Stimuli-responsive clustered nanoparticles for improved tumor penetration and therapeutic efficacy

Hong-Jun Li, Jin-Zhi Du, Xiao-Jiao Du, Cong-Fei Xu, Chun-Yang Sun, Hong-Xia Wang, Zhi-Ting Cao, Xian-Zhu Yang, Yan-Hua Zhu, Shuming Nie, and Jun Wang
PNAS published ahead of print March 28, 2016 https://doi.org/10.1073/pnas.1522080113

  1. Edited by Mark E. Davis, California Institute of Technology, Pasadena, CA, and approved March 3, 2016 (received for review November 8, 2015)

Significance

Successively overcoming a series of biological barriers that cancer nanotherapeutics would encounter upon intravenous administration is required for achieving positive treatment outcomes. A hurdle to this goal is the inherently unfavorable tumor penetration of nanoparticles due to their relatively large sizes. We developed a stimuli-responsive clustered nanoparticle (iCluster) and justified that its adaptive alterations of physicochemical properties (e.g. size, zeta potential, and drug release rate) in accordance with the endogenous stimuli of the tumor microenvironment made possible the ultimate overcoming of these barriers, especially the bottleneck of tumor penetration. Results in varying intractable tumor models demonstrated significantly improved antitumor efficacy of iCluster than its control groups, demonstrating that overcoming these delivery barriers can be achieved by innovative nanoparticle design.

Abstract

A principal goal of cancer nanomedicine is to deliver therapeutics effectively to cancer cells within solid tumors. However, there are a series of biological barriers that impede nanomedicine from reaching target cells. Here, we report a stimuli-responsive clustered nanoparticle to systematically overcome these multiple barriers by sequentially responding to the endogenous attributes of the tumor microenvironment. The smart polymeric clustered nanoparticle (iCluster) has an initial size of ∼100 nm, which is favorable for long blood circulation and high propensity of extravasation through tumor vascular fenestrations. Once iCluster accumulates at tumor sites, the intrinsic tumor extracellular acidity would trigger the discharge of platinum prodrug-conjugated poly(amidoamine) dendrimers (diameter ∼5 nm). Such a structural alteration greatly facilitates tumor penetration and cell internalization of the therapeutics. The internalized dendrimer prodrugs are further reduced intracellularly to release cisplatin to kill cancer cells. The superior in vivo antitumor activities of iCluster are validated in varying intractable tumor models including poorly permeable pancreatic cancer, drug-resistant cancer, and metastatic cancer, demonstrating its versatility and broad applicability.

Over the past few decades, nanomedicine has emerged as a promising means to deliver anticancer therapeutics to tumors as a result of its preferential and selective accumulation at tumor sites via the enhanced permeability and retention (EPR) effect (1⇓⇓–4). However, cancer nanomedicine encounters a series of biological barriers from the site of i.v. injection to the site of action (5). These challenges can be briefly summarized as circulation in the blood stream, extravasation from blood vessels and accumulation at tumor sites, deep penetration into the tumor interstitium, internalization by cancer cells, and intracellular drug release (6). These obstacles existing in the body could considerably prevent nanomedicine from reaching its targets in a sufficient drug concentration (7, 8). To overcome these barriers, a variety of strategies have been envisioned (9⇓⇓⇓⇓⇓⇓⇓⇓–18). Despite great advances, these strategies have mainly focused on one or a few biological barriers and led to suboptimal therapeutic effect.

It is known that physical properties of nanoparticles such as size, shape, and surface charge have profound effects on systemic transport of the nanoparticles in solid tumors (19⇓–21). Due to aberrant vasculature, elevated interstitial fluid pressure, and dense extracellular matrix in the tumor microenvironment, nanoparticles have to overcome considerable interstitial transport hindrance to achieve deep and uniform tumor penetration (7). Such a transport process highly relies on much slower diffusion rather than faster convective transport because of the inherently large sizes of nanoparticles compared with small therapeutics (22). Particle size plays a vital role in dominating the penetration of nanoparticles into tumor tissue as diffusion scales inversely with particle size (23⇓–25). Larger nanoparticles, despite being more advantageous for improved pharmacokinetics and high propensity of vascular extravasation (26), are inherently unfavorable for tumor penetration due to their huge diffusional hindrance in the tumor interstitial space (23, 24). This is also recognized as a reason for the clinically approved Doxil (∼90 nm) only showing modest therapeutic benefits (27). In contrast, smaller nanoparticles show much better tumor penetration (27⇓⇓–30), but very small particles typically suffer from short half-life time and insufficient tumor accumulation because of their rapid clearance (31, 32). Therefore, an ideal delivery system should be relatively larger in its initial size to achieve longer circulation and selective extravasation (33), but once “docking” at tumor sites, it should be switchable to small particles to facilitate tumor penetration. Such a requirement has promoted the recent development of stimuli-responsive nanoparticles that are able to shrink their sizes by responding to enzymes or UV light (15, 34). Although conceptually impressive, those systems are still at an early stage of development. Developing more sophisticated nanomedicines that are capable of systematically overcoming the sequential barriers in a concerted fashion, and are also applicable to a broad range of cancer types, remains formidably challenging.

Here we report the development of stimuli-responsive clustered nanoparticles to systematically overcome these multiple barriers to cancer chemotherapy. The nanoparticles were constructed through molecular assembly of platinum (Pt) prodrug-conjugated poly(amidoamine)-graft-polycaprolactone (PCL-CDM-PAMAM/Pt) with PCL homopolymer and poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-b-PCL) copolymer (Fig. 1 A and B). PEG-b-PCL is used to offer the stealth layer, whereas PCL is chosen to control the size and stability. The nanoparticles are able to function adaptively in the body through precisely responding to the physiological pH, tumor extracellular acidity, and intracellular reductive environment, respectively (Fig. 1B). At physiological pH, the clustered nanoparticles hold the size around 100 nm and have high propensity for long blood circulation and enhanced tumor accumulation through the EPR effect. Then, the acidic tumor extracellular pH (pHe ∼6.5–7.2) (35, 36) triggers the release of small PAMAM prodrugs (∼5 nm) that enable deep and uniform tumor penetration to reach more cancer cells. Finally, the PAMAM prodrugs can be rapidly reduced in the reductive cytosol to release active and potent cisplatin to kill cancer cells and lead to robust antitumor efficacy (37).

Fig. 1.
Fig. 1.

Preparation and physicochemical properties of the clustered nanoparticles. (A) Chemical structure of PCL-CDM-PAMAM/Pt. (B) Self-assembly and structural change of iCluster/Pt in response to tumor acidity and intracellular reductive environment. (C and E) DLS distributions of iCluster/Pt (C) and Cluster/Pt (E). (D and F) TEM images of iCluster/Pt (D) and Cluster/Pt (F) treated in PB at pH 6.8 for 0, 4, and 24 h, respectively. (Scale bar, 100 nm and for the Inset images, 50 nm.) (G and H) PAMAM (green line) and platinum drug (red line) release from iCluster (G) and Cluster (H) under three different conditions, which include PB at pH 7.4 to mimic a neutral environment, PB at pH 6.8 to mimic a tumor extracellular environment, and ascorbic acid solution (5 mM, pH 7.4) to mimic an intracellular redox environment. PAMAM release was quantified by HPLC, whereas platinum release was determined by ICP-MS.

Results

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 pHe 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 pHe and elevated intracellular redox milieu. To test its response to pHe, 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 (iClusterFlu and ClusterFlu). At pH 7.4, iClusterFlu 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, ClusterFlu 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 RhBiClusterFlu and RhBClusterFlu), 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 RhBiClusterFlu or RhBClusterFlu 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 RhBiClusterFlu 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 RhBClusterFlu 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 RhBiClusterFlu-treated MCSs than in RhBClusterFlu-treated ones (Fig. 2B). These results provide clear evidence that the size-changeable iCluster has better tumor penetration.

Fig. 2.
Fig. 2.

In vitro penetration and cell killing efficacy of clustered nanoparticles in BxPC-3 MCSs. (A) Penetration of RhBiClusterFlu and RhBClusterFlu 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 iClusterFlu, ClusterFlu, or PAMAMFlu at pH 6.8 for 4 h and 24 h. Compared with ClusterFlu treatment, the population of positive cells treated with iClusterFlu 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 (T1/2 > 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).

Fig. 3.
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 RhBiClusterFlu and RhBClusterFlu. In the immunofluorescence staining study, for RhBiClusterFlu 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 RhBiClusterFlu 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 RhBClusterFlu, 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 RhBiClusterFlu 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 RhBClusterFlu were confined to the blood vessels over 240 min, suggesting poor tumor penetration of large RhBClusterFlu nanoparticles (SI Appendix, Fig. S17).

Fig. 4.
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 RhBiClusterFlu and RhBClusterFlu 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 RhBiClusterFlu in BxPC-3 tumor at 10, 90, and 240 min postinjection. (Scale bar, 100 μm.) (C) Time and penetration depth-dependent distribution of RhBiClusterFlu. 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).

Fig. 5.
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 RhBiClusterFlu and RhBClusterFlu in A549R and 4T1 tumor tissues also demonstrated that iCluster showed much better tumor penetration than Cluster because of their pHe-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.

Discussion

Despite the fact that nanoparticle-based therapeutics are amenable to preferential accumulation in solid tumors by taking advantage of the EPR effect, they encounter a series of sequential biological barriers upon i.v. administration, which severely impede the achievement of optimal therapeutic outcomes. To adequately address these barriers and achieve effective therapy, nanoparticles must be rationally designed to overcome substantial interstitial transport hindrance brought about by their inherently large sizes to realize deep and uniform tumor penetration (7). In this study, our iCluster system enables its basic physicochemical properties to adaptively change in response to the endogenous stimuli of the tumor microenvironment to accomplish improved therapeutic efficacy by successively increasing blood circulation and tumor vascular extravasation, improving tumor penetration, facilitating cell internalization, and accelerating intracellular drug release.

Our results demonstrate that the decisive step for the effectiveness of iCluster is its robust tumor penetration achieved through pHe-triggered shattering of small PAMAM dendrimers at tumor sites (Figs. 3 and 4). It has been validated that the penetration of nanoparticles in tumor space relies heavily on particle size, with the consensus that smaller particles have improved tissue penetration (26, 33, 44). Such progress has recently inspired interest in developing size-shrinkable anticancer drug delivery systems (15, 34, 45, 46). Compared with previous studies, our strategy has several unique features. First, previous delivery systems simply focused on size-shrinkage medicated tumor penetration, whereas our system is devised to systematically overcome a series of barriers including tumor penetration. Achieving this goal is vitally important because these barriers are interconnected, and simply overcoming one individual barrier is not adequate to produce proper therapeutic outcomes (42, 47). Second, the stimuli that were used to trigger size shrinkage previously were either by enzyme or UV light, whose applicability, to a certain extent, would be restricted to only a subset of cancer types owing to either the heterogeneous expression levels of target enzymes in a specific cancer type or the superficial penetration depth of UV light (14). In contrast, we used acidic extracellular pH, a more general hallmark of the microenvironment of most solid tumors (36, 48), as the stimulus to activate the release of small particles at tumor sites. This endows our strategy with much broader specificity and applicability, which has been partially justified by the superior antitumor activities in multiple tumor models. Third, it should be noted that it is the postshrinkage size rather than the property of size shrinkage that is the pivotal determinant of therapeutic efficacy. This is particularly important for treating poorly permeable solid tumors, because only the sub–30-nm micelles could penetrate the poorly permeable pancreatic tumors to achieve an antitumor effect (27). For previous systems, their final sizes after shrinkage were still as large as 40–70 nm (34, 45, 46), which is likely to offset the usefulness of size shrinkage for treating poorly permeable tumors. In our system, the released small PAMAM dendrimer prodrugs have a size of around 5 nm, which is highly potent in penetrating the intractable tumors to reach cancer cells that are far away from the blood vessels. In essence, our design strategy may open up a new avenue for the creation of the next generation of nanotherapeutics, representing a paradigm shift in nanoparticle-based drug delivery.

Materials and Methods

Detailed materials and methods are provided in SI Appendix, including the synthesis of polymers, preparation and characterization of the clustered nanoparticles, stimuli-responsive PAMAM and platinum drug release from nanoparticles, in vitro penetration, cell internalization, apoptosis in multicellular spheroids, in vivo real-time tumor penetration, immunofluorescent staining, antitumor activities in varying tumor models, histological studies, and statistics. The animal study procedures were approved by the Animal Care and Use Committee of University of Science and Technology of China.

Acknowledgments

The authors thank Dr. Xiao-Dong Ye and Miss Jin-Xian Yang for their assistance with static light scattering measurement. This work was supported by the National Basic Research Program of China (973 Programs, 2012CB932500, 2015CB932100, and 2013CB933900) and the National Natural Science Foundation of China (51125012, 51390482, and 51503195).

Footnotes

  • ↵1H.-J.L., J.-Z.D., and X.-J.D. contributed equally to this work.

  • ↵2To whom correspondence may be addressed. Email: jwang699{at}ustc.edu.cn or snie{at}emory.edu.

  • Author contributions: H.-J.L., J.-Z.D., X.-J.D., S.N., and J.W. designed research; H.-J.L., J.-Z.D., X.-J.D., C.-F.X., C.-Y.S., H.-X.W., Z.-T.C., X.-Z.Y., and Y.-H.Z. performed research; C.-Y.S. contributed new reagents/analytic tools; H.-J.L., J.-Z.D., X.-J.D., S.N., and J.W. analyzed data; and H.-J.L., J.-Z.D., S.N., and J.W. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1522080113/-/DCSupplemental.

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Smart nanomedicine improves tumor penetration
Hong-Jun Li, Jin-Zhi Du, Xiao-Jiao Du, Cong-Fei Xu, Chun-Yang Sun, Hong-Xia Wang, Zhi-Ting Cao, Xian-Zhu Yang, Yan-Hua Zhu, Shuming Nie, Jun Wang
Proceedings of the National Academy of Sciences Mar 2016, 201522080; DOI: 10.1073/pnas.1522080113
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