring the uptake of MCNP constructs using fluorescence microscopy.

A standard curve of ATAP (in 8 M urea) was constructed by measuring the absorbance of different concentrations of ATAP solutions at 280 nm using a UV–visible spectrometer (Cary US). The amount (in milligrams) of ATAP present per milliliter of solution was then calculated by using the following equation:

In order to calculate the amount of ATAP conjugated per milligram of MCNP, the MCNP-ATAP constructs were incubated with 0.1 M DTT solution to cleave the disulfide bonds between MCNP and ATAP. The solution was centrifuged, and the supernatant collected. The amount of ATAP per milligram of MCNP was then calculated using the above equation by measuring the absorbance at A280 nm.

Dynamic light scattering (DLS) and zeta potential analyses were performed using a Malvern Instruments Zetasizer Nano ZS-90 instrument (Southboro, MA, USA) with reproducibility being verified by collection and comparison of sequential measurements. Complexes (MCNP, MCNP-PEI, and MCNP-ATAP) for DLS and zeta potential measurements were prepared using purified water (resistivity = 18.5 MΩ-cm). DLS measurements were performed at a 90° scattering angle at 25 °C. Z-Average sizes of three sequential measurements were collected and analyzed. Zeta potential measurements were collected at 25 °C, and the Z-average potentials following three sequential measurements were collected and analyzed.

U87-EGFRvIII cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium) with high glucose (Invitrogen), 10% fetal bovine serum (FBS), 1% streptomycin–penicillin, 1% Glutamax (Invitrogen), and hygromycin B (30 μg/mL), while MDA-MB-231 and MCF-7 cells were cultured in DMEM/F-12 with 10% FBS, 1% streptomycin–penicillin, and 1% Glutamax.

Twenty-four hours before the magnetically facilitated delivery of MCNPs, 2 × 10⁵ cells in a volume of 500 μL were seeded into each well of a 24-well plate, so as to attain 80–90% confluency at the time of transfection. For the transfection of MCNP-ATAP constructs, the varying amounts of MCNP-ATAP constructs were gently mixed with OptiMEM and then were added to each well to obtain the desired MCNP-ATAP concentration per well. Subsequently, the cell culture plates were placed on the Nd–Fe–B magnetic plates (OZ Biosciences, France) for 15 min. Following this, the cells were transferred back to the incubator and the media was replaced with growth medium after 1–2 h of additional incubation.

The percentage of viable cells was determined by MTS assay following standard protocols described by the manufacturer. All experiments were conducted in triplicate and averaged. Forty-eight hours after initial transfection, the MTS data are represented as formazan absorbance at 490 nm, considering the control (untreated) cells as 100% viable.

Highly tumorigenic U87-EGFRvIII cells and low-tumorigenic MCF-7 cells were cultured in 24-well plates, at a density of 5 × 10⁴ cells per well. For MCF-7 cells, the normal growth media was DMEM/F-12 (with high glucose, Invitrogen), 10% FBS, 1% Glutamax, and 1% penicillin–streptomycin. For the delivery of iRGD-conjugated MCNPs, media was exchanged with serum-free DMEM media, and the cells were incubated with iRGD-MCNPs for 6–8 h. Fluorescence images were taken after replacing the serum-free media with regular media.

Twenty-four hours after seeding cells as described above, varying concentrations (5–20 μg/mL) of MCNP-ATAP constructs were prepared in OptiMEM (Life Technologies) and added to each well. Subsequently, the cell culture plates were exposed to magnetofection for 15 min as described above. The culture plates were placed back into the incubator for 1 h, and afterward, the cells were washed with DPBS and the transfection medium was replaced with fresh growth medium. Twenty-four hours after transfection, cells were washed with DPBS, trypsinized, and exposed to an alternating magnetic field (5 kA/m, 300 kHz) for the desired amount of time. Thereafter, fresh media was added to the treated cells and the cells were plated back into 12-well plates.

Mitochondrial stability was assessed by flow cytometry after incubation with 5,59,6,69-tetrachloro-1,19,3,39-tetraethylbenzimidoazolylcarbocyanino iodide (JC-1; Molecular Probes, Eugene, OR, USA) as per the manufacturer’s recommended protocol. Briefly, the cells were analyzed using flow-cytometry-based JC-1 assay 48 h after initial transfection. The cells were trypsinized, resuspended in warm DPBS, and incubated with JC-1 (2 μM) for 15–30 min at 37 °C and 5% CO₂. Thereafter the cells were centrifuged, resuspended in 500 μL of PBS, and analyzed immediately on a flow cytometer (Gallios, Beckman Coulter, Inc.) with 488 nm excitation using emission filters appropriate for Alexa Fluor 488 dye (520 nm) and R-phycoerthyrin (590 nm). Standard compensation was performed using the carbonyl cyanide 3-chlorophenylhydrazone (CCCP)-treated cells as positive control. Untreated cells (no MCNP and no ATAP) were used as negative controls.

To assay apoptosis using annexin V-FLUOS and propidium iodide staining (Roche), 48 h after initial transfection, 10⁶ cells were prepared in 1 mL of PBS with 10% FBS in each test tube. After centrifugation, cells were resuspended in 100 μL of annexin V binding buffer (ice-cold), and annexin V-FLUOS and propidium iodide (PI) were added following the manufacturer’s recommendation. Samples were incubated in the dark for 15 min at room temperature. Finally, 400 μL of additional ice-cold annexin V binding buffer was added, and the samples were kept on ice under foil until analysis using flow cytometry (Gallios, Beckman Coulter, Inc.). Early apoptotic cells with exposed phosphatidylserine but intact cell membranes bound to annexin V-FITC but excluded propidium iodide. Cells in necrotic or late apoptotic stages were labeled with both annexin V-FITC and propidium iodide.

Handling of animals was performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the The Ohio State University. Five-week-old NCR nude mice (Taconic Farms, Germantown, NY, USA) were implanted subcutaneously in both flanks with 2 × 106 KYSE cells. After tumors reached 4 to 7 mm in diameter (about 9–14 days after implantation), the mice were randomly divided into two groups so that both the mean and the variance of the tumor diameters are of no significant difference among the groups prior to treatment. The ATAP-iRGD-M8 peptide was injected through the tail vein once every 2 days during the whole procedure. Tumor volume was measured by a digital caliper (Thermo Fisher Scientific, Waltham, MA, USA). Tumor volume was determined from the orthogonal dimensions (length, width) using the following formula: tumor volume = ¹/₂(length × width²). According to the IACUC guideline, the experiments were terminated when tumors reached 1.5 cm in diameter. At the end of the experiment, mice were sacrificed and xenografts were removed and weighed. For toxicological evaluation of peptide treatments, mouse body weights were measured every 4 days. In addition, after euthanizing animals, organs (kidney, heart, liver, lung, and spleen) were expanded, fixed in 10% neutral buffered formalin, paraffin embedded, and stained with hematoxylin-eosin (H&E) for pathological analysis.