Smart Superstructures with Ultrahigh pHSensitivity for Targeting Acidic Tumor Microenvironment: Instantaneous Size Switching and Improved Tumor Penetration Hong-Jun Li,†,∥ Jin-Zhi Du,*,‡,∥ Jing Liu,† Xiao-Jiao Du,† Song Shen,† Yan-Hua Zhu,† Xiaoyan Wang,§ Xiaodong Ye,§ Shuming Nie,*,‡ and Jun Wang*,†,§,⊥ †
CAS Center for Excellence in Nanoscience, School of Life Sciences and Medical Center, and ⊥Innovation Center for Cell Signaling Network, University of Science and Technology of China, Hefei, Anhui 230027, China ‡ Department of Biomedical Engineering, Emory University and Georgia Institute of Technology, Atlanta, Georgia 30322, United States § Hefei National Laboratory for Physical Sciences at the Microscale, Hefei, Anhui 230027, China S Supporting Information *
ABSTRACT: The currently low delivery efficiency and limited tumor penetration of nanoparticles remain two major challenges of cancer nanomedicine. Here, we report a class of pH-responsive nanoparticle superstructures with ultrasensitive size switching in the acidic tumor microenvironment for improved tumor penetration and effective in vivo drug delivery. The superstructures were constructed from amphiphilic polymer directed assembly of platinum-prodrug conjugated polyamidoamine (PAMAM) dendrimers, in which the amphiphilic polymer contains ionizable tertiary amine groups for rapid pH-responsiveness. These superstructures had an initial size of ∼80 nm at neutral pH (e.g., in blood circulation), but once deposited in the slightly acidic tumor microenvironment (pH ∼6.5−7.0), they underwent a dramatic and sharp size transition within a very narrow range of acidity (less than 0.1−0.2 pH units) and dissociated instantaneously into the dendrimer building blocks (less than 10 nm in diameter). This rapid size-switching feature not only can facilitate nanoparticle extravasation and accumulation via the enhanced permeability and retention effect but also allows faster nanoparticle diffusion and more efficient tumor penetration. We have further carried out comparative studies of pH-sensitive and insensitive nanostructures with similar size, surface charge, and chemical composition in both multicellular spheroids and poorly permeable BxPC-3 pancreatic tumor models, whose results demonstrate that the pH-triggered size switching is a viable strategy for improving drug penetration and therapeutic efficacy. KEYWORDS: nanoparticle, drug delivery, particle size, pH-responsive, tumor penetration, tumor microenvironment
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nanotherapeutics into the tumor parenchyma. It has been demonstrated that nanotherapeutics, after extravasation from the tumor vessels, are mainly restricted to the adjacent regions of tumor vasculatures due to the high interstitial fluid pressure (IFP) and dense extracellular matrix, thus greatly compromising their therapeutic effects. 5−7 The enhanced tumor accumulation and poor tumor penetration are the two
he design and development of sophisticated nanoparticles for the targeted delivery of therapeutic drugs to solid tumors holds great promise for improving treatment efficacy and minimizing systemic toxicity. However, the currently low delivery efficiency1 and limited tumor penetration of nanoparticles are the two major challenges. To achieve effective tumor treatment, therapeutic agents must penetrate tumor tissue efficiently to reach as many cancer cells as possible. Compared with conventional small-molecule drugs, nanoparticle-based therapeutics are larger in size, which allows them to preferentially accumulate in solid tumors through the enhanced permeability and retention (EPR) effect.2−4 However, the large size also hinders deep penetration of the © 2016 American Chemical Society
Received: April 6, 2016 Accepted: May 31, 2016 Published: May 31, 2016 6753
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Scheme 1. (a) Structure of PEG-b-PAEMA-PAMAM/Pt. (b) Schematic illustration showing the self-assembly of PEG-bPAEMA-PAMAM/Pt into the pH-sensitive cluster nanobombs (SCNs/Pt) at neutral pH and the disintegration of SCNs/Pt into small particles at tumor acidic pH. (c) Schematic illustration of SCNs/Pt as a robust nanoplatform to overcome biological barriers to in vivo drug delivery in poorly permeably pancreatic tumor models. (I) Large SCNs/Pt superstructures are favorable for prolonged blood circulation. (II) Prolonged circulation increases the propensity of large nanoparticles to extravasate from leaky tumor vasculature to accumulate in the vicinity of blood vessels. (III) Once deposited in the acidic tumor microenvironment (pH ∼6.5−7.0), SCNs/Pt instantaneously switch to small particles with a size less than 10 nm for deep tumor penetration.
improved cancer treatment efficacy. However, the means that were employed to trigger size alterations can be practically problematic. For example, the major concerns for UV light are its poor tissue penetration depth and harmfulness to normal tissues.29 For the existing enzyme- or pH-triggered sizeswitchable systems, their size alterations involve the cleavage of chemical bonds and usually take hours to complete the transition,26,30 which may reduce the penetrating capability of small particles and compromise treatment efficacy. To fully exploit the benefits of size-switchable delivery for cancer therapy, the delivery systems that could switch rapidly to small nanoparticles at tumor sites might be more advantageous for promoting particle penetration into the tumor interstitium and boosting cell internalization. Recently, Gao and co-workers established a series of ultra-pH-sensitive nanoprobes based on tertiary-amine-containing polymers.31,32 Their studies together with another report have confirmed that such series of polymers undergo sharp and superfast pH-responsiveness (on the scale of milliseconds),31,33 which allows for rapid and effective tumor delineation.34 Herein, we developed ultra-pHsensitive cluster nanobombs (SCNs) for improved drug delivery. The platinum (Pt)-prodrug conjugated SCNs (SCNs/Pt) are self-assembled from poly(ethylene glycol)-bpoly(2-azepane ethyl methacrylate)-modified PAMAM dendrimers (PEG-b-PAEMA-PAMAM/Pt) (Scheme 1). The PAEMA block of the polymer is pH-sensitive. At neutral pH, PAEMA is hydrophobic and directs the assembly of PEG-bPAEMA-PAMAM/Pt into SCNs/Pt (∼80 nm in diameter) for prolonged blood circulation, while at acidic tumor pH,35 the PAEMA is rapidly protonated and becomes hydrophilic, leading to instantaneous disintegration of SCNs/Pt into small nanoparticles for effective tumor penetration. The Pt-prodrug is covalently attached to the polymer and can be specifically reduced by intracellular redox to release active cisplatin.36,37
intrinsically conflicting attributes of using nanoparticles for cancer treatment. To address the predicament to improve therapeutic efficacy, various innovative strategies have been reported.8−12 Since tumor penetration is hindered by the elevated IFP and dense matrix, normalization of tumor vessels to restore the pressure gradients13 and degradation of the collagen matrix to reduce transport hindrance14 have been promising choices. However, these approaches have limitations. For example, the normalization of tumor vessels needs delicate balance in order not to dramatically compromise the EPR effect, while uncontrollable degradation of the tumor matrix may lead to unexpected risks such as promoting tumor progression or even metastasis.5 Compared with remodeling of the tumor microenvironment, rationally regulating the physiochemical properties of nanoparticles such as particle size and shape provides an alternative solution to addressing this problem.15−18 It has been demonstrated that large stealth nanoparticles (∼60−100 nm) are usually suitable for operating the EPR effect and hold high propensity of extravasation across tumor vasculature to accumulate in the vicinity of blood vessels, but have poor penetration and distribution in the dense tumor matrix.19,20 On the contrary, smaller nanoparticles typically show greater tumor penetration because of their reduced diffusional hindrance, but generally suffer from inferior circulating half-life time and tumor accumulation.20−23 Such a dilemma has prompted the development of a size-changeable delivery system that could maintain large initial size for prolonged blood circulation and selective extravasation, while transforming to small particles at tumor sites for deep penetration and effective tumor distribution. Typical systems have been reported to be capable of shrinking their sizes by responding to either exogenous (e.g., UV light)24,25 or endogenous stimuli (e.g., enzyme and tumor pH).26−28 These studies are conceptually advanced and show 6754
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size change was measured by DLS (Figure 1b). As shown, the size showed a sharp transition from ∼80 nm at pH 6.8 to ∼10 nm at pH 6.7, indicating the extremely sharp transition kinetics of SCNs/Pt. In addition, the size transition at acidic pH was superfast and can be completed within seconds (Figure S3). In contrast, ICNs/Pt maintained their initial size regardless of pH change (Figure S4). The tumor-pH-triggered size change has also been confirmed by TEM and DLS, in which small particles less than 10 nm were observed at pH 6.7 (Figure 1c). The gel filtration chromatography also showed a distinct peak shift from 6.6 min at pH 7.4 to 11.2 min at pH 6.7, indicating the dissolution of the superstructure into small particles (Figure 1d). Both ICNs/Pt and SCNs/Pt were quite stable in phosphate-buffered saline (PBS) and serum (Figure S5). Penetration and Tumor Cell Killing in Multicellular Spheroids (MCSs). Recent studies have shown that cancer nanomedicines with smaller sizes exhibit enhanced in vivo performance through greater tumor penetration.16,19,39,40 The supersensitivity of SCNs/Pt in response to tumor extracellular pH to release smaller particles motivated us to investigate its potential for drug delivery. In our study, BxPC-3 human pancreatic cancer cell derived MCSs were incubated and employed as an in vitro model to assess penetration capability and cell proliferation inhibition of our system. As demonstrated, MCSs are versatile three-dimensional models for studying tumor biology as well as screening cancer therapeutics due to their similarity in morphology and biological microenvironment to solid tumors.41 The penetration activities of Cy5-labeled SCNs and ICNs (denoted as SCNs/Cy5 and ICNs/Cy5) were monitored by confocal laser scanning microscopy (CLSM) (Figure 2). MCSs were incubated with SCNs/Cy5 or ICNs/Cy5 for 4 h at pH 6.7 and 7.4, respectively, and subjected to CLSM Z-stack scanning. The surface of the MCSs was defined as 0 μm. For ICNs/Cy5 treatment, we found that the red fluorescence of Cy5 was mostly located on the periphery of the MCSs at both pH values. At the scanning depth of 25 μm, the fluorescence intensity in the interior area of the MCSs has dropped considerably. SCNs/Cy5 incubated at neutral pH showed comparable penetration behavior to ICNs/Cy5. However, the penetration capability of SCNs/Cy5 at tumor pH (pH 6.7) improved significantly. The red fluorescence inside the MCSs was clearly observed even at the scanning depth of 85 μm, demonstrating the deep penetration and uniform distribution of SCNs/Cy5 at acidic pH because of their rapid disintegration into small particles. In contrast, both SCNs/Cy5 and ICNs/ Cy5 showed limited penetration at pH 7.4, presumably because of their large sizes. The robust penetration of SCNs/Cy5 at acidic pH endowed them with a higher opportunity to access more cancer cells to boost cellular uptake and cell killing efficacy. The cellular uptake was studied by flow cytometry (FACS) through analyzing Cy5-positive cells after incubating the MCSs with SCNs/Cy5 or ICNs/Cy5 at pH 7.4 and 6.7 for 4 and 12 h, respectively (Figure 3a and Figure S6). At pH 7.4, both SCNs/ Cy5 and ICNs/Cy5 treatments exhibited low levels of cell internalization (∼20% positive cells for 4 h and ∼30% for 12 h). In contrast, at pH 6.7, the portion of positive cells treated with SCNs/Cy5 was significantly higher than that incubated with ICNs/Cy5 after 4 h (2.9-fold, p < 0.001) and 12 h (2.6fold, p < 0.001), respectively (Figure 3a). The mean fluorescence intensity inside the positive cells also showed that SCNs/Cy5 could dramatically enhance cellular uptake at
The release profile of cisplatin from PAMAM has been studied in our previous work.28 The penetration capability and therapeutic efficacy of the system were examined in threedimensional multicellular spheroids and a poorly permeable BxPC-3 pancreatic tumor model.
RESULTS AND DISCUSSION Preparation and Tumor-pH-Triggered Size Transition of SCNs/Pt. SCNs/Pt were self-assembled from PEG-bPAEMA-PAMAM/Pt. PAMAM/Pt was prepared according to a previous description.28 The pH-sensitive PEG-b-PAEMA amphiphilic block copolymer was obtained by reversible addition−fragmentation chain transfer (RAFT) polymerization (Figure S1a and b). PEG-b-PAEMA was reduced by 2aminoethanol to produce sulfhydryl-terminated PEG-bPAEMA, which was modified with N-(ε-maleimidocaproyloxy)succinimide ester. The resulting PEG-b-PAEMA-NHS was further coupled with PAMAM/Pt to obtain PEG-b-PAEMAPAMAM/Pt (Scheme S1), which was confirmed by left shift of the elution time in gel filtration chromatography (Figure S1c).38 Through determination of Pt content in PAMAM/Pt and PEG-b-PAEMA-PAMAM/Pt, we learned that one PAMAM carries two PEG-b-PAEMA chains on average. For comparison, we prepared pH-insensitive amphiphilic block polymers poly(ethylene glycol)-b-poly(2-cyclohexylethyl methacrylate) (PEG-b-PCHMA) and PEG-b-PCHMA-PAMAM/Pt following similar procedures (Scheme S2 and Figure S2). SCNs/Pt were prepared by self-assembly of PEG-b-PAEMAPAMAM/Pt through dialysis. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements showed that the diameter of SCNs/Pt was around 80 nm (Figure 1a). The pH-insensitive cluster nanostructures (ICNs/
Figure 1. Tumor-pH-triggered size transition of SCNs/Pt. (a) DLS (left panel) and TEM (right panel) measurements of SCNs/Pt in phosphate buffer (PB) at pH 7.4. (b) pH-Dependent size change of SCNs/Pt analyzed by DLS. (c) DLS (left panel) and TEM (right panel) measurements of SCNs/Pt at pH 6.7. (d) Gel filtration chromatography analysis of SCNs/Pt treated at pH 6.7 and 7.4. The gel filtration chromatography system was equipped with a size exclusion column and evaporative light-scattering detector.
Pt) were obtained from self-assembly of PEG-b-PCHMAPAMAM/Pt under identical conditions. ICNs/Pt showed similar size, zeta potential, and Pt drug content to SCNs/Pt (Table S1). A key feature of SCNs/Pt is their ultrasensitive responsiveness to tumor acidic pH. To test their pH-sensitivity, SCNs/Pt were incubated in a series of phosphate buffer (PB) solutions with pH values in the range of 6.3 to 7.5, and their 6755
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Figure 2. CLSM images showing in vitro penetration of fluorescence-labeled SCNs/Cy5 and ICNs/Cy5 in BxPC-3 multicellular spheroids (MCSs). The MCSs were incubated with SCNs/Cy5 or ICNs/Cy5 for 4 h at designated pH and measured by CLSM Z-stack scanning. The surface of the MCSs was defined as 0 μm. Scale bar = 100 μm.
Figure 3. Internalization and cell apoptosis of SCNs and ICNs in BxPC-3 MCSs. (a) FACS analysis of Cy5-positive MCS cells after incubation with SCNs/Cy5 and ICNs/Cy5 at pH 7.4 or 6.7 for 4 or 12 h, respectively. (b) Mean fluorescence intensity (MFI) quantification of MCS cells incubated with SCNs/Cy5 and ICNs/Cy5 at pH 7.4 or 6.7 for 4 or 12 h, respectively. (c) Cell apoptosis was analyzed after 24 h treatments with different formulations at pH 7.4 or 6.7. Data are presented as mean ± SD (n = 3). **p < 0.01, ***p < 0.001.
Pharmacokinetics, Tumor Accumulation, and Tumor Penetration. The pharmacokinetics of SCNs/Cy5, ICNs/ Cy5, and PAMAM/Cy5 were evaluated in tumor-free ICR mice. As shown in Figure 4a, both SCNs/Cy5 and ICNs/Cy5 exhibited prolonged circulation time (T1/2 > 7 h) in the bloodstream, whereas PAMAM/Cy5 was eliminated rapidly. Other pharmacokinetic data also demonstrated that the two superstructures outperform the control group (Table S2). The deposition of the Pt drug in tumor tissue was evaluated by inductively coupled plasma mass spectrometry (ICP-MS) at 4, 12, and 24 h after systemic injection of the formulations. It was clearly seen that SCNs/Pt treatment significantly enhanced Pt deposition in tumor tissue in comparison with ICNs/Pt, free cisplatin, and PAMAM/Pt treatments at all the time points, at 2.5−3.5-fold higher than ICNs/Pt and at least 4.5−7.0-fold
pH 6.7 (Figure 3b and Figure S7). Facilitated cellular uptake was expected to lead to enhanced cell apoptosis. To test this, the MCSs were incubated with SCNs/Pt or ICNs/Pt for 24 h, and cell apoptosis was analyzed by flow cytometry (Figure 3c and Figure S8). At pH 7.4, SCNs/Pt and ICNs/Pt resulted in similar total cell apoptosis (∼20%), while at pH 6.7 SCNs/Pt treatment led to significantly higher total cell apoptosis than ICNs/Pt treatment (45.2% for SCNs/Pt versus 18.9% for ICNs/Pt, p < 0.001). These in vitro results suggest that the ultrasensitive disintegration of SCNs into small nanoparticles enables penetration enhancement, facilitates cell internalization, and promotes cell death. These observations provide evidence that the instantaneous size switching of SCNs at tumor pH is probably advantageous for in vivo tumor penetration. 6756
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Figure 4. In vivo blood circulation, tumor accumulation, and penetration of SCNs and ICNs. (a) Pharmacokinetics studies of the various formulations. Data are presented as mean ± SD (n = 3). (b) Quantitative analysis of Pt content in tumor tissue. (c) Measurement of the content of Pt in tumor tissue cells. The cells were harvested by digesting tumor mass with Collagen I. For b and c, data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01. (d) Immunofluorescence staining images of the intratumor distribution of SCNs/Cy5 and ICNs/Cy5 4 h after a single injection. Nanoparticles were tagged with Cy5 (red), blood vessels were stained with platelet endothelial cell adhesion molecule1 (PECAM-1) and FITC-labeled secondary antibody (green), and cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar = 200 μm.
Figure 5. In vivo real-time observation of the microdistribution of (a) SCNs/Cy5 and (b) ICNs/Cy5 in BxPC-3 xenografts after intravenous administration. Scale bar = 100 μm. Time-penetration average fluorescence intensity evolution of the intravascular compartments (c) and extravascular compartments (d). Three intravascular compartments (1, 2, and 3) and extravascular compartments (i, ii, and iii) were marked as shown. The intensity profiles were obtained by the normalization of the fluorescence intensity of selected areas to the intravascular intensity at 10 min.
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ACS Nano higher than the other two treatments (Figure 4b, p < 0.05). The tumor tissue was further digested into individual cells, and the content of Pt in these cells was measured. SCNs/Pt still showed 2−3 times higher Pt deposition than ICNs/Pt (Figure 4c, p < 0.05 for 4 h, p < 0.01 for 12 and 24 h, respectively). The biodistributions of these formulations in other major organs were also measured (Figure S9). Since SCNs/Pt and ICNs/Pt have comparable surface charge, size, and pharmacokinetics, they are expected to show a similar EPR effect and tumor vessel extravasation. The significantly improved tumor deposition of SCNs/Pt is likely attributed to their better intratumor diffusional capability. To test this, we studied the intratumoral distribution of SCNs/Cy5 and ICNs/Cy5 with immunofluorescence staining (Figure 4d). For the SCNs/Cy5 group, the red fluorescence perfused uniformly throughout the tumor interstitium until several hundred micrometers away from tumor vessels, indicating the robust penetrating capability of SCNs/Pt. However, for the insensitive ICNs/Cy5 treatment, the red signals were more colocalized with the blood vessels, suggesting that ICNs/Cy5 were incompetent to penetrate effectively in the tumor space. These results visually demonstrated that the triggered release of small nanoparticles in response to tumor acidity is a feasible solution to improve particle transport within the tumor matrix. Next, intravital CLSM was employed to monitor the realtime tumor penetration behaviors of SCNs/Cy5 and ICNs/ Cy5 in vivo. For SCNs/Cy5 injection (Figure 5a), even at 10 min postinjection, considerable red fluorescence could already be observed in the surroundings of the blood vessels. With a time extension to 30 min postinjection, extensive fluorescence was observed in the tumor interstitial space. In contrast, the fluorescent ICNs/Cy5 were still confined to the lumen of the blood vessels and failed to penetrate deeply into tumor parenchyma until 150 min postinjection (Figure 5b). To quantify the spatiotemporal evolution of the fluorescence, we chose three intravascular and three extravascular areas for analysis. The fluorescence intensities were separately averaged and normalized to the initial intravascular intensities at 10 min. In the intravascular compartments (Figure 5c), both fluorescence intensities of SCNs/Cy5 and ICNs/Cy5 decreased gradually, but SCNs/Cy5 decreased more rapidly than ICNs/ Cy5, while in the extravascular compartments, the fluorescence intensity of SCNs/Cy5 increased quickly and reached a plateau of ∼45% of the initial intensity by 70 min postinjection. In contrast, the extravascular fluorescent intensity of ICNs/Cy5 remained less than 10% of the initial intensity by 150 min (Figure 5d). Altogether, these results demonstrate that the ultrasensitive size shrinkage of the SCNs superstructure greatly improved the tumor penetration and distribution in the tumor interstitial space. Antitumor Activity Study in BxPC-3 Pancreatic Xenograft. Next, the antitumor activity of SCNs/Pt was evaluated in nude mice bearing a poorly permeable BxPC-3 tumor xenograft.16 As depicted in Figure 6a, PAMAM/Pt and free cisplatin treatments only moderately inhibited tumor growth, with approximately 22% and 24% inhibition rate versus PBS treatment. ICNs/Pt with a Pt injection dose of 2 mg/kg caused 41% tumor inhibition and was slightly better than PAMAM/Pt and cisplatin. By contrast, SCNs/Pt presented 62% tumor suppression at the injection dose of 1 mg/kg (p < 0.05 versus ICNs/Pt treatment) and 82% tumor suppression at 2 mg/kg injection dose (p < 0.001 versus ICNs/Pt). We also measured the weight of each tumor mass at the end of the
Figure 6. In vivo tumor growth inhibion study of SCNs/Pt and ICNs/Pt in BxPC-3 tumor xenograft. (a) Tumor growth inhibition with varying treatments. Cisplatin, PAMAM/Pt, and ICNs/Pt were intravenously injected at a Pt dose of 2 mg/kg, while SCNs/Pt was injected at doses of 1 and 2 mg/kg. (b) Weight of BxPC-3 tumors at the end of treatment. (c) Quantification of proliferative cells in tumor tissue after the treatment, which was obtained through proliferating cell nuclear antigen (PCNA) staining. (d) Quantification of apoptotic cells in tumor tissue after the treatment, which was obtained through terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick end (TUNEL) staining.
treatment, which also confirmed that SCNs/Pt was the most effective in suppressing tumor growth (Figure 6b and Figure S10). Histological analyses (Figure S11) also indicated that SCNs/Pt was able to markedly reduce cell proliferation (Figure 6c, p < 0.05 versus ICNs/Pt treatment) while increasing cell apoptosis (Figure 6d, p < 0.01 versus ICNs/Pt treatment). Hematoxylin and eosin (H&E) staining of major organs after the treatment indicated that no obvious toxicities were observed (Figure S12).
CONCLUSIONS We have developed ultra-pH-sensitive size-switchable SCNs/Pt for improved tumor penetration and cancer treatment. A unique feature of SCNs/Pt is that they are ∼80 nm at neutral pH, but are able to switch instantaneously to less than 10 nm in response to tumor acidity. This characteristic not only facilitates tumor extravasation and accumulation by taking advantage of the long-circulating benefit of large nanoparticles but also enables deep tumor penetration by making use of the penetrating superiority of small nanoparticles, which collectively contribute to the ultimate improved antitumor effect in a poorly permeable BxPC-3 pancreatic tumor model. The size transition can be achieved on the scale of seconds in the acidic tumor microenvironment, which allows instantaneous release of small particles for prompt tumor penetration and cellular uptake. Such a rapid size alteration at tumor pH has not been achieved previously26−28 and is believed to probably be able to maximize the merits of the size-switchable delivery strategy. In addition, the superstructures are prepared through the selfassembly of a single component, which possibly makes the 6758
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PBS, and subjected to FACS analyses on a BD FACS Calibur flow cytometer (BD Bioscience, Bedford, MA, USA). Apoptosis Analysis of MCS Cells. MCSs were incubated with SCNs/Pt and ICNs/Pt at the Pt drug concentration of 50 μM in PBS at pH 6.7 or 7.4 for 24 h, then washed by PBS, carefully collected by centrifugation, and further incubated in RPMI 1640 medium. After 48 h, the spheroids were washed, trypsinized into individual cells, and measured by FACS after staining with annexin V-FITC and propidium iodide (PI) using the AannexinV-FITC apoptosis detection kit I (BD Bioscience). Pharmacokinetics Study. Female ICR mice were randomly divided into three groups (n = 3 per group). PAMAM/Cy5, SCNs/ Cy5, and ICNs/Cy5 were intravenously injected. At predetermined time points (0.083, 0.5, 1, 2, 4, 8, 16, and 24 h), blood samples were collected from the retro-orbital plexus of the eye, placed in heparinized tubes, and centrifuged to obtain plasma. An equal volume of plasma was withdraw and mixed with the same volume of acetonitrile to precipitate protein. The supernatant was collected after centrifugation. Cy5-labeled nanoparticles in the plasma were quantified by HPLC using a Waters 2475 fluorescence detector. The fluorescence detector was set at 630 nm for excitation and 670 nm for emission. HPLC grade acetonitrile/water (50/50, v/v) was used as the mobile phase at 30 °C with a flow rate of 1.0 mL/min. Breeze software was used for data analysis. Drug Deposition in Tumor Tissue and Major Organs. Free cisplatin, ICNs/Pt, SCNs/Pt, and PAMAM/Pt were administered intravenously at an equivalent dose of 40 μg of platinum per mouse bearing a BxPC-3 xenograft tumor (n = 3 per group). The mice were sacrificed at 4, 12, or 24 h postinjection. Then the tumors were excised, washed with cold saline, dried on filter paper, weighed, and cut into two parts. One part was cut into pieces and nitrated with nitric acid; the other part was digested with 0.1% collagenase I (Worthington Biochemical Corporation, Lakewood, CA, USA) into single cells. The cells were counted and nitrated, and the content of platinum was determined by ICP-MS. For the biodistribution study, the mice were sacrificed at 12 h postinjection, and the major organs including heart, liver, spleen, lung, and kidney were excised, washed with cold saline, dried on filter paper, weighed, and cut into small pieces. The amounts of platinum in these organs were determined by ICP-MS. Tumor Penetration Study in Vivo. For the immunofluorescence assay, mice bearing BxPC-3 xenografts were intravenously injected with SCNs/Cy5 and ICNs/Cy5. Two hours later, the mice were sacrificed and tumors were excised and fixed in 4% paraformaldehyde overnight at 4 °C and then immersed overnight in 30% sucrose solution. The samples were sectioned into slides of 8 μm thickness in a cryostat, incubated with platelet endothelial cell adhesion molecule-1 (PECAM-1) antibody, and then incubated with FITC-conjugated secondary antibody. Samples were observed under a Zeiss LSM710 Meta confocal microscope with a 20× objective. For real-time observation, mice bearing BxPC-3 xenografts were intravenously injected with SCNs/Cy5 and ICNs/Cy5. The mice were then anaesthetized with 2.5% isoflurane (Linuo Pharm., Shandong, China) using a Matrx VMR tabletop with an isoflurane well-fill vaporizer (Midmark Corp., Dayton, OH, USA). An arc-shaped incision was made around the subcutaneous tumor, and the skin flap was elevated without injuring the feeding vessels. The coverslip was attached with just enough pressure to flatten the tumor surface. All in vivo picture acquisitions were performed using a Zeiss LSM710 inverted confocal microscope with a 20× objective. The Cy5 signals were detected using 630/670 nm excitation/emission filters. Tumor Suppression Study. For the tumor suppression study, the mice were randomly divided into six groups (n = 5 per group). PBS, free cisplatin, PAMAM/Pt, and ICNs/Pt were intravenously injected into the mice via tail vein at an equivalent Pt dose of 2.0 mg per kg of mouse body weight, while SCNs/Pt were injected at a Pt dose of 1.0 or 2.0 mg per kg of mouse body weight. The mice received injections on days 16, 19, and 22 after tumor cell implantation. Tumor growth was monitored by measuring the perpendicular diameter of the tumor using calipers. Tumor volume (mm3) was calculated as V = lw2/2, in
quality control of the particles easier than other multicomponent systems.28,42 Lastly, the superstructures can be a versatile platform for the delivery of other anticancer drugs such as doxorubicin, paclitaxel, camptothecin, etc. These drug molecules can be attached to the polymer through delicately designed intracellular responsive bonds such as a hydrazone43 or disulfide bond44 to achieve on-demand intracellular drug release to reduce potential side effects in normal tissues.
EXPERIMENT SECTION Preparation of SCNs/Pt and ICNs/Pt. PEG-b-PAEMA-PAMAM/ Pt (50 mg) was dissolved in 5 mL of DMF and stirred for 10 min at room temperature. Then, 15 mL of ultrapure water was added to the mixture and stirred for 30 min. Subsequently, the nanoparticle solution was dialyzed against water to remove organic solvent. ICNs/Pt were prepared from PEG-b-PCHMA-PAMAM/Pt following the same procedures. Preparation of Fluorescence-Tagged SCNs/Cy5, ICNs/Cy5, and PAMAM/Cy5. Pt-free PEG-b-PAEMA-PAMAM and PEG-bPAEMA-PAMAM were obtained by conjugating PAMAM with PEGb-PAEMA-NHS and PEG-b-PAEMA-NHS, respectively, following the same method described as the preparation of PEG-b-PAEMAPAMAM/Pt. The Pt-free SCNs were obtained with the same method described for the preparation of SCNs/Pt. SCNs/Cy5 were prepared by conjugating the sulfo-Cy5 NHS ester to SCNs with an amidation reaction. Briefly, 10 mg of SCNs was dispersed in 5 mL of pure water, and 1 mg of sulfo-Cy5 NHS ester was added to the solution and reacted overnight at room temperature. Subsequently, the nanoparticle solution was dialyzed against water to remove unconjugated dye. ICNs/Cy5 and PAMAM/Cy5 were prepared similarly. Cell Lines and Animals. The human pancreatic cancer BxPC-3 cell line was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). BxPC-3 cells were maintained in RPMI 1640 medium, containing 10% FBS in a humidified atmosphere containing 5% CO2 at 37 °C. BALB/c nude mice (female, 18−20 g, 5−6 weeks) and ICR mice (female, 25−28 g, 5−6 weeks) were purchased from the Beijing HFK Bioscience Co., Ltd. (Beijing, China). The BxPC-3 xenograft tumor model was generated by injecting 1 × 107 cells in 100 μL of PBS into the right flank of the BALB/c nude mice. The treatment experiments were conducted when the tumor volume was ∼100 mm3. All animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. The procedures were approved by the Animal Care and Use Committee of University of Science and Technology of China. BxPC-3 Multicellular Spheroids. The three-dimensional MCSs were cultured according to the method previously described45 with minor modifications. Briefly, a T75 flask (Corning, USA) was covered by 10 mL of hot agarose solution (1.5 w/v %) and then cooled to room temperature. BxPC-3 cells were seeded at a density of 1 million cells per flask in 15 mL of RPMI 1640 medium containing 1% penicillin−streptomycin and incubated for 4 days to grow into spheroids. Penetration of SCNs/Cy5 and ICNs/Cy5 in MCSs. MCSs were transferred to ultralow-attachment 24-well plates (Corning Incorporated, USA) and incubated in RPMI 1640 medium at pH 6.7 or 7.4. The fluorescence-labeled SCNs/Cy5 and ICNs/Cy5 were added to the medium at a final concentration of 0.2 mg/mL. After 4 h of incubation, the spheroids were harvested and washed with PBS (pH 7.4, 0.01 M) three times and observed with a Zeiss LSM 710 inverted laser confocal scanning microscope imaging system (Germany). Internalization of SCNs/Cy5 and ICNs/Cy5 by MCSs Cells. MCSs were transferred to ultralow-attachment 24-well plates and incubated in RPMI 1640 medium at pH 6.7 or 7.4. The ICNs/Cy5 and SCNs/Cy5 were added to the media at a final concentration of 0.2 mg/mL. After 4 or 12 h of incubation, the spheroids were harvested and washed with PBS three times and trypsinized into individual cells. The cells were washed with PBS three times, resuspended in 200 μL of 6759
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ACS Nano which l and w indicate the length and width of the tumor. The inhibition rate of each treatment was calculated according to the equation [1 − (Vtf − Vti)/(Vpf − Vpi)] × 100%, where Vtf and Vti represent the final and initial tumor volume of the treatment group, while Vpf and Vpi represent the final and initial tumor volume of the PBS group. Immunohistochemical Analysis. Twenty-four hours after the last measurement of tumor volume, the mice were sacrificed. Tumor tissues were excised and fixed in 4% formaldehyde and embedded in paraffin. Then these tissues were cut into tumor slides in 5 μm thickness for immunohistochemical analysis. H&E and PCNA staining were performed according to previous procedures.46 All sections were examined under a Nikon TE2000 microscope (Tokyo Prefecture, Japan). Apoptotic cells were identified using a terminal transferase dUTP nick-end labeling (TUNEL) assay kit (in situ cell death detection kit, Roche, USA) following the manufacturer’s protocol. Deparaffinized sections were first treated with freshly prepared 3% H2O2 for 10 min at room temperature and washed three times with ddH2O for 5 min. Samples were incubated with a 2.0 mg/mL proteinase K working solution (Invitrogen, Carlsbad, CA, USA) for 15 min at 37 °C and washed three times with PBS for 5 min. Then, the tissues were incubated with the TUNEL reaction mixture for 1 h at 37 °C and washed three times with PBS for 5 min. DAPI solution was added to the tissue and incubated for 2 min at room temperature, and after three washings in PBS, the tissues were mounted with antifade mounting solution (Sigma-Aldrich, USA) to reduce fluorescence photobleaching. Then the samples were determined by a Zeiss LSM 710 inverted laser confocal scanning microscope imaging system (Germany). The tumor tissues and major organs of the mice after treatment were collected and stained by hematoxylin and eosin (H&E) to evaluate the toxicities. Statistical Analysis. Statistical analyses were performed using oneway ANOVA in SPSS Statistics; p < 0.05 was considered significant. Data were expressed as mean ± SD.
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ASSOCIATED CONTENT S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b02326. General information and synthetic procedures of the polymers (PDF)
AUTHOR INFORMATION Corresponding Authors
*E-mail:
[email protected] (J. Du). *E-mail:
[email protected] (S. Nie). Tel: 1 404 712 8595. Fax: 1 404 727 3567. *E-mail:
[email protected] (J. Wang). Tel: 86 551 63600335. Fax: 86 551 63600402. Author Contributions ∥
H.-J. Li and J.-Z. Du contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (2013CB933900, 2015CB932100, and 2012CB932500), the National Natural Science Foundation of China (51390482), and the United States National Institutes of Health (Grant R01CA163256). REFERENCES (1) Wilhelm, S.; Tavares, A. J.; Dai, Q.; Ohta, S.; Audet, J.; Dvorak, H. F.; Chan, W. C. Analysis of Nanoparticle Delivery to Tumours. Nat. Rev. Mater. 2016, 110.1038/natrevmats.2016.14. 6760
DOI: 10.1021/acsnano.6b02326 ACS Nano 2016, 10, 6753−6761
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