Preparation and Characterization of the Clustered Nanoparticles.

To prepare the clustered nanoparticles, the polymer components PCL, PEG-b-PCL, and PCL-CDM-PAMAM/Pt were synthesized. The structures and molecular weights of PEG-b-PCL and PCL were characterized (SI Appendix, Figs. S1 and S2). For PCL-CDM-PAMAM/Pt synthesis, PCL was first reacted with 2-propionic-3-methylmaleic anhydride (CDM) to produce PCL-CDM (SI Appendix, Fig. S3). A Pt prodrug c,c,t-[Pt(NH₃)₂Cl₂(OH)(O₂CCH₂CH₂CO₂H)] was conjugated to PAMAM to afford PAMAM/Pt, which was further coupled to PCL-CDM through the reaction of the amino groups of PAMAM with the CDM anhydride residue (SI Appendix, Scheme S1). The resultant amide bond is acid labile (38) and will be cleaved at pH_e to release PAMAM at tumor sites.

The Pt-containing pH-instable clustered nanoparticle (iCluster/Pt) was prepared from coassembly of PCL-CDM-PAMAM/Pt, PEG-b-PCL, and PCL by nanoprecipitation method. The weight ratio of PCL-CDM-PAMAM/Pt:PEG-b-PCL:PCL was optimized as 1:1:1 based on size and size distribution (SI Appendix, Table S1 and Fig. S4). Dynamic light scattering (DLS) indicated that the diameter of iCluster/Pt was around 104.1 nm (Fig. 1C), which was consistent with the transmission electron microscopy (TEM) observation (Fig. 1D). TEM images showed clearly that iCluster/Pt had a raspberry-like structure, presumably due to the presence of PAMAM dendrimers surrounding the hydrophobic core. Each iCluster/Pt contained 108 PAMAM and 719 platinum drugs, as estimated by static light scattering. For comparison, we prepared a pH-stable clustered nanoparticle (Cluster/Pt) by replacing PCL-CDM-PAMAM/Pt with its nonresponsive counterpart PCL-PAMAM/Pt (SI Appendix, Scheme S2). Each Cluster/Pt contained 90 PAMAM and 573 platinum drugs and showed similar morphology, size, and zeta potential as iCluster/Pt (Fig. 1 E and F and SI Appendix, Table S2).

One key design of iCluster/Pt is its sensitivity to biological stimuli such as the acidic pH_e and elevated intracellular redox milieu. To test its response to pH_e, we incubated iCluster/Pt in a tumor-acidity mimic phosphate buffer (PB) solution at pH 6.8 over predetermined time and observed its morphological evolution under TEM. After a 4-h incubation, the raspberry-like morphology of iCluster/Pt was partially deformed and small particles appeared in the solution (Fig. 1D, Middle). After an additional incubation for 24 h, the raspberry structure was almost completely disintegrated (Fig. 1D, Right and SI Appendix, Fig. S5) and changed to a smooth surface that was analogous to nanoparticles formed by PEG-PCL and PCL (SI Appendix, Fig. S6A). Meanwhile, plenty of small nanoparticles were observed in the surroundings, with size comparable to PAMAM (SI Appendix, Fig. S6B), suggesting that small PAMAM dendrimers were released upon cleavage of the amide bond at pH 6.8. In contrast, Cluster/Pt maintained its initial morphology and size under identical conditions (Fig. 1F).

Next, high-performance liquid chromatography (HPLC) was used to monitor and compare the release kinetics of PAMAM from fluorescein (Flu)-labeled clustered nanoparticles (iCluster_Flu and Cluster_Flu). At pH 7.4, iCluster_Flu could generally maintain its stability with ∼12% PAMAM release after a 4-h incubation. However, incubation at pH 6.8 resulted in ∼60% cumulative release by another 4 h (Fig. 1G) and ∼90% release by 24 h (SI Appendix, Fig. S6C), indicating much faster release of PAMAM at acidic pH. In contrast, Cluster_Flu showed minimal release at either pH 7.4 or 6.8 (Fig. 1H). The nanoparticles are designed to release cisplatin specifically in the redox environment (39). To test the release of cisplatin, we incubated the nanoparticles in PB solutions (pH 7.4 and 6.8), and ascorbic acid solution (5 mM, pH 7.4), an intracellular redox environment mimic solution (40), respectively, and quantified Pt drug release via inductively coupled plasma mass spectrometry (ICP-MS). Both nanoparticles showed minimal drug release in PB solutions regardless of pH, but rapid release in the redox environment (Fig. 1 G and H). Blood plasma showed minimal effect on the stability and pH responsiveness of iCluster (SI Appendix, Fig. S7).

Penetration and Cell Apoptosis in Multicellular Spheroids.

We chose multicellular spheroids (MCSs) derived from BxPC-3 human pancreatic cancer cells as an in vitro tumor model to evaluate the penetration and cell killing efficacy of the clustered nanoparticles. Compared with single-layer adherent cells, MCSs have been proposed as a versatile 3D model to study tumor biology and to screen therapeutic agents (41). iCluster and Cluster were dual labeled with two dyes (denoted as ^RhBiCluster_Flu and ^RhBCluster_Flu), in which PAMAM was labeled with fluorescein (Flu, green), whereas the PCL component of the hydrophobic core was labeled with rhodamine B (RhB, red). MCSs were incubated with ^RhBiCluster_Flu or ^RhBCluster_Flu for 15 min, 4 h, and 24 h at pH 6.8, then washed and observed under confocal laser scanning microscopy (CLSM, Fig. 2A and SI Appendix, Fig. S8). For ^RhBiCluster_Flu treatment, the red fluorescence from the hydrophobic core attached to the periphery of MCSs, and no noticeable fluorescence was detected in the internal area by 24 h. Instead, the green signals from PAMAM inside MCSs clearly increased over time, demonstrating the improved penetration of PAMAM. By contrast, both red and green fluorescence signals from nonresponsive ^RhBCluster_Flu were on the peripheral region of the MCSs by 24 h, revealing limited penetration of larger particles. Quantitative analysis indicated that more than 10-fold higher green fluorescence was detected in ^RhBiCluster_Flu-treated MCSs than in ^RhBCluster_Flu-treated ones (Fig. 2B). These results provide clear evidence that the size-changeable iCluster has better tumor penetration.

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Fig. 2.

In vitro penetration and cell killing efficacy of clustered nanoparticles in BxPC-3 MCSs. (A) Penetration of ^RhBiCluster_Flu and ^RhBCluster_Flu in MCSs at pH 6.8 after a 4-h or 24-h incubation. The area marked with white circles was considered the inside area. (Scale bar, 200 μm.) (B) Mean fluorescence intensity (MFI) of green signals in the inside area of MCSs. (C) Quantification of fluorecein-positive MCS cells after incubation with different formulations at pH 6.8 for 4 h or 24 h. (D) Quantification of Pt content in MCSs after incubation with various formulations at pH 6.8 for 24 h. (E) Quantification of Pt content in DNA of MCS cells after various treatments at pH 6.8 for 24 h. (F) Apoptotic cells induced by different treatments at pH 6.8 for 24 h. All data are presented as mean ± SD (n = 3). *P < 0.05, ***P < 0.001.

Next, cell internalization of both nanoparticles was studied by flow cytometry (FACS) and ICP-MS, respectively. In FACS analysis, MCSs were treated with iCluster_Flu, Cluster_Flu, or PAMAM_Flu at pH 6.8 for 4 h and 24 h. Compared with Cluster_Flu treatment, the population of positive cells treated with iCluster_Flu was 1.7-fold higher and 3-fold higher at 4 h (P < 0.05) and 24 h (P < 0.001), respectively (Fig. 2C and SI Appendix, Fig. S9). For ICP-MS analysis, Fig. 2D shows that iCluster/Pt treatment resulted in significantly higher intracellular accumulation of Pt than Cluster/Pt treatment (2.6-fold, P < 0.001). Moreover, DNA of MCS cells were isolated after a 24-h incubation, and the amount of Pt binding to these DNA in MCS cells receiving iCluster/Pt treatment was significantly higher than that receiving Cluster/Pt treatment (3.6-fold, P < 0.001, Fig. 2E). Apoptosis results indicated that iCluster/Pt treatment showed 38.6% total cell apoptosis, which was significantly higher than Cluster/Pt treatment (14.3%, P < 0.001, Fig. 2F and SI Appendix, Fig. S10). Of note, in all experiments, iCluster treatment showed comparable efficacy to PAMAM treatment.

Antitumor Activity and Tumor Penetration in a BxPC-3 Pancreatic Tumor Model.

The superior performance of iCluster/Pt in MCSs further compelled us to study its in vivo activities. We first studied the pharmacokinetic profiles of the clustered nanoparticles. As shown in Fig. 3A, both iCluster/Pt and Cluster/Pt exhibited prolonged half-life time (T_1/2 > 10 h) in the bloodstream compared with PAMAM/Pt and cisplatin, which is reasonable because iCluster/Pt and Cluster/Pt are PEGylated nanoparticles. Other pharmacokinetic parameters also revealed better performance of the two PEGylated nanoparticles (SI Appendix, Table S3). Next, we studied the antitumor activity of iCluster/Pt in nude mice bearing BxPC-3 human pancreatic tumors, which is recognized as a notoriously poorly permeable and intractable tumor model (27). As shown in Fig. 3B, free cisplatin and PAMAM/Pt displayed modest therapeutic efficacy, with 38% and 45% tumor inhibition versus PBS control, whereas Cluster/Pt showed 57% tumor inhibition. In contrast, iCluster/Pt exhibited significant suppression of tumor growth, reaching 88% tumor suppression (P < 0.001, versus Cluster/Pt treatment). The average weight of the tumor mass excised at the end of treatment also demonstrated the same trend (SI Appendix, Fig. S11A). Histological analyses, including hematoxylin and eosin (H&E), proliferating cell nuclear antigen (PCNA), and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end (TUNEL) staining, also indicated that iCluster/Pt treatment markedly reduced proliferative cells while increasing apoptotic cells (SI Appendix, Fig. S12). To investigate dose effect of iCluster/Pt on the inhibition of BxPC-3 tumors, a separate set of in vivo studies with injection dose varying from 1.5 to 6.0 mg/kg was performed. As shown in Fig. 3C, compared with PBS control, all of the iCluster/Pt treatments showed dramatic tumor growth suppression, with inhibition rate of 71%, 92%, and 107% for the dose of 1.5, 4.5, and 6.0 mg/kg, respectively. It is noteworthy that tumor shrinkage was observed for the 6.0 mg/kg treatment. Slight weight loss was observed for mice receiving free cisplatin and the 6.0 mg/kg treatments, whereas no obvious weight loss from other treatments was observed (SI Appendix, Fig. S11 B and C). Additionally, tissue staining and blood tests revealed that the clustered nanoparticles did not cause detectable toxicities to the mice (SI Appendix, Figs. S13 and S14).

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Fig. 3.

In vivo antitumor activity of iCluster/Pt in a BxPC-3 human pancreatic tumor model. (A) Pharmacokinetics of the formulations. These formulations were i.v. injected into ICR mice at a Pt dose of 60 µg per mouse. (B) Growth inhibition of BxPC-3 tumors by different treatments. Mice were i.v. administered at a Pt dose of 3 mg/kg on days 0, 2, and 4. ***P < 0.001. (C) Dose effect of iCluster/Pt on tumor growth inhibition. Mice were i.v. administered on days 0, 2, and 4. (D) Quantification of Pt content in tumor tissue. Formulations were administered i.v. at a Pt dose of 60 µg per mouse. Mice were killed at 12 h and 24 h postinjection, and tumors were excised. *P < 0.05, **P < 0.01. (E and F) Quantification of Pt content in tumor tissue cells (E) and GFP-positive tumor cells (F). For E and F, the tumor was established by s.c. injecting green fluorescent protein (GFP)-expressing BxPC-3 cells. At 12 h and 24 h after injection of the formulations, the tumors were excised, digested, and subjected to FACS to sort the total tumor cells and the GFP⁺ tumor cells. **P < 0.01, ***P < 0.001. Data are presented as mean ± SD n = 3 for A and D–F; n = 5 for B and C.

The distribution of the Pt drug in other major organs was not significantly different between iCluster/Pt and Cluster/Pt treatments (SI Appendix, Fig. S15). However, iCluster/Pt was able to significantly enhance Pt drug accumulation in tumor tissues compared with other treatments, with ∼2-fold higher drug concentration than Cluster/Pt (P < 0.01 for 12 h, and P < 0.05 for 24 h) and at least 7-fold higher than free cisplatin and PAMAM/Pt (Fig. 3D). To get more details, we digested the tumor mass into individual cells and quantified Pt content in these cells by ICP-MS. iCluster/Pt showed two to three times higher internalization of Pt than Cluster/Pt (P < 0.05 for 12 h and P < 0.001 for 24 h; Fig. 3E). To further distinguish tumor cells from other stromal cells in the heterogeneous tumor tissue, we established tumor models by subcutaneously injecting green fluorescent protein (GFP)-expressing BxPC-3 cells in nude mice. After injecting these drug-containing formulations, GFP-positive tumor cells were isolated and sorted by FACS and subjected to ICP-MS to determine Pt content. In these GFP-positive tumor cells, iCluster/Pt still showed significantly higher Pt content than Cluster/Pt at 12 h (2.7-fold, P < 0.05) and 24 h (3-fold, P < 0.001) (Fig. 3F).

iCluster/Pt and Cluster/Pt have similar size, surface property, and pharmacokinetics in the bloodstream. The enhanced deposit of iCluster/Pt in tumor tissues and tumor cells is presumed to be associated with its superior tumor penetration. To test our hypothesis, we used immunofluorescence staining and real-time CLSM observation to study the intratumoral microdistribution of ^RhBiCluster_Flu and ^RhBCluster_Flu. In the immunofluorescence staining study, for ^RhBiCluster_Flu treatment, the green fluorescence from PAMAM showed a uniform perfusion in the tumor interstitium, whereas the red fluorescence was highly colocalized with the blood vessels (yellow). This suggests that ^RhBiCluster_Flu can release PAMAM at tumor sites and the released PAMAM enables efficient extravasation and penetration into deep tumor space, whereas the larger residual nanoparticles are mainly restricted in the blood vessels or the peripheral areas (Fig. 4A, Upper). For the nonresponsive ^RhBCluster_Flu, both green and red signals were colocalized with the blood vessels, indicating its inability to diffuse into tumor space (Fig. 4A, Lower). The quantitative analysis of overlap coefficient of red and yellow, as well as green and yellow also showed the same trend (SI Appendix, Fig. S16). These results substantiate previous observations that smaller nanoparticles are more advantageous for deep tumor penetration than larger ones because of their reduced diffusional hindrance (23, 42). To further visualize real-time extravasation and tumor penetration of these nanoparticles, intravital CLSM was used. For ^RhBiCluster_Flu injection (Fig. 4B), strong green and red fluorescence signals were confined in the blood vessels at 10 min postinjection. By 90 min, the green fluorescence in the blood vessels weakened, whereas more green signals extravasated from the blood vessels and distributed dispersedly in the surroundings. In contrast, the red fluorescence still remained within the blood vessels. To quantitatively analyze the spatiotemporal evolution of the fluorescence, we normalized the fluorescence intensity of each color to its initial intensity at 10 min to afford the time and penetration depth-dependent profiles (Fig. 4C). At 90 min postinjection, nearly 50% of the original intensity of green fluorescence from PAMAM could be detected until 70 μm from the blood vessels, whereas red signals from the larger residual nanoparticles were not detectable beyond the blood vessels. This trend became even more evident by 240 min postinjection. The green fluorescence inside blood vessels diminished markedly while spreading more uniformly in the tumor interstitial space. The quantitative data showed that 25% of the original intensity was still detectable at 160 μm from the blood vessels. The red fluorescence inside the blood vessels weakened as well, but no red fluorescence was observed beyond the vascular wall. In contrast, both the green and red signals from the ^RhBCluster_Flu were confined to the blood vessels over 240 min, suggesting poor tumor penetration of large ^RhBCluster_Flu nanoparticles (SI Appendix, Fig. S17).

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Fig. 4.

Microdistribution of iCluster and Cluster in BxPC-3 xenograft tumor after i.v. injection. (A) CLSM images of immunofluorescence showing the microdistribution of ^RhBiCluster_Flu and ^RhBCluster_Flu in tumor tissue at 4 h postinjection. PAMAM was labeled with Flu (green), whereas the core of the nanoparticles was labeled with RhB (red), and blood vessels were marked with platelet endothelial cell adhesion molecule 1 (PECAM-1) and CFL-647 secondary antibody (yellow). (Scale bar, 50 μm.) (B) Real-time microdistribution of ^RhBiCluster_Flu in BxPC-3 tumor at 10, 90, and 240 min postinjection. (Scale bar, 100 μm.) (C) Time and penetration depth-dependent distribution of ^RhBiCluster_Flu. A region marked with the rectangular frame was selected for the analysis. The intensity profiles were obtained by normalizing the fluorescence intensity of each color to its initial intenstiy at 10 min.

Antitumor Activity in Drug-Resistant and Metastatic Tumor Models.

To verify the broad applicability of iCluster/Pt in cancer chemotherapy, we further investigated its antitumor activities in cisplatin-resistant A549R human lung cancer and 4T1 metastatic murine breast cancer models. In the A549R model, different formulations showed remarkable differences in antitumor activities. Compared with PBS control, free cisplatin exhibited minimal tumor growth inhibition (only 10% inhibition), whereas PAMAM/Pt showed a slightly better effect (∼20% inhibition). In comparison, the long-circulating Cluster/Pt showed considerable enhancement in tumor growth inhibition (60% inhibition). However, the most effective antitumor effect was achieved by iCluster/Pt treatment, reaching 95% inhibition (Fig. 5A, P < 0.001). Of note, the significance between iCluster/Pt and Cluster/Pt appeared as early as day 9 postinjection. No obvious body weight loss was observed for the nanoparticle formulations (SI Appendix, Fig. S18), and histological staining also confirmed the improved therapeutic effect of iCluster/Pt, showing reduction in proliferation while increasing the apoptosis of tumor cells (SI Appendix, Fig. S19).

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Fig. 5.

In vivo antitumor activity in drug-resistant and metastatic tumor models. (A) Inhibition of tumor growth of a A549R cisplatin-resistant human lung cancer model. Mice were i.v. administered an equivalent platinum dose of 1.5 mg/kg on days 0, 3, and 6. Data are presented as mean ± SD (n = 5). **P < 0.05, ***P < 0.001. (B) Kaplan–Meier plots of the animal survival in 4T1 tumor models (n = 10). Mice were treated at a platinum dose of 3 mg/kg via i.v. administration on days 10, 15, and 20 after tumor inoculation.

To further extend the applicability of our strategy to combat metastatic cancer, we established a highly invasive and metastatic 4T1 orthotopic tumor model, which is known to be more aggressive and more refractory to chemotherapy than a s.c. tumor model (43). The mice were treated with varying Pt-containing formulations, and their survival curves were recorded. Compared with PBS and blank iCluster control groups, all other treatments showed improved median survival time. In particular, the iCluster/Pt treatment improved survival time by 74.2%, with significantly longer time to end point than that of Cluster/Pt (Fig. 5B and SI Appendix, Table S4). The comparison of intratumoral microdistribution of ^RhBiCluster_Flu and ^RhBCluster_Flu in A549R and 4T1 tumor tissues also demonstrated that iCluster showed much better tumor penetration than Cluster because of their pH_e-activated PAMAM release at the tumor site (SI Appendix, Figs. S20 and S21), once again indicating that the improved antitumor activities of iCluster are highly associated with enhanced tumor penetration.